Lipid nanoparticles for delivery of therapeutic payloads to cells

Abstract

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

Description

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

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63 / 582,453, filed on September 13, 2023, the contents of which are hereby incorporated herein by reference in their entirety. BACKGROUND

[0002] The development of lipid nanoparticles (LNPs) has recently made significant advances towards 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 effectively delivering the payload to diseased tissue.

[0003] Despite advances in the development of LNPs, substantial challenges still remain. Specifically, LNPs that provide a sufficient quantity of a payload to target cells has been challenging. In particular, development of LNPs that can deliver larger payloads and / or multiple payloads, such as those used in gene editing, is an important goal.

[0004] Additionally, in vivo delivery of therapeutic payloads to cells, such as to hematopoietic stem cells (HSCs) and immune cells such as T cells, is currently being investigated but can be challenging. For example, the specific delivery of a genetic medicine payload, such as a gene therapy or gene editor agent, to the rare long-term HSC (LT-HSC) population or to T cells be difficult as any therapeutic agent delivered systemically must evade or overwhelm myriad mechanisms that will prevent its delivery to the relevant microenvironment (for example, liver uptake, phagocytic cell uptake, complement activation, opsonization, phagocytic cell uptake, etc.). Furthermore, after delivery to the target cell, the payload(s) must be able to disassociate from the endosome so it can be expressed in sufficient quantities in the targeted cell.

[0005] Moreover, LNPs often exhibit unfavorable toxicological profiles, that precludes their use as therapeutic delivery vehicles. LNPs may accumulate and deliver payloads tocells other than the intended target, which results in potential toxicity. The unfavorable safety profile often stems from the ionizable lipid used in the LNP.

[0006] Accordingly, there exists a need to develop LNPs with ionizable lipids that have favorable safety profiles and that can be used to promote cell-type specificity, high transfection efficiency, and a high degree of endosomal escape. SUMMARY OF THE INVENTION

[0007] The disclosure provides lipid nanoparticles (LNPs) or conjugates comprising a lipid nanoparticle (LNP) encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or reporter construct, for delivery to cells. In some embodiments, the cells are 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). In some cases, the LNPs and conjugates described herein can deliver the payload to cells (e.g., T cells or HSCs) more effectively than a baseline conjugate or baseline LNP. In some embodiments, the disclosure also provides conjugates comprising a lipid nanoparticle (LNP) encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or reporter construct, for delivery to cells (e.g., T cells or HSCs) that have a targeting moiety on its surface. The conjugates having at least one targeting moiety described herein will also be referred to as targeted LNPs or tLNPs.

[0008] In one aspect, the disclosure provides an ionizable lipid that is a compound of formula (I):formula (I), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3;n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1is C4-10alkyl; R2is C1-4alkyl; R3and R4are independently C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0009] In some embodiments of the formula (I), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y issome embodiments, Y isIn some embodiments, Y is . In some embodiments, Y is . In some embodiments, Y is . In some embodiments, Y is . In some embodiments, Y is In some embodiments, X is O. In some embodiments, X ismethylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they areattached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0010] In some embodiments, the compound of the formula (I) has a structure as set forth in Table 1, as disclosed herein.

[0011] In some embodiments, the ionizable lipid has one of the structures depicted below:Lipid 178Lipid 235

[0012] In another aspect, the disclosure provides a lipid nanoparticle (LNP) for delivery of a therapeutic agent encapsulated in the LNP (to, e.g., 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 ionizable lipid is selected from Lipid 093, Lipid 176, Lipid 178, Lipid 183, and Lipid 235. It will be understood that any reference to an ionizable lipid in an LNP as set forth herein also includes pharmaceutically acceptable salts of the lipids.

[0013] In some embodiments, the LNP is delivered in vivo to a patient in need thereof. In some embodiments, the LNPs are delivered ex vivo to the isolated cells (e.g., T cells or HSCs) of a subject (e.g., human patient) in need thereof. In some such embodiments, HSCs are first collected from the bone marrow of a subject. Following isolation of the desired cell subpopulation, the targeted LNPs of the disclosure can be mixed with the isolated cells, thereby resulting in effective transduction of the payload(e.g., a gene modifying system) into the cells The modified cells can be administered to the patient for engraftment, as described in the art.

[0014] 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. In some embodiments, the cells are T cells. In other 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 or LT- HSCs of the subject). Alternatively, the conjugates can be administered to isolated cells such as T cells or HSCs (e.g., LT-HSCs) ex vivo.

[0015] In some embodiments, the targeting moiety of the conjugate is an antibody, Fab fragment or single chain variable fragment (scFv), a DARPIN, a VHH domain antibody, a FN3 domain, a nanobody, a single domain antibody or a Centyrin. In other embodiments, the targeting moiety is a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin or a N-Acetylgalactosamine (GalNac). In some embodiments, the targeting moiety is a peptide or protein, such as a ligand or part of a ligand, that binds to a receptor (e.g., a receptor on the surface of a T cell or HSC). 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 or LT-HSC, wherein the affinity of the peptide or protein targeting moiety for the receptor or ligand on the surface of the T cell, HSC, HPC or LT-HSC is modulated via phage display.

[0016] 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 093, Lipid 176, Lipid 178, Lipid 183, and Lipid 235.

[0017] 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.

[0018] The disclosure further provides delivery of particular payloads to cells, wherein the payloads are encapsulated within LNPs or conjugates of the disclosure. Insome 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.

[0019] 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 a gene modifying system, as described herein.

[0020] 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 or LT-HSCs 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 delivery 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 LNPcomprising a baseline lipid (such as V003) in an animal model, as described herein. In some embodiments, the LNP is a conjugate.

[0021] In some embodiments, a payload (e.g., a therapeutic payload) delivered to T cells, HSCs, HPCs or LT-HSCs by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as V003). In some embodiments, a payload (e.g., a therapeutic payload) delivered to T cells, HSCs, HPCS or LT-HSCs by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as V003). In some embodiments, the LNP is a conjugate.

[0022] 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.

[0023] 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 about 20% to about 50%, from about 20% to about 40%, or from about 20% to about 30% of HSCs in a subject comprise a genome that has been modified or altered following administration of an LNP described herein, such as a conjugate.

[0024] In some embodiments, the LNPs of the present disclosure, e.g., conjugates, comprise one or more pegylated lipid molecules. In some embodiments, the targeting moiety (e.g., antibody, Fab fragment or scFv) of a conjugate 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. In some embodiments, the PEG spacer between the lipid and the targeting moiety comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the targeting moiety is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the targeting moiety is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the lipid 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.

[0025] In some embodiments, an LNP of the present disclosure, e.g., conjugate, comprises one or more non-pegylated lipids (e.g. non-pegylated phospholipids). Such lipids are often referred to as helper lipids. In some embodiments, the targeting moiety (e.g., antibody, Fab fragment or scFv) is associated with or chemically bonded to at least one of the non-pegylated lipids. In some embodiments, the non-pegylated lipid (helper lipid) is selected from POPC, DOPC, DOPE, and DSPC. In some embodiments, the non- pegylated lipid (helper lipid) is a sphingolipid. In some such embodiments, the non- pegylated lipid is a sphingomyelin. In some embodiments, the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0026] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., conjugate, is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the conjugate is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the conjugate is about 20%, about 21%, about 22%, about 23%, about24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%. As set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs with these percentages of helper lipids provides enhanced transduction and / or expression of the payloads relative to LNPs with smaller or larger quantities of helper lipid. It will be understood that mol% of the helper lipid as used herein refers to the mol% of the total lipid component of the LNP, which does not include the therapeutic agent (i.e., payload) or the targeting moiety

[0027] In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the 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, 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.

[0028] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0029] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 176 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 176 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC orsphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0030] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 178 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 178 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0031] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 183 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 183 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0032] In any of the foregoing embodiments, the 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.

[0033] In some embodiments, the LNP comprises a targeting moiety that targets a receptor on the surface of a T cell selected from CD2, CD3, CD4, CD5, CD6 and CD7.

[0034] In some embodiments, the LNP comprises a targeting moiety that targets a receptor on the surface of a HSC selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, and CD338. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD33. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD34. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD38. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD43. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD46. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD49d. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to C503. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD59. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD71. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD105. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD117. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD123. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD164. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD184. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that binds to CD338.

[0035] In some embodiments, a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs, as described herein, comprises: a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties bind to CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4); and a therapeutic agent encapsulated within the LNP.

[0036] In some embodiments, the conjugates are formed via a Click reaction between a first Click handle on the targeting moiety (e.g., antibody, Fab fragment, or scFv) and a second Click handle on the LNP, thereby generating a Click product. The second Clickhandle can be on one or more of the lipids comprising the LNP. In some embodiments, the second Click handle is covalently bonded to a pegylated lipid comprising the LNP.

[0037] In one embodiment, the Click product can be formed using a copper-catalyzed Click reaction. One such copper-catalyzed Click reaction is a Huisgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. In some embodiments, the first or second Click handle comprises a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne. cyclooctyne, and difluorinated cyclooctyne, In some embodiments, the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes. In another embodiment, the Click product can be formed using copper-free Click chemistry. For example, the Click product can be formed between an azide and dibenzocyclooctene (DBCO). Alternatively, the Click product can be formed using a Staudinger reaction between an azide and a phosphine, hence producing an aza-ylide. In some embodiments, the Click product can be formed from an inverse electron demand Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the first or second Click handle. In some embodiments, the first Click handle comprises a tetrazine (Tz) ring and the second Click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is unsubstituted. In some such embodiments, the tetrazine rung is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl substituted tetrazine.

[0038] In some embodiments, the Click product is formed by conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence. For instance, an antibody, Fab fragment or single chain variable fragment (scFv), can be covalently linked to a first Click handle through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle on the LNP, thereby generating a Click product. In some embodiments, the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide. In some embodiments, the antibody Fab fragment or scFv is directly bonded to the enzyme recognition sequence. In some embodiments, the antibody Fab fragment or scFv is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C-terminus ofthe 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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.

[0040] FIG.1 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.

[0041] FIG.2 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.

[0042] FIG.3 shows an enzymatic approach of site-specifically introducing a Tz ring onto a sugar moiety of an antibody.

[0043] FIG.4 shows an example of a light-induced crosslinking approach of site- specifically introducing a Tz ring onto an antibody.

[0044] FIG.5 shows an example of using a non-natural amino acid to site- specifically introduce a Tz ring onto an antibody or Fab fragment.

[0045] FIG.6 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).

[0046] FIG.7 shows a Fab fragment modified with a free cysteine designed to react with a maleimide group.

[0047] FIG.8A 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.8B shows a schematic of producing a conjugate through the reaction ofa Fab fragment that has been modified using a random approach (NHS modification) and an LNP.

[0048] FIG.9 shows an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).

[0049] FIG.10A 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.10B shows assembly of an LNP encapsulating a therapeutic payload using a post-insertion technique.

[0050] FIG.11 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.

[0051] FIG.12 shows an exemplary schematic of conjugate formation. 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.

[0052] FIG.13 shows an exemplary schematic of conjugate formation. 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.

[0053] FIG.14 shows an exemplary schematic of conjugate formation using DBM as a bridging agent.

[0054] FIG.15 shows an exemplary schematic of conjugate formation. In FIG.7, a first Fab fragment (Fab1) and a second Fab fragment (Fab2) are each contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide between the heavy and light chain of each Fab. After step (i), each Fab comprises two free cysteine residues with one free cysteine residue on the heavy chain and one free cysteine residue on the light chain. In step (ii), the first Fab fragment and the second Fab fragment are contacted with an 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.

[0055] FIG.16 shows some examples of a pegylated lipid bonded to a maleimide moiety.

[0056] FIG.17 shows some examples of non-pegylated lipid bonded to a maleimide moiety.

[0057] FIG.18 shows some examples of ionizable lipid bonded to a maleimide moiety.

[0058] FIG.19 shows some examples of sterols bonded to a maleimide moiety.

[0059] FIGs.20A-20D show the synthesis of Lipid 093.

[0060] FIGs.21A-21F show the synthesis of Lipid 176.

[0061] FIG.22A provides a bar graph showing enhanced transduction of GFP in LSK cells using LNPs containing ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in an in vivo mouse model. FIG.22B provides a bar graph showing enhanced expression levels of GFP in LSK cells using LNPs containing ionizable Lipid 093.

[0062] FIG.23A provides a bar graph showing enhanced transduction of GFP in LT- HSCs using LNPs containing ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model. FIG.23B provides a bar graph showing enhanced expression of GFP in LT-HSCs using LNPs containing ionizable Lipid093 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model.

[0063] FIG.24 provides a bar graph showing the results of screening targeted LNPs (tLNPs) in humanized NBSGW mice with increased quantities of helper lipid. FIGs. 24A and 24B show the results for transduction and expression in HSPCs, respectively. FIGs.24C and 24D show the results for transduction and expression in LT-HSCs, respectively.

[0064] FIG.25 provides a bar graph showing the results of screening targeted LNPs (tLNPs) in nonhuman primates. DETAILED DESCRIPTION I. DEFINITIONS

[0065] 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.

[0066] Expression cassette: The term “expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention.

[0067] 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.

[0068] 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.

[0069] 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, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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 itsencoding 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).

