Site-specific conjugation of targeting moieties to lipid nanoparticles

Abstract

The disclosure provides Fab fragments that are site-selectively conjugated to the surface of a lipid nanoparticle (LNP) through the natural interchain disulfide bond between the heavy chain and the light chain (i.e., the CL-CH1 disulfide bond) to make targeted LNPs (also referred to herein as conjugates). The targeted LNPs (conjugates) produced by the methods disclosed herein can transduce specific targeted cells. Hence, the targeted LNPs are capable of delivering therapeutic payloads to cells to treat disease in a subject.

Description

SITE-SPECIFIC CONJUGATION OF TARGETING MOIETIES TO LIPID NANOPARTICLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims priority to U.S. Provisional Patent Application No. 63 / 562,933, filed March 8, 2024, which is herein incorporated by reference in its entirety.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (252052002040seqlist.xml; Size: 94,626 bytes; and Date of Creation: March 5, 2025) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates generally to the field of lipid nanoparticles (LNPs) for drug delivery and more specifically LNPs with Fab fragments conjugated to the LNP surface.BACKGROUND

[0004] 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. Nonetheless, substantial safety issues still remain. For instance, LNPs may accumulate and deliver payloads to cells other than the intended target, which results in potential toxicity. Accordingly, an important goal is to develop LNPs that target diseased tissue and that can be administered at nontoxic doses.

[0005] One approach to improve the toxicological profile and increase the efficacy of LNPs is to modify the surface of the LNP with an antibody or functional fragment thereof, e.g., a Fab fragment or VHH domain, that targets specific cells, such as diseased cells. For instance, LNPs can be coated with Fab fragments that bind to particular cellular receptors on target cells, resulting in accumulation of a pay load, such as a therapeutic agent, in the targeted tissue relative to other tissue in the body. Different approaches have been used to introduce Fab fragments onto the surface of an LNP. For example, one approach relies on functionalizing a pre-formed LNP with an antibody or antigen-binding fragment thereof. 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 acomplementary group that is covalently bonded to the antibody or antigen-binding fragment thereof, hence generating a conjugate of the LNP and the antibody or antigen-binding fragment thereof. However, many of these approaches produce LNPs that are conjugated to the Fab fragments in a nonhomogeneous manner. Generally, when an antibody or antigen-binding fragment thereof (e.g., a Fab fragment) is functionalized with a reactive group, such functionalization occurs in a random manner, resulting in a heterogeneous population of antibodies or fragments thereof (e.g., Fab fragments) that shows significant batch-to-batch variability. The randomly modified antibodies or fragments thereof (e.g., Fab fragments) produce LNPs that have their surfaces modified in a random manner. The reaction between the randomly modified antibodies or fragments thereof (e.g., Fab fragments) with the LNPs may not occur with optimal efficiency. Moreover, LNPs decorated in this manner may contain a proportion of antibodies or fragments thereof (e.g., Fab fragments) that are incapable of efficiently binding to their target receptors on cells because of the ineffective way they were orientated on the surface of the LNP following conjugation.

[0006] Therefore, there exists a need to develop LNPs that have surfaces modified with antibodies or antigen-binding fragments thereof (e.g., Fab fragments and / or VHH domains) wherein the antibodies or antigen-binding fragments thereof are linked to the LNP in a site-specific manner.BRIEF SUMMARY

[0007] It has been surprisingly found that antigen-binding fragments of antibodies (e.g., Fab fragments) can be site-selectively conjugated to the surface of a lipid nanoparticle (LNP) to make targeted LNPs (also referred to herein as conjugates). For instance, Fab fragments and related structures can be covalently bound to an LNP through the natural interchain disulfide bond between the heavy chain and the light chain (i.e., the CL-CH1 disulfide bond) of the antibody fragment and a lipid molecule on the LNP. Likewise, VHH domains can be covalently bound through one or more cysteine residues of a chemically reduced intramolecular disulfide bond of the VHH domain and a lipid molecule on the LNP. The targeted LNPs (conjugates) produced by the methods disclosed herein have the ability to transduce specific targeted cells. Hence, the targeted LNPs are capable of delivering therapeutic payloads to cells and can be used to treat a multitude of diseases and other disorders.

[0008] In some embodiments, the antigen-binding fragment is a Fab fragment. In some embodiments, the antigen-binding fragment is a F(ab')2 fragment. In some embodiments, the antigen-binding fragment is a Fab' fragment.

[0009] In some embodiments, the antigen-binding fragment is a VHH domain. For instance, in some embodiments, the disulfide bond can be recombinantly introduced at the C-terminus of the VHH domain. In some embodiments, the antigen-binding fragment is a VHH domain where a cysteine has been introduced (e.g., recombinantly added) to the VHH domain. In some embodiments, a cysteine is recombinantly introduced to the C-terminus of a VHH.

[0010] In some embodiments, the antigen-binding fragment is a single-chain fragment variable (scFv) where a disulfide bond has been introduced between the two variable domains (VL and VH). For instance, in some embodiments, the disulfide bond can be recombinantly introduced at the C-terminus of the scFv. In some embodiments, the antigen-binding fragment is a single-chain fragment variable (scFv) where a cysteine has been introduced (e.g., recombinantly added) to the scFV, for example, to the C-terminus of the scFv.

[0011] In one aspect, the disclosure provides a method of making a targeted LNP, said method comprising:(i) contacting 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 (CHI), 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.

[0012] In some aspects, the disclosure provides a conjugate produced by a method comprising:(i) contacting 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 (CHI), 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.