[0074] 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.

[0075] 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, thechoice 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).

[0076] It is understood that aspects and embodiments described herein as “comprising” include “consisting of” and “consisting essentially of” embodiments.

[0077] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0078] In some embodiments, reference to about a measurable parameter value encompasses variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value. II. LIPID NANOPARTICLES

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] In some embodiments, the lipid particle comprises an ionizable lipid, a non- cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non- cationic lipid in an amount from about 5 mol % to about 35 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid (e.g., comprising the therapeutic agent and / or encoding the gene modifying polypeptide, template nucleic acid, or gene modifying system) can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1.

[0084] In some embodiments, the average LNP diameter of the targeted LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm toabout 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.

[0085] An LNP described herein, e.g., a targeted LNP, may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20.

[0086] 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.

[0087] 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 some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0088] 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.

[0089] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.

[0090] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, targeted LNPs of the disclosure are formulated using 2,2‐dilinoleyl‐4‐dimethylaminoethyl‐[1,3]‐dioxolane (DLin‐KC2‐DMA) or dilinoleylmethyl‐4‐dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. AngewChem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.

[0091] Additional specific LNP formulations useful for delivery of nucleic acids are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO. Ionizable Lipids

[0092] 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) 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 agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described hereinencapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule (e.g., an mRNA molecule).

[0093] 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.

[0094] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (I):formula (I), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1is C4-10alkyl; R2is C1-4alkyl; R3and R4are independently C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0095] In some embodiments of the formula (I), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y issome embodiments, Y is. In some embodiments, Y is. , . In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0096] In some embodiments, compounds of formula (I) are compounds of formulaformula (I-A), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle; and Y is a lipophilic tail, branched or unbranched.

[0097] In some embodiments of the formula (I-A), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, Y is. In some embodiments, Y is. In some embodiments, Y is. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0098] In some embodiments, compounds of formula (I) are compounds of formula (I-B):formula (I-B), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0099] In some embodiments of the formula (I-B), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0100] In some embodiments, compounds of formula (I) are compounds of formula (I-C):formula (I-C), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0101] In some embodiments of the formula (I-C), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0102] In some embodiments, compounds of formula (I) are compounds of formula (I-D):formula (I-D), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0103] In some embodiments of the formula (I-D), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0104] In some embodiments, compounds of formula (I) are compounds of formula (I-E):formula (I-E), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0105] In some embodiments of the formula (I-E), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0106] In some embodiments, compounds of formula (I) are compounds of formula (I-F):formula (I-F), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0107] In some embodiments of the formula (I-F), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0108] In some embodiments, compounds of formula (I) are compounds of formula (I-G):formula (I-G), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0109] In some embodiments of the formula (I-G), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0110] In some embodiments, compounds of formula (I) are compounds of formula (I-H):formula (I-H), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0111] In some embodiments of the formula (I-H), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0112] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (II):formula (II), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; and Y is a lipophilic tail, branched or unbranched.

[0113] In some embodiments of the formula (II), Y is. , . n some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.

[0114] In some embodiments, compounds of formula (II) are compounds of formula (II-A):formula (II-A), or a pharmaceutically acceptable salt thereof, wherein: Y is a lipophilic tail, branched or unbranched.

[0115] In some embodiments of the formula (II-A), Y is.

[0116] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (III):formula (III), or a pharmaceutically acceptable salt thereof, wherein: R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle; and Y is a lipophilic tail, branched or unbranched.

[0117] In some embodiments of the formula (III), Y is. , ropyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N towhich they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0118] In some embodiments, compounds of formula (III) are compounds of formulaformula (III-A), or a pharmaceutically acceptable salt thereof, wherein: Y is a lipophilic tail, branched or unbranched.

[0119] In some embodiments of the formula (III-A), Y is.

[0120] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 1. Table 1: Exemplary Ionizable Lipids

[0121] In some embodiments, the ionizable lipid has one of the structures depictedLipid 235

[0122] In some cases, an LNP containing an ionizable lipid of formula (I), formula (II) or Formula (III) or an ionizable lipid shown in Table 1 exhibits higher levels of transduction in HSCs and / or higher expression of a payload protein in HSCs relative to an LNP that contains V003 as the ionizable lipid. In some cases, an LNP containing an ionizable lipid of formula (I), formula (II) or Formula (III) or an ionizable lipid shown in Table 1 exhibits higher levels of transduction in T cells and / or higher expression of a payload protein in T cells relative to an LNP that contains V003 as the ionizable lipid. V003, depicted below is described in U.S. Patent No.10,059,655.Lipid V003

[0123] In some embodiments, Lipid 093 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells or HSCs). In some embodiments, Lipid 176 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells or HSCs). In some embodiments, Lipid 178 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells or HSCs).

[0124] In some embodiments, Lipid 183 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells or HSCs).

[0125] 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%.

[0126] The compounds disclosed herein (e.g., lipids in Table 1), 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

[0127] The LNPs, e.g., targeted LNPs, of the disclosure comprise one or more ionizable lipids. Exemplary helper lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O- dimethyl PE), l8-l-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0128] Other examples of non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate,isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl- aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017 / 099823 or US patent publication US2018 / 0028664, the contents of which is incorporated herein by reference in their entirety. In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety.

[0129] 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.

[0130] In some embodiments, the helper lipid comprises 5-40% (mol) , 8%-30%, 10%- 28%, 20%-36%, 22%-32%, or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0131] 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 transductionand expression of the payloads relative to targeted LNPs with smaller or larger quantities of helper lipid.

[0132] 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.

[0133] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table 1 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table 1 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0134] 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 about20% 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%.

[0135] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 20%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0136] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 176 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 176 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0137] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 178 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 178 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, themol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0138] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 183 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 183 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. Sterols

[0139] 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.

[0140] 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%) ofthe total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0141] 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

[0142] 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.

[0143] 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 Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018 / 0028664, the 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: ,,.

[0144] In some embodiments, lipids conjugated with a molecule other than a PEG can also be 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.

[0145] Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA- lipid conjugates 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.

[0146] In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (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. Additional components

[0147] 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 at least a first nucleic acid and a second nucleic acid, whereinthe second nucleic acid is different than the first nucleic acid.. Without limitations, other additional compounds can be selected from the group 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.

[0148] 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.

[0149] 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 lipidreagent 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.

[0150] 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.

[0151] 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.

[0152] 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 by determining the presence of one or more modified nucleotides or nucleosides, e.g., using LC-MS / MS analysis. III. CONJUGATES

[0153] In one aspect, the disclosure provides an LNP (conjugate) comprising an ionizable lipid as described herein (e.g., in Table 1), wherein the LNP can deliver a payload, such as a therapeutic agent (e.g., a gene modifying system) 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.

[0154] In another aspect, the LNP (conjugate) comprises a targeting moiety that binds to a protein (e.g., a protein receptor) on a cell such as an HSC (e.g., a LT-HSC) HPC or T cell, as described herein. In some embodiments, an LNP (conjugate) comprises both an ionizable lipid and a targeting moiety, as described herein.

[0155] In one aspect, the disclosure provides targeted LNPs (conjugates) comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system), wherein the targeting moiety binds to a protein (e.g., protein receptor) on an HSC (e.g., LT-HSC), HPC or T cell. In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In some instances, the targeting moiety is an antibody, a Fab fragment, a 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 aDNA or RNA apatamer), a carbohydrate, a vitamin or a N-Acetylgalactosamine (GalNac). In some embodiments, the payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system) is capable of modifying one or more genes of the target HSC.

[0156] 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 targets T cell receptors including, but not limited to, CD2, CD3, CD4, CD5, CD6 or CD7. In other embodiments, the targeting moiety component of a conjugate of the disclosure targets hematopoietic stem cells (HSCs). In some embodiments, the targeting moiety component of a conjugate of the disclosure targets HSC receptors including, but not limited to, CD90 or CD117. In other embodiments, the targeting moiety component of a conjugate binds to CD8, TCR alpha, TCR beta, CD10, CD33, CD34, CD68, CD19, CCR7, CD62L, CD25, CXCR3, CCR2, CCR3, CCR4, CCR5, CCR, or CCR7.

[0157] 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.

[0158] In some embodiments, the LNP comprises a targeting moiety that targets a receptor on the surface of the HSC selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, and CD338. In some embodiments, the targeting moiety on the LNP binds to CD33. In some embodiments, the targeting moiety on the LNP binds to CD34. In some embodiments, the targeting moiety on the LNP binds to CD38. In some embodiments, the targeting moiety on the LNP binds to CD43. In some embodiments, the targeting moiety on the LNP binds to CD46. In some embodiments, the targeting moiety on the LNP binds to CD49d. In some embodiments, the targeting moiety on the LNP binds to CD50. In some embodiments, the targeting moiety on the LNP binds to CD59. In some embodiments, thetargeting 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.

[0159] 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.

[0160] In some embodiments, the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some such embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In other such embodiments, the targeting moiety is a scFv. In some embodiments, the targeting moiety is a Fab fragment.

[0161] In one embodiment, the Click product can be formed using a copper-catalyzed Click reaction. One such copper-catalyzed Click reaction is a Huisgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. In some embodiments, the first or second Click handle comprises a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne. cyclooctyne, and difluorinated cyclooctyne. In some embodiments, the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes.

[0162] 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 anddibenzocyclooctene (DBCO). Alternatively, the Click product can be formed using a Staudinger reaction between an azide and a phosphine, hence producing an aza-ylide.

[0163] 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.

[0164] 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)v and A(EAAAK)u ALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0165] 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., sortaserecognition 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%.

[0166] 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.

[0167] 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.

[0168] In some embodiments, the first or second Click handle is a TCO derivative having one of the following structures:

[0169] In some embodiments, the spacer that links the targeting moiety to the Tz ring is an enzyme recognition sequence. Accordingly, the disclosure provides methods of conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence, wherein said conjugating is accomplished via a Click reaction between a Tz ring covalently bound to the targeting moiety and a TCO moiety bound to the LNP. For instance, the disclosure provides methods of conjugating an LNP to an antibody, Fab fragment or single chain variable fragment (scFv), wherein the antibody, Fab fragment or scFv is covalently linked to a first Click handle (Tz ring) through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle (TCO moiety), said method comprising contacting the LNP with an antibody, Fab fragment or scFv such that first Click handle reacts with the second Click handle to form a Click reaction product (dihydropyridazine) that conjugates the antibody, Fab fragment or scFv to the LNP. In some embodiments, the antibody, Fab fragment or scFv is directly bonded to the enzyme recognition sequence. In some embodiments, the antibody Fab fragment or scFv is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C-terminus of the antibody, Fab fragment or scFv include, but are not limited to (GGGGS)v, (G)v, (EAAAK)v, (PAPAP)v, (AP)v and A(EAAAK)u ALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0170] 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 toan 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 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%.

[0171] 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.

[0172] 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 scFvinclude, but are not limited to (GGGGS)v, (G)v, (EAAAK)v, (PAPAP)v, (AP)v and A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0173] 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.1) The glycine and the histidine residues are removed during the transpeptidation reaction.

[0174] 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.

[0175] 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.

[0176] FIG.1 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 handleis 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.

[0177] In one embodiment, the conjugate comprises a protein targeting moiety as set forth above (e.g., antibody, Fab fragment or scFv) and a lipid nanoparticle (LNP), wherein a C-terminus of the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a sortase recognition motif and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some embodiments, the C-terminus of one or more of the heavy or light chains of the antibody, the C-terminus of the heavy or light chain of the Fab fragment, or C-terminus of the scFv is conjugated to the lipid nanoparticle through a linker, 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.

[0178] 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.

[0179] 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)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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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)v and A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] FIG.2 depicts the formation of an LNP conjugated to the C-terminus of a Fab fragment through a LplA ligation followed by a Click reaction. In the schematic, a C- terminus of the Fab fragment is first covalently modified with a LplA acceptor peptide. Specifically, a first Click handle (Click handle 1) comprising a Tz ring is first introduced by chemically reacting a carboxylic acid to the lysine residue of the LplA acceptor peptide. The resultant Fab fragment modified with the first Click handle is then reacted with an LNP that comprises at least one lipid that is covalently bonded to a second Clickhandle comprising a TCO moiety (Click handle 2), thus affording an LNP site- specifically conjugated to the Fab fragment.

[0188] 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.

[0189] 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.

[0190] 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 carboxylic acid compound that includes the first Click handle. The resultant modified targeting moieties (antibodies or Fab fragments or scFvs) are reacted with an LNP that has been modified with a second Click handle, as disclosed herein, thereby generating a Click product. An LNP surface modified with a Fab fragment is depicted below, where R is a lipid group (e.g., C2-C30 alkyl group). Fab-Z-GFEIDKVWYDLDA

[0191] 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.

[0192] 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.

[0193] 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.3 shows one particular embodiment of introducing a Tz Click handle through a sugar moiety on an antibody. In step 1, an azide moiety is site-specifically introduced into a sugar moiety of the targeting antibody catalyzed by the enzymes galactosidase and transferase. The azide functionality then reacts with a dibenzocyclooctene (DBCO)-MeTz via a Click reaction. The Click reaction is used 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.

[0194] 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.4 shows one particular embodiment using oYo-Link Tz. In FIG.4, 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.