[0013] In one aspect, the disclosure provides a method of making a targeted LNP, said method comprising:(i) introducing a cysteine residue to a VHH domain or scFv;(ii) contacting the scFv or VHH domain with a reducing reagent, whereby the reducing reagent reduces any intermolecular disulfide bonds between pairs of scFvs or pairs of VHH domains (e.g., in a solution or mixture comprising a plurality of the scFvs and / or VHH domains), thereby generating two free cysteine residues; and(iii) contacting the product of step (ii) 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.

[0014] In some embodiments, step (i) involves introducing the cysteine residue at the C-terminus of the scFV or VHH domain. In some embodiments, a linker can also be introduced (e.g. recombinantly added) at the C-terminus between the scFv or VHH domain sequence and the engineered cysteine residue. In such embodiments, the scFv or VHH domain following step (i) would have the structure scFv-linker-cysteine residue or VHH domain-linker-cysteine. In some embodiments, the linker between the scFv (or VHH domain) and the cysteine residue is an amino acid linker. In some such embodiments, the amino acid linker is a flexible amino acid linker. In some embodiments, the linker is a rigid amino acid linker. In some embodiments, the linker can comprise a glycine (G) and a serine residue (S). In some embodiments, the linker can comprise GS residues. In some embodiments, the linker can comprise GGGS or GGS residues. In some embodiments, the linker can comprise repeats of GGGS, GGCS, GGS, or GS residues.

[0015] In other embodiments, a portion of a hinge region of an antibody can be introduced (e.g. recombinantly added) at the C-terminus between the scFv or VHH domain sequence and the engineered cysteine residue. In such embodiments, the scFv or VHH domain following step (i) would have the structure scFv-hinge portion-cysteine residue or VHH domain-hinge portion- cysteine residue. In some such embodiments, DKTHT can be recombinantly added at the C- terminus of the scFv / VHH, followed by a cysteine.

[0016] It will be understood that introducing a cysteine residue into a scFv or VHH domain, particularly at the C-terminus, can potentially result in dimerization of the scFv or VHH domainsvia formation of an intermolecular disulfide bond. Reduction of the disulfide bond in step (ii) through methods disclosed herein regenerates the free cysteine residues, which can then react with a thiol-reactive group in step (iii), hence generating a targeted LNP.

[0017] In some aspects, the disclosure provides a conjugate produced by a method comprising:(i) contacting a scFv or VHH domain that comprises an engineered cysteine at the C- terminus of the scFv or VHH domain with a reducing reagent; 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.

[0018] In another aspect, the disclosure provides a method of making a targeted LNP, said method comprising:(i) contacting a VHH domain with a reducing reagent, wherein the VHH domain comprises an intramolecular disulfide bond, whereby the reducing reagent reduces the intramolecular disulfide bond of the VHH domain 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.

[0019] In some aspects, the disclosure provides a conjugate produced by a method comprising:(i) contacting a VHH domain with a reducing reagent, wherein the VHH domain comprises an intramolecular disulfide bond, whereby the reducing reagent reduces the intramolecular disulfide bond of the VHH domain 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.

[0020] In some embodiments, the intramolecular disulfide bond in the VHH domain that is reduced is between Cys 22 and Cys 92 of the VHH A3 domain defined in the Kabat numbering scheme. In some embodiments, the free Cys 22 residue, following the reduction reaction in step (i), covalently binds to the precursor LNP in step (ii), thereby forming the targeted LNP. In some embodiments, the free Cys 92 residue, following the reduction reaction in step (i), covalently binds to the precursor LNP in step (ii), thereby forming the targeted LNP. In some embodiments, thefree Cys 22 and the free Cys 92 residue, following the reduction reaction in step (i), covalently bind to the precursor LNP in step (ii), thereby forming the targeted LNP.

[0021] 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 DTT, TCEP, and DTE. 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 remove following step (i), prior to conjugation.

[0022] In some embodiments, the thiol-reactive group is maleimide. In some embodiments, maleimide reacts with one of the two free cysteine residues of the antigen-binding fragment of an antibody. In some embodiments, maleimide reacts with one of the two free cysteine residues of a Fab fragment, F(ab')2 fragment, or Fab' fragment to form a thiosuccinimide moiety. In some embodiments, maleimide reacts with one of the two free cysteine residues of the VHH domain to form a thiosuccinimide moiety.

[0023] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM). Following reduction of the disulfide bond, the reduced Fab fragment, F(ab')2 fragment, Fab' fragment or VHH domain is added to DBM covalently bonded to a lipid. In some embodiments, 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.

[0024] In some embodiments, the thiol-reactive group can be introduced onto any of the lipids comprising the ENP. 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

[0025] In some aspects, the disclosure provides a conjugate comprising an ENP and a Fab fragment, wherein the LNP is covalently bonded to either or both of a first cysteine residue in the constant region of the heavy chain of the Fab fragment and / or a second cysteine residue in the constant region of light chain of the Fab fragment. In some embodiments, the Fab fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Fab fragment and the constant region of the light chain of the Fab fragment. In some embodiments, both theconstant region of the heavy chain and the constant region of the light chain of the Fab fragment are covalently bonded to the LNP. In some embodiments, only the constant region of the heavy chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the light chain remains associated with the covalently bound heavy chain on the surface of the LNP. In some embodiments, only the constant region of the light chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the heavy chain remains associated with the covalently bound light chain on the surface of the LNP. In some of the foregoing embodiments, the Fab fragment is linked to the LNP through a thio succinimide moiety. In other of the foregoing embodiments, the Fab fragment is linked to the LNP through a dithiomalemide moiety (see FIG. 6). In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through a maleamic acid moiety (see FIG. 6).