[0195] 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.5 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.

[0196] 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.6 shows one particular embodiment of introducing a first Click handle (e.g., Tz) onto a Fab fragment. In FIG.6, a hinge with afree 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.7). 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,

[0197] 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).

[0198] In some embodiments, the Click product can be formed using oxime and hydrazone ligations. In some embodiments, a ketone or aldehyde can react with α effect amine, such as hydroxylamine, hydrazine and hydrazide (see, e.g., Agten et al., ChemBioChem 2013, 14 (18), 2431-2434 and Dirksen et al., J. Am. Chem. Soc.2006, 128 (49), 15602-15603).

[0199] Exemplary lipids and cholesterol molecules bonded to Click handles that can be used to make targeted LNPs are depicted below.

[0200] Conjugates prepared by site-specific methods (e.g., FIGs.1-6) 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 150targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 targeting moieties (e.g., antibodies, Fab fragments or scFvs) per LNP.

[0201] 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

[0202] 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.

[0203] Proteolytic cleavage of an IgG antibody results in the formation of a Fab fragment known as a F(ab’)2fragment. The F(ab’)2fragment does not include the CH2domain or the CH3 domain. However, the hinge region of the antibody is retained in a F(ab’)2fragment. The F(ab’)2fragment includes disulfide bonds that covalently link two Fab fragments. Reduction of the disulfide bond in the F(ab’)2generates two F(ab’) fragments. The sulfhydryl (thiol) groups of the F(ab’) could potentially react with a thiol- reactive group on the surface of an LNP, hence generating a conjugate. However, owing to the presence of multiple sulfhydryl groups in the hinge region of the F(ab’) fragment, site-specific conjugation is challenging. Moreover, the reduction of the F(ab’)2 to the F(ab’) fragments could also disrupt the natural interchain disulfide bonds between the CL and CH1 regions of the Fab fragments, hence further compromising site-specific conjugation.

[0204] As set forth herein, Fab fragment 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).

[0205] 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 CLand 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.9.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.10A, 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.10A). 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.10B, 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.

[0211] 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 someembodiments, 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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 ambienttemperature (e.g., about 23 to about 25° C). In some embodiments, the reaction is performed at about 0° C to about 4° C.

[0216] A schematic showing the reduction of an interchain disulfide bond in a Fab fragment is depicted in FIG.11. In some embodiments, the Fab fragment depicted in FIG.11 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.11, 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.

[0217] FIG.12 shows an exemplary schematic of conjugate formation, as described herein. In FIG.12, 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.

[0218] 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.13. In some embodiments, the conjugates formed by methods disclosed herein include at least one, at least two or all three orientations shown in FIG.13. 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-reactivegroups. 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.

[0219] In some embodiments, the thiol-reactive group is maleimide, as shown in FIG. 13. 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.

[0220] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM) as shown in FIG.14. 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.14). As set forth above, the lipid may be part of an LNP or may be post-inserted into an LNP following reaction with the Fab fragment. Both of the free cysteine residues displace the two bromine groups of DBM, hence generating a dithiomalemide. The dithiolmalemide can be converted to the corresponding maleamic acid via hydrolysis. DBM reacts with a free cysteine residue on the heavy chain and a free cysteine residue on the light chain of the Fab fragment to form a bridge between the cysteine residues. Accordingly, the heavy and light chain of the Fab fragment are effectively bridged together following reaction with DBM.

[0221] In all embodiments discussed above, the thiol-reactive group can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, the thiol-reactive group is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid-PEGx-thiol-reactive group, wherein x is 2-120 ethylene glycol units. In such embodiments, at least one free cysteine residue of anFab fragment reacts with the thiol-reactive group bonded to the one or more of the pegylated lipids comprising the LNP. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the thiol-reactive group. In some embodiments, the PEG spacer between the lipid and the thiol-reactive group comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the thiol-reactive group is bonded to at least one of the non-pegylated phospholipids comprising the LNP. In some embodiments, the thiol- reactive group is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, the lipid portion of the pegylated lipid bonded to the thiol-reactive group is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid. FIG.16 shows some examples of a pegylated lipid bonded to a thiol-reactive group, in this case a maleimide moiety.

[0222] In some embodiments, the thiol-reactive group is bonded to at least one of the non-pegylated phospholipids (helper lipids) comprising the LNP. FIG.17 shows some examples of a non-pegylated lipid bonded to a maleimide moiety.

[0223] In some embodiments, the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP. FIG.18 shows some examples of an ionizable lipid bonded to a maleimide moiety.

[0224] In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. FIG.19 shows some examples of sterols bonded to a maleimide moiety.

[0225] 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.14). In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through a maleamic acid moiety (see FIG.14).

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.15. In FIG.15, 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.

[0230] 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 eachcontacted with a reducing reagent in separate reaction vessels). In some embodiments, the first Fab fragment is contacted with the reducing reagent prior to step (ii) (wherein the reduced first Fab fragment is conjugated to the LNP surface). In some embodiments, the second Fab fragment is contacted with the reducing reagent after step (ii) (wherein the reduced second Fab fragment is conjugated to the LNP surface). In some embodiments, the reduced first Fab fragment and the reduced second Fab fragment are contacted with the LNP simultaneously. In some embodiments, the reduced first Fab fragment thereof and the reduced second Fab fragment are contacted with the LNP sequentially (in either order). It can be contemplated that any number of Fab fragments thereof can be implemented in the method or process (e.g., a third, fourth, fifth, etc. Fab fragment). In some embodiments, a total of three different Fab fragments can be conjugated to the surface of the LNP. In some embodiments, a total of four different Fab fragments can be conjugated to the surface of the LNP.

[0231] 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.

[0232] In some embodiments, conjugates prepared by the method or process disclosed herein have a high density of the Fab fragment on the surface of the LNP. For instance, the conjugate can comprise a plurality of Fab fragments conjugated to the LNP surface. In some embodiments, the conjugate can comprise more than 10 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 20 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 30 Fab fragments. In some embodiments, the conjugate can comprise more than 50 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 75 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 100 Fab fragments. In some embodiments, the conjugate can comprise from about 50 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 Fab fragments per LNP. IV. PAYLOADS

[0233] The LNPs, e.g., conjugates (targeted LNPs), of the disclosure can be used to deliver payloads to cells, e.g., T cells, HSCs or HSC progenitors. 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 or nanobody component, of the targeted LNP (conjugate).

[0234] The conjugates disclosed herein can be used to deliver a therapeutic of interest to a cell. For instance 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.

[0235] 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 / DNA molecules. 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.

[0236] 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. 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., an HSC (e.g., LT-HSC) or HSC progenitor in the subject). In some embodiments, the therapeutic agent is a gene therapy agent or gene editing agent. In some embodiments, the therapeutic agent is a gene modifying polypeptide, as described herein. In some embodiments, the therapeutic agent is a gene modifying system, as described herein.

[0237] 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 or HSC progenitor 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 or HSC progenitor 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.

[0238] 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 or HSC progenitor. 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, helicase, 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., targetedLNP, 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, In some embodiments, the LNPs, e.g., targeted LNPs, of the disclosure can be used to deliver gene editing components into T cells, HSCs or HSC progenitors. In some embodiments, the LNPs, e.g., targeted LNPs, can be used to deliver a CRISPR-Cas system into T cells or HSCs. 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 or HSCs. 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 or HSCs. 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 or HSCs (e.g., LT-HSCs). 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 or HSCs (e.g, LT-HSCs). 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.

[0239] 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).

[0240] In some embodiments, multiple components of a gene modifying system may be prepared as a single LNP formulation, e.g., an LNP formulation comprises mRNAencoding for 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.

[0241] 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 embodiment, the target gene is different from the gene containing a disease-causing mutation. For example, the systems or methods provided 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.

[0242] Table 2 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 2. 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

[0243] 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).

[0244] 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.

[0245] 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)β,TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor.V. METHODS OF TREATMENT

[0246] In some embodiments, a conjugate described herein is used to treat a disease, disorder, or condition. In some embodiments, a conjugate 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, a conjugate described herein is used to treat a hematopoietic stem cell (HSC) disease, disorder, or condition, e.g., as listed in Table 3. In some embodiments, a conjugate described herein is used to treat a kidney disease, disorder, or condition, e.g., as listed in Table 4. In some embodiments, a conjugate described herein is used to treat a liver disease, disorder, or condition, e.g., as listed in Table 5. In some embodiments, a conjugate described herein is used to treat a lung disease, disorder, or condition, e.g., as listed in Table 6. In some embodiments, a conjugate described herein is used to treat a skeletal muscle disease, disorder, or condition, e.g., as listed in Table 7. In some embodiments, a conjugate described herein is used to treat a skin disease, disorder, or condition, e.g., as listed in Table 8.Table 3: Exemplary diseases treated with LNPs of the disclosureTable 4: KidneyTable 5: LiverTable 6: LungTable 7: Skeletal muscleTable 8: Skin

[0247] 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.

[0248] In some embodiments, an LNP, e.g., targeted LNP, disclosed herein is delivered ex vivo to isolated cells (e.g., T cells or HSCs) 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.

[0249] 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.

[0250] 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.

[0251] 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).

[0252] 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 3- 8.

[0253] 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 such embodiments, the LNP, e.g., targeted LNP, is administered by parenteral 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).

[0254] In some embodiments, an LNP, e.g., targeted LNP, of the disclosure delivers a therapeutic agent, as described above, to T cells or to HSCs (e.g., LT-HSCs) 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 or to HSCs (e.g., LT-HSCs) of a patient following invivo administration. In some embodiments, an LNP, e.g., targeted LNP, of the disclosure delivers a gene modifying system to T cells or to HSCs (e.g., LT-HSCs) 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 or to HSCs (e.g., LT-HSCs) 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 or HSC (e.g., LT-HSC).

[0255] It has surprisingly been discovered that particular combinations of targeting moieties (e.g., antibodies, Fab fragments or scFvs), and ionizable lipids in particular LNP formulations (e.g., comprising particular amounts of helper lipid)) are capable of delivering therapeutic agents to more HSCs (e.g., LT-HSCs) and / or delivering greater amounts of therapeutic agents to T cells or to HSCs (e.g., LT-HSCs) following in vivo administration to a subject. In some cases, the LNPs described herein result in enhanced levels of modification or alteration of the genomic DNA of the T cells or HSCs, e.g., enhanced levels of gene editing in the T cells or HSCs. In some embodiments, the percentage of T cells or HSCs exhibiting modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 20%. In some cases, the LNPs described herein result in enhanced levels of modification or alteration of the genomic DNA of T cells or HSCs, e.g., enhanced levels of gene editing in the T cells or HSCs. In some embodiments, the percentage of T cells or HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 30%. In some embodiments, the percentage of T cells or HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 40%. In some embodiments, the percentage of T cells or HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 50%. In some embodiments, the percentage of T cells or HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 60%. In some embodiments, the percentage of T cells or HSCs having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is at least 70%. In some embodiments, the percentage of Tcells or HSCs (e.g., LT-HSCs) having modified or altered (e.g., edited) genomic DNA in the subject after administration of the LNPs, e.g., targeted LNPs, is from about 20% to about 50%, from about 20% to about 40%, or from about 20% to about 30%. In some embodiments, 10% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 15% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 20% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 25% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 30% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 35% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to abaseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 40% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 45% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, 50% more T cells or HSCs have modified or altered (e.g., edited) genomic DNA in the subject after administration of an LNP as described herein, e.g., a targeted LNP, comprising a therapeutic agent (e.g., a nucleic acid encoding a gene modifying polypeptide or one or more nucleic acids encoding a gene modifying system) relative to a baseline LNP (e.g., a LNP comprising V003) comprising the same therapeutic agent. In some embodiments, the number of T cells or HSCs comprising modified or altered (e.g., edited) genomic DNA can be determined by DNA sequencing.

[0256] In some embodiments, the LNPs described herein can deliver greater amounts of a payload, e.g., a therapeutic agent or a reporter, to a T cell or an HSC (e.g., an LT-HSC) when administered in vivo to a subject compared to a baseline LNP (e.g., V003). In some embodiments, the targeted LNPs described herein can deliver greater amounts of a payload, e.g., a therapeutic agent or a reporter, to a T cell or an HSC (e.g., an LT-HSC) when administered in vivo to a subject compared to a baseline LNP (e.g., an LNP comprising V003) comprising the same targeting moieties as the targeted LNP. In some embodiments, the LNPs described herein, e.g., targeted LNPs, can deliver greater amounts of a nucleic acid, e.g., a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide or one or more nucleic acids comprising a gene modifying system), to a T cell or an HSC (e.g., an LT-HSC) when administered in vivo to a subject compared to a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., a therapeutic agent such as a nucleic acid (e.g., mRNA) encoding a gene modifyingpolypeptide or one or more nucleic acids encoding a gene modifying system) that is delivered to T cells or HSCs (e.g., LT-HSCs) in vivo using an LNP as described herein (e.g., a targeted LNP) is expressed at higher levels relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 10% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 15% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 20% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 25% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 30% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 35% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 40% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 45% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, a nucleic acid payload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits 50% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some embodiments, a nucleic acidpayload (e.g., mRNA) that is delivered to T cells or HSCs in vivo using an LNP as described herein (e.g., a targeted LNP) exhibits between 10% to 50%, between 20% to 40%, between 10% to 30%, or between 30% to 50% higher expression relative to the same payload delivered using a baseline LNP (e.g., an LNP comprising V003). In some cases, the level of expression of the payload can be determined by measuring protein levels, e.g., by flow cytometry. In some cases, for example, when the payload comprises a gene modifying polypeptide or gene modifying system, the expression level of the payload can be measured by determining the level of modification or alteration of the genomic DNA of the T cells or HSCs, e.g., by DNA sequencing.