[0026] In some aspects, the disclosure provides a conjugate comprising an LNP and a F(ab')2 fragment, wherein the LNP is covalently bonded to either or both of a first cysteine residue in the constant region of the heavy chain of the F(ab')2 fragment and / or a second cysteine residue in the constant region of light chain of the F(ab')2 fragment. In some embodiments, the F(ab')2 fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the F(ab')2 fragment and the constant region of the light chain of the F(ab')2 fragment. In some embodiments, both the constant region of the heavy chain and the constant region of the light chain of the F(ab')2 fragment are covalently bonded to the LNP. In some embodiments, only the constant region of the heavy chain of the F(ab')2 fragment is covalently bonded to the LNP. In some such embodiments, the light chain remains associated with the covalently bound heavy chain on the surface of the LNP. In some embodiments, only the constant region of the light chain of the F(ab')2 fragment is covalently bonded to the LNP. In some such embodiments, the heavy chain remains associated with the covalently bound light chain on the surface of the LNP. In some of the foregoing embodiments, the F(ab')2 fragment is linked to the LNP through a thiosuccinimide moiety. In other of the foregoing embodiments, the F(ab')2 fragment is linked to the LNP through a dithiomalemide moiety. In still other of the foregoing embodiments, the F(ab')2 fragment is linked to the LNP through a maleamic acid moiety.

[0027] In some aspects, the disclosure provides a conjugate comprising an LNP and a Fab' fragment, wherein the LNP is covalently bonded to either or both of a first cysteine residue in the constant region of the heavy chain of the Fab' fragment and / or a second cysteine residue in the constant region of light chain of the Fab' fragment. In some embodiments, the Fab' fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Fab' fragmentand the constant region of the light chain of the Fab' fragment. In some embodiments, both the constant region of the heavy chain and the constant region of the light chain of the Fab' fragment are covalently bonded to the LNP. In some embodiments, only the constant region of the heavy chain of the Fab' fragment is covalently bonded to the LNP. In some such embodiments, the light chain remains associated with the covalently bound heavy chain on the surface of the LNP. In some embodiments, only the constant region of the light chain of the Fab' fragment is covalently bonded to the LNP. In some such embodiments, the heavy chain remains associated with the covalently bound light chain on the surface of the LNP. In some of the foregoing embodiments, the Fab' fragment is linked to the LNP through a thio succinimide moiety. In other of the foregoing embodiments, the Fab' fragment is linked to the LNP through a dithiomalemide moiety. In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through a maleamic acid moiety.

[0028] In some aspects, the disclosure provides a conjugate comprising an LNP and a VHH domain, wherein the LNP is covalently bonded to either or both of a first cysteine residue in the VHH domain and / or a second cysteine residue in the VHH domain, wherein the first cysteine is Cys 22 of the VHH domain defined by Kabat numbering and the second cysteine is Cys 92 of the VHH domain defined by Kabat numbering.

[0029] In some aspects, the disclosure provides a conjugate comprising a plurality of Fab fragments attached to the surface of a lipid nanoparticle (LNP) encapsulating a therapeutic agent, the conjugate comprising: a plurality of thiol reactive groups conjugated to the surface of the LNP, wherein each Fab fragment in the plurality of Fab fragments comprises a reduced interchain disulfide bond such that a cysteine on the heavy chain of the Fab fragment is no longer covalently linked to a cysteine on the light chain of the Fab fragment, and wherein each Fab fragment comprises: the heavy chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups, the light chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups, orthe heavy chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups and the light chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups.

[0030] In some embodiments, the Fab fragment conjugated to the LNP is an IgGl Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl 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 IgGl 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 IgGl Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment are covalently bonded to the LNP.

[0031] In some embodiments, the Fab fragment conjugated to the LNP is an IgG2 Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment are covalently bonded to the LNP.

[0032] In some embodiments, the Fab fragment conjugated to the LNP is an IgG4 Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment are covalently bonded to the LNP.

[0033] In another aspect, the disclosure provides a targeted LNP made by a process comprising contacting a Fab fragment that has been functionalized with a sortase tag with a precursor LNP comprising a plurality of polyglycine molecules covalently bonded to the surface of the LNP in the presence of sortase, whereby the Fab fragment is conjugated to the surface of the precursor LNP through a sortase mediated ligation of the sortase tag and one or more polyglycine molecules, thereby forming a targeted LNP.DESCRIPTION OF THE FIGURES

[0034] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0035] FIG. 1 shows an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).

[0036] FIG. 2A shows assembly of an LNP encapsulating a therapeutic payload with a functional group (e.g., maleimide, 2,3-dibromomaleimide, sortase tag, etc.) to be reacted with an antibody or antigen-binding fragment thereof. FIG. 2B shows assembly of an LNP encapsulating a therapeutic payload using a post-insertion technique.

[0037] FIG. 3 shows an exemplary schematic of the reduction of an interchain disulfide bond in a Fab fragment. After reduction, each free cysteine residue is available to react with a thiolreactive group conjugated to the surface of the LNP.

[0038] FIG. 4 shows an exemplary schematic of conjugate formation. In FIG. 4, a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)). In step (ii), the Fab fragment is contacted with an LNP comprising a plurality of thiol-reactive groups (e.g., a plurality of maleimide or DBM groups) conjugated to the surface of the LNP, whereby a thiol-reactive group of the plurality reacts with one of the two free cysteine residues of the Fab fragment.