[0257] 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 cancer, β-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. EXAMPLES

[0258] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and are not to be construed as limiting the scope or content of the invention in any way. Example 1: Synthesis of ionizable lipids

[0259] A number of ionizable lipids were synthesized for formulation in LNPs that were tested for delivery to HSPCs.

[0260] General: All reactions were run using anhydrous grade solvents under an atmosphere of nitrogen in flasks or vials with magnetic stirring, unless otherwise noted. Anhydrous solvents were from Wuxi EHS and used as received. Column silica gel chromatography using silica gel 100-200 plates, and compounds were visualized using phosphomolybdic acid. Nuclear magnetic resonance (NMR) spectroscopy was performed either using 400 MHz spectrometer; chemical shifts are reported in & parts per million (ppm) referenced to tetramethylsilane at δ = 0.00 ppm for CDCl3 samples, and residual solvent peak (δ = 7.27 ppm) for CDCl3samples. Liquid Chromatography-Mass Spectrum(LC-MS) was performed using a detector (ESI*) using one of the following general methods.

[0261] Method A (20 min run): 60-100AB-20MIN_Phenyl-Hexyl-ELSD-3：LC / MS (The gradient was 60% B in 0-0.4 min, 60-100% B in 0.4-17 min, 100% B in 17-20 min, 60% B in 0.01, the flow rate was 0.8 ml / min. Mobile phase A was 0.05% Trifluoroacetic Acid in water, mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Waters XSelect CSH Phenyl-Hexyl 150*4.6mm, 3.5 um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.

[0262] Method B (22 min run): 50-100AB-22MIN-CSH_Phenyl-Hexyl-ELSD-3M： LC / MS (The gradient was 50% B in 0-0.4 min, 50-100% B in 0.4-17 min, 100% B in 17- 20 min, 50% B in 0.01, the flow rate was 0.8 ml / min. Mobile phase A was 0.05% Trifluoroacetic Acid in water, mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Waters XSelect CSH Phenyl- Hexyl 150*4.6mm, 3.5 um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.

[0263] Method C (10 min run): WUXIAB60-Phenyl-Hexyl-10MIN-1: LCMS (The gradient was 60% B in 0.4 min and 60-100% B at 0.4-8.0 min, hold at 100% B for 1 min, 100-0% B in 0.01 min, and then held at 60% B for 2min. Mobile phase A was 0.05% Trifluoroacetic Acid in water, mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Waters XSelect CSH Phenyl- Hexyl 50*2.1mm, 3.5 um. Detection methods are ELSD. The ESI MS range was 100- 2000.

[0264] Method D (10 min run): WUXIAB40-Phenyl -Hexyl_10min：LC / MS (The gradient was 40% B in 0-0.4 min, 40-100% B in 0.4-8 min, 100% B in 8-9 min, 40% B in 9.01-10 min, the flow rate was 0.8 ml / min. Mobile phase A was 0.05% Trifluoroacetic Acid in water, mobile phase B was 0.05% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Waters XSelect CSH Phenyl-Hexyl 50*2.1mm, 3.5 um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive electrospray ionization. MS range was 100-2000.

[0265] List of abbreviations DCM: dichloromethane DIEA: N,N-diisopropylethylamine. DMAP: 4-(dimethylamino)pyridine EDCI: 3-(ethyliminomethylideneamino)propyl-dimethylazanium;chloride Eq or eqv: equivalents Py: pyridine Lipid 093 General procedure for preparation of compound 13

[0266] Compound 13 was prepared according to the synthetic scheme shown in FIG. 20A. To a mixture of decanedioic acid (40 g, 197.78 mmol, 1.0 eq) in DCM (400 mL) was added DIEA (30.67 g, 237.3 mmol, 41.34 mL, 1.2 eq), DMAP (4.83 g, 39.56 mmol, 0.2 eq) and EDCI (37.91 g, 197.78 mmol, 1.0 eq) at 20°C. Then 2-butyloctan-1-ol (29.48 g, 158.2 mmol, 0.8 eq) was added by one portion at 20°C. The resulting mixture was degassed and purged with N2for 3 times and stirred at 20°C for 12 hr under N2atmosphere. TLC (Petroleum ether: Ethyl acetate = 5:1, the starting material Rf = 0.8, product Rf = 0.5) showed the reaction was completed. The reaction mixture was diluted with H2O (200 mL) and extracted with DCM (200 mL * 2). The combined organic layers were washed with brine (200 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 80:1 to 10:1 remove impurities, 10:1 to 1:1 to get product) to give 10-(2-butyloctoxy)-10-oxo-decanoic acid (compound 13) (50 g, 134.9 mmol, 34.1% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 3.98 (d, J = 5.8 Hz, 2H), 2.33 (td, J = 7.6, 19.2 Hz, 4H), 1.68 - 1.47 (m, 6H), 1.37 - 1.23 (m, 24H), 0.96 - 0.84 (m, 6H). General procedure for preparation of compound 4

[0267] Compound 4 was prepared according to the synthetic scheme shown in FIG.20B. In a 250 mL round-bottom flask equipped with a stir bar, compound 3 (13.0 g, 31.81 mmol, 1.0 eq) was dissolved in isopropanol (65 mL). HCl (1 M, 65 mL) was added to the mixture by dropwise. The mixture was stirred at 15°C for 0.5 hr under N2atmosphere. TLC (Petroleum ether: Ethyl acetate = 1:1, the starting material Rf= 0.7, product Rf= 0.5)showed the reaction was completed. Then saturated NaHCO3 solution (100 mL) was added to the mixture by dropwise (pH = 7) and extracted with DCM (100 mL * 2). The combined organic layers were washed with brine (100 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue (11 g). DCM (60 mL) and 20 g of silica was added to the residue. Solvent was concentrated under reduced pressure again. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 50:1 (800 mL), 30:1 (800 mL, impurity eluted), 10:1, product 5:1 to 1:1 (product eluted)) to give [3-hydroxy-2-(hydroxymethyl)propyl] (9Z,12Z)-octadeca- 9,12-dienoate (compound 4) (9.0 g, 24.42 mmol, 76.8% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 5.42 - 5.30 (m, 4H), 4.26 (d, J = 6.4 Hz, 2H), 3.77 (dq, J = 5.2, 10.9 Hz, 4H), 2.78 (t, J = 6.4 Hz, 2H), 2.34 (t, J = 7.6 Hz, 4H), 2.18 (s, 1H), 2.06 (q, J = 7.0 Hz, 5H), 1.63 (br t, J = 7.2 Hz, 2H), 1.40 - 1.25 (m, 15H), 0.90 (t, J = 6.8 Hz, 3H). General procedure for preparation of compound 14

[0268] Compound 14 was prepared according to the synthetic scheme shown in FIG. 20C. In a 500 mL round-bottom flask equipped with a stir bar, compound 13 (22.0 g, 59.37 mmol, 1 eq) was dissolved in DCM (220 mL). Then DIEA (11.51 g, 89.05 mmol,15.51 mL, 1.5 eq), DMAP (2.18 g, 17.81 mmol, 0.3 eq) and EDCI (17.07 g, 89.05 mmol, 1.5 eq) was added in order at 20°C. Then compound 4 (21.88 g, 59.37 mmol, 1 eq) was added at 20°C. The mixture was degassed and purged with N2for 3 times, and then stirred at 20°C for 16 hr under N2 atmosphere. TLC (Petroleum ether: Ethyl acetate = 3:1, the starting material Rf= 0.65, product Rf= 0.55) showed the reaction was completed. The reaction mixture was diluted with H2O (100 mL) and extracted with DCM (100 mL * 3). The combined organic layers were washed with brine (50 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = from 80:1 to 10:1 to remove impurities; from 10:1 to 5:1 to get product) to give O1-(2-butyloctyl) O10-[2-(hydroxymethyl)-3-[(9Z,12Z)-octadeca-9,12-dienoyl]oxy-propyl] decanedioate (compound 14) (40 g, 55.47 mmol, 93.4% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 5.42 - 5.30 (m, 4H), 4.23 - 4.14 (m, 4H), 3.97 (d, J = 6.0 Hz, 2H), 3.62 (d, J = 5.4 Hz, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.36 - 2.27 (m, 6H), 2.25 - 2.14 (m, 1H), 2.06 (q, J = 6.6 Hz, 4H), 1.62 (br d, J = 4.8 Hz, 8H), 1.49 - 1.36 (m, 3H), 1.34 - 1.25 (m, 35H), 0.94 - 0.86 (m, 9H).General procedure for preparation of Lipid 093

[0269] Lipid 093 was prepared according to the synthetic scheme shown in FIG.20D. In a 500 mL round-bottom flask equipped with a stir bar, compound 14 (10 g, 13.87 mmol, 1.0 eq) was dissolved in DCM (100 mL). Then Py (1.76 g, 22.19 mmol, 1.79 mL, 1.6 eq), DMAP (508.26 mg, 4.16 mmol, 0.3 eq) were added, followed by (4-nitrophenyl) carbonochloridate (4.47 g, 22.19 mmol, 1.6 eq) at 20°C. The mixture was degassed and purged with N2 for 3 times and stirred at 20°C for 1 hr under N2 atmosphere. TLC (Petroleum ether: Ethyl acetate= 3:1, the starting material Rf= 0.5, product Rf= 0.7) showed the reaction was completed.3-(diethylamino)propan-1-ol (5.42 g, 41.30 mmol, 6.16 mL, 3.0 eq) was added to the reaction mixture. The mixture was stirred at 20°C for another 16 hr. TLC (Dichloromethane: Methanol = 10:1, the starting material Cpd.12: Rf= 0.7, product Lipid 093: Rf= 0.5) showed the reaction was completed. The mixture was diluted with H2O (150 mL) and extracted with DCM (100 mL * 2). The organic phase was separated, washed with Na2CO3 solution (100 mL * 5), brine (100 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = from 100:1 to 5:1 to remove impurities, from 5:1 to 3:1 to get product) for two times to give Lipid 093 (15.6 g, 17.76 mmol, 64.5% yield, 100% purity) as colorless oil. LCMS (Method A): Rt 11.80 min, m / z calculated [M+H]+: 878.7, found: 878.7.1H NMR (400 MHz, CDCl3-d) δ 5.42 - 5.30 (m, 4H), 4.23 - 4.12 (m, 8H), 3.97 (d, J = 5.8 Hz, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.62 - 2.49 (m, 6H), 2.49 - 2.38 (m, 1H), 2.35 - 2.27 (m, 6H), 2.06 (q, J = 6.8 Hz, 4H), 1.91 - 1.69 (m, 2H), 1.62 (br s, 8H), 1.38 - 1.24 (m, 40H), 1.04 (br t, J = 6.8 Hz, 6H), 0.94 - 0.86 (m, 9H). Lipid 176 General procedure for preparation of compound 13

[0270] Compound 13 was prepared according to the synthetic scheme shown in FIG. 21A. To a mixture of decanedioic acid (20 g, 98.89 mmol, 1 eq) in DCM (200 mL) was added DIEA (15.34 g, 118.7 mmol, 20.67 mL, 1.2 eq), DMAP (2.42 g, 19.78 mmol, 0.2 eq) and EDCI (18.96 g, 98.89 mmol, 1 eq) in order. Then 2-butyloctan-1-ol (14.74 g, 79.11 mmol, 0.8 eq) was added to the mixture by one portion. The resulting mixture was degassed and purged with N2for 3 times and stirred at 20°C for 12 hr under N2atmosphere. TLC (Petroleum ether: Ethyl acetate = 5:1, product Rf = 0.5) showed the reaction was completed. The residue was extracted with DCM (100 mL * 3) and H2O (100 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = from 80: 1 to 10:1 to remove impurities; from 10:1 to 0:1 and Dichloromethane: Methanol = 50:1 to 5:1 to get product) to give 10-(2-butyloctoxy)-10-oxo-decanoic acid (compound 13) (29 g, 78.26 mmol, 79.1% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 3.98 (d, J = 5.8 Hz, 2H), 2.32 (td, J = 7.4, 14.4 Hz, 4H), 1.68 - 1.63 (m, 2H), 1.40 (br d, J = 6.6 Hz, 2H), 1.35 - 1.24 (m, 24H), 0.95 - 0.83 (m, 6H). General procedure for preparation of compound 38

[0271] Compound 38 was prepared according to the synthetic scheme shown in FIG. 21B. To a mixture of compound 13 (22 g, 59.37 mmol, 1.0 eq) in DCM (250 mL) was added, DIEA (11.51 g, 89.05 mmol, 15.51 mL, 1.5 eq), DMAP (1.45 g, 11.87 mmol, 0.2 eq) and EDCI (17.07 g, 89.05 mmol, 1.5 eq) in order. Then (2,2-dimethyl-1,3-dioxan-5- yl)methanol (10.41 g, 71.24 mmol, 1.2 eq) was added to the mixture. The mixture was degassed and purged with N2 for 3 times and stirred at 20°C for 16 hr under N2 atmosphere. TLC (Petroleum ether: Ethyl acetate = 5:1, the starting material Rf= 0.3, product Rf= 0.5) showed the reaction was completed. The reaction mixture was diluted with H2O 200 mL and extracted with DCM (100 mL * 2). The combined organic layers were washed with brine (100 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 100:1 to 10:1) to give O10-(2-butyloctyl) O1- [(2,2-dimethyl-1,3-dioxan-5-yl)methyl] decanedioate (compound 38) (19 g, 38.10 mmol, 64.2% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 4.17 (d, J = 7.0 Hz, 2H), 4.02 - 3.95 (m, 4H), 3.74 (dd, J = 6.0, 12.0 Hz, 2H), 2.31 (dt, J = 3.6, 7.5 Hz, 4H), 2.01 (br s, 1H), 1.62 (br s, 5H), 1.43 (d, J = 12.9 Hz, 6H), 1.35 - 1.24 (m, 25H), 0.94 - 0.86 (m, 6H). General procedure for preparation of compound 39

[0272] Compound 39 was prepared according to the synthetic scheme shown in FIG. 21C. To a solution of compound 38 (19 g, 38.10 mmol, 1 eq) in IPA (100 mL) was addedHCl (1 M, 100 mL). The mixture was stirred at 15°C for 30 min under N2 atmosphere. TLC (Petroleum ether: Ethyl acetate = 1:1, the starting material Rf= 0.7, product Rf= 0.5) showed the reaction was completed. The reaction mixture was diluted with saturated NaHCO3 solution (100 mL) and extracted with DCM (100 mL * 2). The combined organic layers were washed with brine (100 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = from 50:1 to 10:1 to remove impurities; from 5:1 to 1:1 to get product) to give compound 39 (16 g, 34.88 mmol, 91.6% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 4.27 (d, J = 6.4 Hz, 2H), 3.98 (d, J = 5.8 Hz, 2H), 3.78 (dq, J = 5.2, 10.8 Hz, 4H), 2.32 (td, J = 7.6, 13.6 Hz, 6H), 2.16 - 2.00 (m, 1H), 1.62 (br s, 6H), 1.37 - 1.22 (m, 25H), 0.96 - 0.84 (m, 6H). General procedure for preparation of compound 34

[0273] Compound 34 was prepared according to the synthetic scheme shown in FIG. 21D. To a mixture of octanedioic acid (5.0 g, 28.7 mmol, 1.0 eq) in DCM (50 mL) was added DIEA (5.5 g, 43.06 mmol, 7.5 mL, 1.5 eq), DMAP (701.3 mg, 5.7 mmol, 0.2 eq) and EDCI (5.5 g, 28.7 mmol, 1.0 eq) in order. Then the undecan-3-ol (3.9 g, 22.9 mmol, 0.8 eq) was added to the mixture. The mixture was degassed and purged with N2 for 3 times and stirred at 20°C for 12 hr under N2atmosphere. TLC (Petroleum ether: Ethyl acetate = 2:1, product Rf= 0.3) showed the starting material was consumed completely. The reaction mixture was diluted with H2O (20 mL) and extracted with DCM (10 mL * 2). The combined organic layers were washed with brine (10 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = 20:1, 10:1 to 3:1 and Dichloromethane: Methanol = 30:1, 10:1 to 0:1) to give 8-(1-ethylnonoxy)-8- oxo-octanoic acid (compound 34) (5.6 g, 17.1 mmol, 59.7% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 4.87 - 4.78 (m, 1H), 2.40 - 2.27 (m, 4H), 1.70 - 1.60 (m, 4H), 1.59 - 1.47 (m, 4H), 1.41 - 1.34 (m, 4H), 1.33 - 1.20 (m, 13H), 0.93 - 0.83 (m, 6H). General procedure for preparation of compound 40

[0274] Compound 40 was prepared according to the synthetic scheme shown in FIG. 21E. To a mixture of compound 39 (1.4 g, 3.0 mmol, 1.0 eq) in DCM (10 mL) was added DIEA (786.9 mg, 6.0 mmol, 1.0 mL, 2.0 eq), DMAP (74.3 mg, 608.8 μmol, 0.2 eq) andEDCI (875.3 mg, 4.5 mmol, 1.5 eq) in order. Then compound 34 (1.0 g, 3.0 mmol, 1.0 eq) was added to the mixture. The mixture was degassed and purged with N2for 3 times and stirred at 20°C for 12 hr under N2atmosphere. TLC (Petroleum ether: Ethyl acetate = 3:1, the starting material Rf = 0.5, product Rf = 0.4) showed the starting material was consumed completely and the reaction was completed. The reaction mixture was diluted with H2O (20 mL) and extracted with DCM (10 mL * 2). The combined organic layers were washed with brine (10 mL * 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = from 20:1 to 3:1 to remove impurities; from 3: 1 to 0:1 and Dichloromethane: Methanol = 50:1 to 3:1 to get product) to give compound 40 (2.3 g, 2.9 mmol, 98.2% yield) as colorless oil.1H NMR (400 MHz, CDCl3-d) δ 4.87 - 4.76 (m, 1H), 4.28 - 4.12 (m, 4H), 4.02 - 3.94 m, 2H), 3.67 - 3.57 m, 2H), 2.39 - 2.14 (m, 10H), 1.67 - 1.58 (m, 10H), 1.57 - 1.49 (m, 4H), 1.38 - 1.35 (m, 3H), 1.33 - 1.23 (m, 36H), 0.93 - 0.85 (m, 12H). General procedure for preparation of Lipid 176

[0275] Lipid 176 was prepared according to the synthetic scheme shown in FIG.21F. To a solution of compound 40 (1.0 g, 1.3 mmol, 1.0 eq) in DCM (10 mL) was added Py (164.5 mg, 2.0 mmol, 167.9 μL, 1.6 eq), DMAP (47.6 mg, 390.0 μmol, 0.3 eq) and (4- nitrophenyl)carbonochloridate (419.3 mg, 2.0 mmol, 1.6 eq) in order. The mixture was stirred at 20°C for 16 hr. TLC (Petroleum ether: Ethyl acetate = 3:1, the starting material Rf= 0.3, product (Cpd.8) Rf= 0.5) showed the starting material was consumed completely and the reaction was completed. Then 3-(diethylamino)propan-1-ol (509.8 mg, 3.8 mmol, 579.3 μL, 3.0 eq) was added to the mixture by one portion and the mixture was stirred at 20°C for 16 hr. TLC (Dichloromethane: Methanol = 10:1, product Rf = 0.5) showed the reaction was completed. The reaction mixture was quenched by addition H2O (20 mL) at 20°C, and then diluted with Na2CO3 (50 mL) and extracted with DCM (10 mL * 3) and H2O (10 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = from 20:1 to 10:1 to remove impurities; from to 5:1 to 0:1 and Dichloromethane: Methanol = 50:1 to 3:1 to get product) for 2 times to give Lipid 176 (316.8 mg, 342.0 μmol, 26.4% yield) as colorless oil. LCMS (Method D): Rt 5.44 min,m / z calculated [M+H]+: 926.7, found: 926.8.1H NMR (400 MHz, CDCl3-d) δ 4.87 - 4.73 (m, 1H), 4.23 - 4.18 (m, 4H), 4.16 - 4.13 (m, 3H), 4.02 - 3.91 (m, 2H), 2.61 - 2.50 (m, 5H), 2.48 - 2.39 (m, 1H), 2.35 - 2.25 (m, 8H), 1.90 - 1.79 (m, 2H), 1.69 - 1.47 (m, 14H), 1.38 - 1.21 (m, 41H), 1.08 - 1.00 (m, 6H), 0.95 - 0.82 (m, 12H). Example 2: Screening of Base LNPs comprising novel ionizable lipids for HSPC delivery in C57BL / 6 Mice

[0276] A general screening paradigm was developed to facilitate the identification of ionizable lipids that can be formulated in LNPs, including targeted LNPs (tLNPs), to enhance delivery of a payload to HSPCs in vivo in humans. The paradigm is as follows: a) screen “base LNPs” that each have an identical formulation except for the ionizable lipid (selected from the ionizable lipids in Table 1) incorporated in the formulation in C57BL / 6 mice to identify those that exhibit enhanced delivery of a reporter GFP mRNA to mouse LSK cells (HSCs) and LT-HSCs relative to a base LNP comprising a baseline control ionizable lipid (the V003 lipid); b) conjugate a targeting moiety (as described herein) that can bind to a protein on the surface of human HSPCs / LT-HSCs to those base LNPs exhibiting higher HSC delivery in C57BL / 6 mice relative to the baseline control LNP to create and screen for targeted LNPs (tLNPs comprising the base LNPs plus targeting moiety) exhibiting higher delivery of a reporter GFP mRNA to human HSPCs / LT-HSCs engrafted in NBSGW mice; c) optionally optimize the LNP formulations (e.g., the relative proportions of LNP lipid components) of those tLNPs exhibiting higher delivery to human HSPCs / LT-HSCs in NBSGW mice to further enhance delivery of the reporter to human HSPCs / LT-HSCs engrafted in NBSGW mice; and d) then test those optimized tLNPs exhibiting the highest HSPC / LT-HSC delivery in the mouse models in nonhuman primates (NHPs) to evaluate their ability to deliver GFP mRNA to primate HSPCs / LT-HSCs in vivo.

[0277] Ionizable Lipid Synthesis: Ionizable lipids were produced according to or by methods similar to those described in Example 1.

[0278] Base LNP Production: Base LNPs were formulated with an ionizable lipid from Table 1. or with the V003 ionizable lipid. LNPs were prepared using molar ratios of either 47% ionizable lipid: 42.5% cholesterol: 8.0% DSPC: 2.0% DMG-PEG2000: 0.5% DSPE-PEG2000-TCO or 47% ionizable lipid: 43.5% cholesterol: 8.0% DSPC: 1.0%DMG-PEG2000: 0.5% DSPE-PEG2000-TCO using a N:P of 6. LNPs were formulated using microfluidic mixing using a Precision Nanosystems NanoAssemblr Ignite according to the manufacturer’s protocol. A ratio of aqueous: organic solvent of 3:1 was maintained during mixing by using differential flow rates. After mixing, the LNPs were collected, diluted in citrate buffered solution pH = 6.0 (approximately 3:1 v / v). After 15 minutes, the diluted LNP solution was neutralized using 1 vol% Tris HCl pH = 8.0. After neutralization each LNP formulated was characterized by dynamic light scattering (DLS) to measure the size (e.g., diameter) and polydispersity (PDI). Following neutralization, buffer exchange was conducted on the solution using dialysis in sucrose buffer (pH = 7.6, 9.0 wt% sucrose, 50 mM Tris, 50 mM salt) at 4°C. The buffer was replaced at 2 hours and dialysis continued overnight against a 10kDa filter. Following removal from the dialysis cassettes, each LNP formulated was characterized by DLS to measure the size and PDI. Each LNP formulation was concentrated using 100kDa Amicon Filters at 4C and 1500 RCF. The concentrated solution was then sterile filtered using a 0.2 µm syringe filter and measured using DLS. The filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) assay. LNP solutions were diluted to the desired concentration and stored at -80°C for future use.

[0279] Characterization by DLS: LNP hydrodynamic diameter and polydispersity index (PDI) were measured using single cell dynamic light scattering (DLS) (Malvern Zetasizer Pro Red Label), high throughput DLS (Wyatt DynaPro III), or high resolution DLS (Malvern NanoSight NS300). Samples were prepared by mixing LNP mixture with 1X PBS (GIBCO 10010-023) at a 1:5 ratio before LNP concentration or a 1:20 ratio after LNP concentration and then analyzed.

[0280] Characterization by RiboGreen: Concentration and encapsulation efficiency of the LNPs were measured using the Quant-iT RiboGreen RNA Kit. In brief, the LNP sample, mRNA standard, RiboGreen reagent, and Triton X-100 were all diluted in TE buffer to the appropriate concentration. Each sample well was comprised of 15 uL of LNP sample, 100 uL of RiboGreen reagent, 50 uL of Triton X-100, and the remaining volume being TE buffer up to 200 uL total. Samples were incubated in the dark for 30 minutes and the fluorescence was measured using an excitation wavelength of 485 nmand an emission wavelength of 525 nm. Nucleic acid concentration for unlysed and lysed LNPs were found as per manufacturer’s instruction with the encapsulated mRNA used as the known standard. The concentration of the mRNA in the lysed LNP was used as the concentration of the LNP, and the encapsulation was given as the ratio of the difference in lysed and unlysed LNPs to the lysed LNPs.

[0281] Characterization by TNS: To determine the pKa of the LNPs, stock buffers of 25 mM PBS and 125 mM sodium chloride were prepared. The pH of the various buffers was adjusted to roughly pH 4 – 9.5 using hydrogen chloride and sodium hydroxide. Using 3 replicates of each pH value for each LNP sample, 386 uL of pH buffer, 10 uL of LNP sample (0.04 mg / mL mRNA), and 4 uL of TNS sample (0.6 mM) were added to each well and incubated in the dark for 30 minutes. Fluorescence of each sample was taken (Molecular Devices SpectraMax i3x) using an excitation wavelength of 325 nm and an emission wavelength of 435 nm.