[0039] FIG. 5 shows an exemplary schematic of conjugate formation. In FIG. 5, a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues. The reduced Fab fragment is then 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. In some cases, a maleimide group of the plurality reacts with the free cysteine residue of the heavy chain of the Fab fragment. In some cases, a maleimide group of the plurality reacts with the free cysteine residue of the light chain of the Fab fragment. In other cases, a maleimide group of the plurality reacts with the free cysteine residue of the heavy chain of the Fab fragment and a different maleimide group of the plurality reacts with the free cysteine residue of the light chain of the Fab fragment.

[0040] FIG. 6 shows an exemplary schematic of conjugate formation using DBM as a bridging agent.

[0041] FIG. 7 shows an exemplary schematic of conjugate formation. In FIG. 7, a first Fab fragment (Fabl) 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. Fab 1 and Fab 2 can be reduced in the same solution or in separate solutions. 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 one of the two free cysteine residues of the first Fab Fragment and another thiol-reactive group of the plurality reacts with one of the two free cysteine residues of the second Fab Fragment. Fab 1 and Fab 2 can be contacted with the LNP comprising the plurality of the thiol-reactive groups at the same time or concurrently. In some cases, a thio-reactive group reacts with each of the free cysteines of Fab 1 and / or Fab 2.

[0042] FIG. 8 shows an exemplary schematic of conjugate formation. In FIG. 8, a Fab fragment comprises a sortase tag, in this case having the sequence: LPETG (SEQ ID: 63). The Fab fragment is contacted with an LNP in the presence of sortase, wherein the LNP comprises a plurality of polyglycine molecules conjugated to the surface of the LNP, whereby the Fab fragment is conjugated to the surface of the LNP through a sortase mediated ligation of the sortase tag and a poly glycine molecule of the plurality.

[0043] FIGS 9A and 9B show the sizes (kDa) of anti-CD117 and anti-CD3 Fab fragments on SDS-PAGE protein gels wherein the Fab fragments were either reduced using TCEP or were not reduced prior to running on the gel. The results demonstrate that the reduction methods described herein completely reduce the interchain disulfide bond of Fab fragments, hence generating free cysteine residues. For all Fab fragments, the reduced samples show either one or two bands, dependent on sequence, around 25 kDa, with no detectable signal at the initial 50 kDa, indicating complete reduction of the interchain disulfide bond. FIGs 9C-9H show that the reduced Fab fragment samples retained their binding affinity, with on-rates and off-rates that were very similar to the non-reduced Fab fragments.

[0044] FIG. 10A provides a bar graph showing the percentage of CD34+ cells that express GFP following incubation with anti-CD117 tLNPs produced using the reduction / conjugation processdescribed herein compared to cells incubated with a base non-targeted LNP. FIG. 10B provides a bar graph showing the GFP expression levels in the CD34+ cells of FIG. 10A.

[0045] FIG. 11 provides a schematic showing the process for testing whether complete reduction of two different Fab fragments results in chain swapping between the Fab fragments following oxidation.

[0046] FIG. 12 provides a bar chart showing the amount of GFP expression following administration of dual tLNPs conjugated to either an anti-CD117 Fab and anti-CD34 Fab, or an anti-CDl 17 VHH domain and an anti-CD34 fab, at different doses.

[0047] FIGS. 13A and 13B provide bar charts showing the level of B2M knockout in human HSPCs (FIG. 13A) and LT-HSCs (FIG. 13B) engrafted in a mouse model following delivery of a gene modifying system using anti-CDl 17 tLNPs or anti-CDl 17 / anti-CD34 dual tLNPs created using the reduction / conjugation processes described herein. One of the anti-CDl 17 tLNPs was produced using a process wherein the reducing agent (TCEP) was removed prior to conjugation (- TCEP) while the other tLNPs were produced using a process wherein the reducing agent (TCEP) was not removed (+ TCEP) prior to conjugation.

[0048] FIG. 14 shows some examples of a pegylated lipid bonded to a maleimide moiety.

[0049] FIG. 15 shows some examples of non-pegylated lipid bonded to a maleimide moiety.

[0050] FIG. 16 shows some examples of ionizable lipid bonded to a maleimide moiety.

[0051] FIG. 17 shows some examples of sterols bonded to a maleimide moiety.DETAILED DESCRIPTION

[0052] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.I. Definitions

[0053] Before describing the invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0054] As used herein, the terms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.

[0055] As used here, the term “about” refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0056] As used herein, the term "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0057] As used herein, the terms “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” can be interchanged and are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0058] Although the following description uses terms “first”, “second”, etc. to describe various elements, these elements should not be limited by the terms. These terms are only used to distinguish one element from another. For example, a first antibody could be termed a second antibody, and, similarly, a second antibody could be termed a first antibody, without departing from the scope of the various described embodiments. The first antibody and the second antibody are both antibodies, but they are not the same antibody.

[0059] Antigen binding domain: The term “antigen binding domain” as used herein refers to that portion of a targeting moiety, e.g., an 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 self-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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] Gene modifying polypeptide: A “gene modifying polypeptide,” and “retrotransposon gene modifying polypeptide” as used herein interchangeably to refer to a polypeptide comprising a retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain, 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 said domains, 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 endonuclease domain is a catalytically inactive endonuclease domain. In some embodiments, the retrotransposase reverse transcriptase domain and a retrotransposase endonuclease domain are derived from the same retrotransposase. In some embodiments, the gene modifying polypeptide is capable of integrating the sequence substantially without relying on hostmachinery. 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 WO / 2021 / 178717, which is incorporated herein by reference, including Tables 10, 11, X, 3A, 3B, and Z1 therein. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid.