[0282] Base Lipid (LNP) Screening in C57BL / 6 mice: A series of studies was conducted to compare base LNPs comprising different ionizable lipids selected from Table 1 (above) to a base LNP comprising the V003 ionizable lipid (baseline LNP). The V003 ionizable lipid was used as a standard control to set a baseline delivery level to C57BL / 6 LSK cells and LT-HSCs by which to compare the delivery levels of the test ionizable lipids in each study. Those base LNPs comprising an ionizable lipid from Table 1 that demonstrated an increase in the percent delivery to and / or GFP expression in LSK cells and / or LT-HSCs relative to the baseline LNP comprising the V003 ionizable lipid were selected for further testing in humanized NBSGW mice.

[0283] Female C57BL / 6 mice 6-8 weeks of age were purchased from Jackson Labs (Bar Harbor, ME; Catalog #000664). Following acclimation to the vivarium for 3-5 days, animals were randomized into groups of 4-5 and baseline weights were obtained. Animals were then treated via intravenous tail vein injection with 200 ul of candidate base lipids formulated with GFP mRNA at a dose of 2 mg / kg. Following a 16-hour incubation, animals were euthanized via CO2 asphyxiation, weighed and their femurs were collected and then used to isolate bone marrow cells. In some cases, serum samples were collected to measure ALT and AST levels as biomarkers for liver damage.

[0284] Flow cytometric analysis of C57BL / 6 mouse bone marrow: Bone marrow cells (1 X 106in 0.1 ml PBS) from each test mouse were subsequently aliquoted to individual wells in 96 well plates and antibody fluorochrome conjugate mixtures (Table 9) were added to the cells in a total volume of 0.1 ml. Following a 30-minute incubation at room temperature, the plates were centrifuged for 5 minutes at 500G and supernatants were removed. The cells were then resuspended in 0.2 ml PBS and evaluated on an Aligent NovoCyte Quanteon (2010098) or Cytek Northern Lights (N7-00008) flow cytometer using FlowJo software (Ashland, OR). Cells stained with relevant isotype control fluorochrome conjugated antibodies were used to set the negative control gates. HSCs and early progenitor cells were defined as hCD45+ / Lin- / CD34+ / CD38-, while long-term HSCs (LT-HSCs) were defined as hCD45+ / Lin- / CD34+ / CD38- / CD90+ / CD45RA-.

[0285] Table 9. Flow cytometry Antibodies

[0286] Summary of Base Lipid Screening Studies: Table 10 provides the results of the screening studies. Base LNPs containing Lipid 093, Lipid 176, Lipid 178, and Lipid 183 on average exhibited increased delivery to and / or increased GFP expression in LSK cells and / or LT-HSCs in C57BL / 6 mice relative to a base LNP containing the V003 ionizable lipid (baseline LNP). FIGs.22A and 22B show a subset of the results for Base LNPs containing high performing ionizable lipids. FIG.22A shows enhanced transduction of GFP in LNPs using ionizable Lipid 093 relative to ionizable Lipid V003 (Study 1 inTable 10). FIG.22B shows GFP expression levels about equivalent to V003 LNP in this mouse model. Table 10: Results of Base LNP screening in C57BL / 6 Mice.Example 3: Screening of targeted LNPs (tLNPs) comprising novel ionizable lipids for HSPC delivery in a humanized mouse model

[0287] A subset of the base LNPs exhibiting enhanced delivery to HSCs relative to the baseline control LNP in Example 2 were modified to try to increase their ability to target human HSPCs in vivo, and then evaluated for their delivery GFP mRNA to HSPCs / LT- HSCs in a humanized mouse model in which human HSPCs were engrafted.

[0288] Targeted LNP (tLNP) production: Human HSCs express CD117 on their surface. Therefore, a targeting moiety comprising an antibody Fab that binds specifically to CD117 was conjugated to the surface of the best performing base LNPs of Example 2 to further enhance their delivery potential for human HSPCs in vivo. Specifically, the anti-CD117 targeting moiety was conjugated to the surface of the base LNPs of Example 2 formulated with the ionizable lipid Lipid 093, to produce targeted LNPs (tLNPs) to be tested in an NBSGW mouse model engrafted with human HSPCs.

[0289] Base LNPs were prepared using molar ratios of 47% ionizable lipid: 42.5% cholesterol: 8.0% DSPC: 2.0% DMG-PEG2000: 0.5% DSPE-PEG2000-TCO using a N:P of 6. LNPs were formulated using microfluidic mixing using a Precision Nanosystems NanoAssemblr Ignite according to the manufacturer’s protocol. A ratio of aqueous: organic solvent of 3:1 was maintained during mixing by using differential flow rates. After mixing, the LNPs were collected, diluted in citrate buffered solution pH = 6.0 (approximately 3:1 v / v). After 15 minutes, the diluted LNP solution was neutralized using 1 vol% Tris HCl pH = 8.0. After neutralization each LNP formulated was characterized by dynamic light scattering (DLS) to measure the size (e.g., diameter) and polydispersity (PDI). Following neutralization, buffer exchange was conducted on the solution using dialysis in sucrose buffer (pH = 7.6, 9.0 wt% sucrose, 50 mM Tris, 50 mM salt) at 4°C. The buffer was replaced at 2 hours and dialysis continued overnight against a 10kDa filter. Following removal from the dialysis cassettes, each LNP formulated was characterized by DLS to measure the size and PDI. Each LNP formulation was concentrated using 100kDa Amicon Filters at 4C and 1500 RCF. The concentrated solution was then sterile filtered using a 0.2 µm syringe filter and measured using DLS.The filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p-toluidinyl)naphthalene-6- sulfonic acid (TNS) assay. LNP solutions were diluted to the desired concentration and stored at -80°C for future use.

[0290] An anti-CD117 Fab was produced with an additional sortase tag (LPETG) on the C terminus of the heavy chain.10uM CD117 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with gentle mixing. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 10K MWCO Amicon centrifugal UF device. To confirm a DOL (degree of labeling) of close to 1 meTz per antibody, sortase modified anti-CD117 Fab was allowed to react with 5eq AF594 containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed via Zeba desalting columns. DOL was determined by first measuring absorbances at A280 and A590 using a Nanodrop, and then calculating the DOL using extinction coefficients and correction factors. The resulting meTz- modified anti-CD117 Fab was added to the TCO modified-LNP solution described above for antibody-LNP conjugation, with a 1:1 mass ratio between antibody and mRNA (eGFP). The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD117 Fab was removed by a 300K mPES TFF membrane. The resulting antibody-LNP product was further concentrated using a 100k MWCO Amicon centrifugal UF device. A ribogreen assay was then performed to quantify mRNA content and encapsulation efficiency.

[0291] Characterization: LNPs were characterized by DLS, RiboGreen, and TNS as described in Example 2.

[0292] Targeted Lipid (tLNP) screening in “humanized” NBSGW mice: A study was conducted to compare anti-CD117 tLNPs formulated with Lipid 093 to a targeted LNP (tLNP) formulated with the V003 ionizable lipid. The V003 ionizable lipid was used as a standard control to set a baseline delivery level to human HSPCs / LT-HSCs engrafted in NBSGW mice by which to compare the delivery levels of the test ionizable lipids in eachstudy. Those tLNPs demonstrating an increase in the percent delivery to and / or GFP expression in human HSPCs and / or LT-HSCs relative to the tLNP comprising the V003 ionizable lipid (baseline tLNP) were selected for further testing in nonhuman primates (NHPs) (see Example 5).

[0293] Briefly, female NBSGW mice 6-8 weeks of age (Catalog #026622) were purchased from Jackson Labs (Bar Harbor, ME). Following acclimation to the vivarium for 3-5 days, animals received a tail vein injection of 2 X 106human CD34+ HSPCs (HemaCare Division of Charles River Labs, Wilmington, MA) and the human cells were allowed to engraft for 12 weeks before the animals were used in screening studies. For each tLNP screening study, “humanized” NBSGW mice were randomized into groups of 4-5 animals and baseline weights were obtained. Animals were then treated via intravenous tail vein injection with 100 ul of candidate tLNPs formulated with GFP mRNA at a dose of 2 mg / kg. Following a 16-hour incubation, animals were euthanized via CO2 asphyxiation, weighed and their femurs and tibias were collected and the bone marrow was isolated. In some cases, serum samples were collected to measure ALT and AST levels as biomarkers for liver damage.

[0294] Flow cytometric analysis of “humanized” NBSGW mouse bone marrow: Bone marrow cells (1 X 106in 0.1 ml PBS) from each test mouse were subsequently aliquoted to individual wells in 96 well plates and antibody fluorochrome conjugate mixtures (Table 11) were added to the cells in a total of 0.1 ml. Following a 30-minute incubation at room temperature, the plates were centrifuged for 5 minutes at 500G and supernatants were removed. The cells were then resuspended in 0.2 ml PBS and evaluated on an Aligent NovoCyte Quanteon (2010098) or Cytek Northern Lights (N7-00008) flow cytometer using FlowJo software (Ashland OR). Cells stained with relevant isotype control fluorochrome conjugated antibodies were used to set the negative control gates. HSC and early progenitor cells were defined as hCD45+ / Lin- / CD34+ / CD38-, while long- term HSCs (LT-HSCs) were defined as hCD45+ / Lin- / CD34+ / CD38- / CD90+ / CD45RA-.Table 11. Flow cytometry antibodies

[0295] Summary of Targeted LNP screening studies: Table 12 provides the results of the screening studies. Anti-CD117 targeted LNPs containing Lipid 093 on average exhibited increased delivery to and / or increased GFP expression in human HSPCs and / or LT-HSCs in the humanized NBSGW mouse model relative to the targeted LNP containing the V003 ionizable lipid (baseline tLNP).

[0296] The results from Study 1 in Table 12 are depicted graphically in FIGs.23A and 23B. Notably, targeted LNPs comprising Lipid 093 shows significantly increased transduction (FIG.23A) and GFP expression (FIG.23B) relative to targeted LNPs comprising V003 in LT-HSPCs. Lipid 093 tLNPs also exhibited increased GFP transduction and expression in the HSPCs. Surprisingly, Lipid 093 performed better in the humanized mouse model relative to the mouse model described in Example 2.

[0297] These results indicate that targeted LNPs formulated with a subset of ionizable lipids could exhibit enhanced delivery of a payload gene to HSPCs / LT-HSCs in vivo fortherapeutic applications. Some of these ionizable lipids were selected for testing in nonhuman primates to determine whether they are capable of enhancing delivery in a larger animal model (see Example 5). Table 12. Screening results of targeted LNP (tLNP) screening in humanized NBSGW mice.Example 4: Screening of targeted LNPs (tLNPs) comprising novel ionizable lipids for HSPC delivery in a humanized mouse model

[0298] A base LNP comprising Lipid 093 exhibiting enhanced delivery to HSPCs relative to the baseline control LNP in Example 3 was modified to try to increase their ability to target human HSPCs in vivo, and then evaluated for their delivery of GFP mRNA to HSPCs / LT-HSCs in a humanized mouse model in which human HSPCs were engrafted. Experiments were conducted as described in Example 3, but with increased quantities of helper lipid (22% DSPC or sphingomyelin). Results are shown in FIGs.24A-D. The results show that LNPs comprising 22% helper lipid deliver payload mRNA to a high percentage of HSPCs and LT-HSPCs, leading to GFP expression. Example 5: Targeted LNPs exhibiting enhanced delivery to LT-HSPCs in nonhuman primates (NHPs)

[0299] A subset of the tLNPs exhibiting enhanced delivery to HSPCs and LT-HSCs relative to the baseline control targeted LNPs were evaluated for their ability to deliver GFP mRNA to HSPCs / LT-HSCs in cynomolgus macaques. Specifically, tLNPs formulated with ionizable lipids Lipid 093 was tested and compared to a tLNP formulated with the V003 ionizable lipid in a series of studies.

[0300] Targeted LNP (tLNP) production: Base LNPs were prepared as described in Example 4 using the V003 or Lipid 093 ionizable lipid and either DSPC or egg sphingomyelin as helper lipid. LNPs were formulated using microfluidic mixing using a Precision Nanosystems NanoAssemblr Ignite according to the manufacturer’s protocol. A ratio of aqueous: organic solvent of 3:1 was maintained during mixing by using differential flow rates. After mixing, the LNPs were collected, diluted in citrate buffered solution pH = 6.0 (approximately 3:1 v / v). After 15 minutes, the diluted LNP solution was neutralized using 1 vol% Tris HCl pH = 8.0. After neutralization each LNP formulated was characterized by dynamic light scattering (DLS) to measure the size (e.g., diameter) and polydispersity (PDI). Following neutralization, buffer exchange was conducted on the solution using dialysis in sucrose buffer (pH = 7.6, 9.0 wt% sucrose, 50 mM Tris, 50 mM salt) at 4°C. The buffer was replaced at 2 hours and dialysis continued overnight against a 10kDa filter. Following removal from the dialysis cassettes, each LNP formulated was characterized by DLS to measure the size and PDI. Each LNP formulation was concentrated using 100kDa Amicon Filters at 4C and 1500 RCF. The concentrated solution was then sterile filtered using a 0.2 µm syringe filter and measured using DLS. The filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p- toluidinyl)naphthalene-6-sulfonic acid (TNS) assay. LNP solutions were diluted to the desired concentration and stored at -80°C for future use.

[0301] A targeting moiety comprising an antibody Fab that binds specifically to CD117 was conjugated to the surface of the LNPs as described in prior examples. Specifically, an anti-CD117 Fab was produced with an additional sortase tag (LPETG) on the C terminus of the heavy chain.10uM CD117 Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with gentle mixing. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 10K MWCO Amicon centrifugal UF device. To confirm a DOL (degree of labeling) of close to 1 meTz per antibody, sortase modified anti-CD117 Fab was allowed to react with 5eq AF594 containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed via Zeba desalting columns. DOL was determined byfirst measuring absorbances at A280 and A590 using a Nanodrop, and then calculating the DOL using extinction coefficients and correction factors. The resulting meTz- modified anti-CD117 Fab was added to the TCO modified-LNP solution described above for antibody-LNP conjugation, with a 1:1 mass ratio between antibody and mRNA (eGFP). The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD117 Fab was removed by a 300K mPES TFF membrane. The resulting antibody-LNP product was further concentrated using a 100k MWCO Amicon centrifugal UF device. A ribogreen assay was then performed to quantify mRNA content and encapsulation efficiency.

[0302] Characterization: LNPs were characterized by DLS, RiboGreen, and TNS as described in Example 2.

[0303] tLNP screening in cynomolgus macaques: Sexually mature male or female cynomolgus macaques (Biomere, Worcester, MA) were enrolled in the study and baseline weights and blood samples were collected (see Table 13). Animals were pre-treated with famotidine (0.5 mg / kg, IM) and diphenhydramine (5 mg / kg, IM) on Day -1, and on Day 1 at 30-60 minutes prior to start of treatment. Two of the animals administered tLNPs containing the V003 ionizable lipid pretreated with G-CSF and Plerixafor to mobilize HSCs prior to administering the tLNPs. For these animals, G-CSF was administered daily for 5 consecutive days (Day –4 to Day 0) before the tLNPs were administered. Plerixafor was administered 2 hours before the tLNPs were administered. For each animal, tLNPs were administered intravenously through a saphenous vein, cephalic vein, or tail vein with a temporary IV catheter on Day 1 at a dose of 2 mg / kg via an approximate 60- minute infusion using an infusion pump, followed by about 0.5 mL of saline to flush the dose from the IV catheter. Additional blood samples were collected at 1 hour and 15 to 20 hours post infusion. Lymph node and bone marrow aspirate were also collected at 15- 20 hours after the end of infusion. Separately, bone marrow aspirates from non-treated cynomolgus macaques were collected to establish a negative control for baseline %GFP+ and MFI levels.Table 13. Blood Sample Collection Schedule

[0304] Flow cytometric analysis of NHP bone marrow: Bone marrow cells (3 X 106in 0.1 ml PBS) from each nonhuman primate in the studies were subsequently aliquoted to individual wells in 96 well plates and centrifuged at 500 G for 5 min at RT. A master mix composed of antibody fluorochrome conjugate mixtures (Table 14 ) was added to the cells in a total of 0.1 ml. Following a 30-minute incubation at 4°C, the plates were centrifuged for 5 min at 500G and supernatants were removed. Cells were resuspended in 200 uL of fixation buffer (BD Cytofix / Cytoperm) and incubated for 20 min at 4°C. After fixation, cells were centrifuged for 5 min at 500G and washed once in 200 uL of PBS. After centrifugation, the supernatants were discarded and the cells were resuspended in 0.2 ml PBS and evaluated on an Aligent NovoCyte Quanteon (2010098) or Cytek Northern Lights (N7-00008) flow cytometer. Analysis and quantification of the analyzed BM samples was performed on FlowJo software (Ashland OR). FMO (fluorescence- minus-one) controls for all channels were used to set the negative control gates. HSCs and early progenitor cells were defined as hCD45+ / Lin- / CD34+ / CD38-, while long-term HSCs (LT-HSCs) were defined as hCD45+ / Lin- / CD34+ / CD38- / CD90+ / CD45RA-. Table 14. Flow cytometry antibodies

[0305] Summary of tLNP Screening Studies: Table 15 and FIG.25 provide the results of the NHP studies. Increasing the percentage of helper lipid in the tLNP formulations enhanced the ability of targeted LNPs to deliver GFP mRNA to HSPCs and LT-HSCs across studies. For example, V003 targeted LNPs formulated with 22% DSPC exhibited slightly higher targeting of LT-HSCs relative to V003 tLNPs formulated with 8% DSPC (compare Study 1 results to Study 2 results). Targeted LNPs containing Lipid 093 exhibited increased delivery of GFP mRNA to HSPCs and LT-HSCs relative to tLNPs containing the V003 ionizable lipid when the LNPs were formulated with 22% DSPC helper lipid (see Study 2 and Study 3 results). Targeted LNPs containing Lipid 093 exhibited similarly elevated levels of delivery to HSPCs and LT-HSCs in NHPs when formulated with egg sphingomyelin, an alternative helper lipid (Study 3). In addition, Table 15 shows that the HSPCs and / or LT-HSCs of NHPs that were transduced by the tLNPs formulated with Lipid 093 and 22% helper lipid (DSPC or sphingomyelin) in vivo generally expressed GFP at higher levels than the HSPCs and / or LT-HSCs of NHPs that were transduced with tLNPs formulated with the V003 ionizable lipid formulated with 22% helper lipid (DSPC). Overall, the results show that Lipid 093 tLNPs can deliver a mRNA encoding a payload gene to more HSPCs and LT-HSCs in an NHP model relative to V003 tLNPs, and the cells transduced with Lipid 093 tLNPs can express higher levels of the payload gene relative to the cells transduced with V003 tLNPs. The performance of the Lipid 093 tLNPs increased relative to the V003 tLNPs when the tLNPs are formulated with higher levels of helper lipids.Table 15. Screening results of targeted LNP (tLNP) screening in nonhuman primates.Example 6: LNPs comprising ionizable Lipid 235 suitable for mRNA delivery to HSCs in vivo

[0306] Ionizable Lipid 235 was synthesized, formulated in base LNPs, and then tested in studies to determine if they could deliver an mRNA to HSCs in vivo as effectively as Lipid 093 LNPs.

[0307] Ionizable Lipid Synthesis: Ionizable Lipid 235 were produced according to or by methods similar to those described in Example 1.

[0308] Base LNP Production: Base LNPs were formulated with an ionizable lipid to be tested (Lipid 235) or with the Lipid 093 ionizable lipid. LNPs were prepared as described in Example 1. Briefly, molar ratios of 47% ionizable lipid: 42.5% cholesterol: 8.0% DSPC: 2.0% DMG-PEG2000: 0.5% DSPE-PEG2000-TCO using a N:P of 6. The aqueous phase was composed of mRNA encoding the eGFP in 25 mM acetate buffer.LNPs were formulated using microfluidic mixing using a Precision Nanosystems NanoAssemblr Ignite according to the manufacturer’s protocol. A ratio of aqueous: organic solvent of 3:1 was maintained during mixing by using differential flow rates. After mixing, the LNPs were collected, diluted in citrate buffered solution pH = 6.0 (approximately 3:1 v / v). After 15 minutes, the diluted LNP solution was neutralized using 1 vol% Tris HCl pH = 8.0. Following neutralization, buffer exchange was conducted on the solution using dialysis in sucrose buffer (pH = 7.6, 9.0 wt% sucrose, 50 mM Tris, 50 mM salt) at 4°C. The buffer was replaced at 2 hours and dialysis continued overnight against a 10kDa filter. Each LNP formulation was concentrated using 100kDa Amicon Filters at 4C and 1500 RCF. The concentrated solution was then sterile filtered using a 0.2 µm syringe filter and measured using DLS. The filtered LNP samples were evaluated for mRNA content using the Ribogreen assay. Additionally, the pKa of LNPs was measured using the 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) assay. LNP solutions were diluted to the desired concentration and stored at -80°C for future use.

[0309] Base Lipid (LNP) Screening in C57BL / 6 mice: Base LNPs formulated with Lipid 235 and an mRNA encoding GFP were administered to C57BL / 6 mice. LNPs formulated with Lipid093 were used as a standard to set a mRNA baseline delivery level to C57BL / 6 LSK cells and LT-HSCs by which to compare the delivery levels of the test ionizable lipids in each study. Flow cytometry was then used to determine whether any of the test LNPs demonstrated an equal or greater level of GFP mRNA delivery to LSK cells and / or LT-HSCs relative to the Lipid 093 LNPs.

[0310] Female C57BL / 6 mice 6-8 weeks of age were purchased from Jackson Labs (Bar Harbor, ME; Catalog #000664). Following acclimation to the vivarium for 3-5 days, animals were randomized into groups of 4-5 and baseline weights were obtained. Animals were then treated via intravenous tail vein injection with 200 ul of candidate base lipids formulated with GFP mRNA at a dose of 0.5 mg / kg. Following a 16-hour incubation, animals were euthanized via CO2 asphyxiation, weighed and their femurs were collected and then used to isolate bone marrow cells. In some cases, serum samples were collected to measure ALT and AST levels as biomarkers for liver damage.

[0311] Flow cytometric analysis of C57BL / 6 mouse bone marrow: Bone marrow cells (1 X 106in 0.1 ml PBS) from each test mouse were subsequently aliquoted to individual wells in 96 well plates and antibody fluorochrome conjugate mixtures (Table 16) were added to the cells in a total volume of 0.1 ml. Following a 30-minute incubation at room temperature, the plates were centrifuged for 5 minutes at 500G and supernatants were removed. The cells were then resuspended in 0.2 ml PBS and evaluated on an Aligent NovoCyte Quanteon (2010098) or Cytek Northern Lights (N7-00008) flow cytometer using FlowJo software (Ashland, OR). Cells stained with relevant isotype control fluorochrome conjugated antibodies were used to set the negative control gates. HSCs and early progenitor cells were defined as hCD45+ / Lin- / CD34+ / CD38-, while long-term HSCs (LT-HSCs) were defined as hCD45+ / Lin- / CD34+ / CD38- / CD90+ / CD45RA-. Table 16. Flow cytometry Antibodies

[0312] Summary of Base Lipid Screening Studies: Table 17 provides the results of this study. Base LNPs containing Lipid 235 on average exhibited the same or increased levels of delivery to and / or the same or increased levels of GFP expression in LSK cells and / or LT-HSCs in C57BL / 6 mice relative to a base LNP formulated with Lipid 093.Table 17: Results of Base LNP screening in C57BL / 6 Mice.Example 7 : Targeted LNPs formulated with Lipid 176, or Lipid 178 exhibit delivery of RNA payloads comprising an exemplary gene modifying system to HSPCs in vivo

[0313] LNPs were formulated with the Lipid 176 or Lipid 178 ionizable lipid and an all- RNA payload comprising an exemplary gene editing system, ,a surrogate for a gene modifying system, as described herein, in this case, an mRNA encoding a wild-type Cas9-RT fusion protein and an sgRNA specific for the first exon of the human Beta 2 Microglobulin (B2M) gene. The LNPs were conjugated to the anti-CD117 (Ab67) and anti-CD34-1 (h4C8) targeting moieties to create dual targeted LNPs. The tLNPs were then administered to humanized mice and evaluated for their effectiveness in delivering the RNA payload in vivo as measured by the percentage of B2M indels in HSPCs using Amp-Seq. Targeted LNP (tLNP) production

[0314] Targeted LNPs were generally formulated and then conjugated to targeting moieties.

[0315] Briefly, base LNPs were prepared at a molar ratio of 47% ionizable lipid (Lipid 176 or Lipid 178): 28.5% cholesterol: 22% DSPC: 2.0% DPG-PEG2000: 0.5% DSPE- PEG2000-TCO at an N / P of 6 in the ethanol phase, as described in previous examples. The aqueous phase was composed of mRNA (encoding the gene modifying polypeptide) and an sgRNA at a mass ratio of 1:1 in 25 mM acetate buffer. The two phases were mixed using a microfluidic device to formulate base LNP at a total flow rate of 18 ml / min (aqueous phase: ethanol phase=3:1).3x volume of citrate-buffered saline was added to LNP solution and resulted solution was neutralized by 1 vol% of 1M Tris-HCl, pH 8 after 15 minutes.