[0066] 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.

[0067] 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 heterologousdomain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector). In some embodiments, a domain is heterologous relative to another domain, if the first domain is not naturally comprised in the same polypeptide as the other domain (e.g., a fusion between two domains of different proteins from the same organism).

[0068] Heterologous gene modifying polypeptide: As used herein, the term “heterologous gene modifying polypeptide” refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, the heterologous gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, the heterologous gene modifying polypeptide integrates a sequence into a random position in a genome, and in some embodiments, the heterologous gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, the sequence that is integrated comprises a deletion, substitution, or insertion relative to the target DNA molecule. In some embodiments, a heterologous gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the template nucleic acid into the target DNA. Heterologous gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence. Heterologous gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) ofotherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. Exemplary heterologous gene modifying polypeptides, and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to heterologous gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a heterologous gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a heterologous gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “heterologous gene modifying system,” as used herein, refers to a system comprising a heterologous gene modifying polypeptide and a template nucleic acid.

[0069] 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.

[0070] Nucleic acid molecule: “Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. The nucleic acid molecule can be double- stranded or single-stranded, circular, or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complimentary to SEQ ID NO:1. The choice between the two is dictated by the context in which SEQ ID NO:1 is used. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturallyoccurring 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 chemically modified bases, backbone, and modified caps. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs). Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs). In various embodiments, the nucleic acids are in operative association with additional genetic elements, such as tissue-specific expression-control sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats / direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (57 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).

[0071] Primer Binding Sequence: The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region comprised in a target nucleic acid sequence. In some instances, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. In some embodiments the primer region comprises at least 5, 6, 7, 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments when a template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region comprised in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.10072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.II. Targeted Lipid Nanoparticles (Conjugates)A. Conjugating Lipid Nanoparticles to Targeting Moieties

[0073] In one aspect, the disclosure provides a method of making a targeted LNP, said method comprising:(i) contacting an antigen-binding fragment with a reducing reagent, whereby the reducing reagent reduces an interchain or intramolecular disulfide bond of the antigen-binding 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.

[0074] It has been surprisingly found that an antigen-binding fragment (e.g., Fab fragment or VHH domain) can be site- selectively conjugated to the surface of a precursor lipid nanoparticle (LNP) through methods disclosed herein to make a targeted LNP (also referred to herein as a conjugate). Particular examples of various lipid components that can be used to construct LNPs of the disclosure are discussed in Section III. The targeted LNPs (conjugates) can be used to deliver payloads that that can be used for therapeutic purposes. Particular examples of payloads are discussed in Section IV. Delivery of the payloads to targeted cells finds application in the treatment of particular diseases, some examples which are set forth in Section VII.

[0075] In some embodiments involving reduction of an interchain disulfide bond, the reduced disulfide bond is not part of the hinge region of the antigen-binding fragment.

[0076] In some embodiments, the antigen-binding fragment is a Fab fragment. In some embodiments, the antigen-binding fragment is a F(ab')2 fragment. In some embodiments, the antigen-binding fragment is a Fab' fragment. In some embodiments, the antigen-binding fragment is a VHH domain. In some embodiments, the antigen-binding fragment is a single-chain fragment variable (scFv) where a disulfide bond has been introduced between the two variable domains (VL and VH). For instance, in some embodiments, the disulfide bond can be recombinantly introduced at the C-terminus of the scFv. In some embodiments, the antigen-binding fragment is a single-chain fragment variable (scFv) wherein a cysteine has been introduced into the scFv. In some embodiments, the cysteine has be recombinantly introduced at the C-terminus of the scFv.

[0077] 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 poly glycine) prior to reacting with the antigenbinding fragment (e.g., Fab fragment or VHH domain). The process for conjugating a targeting moiety, as disclosed herein, involves reducing the natural interchain disulfide bond between the CL and CHI domains of a Fab fragment (or F(ab')2 fragment or F(ab') 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.

[0078] 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 antigen-binding 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.

[0079] Any suitable reducing reagent can be used to reduce the interchain disulfide bond of the antigen-binding fragment. Examples of reducing reagents include, but are not limited to, 2- mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP), and combinations thereof. In some embodiments, the reducing reagent is a mild reducing reagent. Examples of mild reducing reagents include, e.g., DTT, TCEP, and DTE. In some embodiments, the reducing reagent is TCEP. Any suitable reaction conditions can be used for the reduction of the interchain disulfide bond in step (i). In some embodiments, the reduction reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reduction reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reduction reaction is performed at physiological temperature (e.g. , about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25 °C. In some embodiments, the reduction reaction is performed at ambient temperature (e.g. , about 23 to about 25° C). In some embodiments, the reduction reaction is performed at about 0° C to about 4° C.

[0080] In some embodiments, the thiol-reactive group is maleimide. In some embodiments, maleimide reacts with one of the two free cysteine residues of the antigen binding fragment to form a thio succinimide moiety. In some embodiments, maleimide reacts with a free cysteine residue on the heavy chain of the antigen binding fragment. In some embodiments, maleimide reacts with a free cysteine residue on the light chain of the antigen binding fragment. In some embodiments, two maleimide groups each react with the antigen-binding fragment (e.g., a 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.