[0316] Each targeting moiety (Fab) was produced with an additional sortase tag (LPETG) on the C terminus of the heavy chain.10uM Fab-LPETG and 1mM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl2, pH 7.4).2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with gentle mixing. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 10K MWCO Amicon centrifugal UF device. To confirm a DOL (degree of labeling) of close to 1 meTz per targeting moiety, sortase modified Fab was allowed to react with 5eq AF594 containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed via Zeba desalting columns. DOL was determined by first measuring absorbances at A280 and A590 using a Nanodrop, and then calculating the DOL using extinction coefficients and correction factors. The resulting meTz-modified Fabs were added to the TCO modified-LNP solution described above for Fab-LNP conjugation, with a 1:1 mass ratio between Fab and total RNA payload for tLNPs or a 1:1:1 mass ratio between Fab1, Fab2 and total RNA payload for dual tLNPs. The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted Fabs were removed by a 300K mPES TFF membrane. The resulting tLNP and dual tLNP products were further concentrated using a 100k MWCO Amicon centrifugal UF device. A ribogreen assay was then performed to quantify RNA content and encapsulation efficiency. Administration of tLNPs in “humanized” NBSGW mice

[0317] Briefly, female NBSGW mice 6-8 weeks of age (Catalog #026622) were purchased from Jackson Labs (Bar Harbor, ME). Following acclimation to the vivarium for 3-5 days, animals received a tail vein injection of 5 X 105human CD34+ HSPCs (HemaCare Division of Charles River Labs, Wilmington, MA) and the human cells were allowed to engraft for 12 weeks before the animals were used in screening studies. Animals were then treated via intravenous tail vein injection with 100 ul of candidate tLNPs formulated with the payload at a dose of 4 mg / kg. Seven days later, animals were euthanized via CO2 asphyxiation, weighed and their femurs, tibias and hips were collected and the bone marrow was isolated. HSCs and early progenitor cells (HSPC) were defined as Lin- / CD34+ / CD38-, while long-term HSCs (LT-HSCs) were defined asLin- / CD34+ / CD38- / CD90+. LT-HSC were sorted as a pool from the mice belonging to the same experimental group. Editing of the B2M locus (indels) was quantified by amplicon-sequencing (Amp-Seq) of the locus near the binding site of the protospacer. Results

[0318] Administering the anti-CD34 / CD117 dual tLNPs formulated with Lipid 176 or Lipid 178 and the exemplary gene editing system resulted in editing of the target locus in human HSPCs and LT-HSCs in vivo. On average the tLNPs formulated with Lipid 176 resulted in an average of 4.02% indels leading to B2M knockout in HSPCs and 3.19% indels leading to B2M knockout in LT-HSCs. On average the tLNPs formulated with Lipid 178 resulted in an average of 5.72% indels leading to B2M knockout in HSPCs and 6.04% indels leading to B2M knockout in LT-HSCs.

Claims

CLAIMS 1. A compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1is C4-10alkyl; R2is C1-4alkyl; R3and R4are independently C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

2. A compound having a structure as set forth in Table 1, or a pharmaceutically acceptable salt thereof.

3. A compound having the structure:or a pharmaceutically acceptable salt thereof.

4. A compound having the structure:or a pharmaceutically acceptable salt thereof.

5. A compound having the structure:or a pharmaceutically acceptable salt thereof.

6. A compound having the structure:or a pharmaceutically acceptable salt thereof; oror a pharmaceutically acceptable salt thereof.

7. A lipid nanoparticle (LNP) comprising a compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof.

8. The lipid nanoparticle of claim 7, further comprising a targeting moiety.

9. The lipid nanoparticle of claim 8, wherein the targeting moiety targets hematopoietic stem cells (HSCs).

10. The lipid nanoparticle of claim 8, wherein the targeting moiety targets immune cells.

11. The lipid nanoparticle of claim 10, wherein the immune cells are T cells.

12. 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.

13. The LNP of clam 12, wherein the ionizable lipid has the structure:.

14. The LNP of clam 12, wherein the ionizable lipid has the structure:.

15. The LNP of clam 12, wherein the ionizable lipid has the structure:.

16. The LNP of clam 12, wherein the ionizable lipid has the structure:.

17. The LNP of any one of claims 12-16, wherein the cells are immune cells.

18. The LNP of claim 17, wherein the immune cells are T cells.

19. The LNP of any one of claims 12-16, wherein the cells are hematopoietic stem cells (HSCs).

20. The LNP of any one of claims 12-19, wherein the therapeutic agent comprises at least one nucleic acid molecule.

21. The LNP of claim 20, wherein the nucleic acid molecule is selected from an mRNA molecule or a DNA molecule.

22. The LNP of claim 20, wherein the nucleic acid molecule is an mRNA molecule.

23. The LNP of claim 20, wherein the nucleic acid molecule is a siRNA or miRNA molecule.

24. The LNP of claim 20, wherein the therapeutic agent comprises two nucleic acid molecules.

25. The LNP of claim 24, wherein the two nucleic acid molecules are RNA molecules.

26. The LNP of claim 25, wherein one nucleic acid comprises an mRNA molecule (e.g., an mRNA encoding a gene modifying polypeptide) and one nucleic acid comprises an RNA molecule (e.g., a guide RNA or template RNA).

27. The LNP of claim 26, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a guide RNA.

28. The LNP of claim 24, wherein one nucleic acid is an mRNA molecule and one nucleic acid is a DNA molecule.

29. The LNP of any one of claims 24-28, wherein the at least one nucleic acid molecules encodes at least one component for altering a genome, e.g., a gene modifying polypeptide..

30. The LNP of claim 29, wherein the at least one component comprises an mRNA encoding a CRISPR-associated nuclease (Cas) and a guide RNA.

31. The LNP of claim 30, wherein the Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).

32. The LNP any one of claims 24-31, wherein the mol% of ionizable lipids in the LNP ranges from about 35% to about 60%.

33. The LNP of claim 32, wherein the mol% of ionizable lipids in the LNP ranges from about 40% to about 50%.

34. The LNP of claim 32, wherein the mol% of ionizable lipids in the LNP ranges from about 45% to about 50%.

35. The LNP of any one of claims 24-34, 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.

36. The LNP of claim 35, wherein the helper lipid is DSPC.

37. The LNP of claim 35, wherein the helper lipid is sphingomeylin.

38. The LNP of claim 37, wherein the sphingomeylin has a head group selected from phosphocholine, phosphoethanolamine and ceramide.

39. The LNP of claim 38, wherein the sphingomeylin is egg sphingomeylin.

40. The LNP of any one of claims 12-39, wherein the mol% of helper lipid is from about 18% to about 32%.

41. The LNP of claim 40, wherein the mol% of helper lipid is from about 20% to about 25%.

42. The LNP of claim 41, wherein the mol% of helper lipid is from about 22% to about 28%.

43. 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 7-42.

44. The method of claim 43, wherein the disease is selected from any one of the diseases in Tables 3-8.

45. A conjugate for targeted 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, wherein the plurality of targeting moieties is configured to target cells.; and a therapeutic agent encapsulated within the LNP.

46. The conjugate of clam 45, wherein the ionizable lipid has the structure:.

47. The conjugate of clam 45, wherein the ionizable lipid has the structure: ..

48. The conjugate of clam 45, wherein the ionizable lipid has the structure:.

49. The conjugate of clam 45, wherein the ionizable lipid has the structure:.

50. The conjugate of any one of claims 45-49, wherein the plurality of targeting moieties is configured to target a receptor on the surface of HSCs.

51. The conjugate of claim 50, wherein the plurality of targeting moieties is configured to target a receptor on the surface of HSCs selected from CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184.

52. The conjugate of claim 51, wherein the plurality of targeting moieties is configured to target CD117.

53. The conjugate of claim 51, wherein the plurality of targeting moieties is configured to target CD71.

54. The conjugate of claim 51, wherein the plurality of targeting moieties is configured to target CD34.

55. The conjugate of any one of claims 45-49, wherein the plurality of targeting moieties is configured to target a receptor on the surface of immune cells.

56. The conjugate of claim 55, wherein the immune cells are T cells.

57. The conjugate of any one of claims 45-56, wherein the plurality of targeting moieties comprises antibodies or fragments thereof.

58. The conjugate of any one of claims 45-56, wherein the plurality of targeting moieties comprises Fab fragments.

59. The conjugate of any one of claims 45-56, wherein the plurality of targeting moieties comprises single chain variable fragments (scFv).

60. The conjugate of any one of claims 45-59, wherein the therapeutic agent comprises at least one nucleic acid molecule.

61. The conjugate of claim 60, wherein the nucleic acid molecule is selected from an mRNA molecule or a DNA molecule.

62. The conjugate of claim 60, wherein the nucleic acid molecule is an mRNA molecule.

63. The conjugate of claim 60, wherein the nucleic acid molecule is a siRNA or miRNA molecule.

64. The conjugate of claim 60, wherein the therapeutic agent comprises two nucleic acid molecules.

65. The conjugate of claim 64, wherein the two nucleic acid molecules are RNA molecules.

66. The conjugate of claim 65, wherein one nucleic acid comprises an mRNA molecule (e.g., an mRNA encoding a gene modifying polypeptide) and one nucleic acid comprises an RNA molecule (e.g., a guide mRNA or template RNA).

67. The conjugate of claim 65, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a guide RNA.

68. The conjugate of claim 65, wherein one nucleic acid is an mRNA molecule and one nucleic acid is a DNA molecule.

69. The conjugate of any one of claims 65-68, wherein the at least one nucleic acid molecules encodes at least one component for altering a genome (e.g., an mRNA encoding a gene modifying polypeptide).

70. The conjugate of claim 69, wherein the at least one component comprises an mRNA encoding a CRISPR-associated nuclease (Cas) and a guide RNA.

71. The conjugate of claim 70, wherein the Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).

72. The conjugate of claim 70 , wherein the guide RNA is a single guide RNA.

73. The conjugate of any one of claims 45-70, wherein the mol% of ionizable lipids in the LNP ranges from about 35% to about 60%.

74. The conjugate of claim 73, wherein the mol% of ionizable lipids in the LNP ranges from about 40% to about 50%.

75. The conjugate of claim 73, wherein the mol% of ionizable lipids in the LNP ranges from about 45% to about 50%.

76. The conjugate of any one of claims 45-75, 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.

77. The conjugate of claim 76, wherein the helper lipid is DSPC.

78. The conjugate of claim 76, wherein the helper lipids is sphingomeylin.

79. The conjugate of claim 78, wherein the sphingomeylin has a head group selected from phosphocholine, phosphoethanolamine and ceramide.

80. The conjugate of claim 79, wherein the sphingomeylin is egg sphingomeylin.

81. The conjugate of any one of claims 45-80, wherein the mol% of helper lipid in the conjugate is from about 18% to about 32%.

82. The conjugate of claim 81, wherein the mol% of helper lipid in the conjugate is from about 20% to about 25%.

83. The conjugate of claim 82, wherein the mol% of helper lipid in the conjugate is from about 21% to about 23%.

84. The conjugate of claim 82, wherein the mol% of helper lipid in the conjugate is from about 22% to about 28%.

85. The conjugate of any one of claims 45-84, wherein the LNP further comprises one or more PEGylated lipids.

86. The conjugate of claim 85, wherein the targeting moieties are conjugated to the LNP through the PEGylated lipids.

87. The conjugate of claim 85 or claim 86, wherein the PEGylated lipids each comprise a lipid component, wherein the lipid component of the PEGylated lipids is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, stearic acid, and any combination thereof.

88. The conjugate of claim 87 wherein the lipid component of the PEGylated lipids is DSPE.

89. The conjugate of any one of claims 85-88 , wherein the molar ratio of PEGylated lipids in the LNP ranges from about 0.05% to about 2%.

90. The conjugate of any one of claims 45-89, wherein the plurality of lipid molecules comprises one or more cholesterol molecules.

91. The conjugate of claim 90, wherein the cholesterol molecules are selected from beta- sitoesterol, hydroxycholesterol, stigmastanol, and any combination thereof.

92. The conjugate of claim 90 or claim 91, wherein the molar ratio of cholesterol molecules in the LNP ranges from about 35% to about 55%.

93. The conjugate of any one of claims 45-92, wherein the plurality of targeting moieties comprises more than 10 targeting moieties.

94. The conjugate of claim 93, wherein the plurality of targeting moieties comprises more than 20 targeting moieties.

95. The conjugate of claim 93, wherein the plurality of targeting moieties comprises more than 50 targeting moieties.

96. The conjugate of claim 93, wherein the plurality of targeting moieties comprises more than 100 targeting moieties.

97. The conjugate of any one of claims 45-96, wherein the conjugate is formed via a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP, thereby generating a Click product.

98. The conjugate of claim 97, wherein the second Click handle is covalently bonded to a pegylated lipid on the LNP.

99. The conjugate of claim 97 or claim 98, wherein the LNP is conjugated to the targeting moiety site specifically.

100. The conjugate of any one of claims 97-99, wherein the Click reaction is 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.

101. The conjugate of claim 100, wherein the first Click handle comprises a tetrazine ring and the second Click handle comprises a TCO moiety.

102. The conjugate of claim 100 or claim 101, wherein the tetrazine ring is unsubstituted.

103. The conjugate of claim 101 or claim 102, wherein the tetrazine ring is methyltetrazine.

104. The conjugate of claim 103, wherein the tetrazine ring is 6-methyltetrazine.

105. The conjugate of claim 101, wherein the tetrazine ring has one of the following structures:N N106. The conjugate of any one of claims 100-105, wherein the first Click handle is conjugated to the targeting moiety through a sortase mediated ligation of a sortase recognition motif of the targeting moiety and a plurality of glycine residues conjugated to the first Click handle.

107. 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 45-106.

108. The method of claim 107, wherein the disease is selected from any one of the diseases in Tables 3-8.

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