[0081] 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 antigen binding fragment in step (ii). In some embodiments, the reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reaction is performed at physiological temperature (e.g., about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reaction is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the reaction is performed at about 0° C to about 4° C.B. Targeted LNP production using interchain CL-CH1 disulfide reduction followed by conjugation (Fab fragments, F(ab')2 fragments or Fab' fragments)

[0082] 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 (CHI, CH2, and CH3). The Fab region of the antibody includes the VL, CL, VH, and CHI domains. The Fc region includes the constant heavy chain domains CH2, and CH3. A hinge region of the IgG antibody covalently links the CHI 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 IgGl possess an intermolecular disulfide bond between Cys233 (Kabat numbering) of the heavy domain andCys214 (Kabat numbering) of the light domain. Therapeutic antibodies of type IgG4 possess an intermolecular disulfide bond between Cysl27 (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 Cysl35 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.

[0083] Proteolytic cleavage of an IgG antibody results in the formation of a Fab fragment known as a F(ab')2 fragment. The F(ab’)2 fragment does not include the CH2 domain or the CH3 domain. However, the hinge region of the antibody is retained in a F(ab')2 fragment. The F(ab’)2 fragment includes disulfide bonds that covalently link two Fab fragments. Reduction of the disulfide bond in the F(ab')2 generates two Fab’ 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 Fab’ fragment, site-specific conjugation is challenging. Moreover, the reduction of the F(ab')2 to the Fab’ fragments could also disrupt the natural interchain disulfide bonds between the CL and CHI regions of the Fab fragments, hence further compromising site-specific conjugation.

[0084] In some aspects of the discloure, a Fab fragment, F(ab')2 fragment, or Fab' fragment can be conjugated to the surface of a precursor LNP through a cysteine residue of a reduced natural interchain disulfide bond between the heavy chain and the light chain (i.e., the CL-CH1 disulfide bond) of the Fab fragment, F(ab')2 fragment or the Fab' fragment to make a targeted LNP.

[0085] In some embodiments where a F(ab')2 fragment is conjugated to the surface of the precursor LNP, the disulfide bond in the hinge region of the F(ab')2 fragment is maintained (i.e., not reduced). In some embodiments where a F(ab')2 fragment is conjugated to the surface of the precursor LNP, the disulfide bond in the hinge region of the F(ab')2 is reduced. In some such embodiments, conjugation of the F(ab')2 fragment to the LNP occurs through both a cysteine residue of a reduced natural interchain disulfide bond between the heavy chain and the light chain (i.e., the CL-CH1 disulfide bond) of the F(ab')2 fragment and through a cysteine residue of a reduced disulfide bond in the hinge region of the F(ab')2 fragment.

[0086] As described herein, despite the removal of the natural interchain disulfide bond linking the heavy and light chains of the Fab fragment, F(ab')2 fragment or 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, F(ab')2 fragments or Fab' fragments for targeting immune cells or hematopoietic stem cells (HSCs) are discussed in Section V and SectionVI, respectively. Accordingly, the methodology disclosed herein provides a means of developing site-specific conjugation of Fab fragments, F(ab')2 fragments or Fab' fragments to the surface of LNPs that can effectively target particular cell types for therapeutic applications. A schematic of an LNP site- specifically conjugated to a Fab fragment is shown in FIG. 1.

[0087] In one embodiment, Fab fragments, F(ab')2 fragments or Fab' fragments used for conjugation may be generated by recombinant methods. In particular embodiments, the Fab fragments, F(ab')2 fragments or Fab' fragments generated recombinantly are designed not to include a hinge region at the C-terminus. Therefore, the recombinantly generated Fab fragments, F(ab')2 fragments or 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, F(ab')2 fragment or Fab' fragmen to the surface of an LNP.

[0088] In some embodiments, the Fab fragment F(ab')2 fragment or Fab' fragment is of the IgG class, the IgM class, or the IgA class. In some embodiments, the Fab fragment, F(ab')2 fragment or Fab' fragment is of the IgG class and has an IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fab fragment, F(ab')2 fragment or Fab' fragment is a native protein. In some embodiments, the Fab fragment F(ab')2 fragment or Fab' fragment is an engineered protein.

[0089] In one aspect, the disclosure provides a method 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 (CHI), 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.

[0090] 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 (CHI),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.

[0091] In another aspect, the disclosure provides a method of making a targeted LNP, said method comprising:(i) contacting a composition comprising a F(ab')2 fragment with a reducing reagent, wherein the F(ab')2 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 (CHI), whereby the reducing reagent reduces the interchain disulfide bond of the F(ab')2 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.

[0092] In some aspects, the disclosure provides a conjugate produced by a method comprising:(i) contacting a composition comprising a F(ab')2 fragment with a reducing reagent, wherein the F(ab')2 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 (CHI), whereby the reducing reagent reduces the interchain disulfide bond of the F(ab')2 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.

[0093] In another aspect, the disclosure provides a method 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 (CHI), 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.

[0094] 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 (CHI), 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.

[0095] In some embodiments, the thiol-reactive group (e.g., maleimide, pyridyl disulfide, 2,3- dibromomaleimide, or haloacetyl) is chemically reacted with a lipid molecule to create a modified lipid wherein the thiol-reactive group is covalently attached to the lipid where it is capable of reacting with at least one free cysteine residue of the reduced Fab fragment (either on the heavy or light chain of the Fab fragment). The reaction between the thiol-reactive group and the at least one free cysteine residue can be completed prior to or after formation of the LNP with the modified lipid. For instance, as shown in FIG. 2A, the various components (e.g., lipids) comprising the LNP and a therapeutic pay load 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 thiolreactive groups (each thiol-reactive group shown schematically as a “functional group” in FIG. 2A). The thiol-reactive group can then be reacted with at least one free cysteine residue of the Fab fragment, hence generating a conjugate. Alternatively, a lipid that has been modified with the thiol-reactive group can be directly reacted with at least one free cysteine residue of a Fab fragment. As depicted in FIG. 2B, the resultant modified lipid attached to the Fab fragment can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.

[0096] Any suitable reducing reagent can be used to reduce the interchain disulfide bond of the Fab fragment, F(ab')2 fragment or Fab' fragment. Examples of reducing reagents include, but are not limited to, 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol(DTE), and tris(carboxyethyl)phosphine (TCEP), and combinations thereof. In some embodiments, the reducing reagent is a mild reducing reagent. Examples of mild reducing reagents include, e.g., DTT, TCEP, and DTE. In some embodiments, the reducing reagent is TCEP. Any suitable reaction conditions can be used for the reduction of the interchain disulfide bond in step (i). In some embodiments, the reduction reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reduction reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reduction reaction is performed at physiological temperature (e.g., about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reduction reaction is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the reduction reaction is performed at about 0° C to about 4° C.

[0097] 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.

[0098] 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.

[0099] 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, F(ab')2 fragment or Fab' fragment. In some embodiments, maleimide reacts with a free cysteine residue on the light chain of the Fab fragment, F(ab')2 fragment or Fab' fragment. In some embodiments, two maleimide groups each react with the Fab fragment, F(ab')2 fragment or 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.

[0100] 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 F(ab')2 fragment or 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). Insome embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reaction is performed at ambient temperature e.g., about 23 to about 25° C). In some embodiments, the reaction is performed at about 0° C to about 4° C.

[0101] A schematic showing the reduction of an interchain disulfide bond in a Fab fragment is depicted in FIG. 3. In some embodiments, the Fab fragment depicted in FIG. 3 can be produced recombinantly without a hinge region at the C-terminus. Accordingly, in such embodiments, the Fab fragment only comprises a single interchain disulfide bond, which is located in the CL-CH1 interface of the Fab fragment. As shown in FIG. 3, 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.

[0102] FIG. 4 shows an exemplary schematic of conjugate formation, as described herein. In FIG. 4, a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)). In step (ii), the reduced Fab fragment is reacted with an LNP comprising a plurality of thiol-reactive groups (e.g., maleimide or DBM) conjugated to the surface of the LNP, whereby the thiol-reactive groups react with the free cysteine residues of the reduced Fab fragment. The Fab fragment is site-specifically conjugated to the surface of the LNP via a linkage through at least one of the free cysteine residues of the Fab fragment.

[0103] Following reaction of the reduced Fab fragment with the thiol-reactive group of the LNP, either the heavy chain, light chain or both the heavy chain and light chain of the Fab fragment are conjugated to the surface of the LNP. The various orientations are depicted in FIG. 5. In some embodiments, the conjugates formed by methods disclosed herein include at least one, at least two or all three orientations show in FIG. 5. The concentration of the thiol-reactive group (e.g., maleimide) will likely determine which orientation is dominant. In some embodiments, increasing the number of thiol-reactive groups on the LNP increases the number of Fab fragments conjugated to two thiol-reactive groups. In some embodiments, decreasing the number of thiol-reactive groups on the LNP decreases the number of Fab fragments conjugated to two thiol-reactive groups. Regardless of the orientation, the heavy chain and the light chain remain intact on the surface of the LNP, thus forming a functional Fab fragment that is capable of engaging with a receptor on a targeted cell.

[0104] In some embodiments, the thiol-reactive group is maleimide, as shown in FIG. 5. In some embodiments, maleimide reacts with one of the two free cysteine residues of the antibody or antigen-binding fragment thereof. In some embodiments, maleimide reacts with one of the two free cysteine residues of the Fab fragment to form a thio succinimide 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.

[0105] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM) as shown in FIG. 6. Following reduction of the disulfide bond, the reduced Fab fragment is added to DBM covalently bonded to a lipid (represented by squiggly line in FIG. 6). As set forth above, the lipid may be part of an LNP or may be post-inserted into an LNP following reaction with the Fab fragment. Both of the free cysteine residues displace the two bromine groups of DBM, hence generating a dithiomalemide. The dithiolmalemide can be converted to the corresponding maleamic acid via hydrolysis. DBM reacts with a free cysteine residue on the heavy chain and a free cysteine residue on the light chain of the Fab fragment to form a bridge between the cysteine residues. Accordingly, the heavy and light chain of the Fab fragment are effectively bridged together following reaction with DBM.

[0106] In all embodiments discussed above, the thiol-reactive group can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, the thiol-reactive group is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid-PEGx-thiol- reactive group, wherein x is 2-120 ethylene glycol units. In such embodiments, at least one free cysteine residue of a Fab fragment reacts with the thiol-reactive group bonded to the one or more of the pegylated lipids comprising the LNP. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the thiol-reactive group. In some embodiments, the PEG spacer between the lipid and the thiol-reactive group comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 1,000 (i.e., PEG1000) to about3,000 (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 component 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. 14 shows some examples of a pegylated lipid bonded to a thiol-reactive group, in this case a maleimide moiety.

[0107] In some embodiments, the thiol-reactive group is bonded to at least one of the non- pegylated phospholipids (helper lipids) comprising the LNP. FIG. 15 shows some examples of a non-pegylated lipid bonded to a maleimide moiety.

[0108] In some embodiments, the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP. FIG. 16 shows some examples of an ionizable lipid bonded to a maleimide moiety.

[0109] In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. FIG. 17 shows some examples of sterols bonded to a maleimide moiety.

[0110] 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 Lab fragment, P(ab')2 fragment or Lab' 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 Lab fragment and a second cysteine residue in the constant region of light chain of the Lab fragment. In some embodiments, the Lab fragment P(ab')2 fragment or Lab' fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Lab fragment P(ab')2 fragment or Lab' fragment and the constant region of the light chain of the Lab fragment P(ab')2 fragment or Lab' 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 Lab fragment, P(ab')2 fragment or Lab' fragment are covalently bonded to the LNP. In some embodiments, only the constant region of the heavy chain of the Lab fragment, P(ab')2 fragment or Lab' 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 Lab fragment, P(ab')2 fragment orFab' 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, F(ab')2 fragment or Fab' fragment is linked to the LNP through a thio succinimide moiety. In other of the foregoing embodiments, the Fab fragment, F(ab')2 fragment or Fab' fragment is linked to the LNP through a dithiomalemide moiety (see FIG.6). In still other of the foregoing embodiments, the Fab fragment, F(ab')2 fragment or Fab' fragment is linked to the LNP through a maleamic acid moiety (see FIG. 6).

[0111] In some embodiments, the Fab fragment, F(ab')2 fragment or Fab' fragment conjugated to the LNP is an IgGl Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment, F(ab')2 fragment or 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 IgGl Fab fragment, F(ab')2 fragment or 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 IgGl Fab fragment, F(ab')2 fragment or Fab' fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment, F(ab')2 fragment or Fab' fragment are covalently bonded to the LNP.

[0112] In some embodiments, the Fab fragment conjugated to the LNP is an IgG2 Fab fragment, F(ab')2 fragment or Fab' fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment, F(ab')2 fragment or 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, F(ab')2 fragment or 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, F(ab')2 fragment or Fab' fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment, F(ab')2 fragment or Fab' fragment are covalently bonded to the LNP.

[0113] In some embodiments, the Fab fragment conjugated to the LNP is an IgG4 Fab fragment, F(ab')2 fragment or Fab' fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment, F(ab')2 fragment or 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, F(ab')2 fragment or 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, F(ab')2 fragment or Fab' fragment and the cysteine at position 214(Kabat numbering) of the light chain of the IgG4 Fab fragment, F(ab')2 fragment or Fab' fragment is covalently to the LNP.

[0114] The processes described herein also enable the ability to conjugate two or more different Fab fragments, F(ab')2 fragment or Fab' fragments to the surface of an LNP. An embodiment showing the conjugation of two different Fab fragments (which can bind to the same or different antigen) to the surface of an LNP is shown in FIG. 7. In FIG. 7, two Fab fragments (Fabl 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 Fabl and Fab2 are conjugated to the surface of the LNP. Despite reduction of the disulfide bonds in Fabl and Fab2, the heavy chain and light chain in Fabl 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 Fabl associate with the heavy or light chain of Fab2 on the surface of the LNP.

[0115] In some embodiments involving conjugating two Fab fragments (i.e., a first Fab fragment and a second Fab fragment) to the surface of an LNP, the first Fab fragment and the second Fab fragment can be reduced in the same reaction (e.g., the first and second Fab fragments are mixed in a reaction vessel and contacted with the same reducing reagent). In some embodiments, the first Fab fragment and the second Fab fragment are reduced separately (e.g., the first and second Fab fragments thereof are each contacted with a reducing reagent in separate reaction vessels). In some embodiments, the first Fab fragment is contacted with the reducing reagent prior to step (ii) (wherein the reduced first Fab fragment is conjugated to the LNP surface). In some embodiments, the second Fab fragment is contacted with the reducing reagent after step (ii) (wherein the reduced second Fab fragment is conjugated to the 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.

[0116] 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.

[0117] In some embodiments, conjugates prepared by the method or process disclosed herein have a high density of the Fab fragment, F(ab')2 fragment or Fab' fragment on the surface of the LNP. For instance, the conjugate can comprise a plurality of Fab fragments, F(ab')2 fragment or Fab' fragments conjugated to the LNP surface. In some embodiments, the conjugate can comprise more than 10 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise more than 20 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise more than 30 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise more than 50 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise more than 75 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise more than 100 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise from about 50 to about 200 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 Fab fragments, F(ab')2 fragments or Fab' fragments per LNP.C. Targeted LNPs comprising scFv or VHH domains

[0118] In one aspect, the disclosure provides a method of making a targeted LNP, said method comprising:(i) introducing a cysteine residue to a VHH domain or scFv;(ii) contacting the scFv or VHH domain with a reducing reagent, whereby the reducing reagent reduces any intermolecular disulfide bonds between pairs of scFvs or pairs of VHH domains (e.g., in a solution or mixture comprising a plurality of the scFvs and / or VHH domains), thereby generating two free cysteine residues; and(iii) contacting the product of step (ii) 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.

[0119] In some embodiments, step (i) involves introducing the cysteine residue at the C-terminus of the scFV or VHH domain, e.g., by recombinant engineering. In some embodiments, a linker can also be introduced (e.g. recombinantly added) at the C-terminus between the scFv or VHH domain sequence and the engineered cysteine residue. In such embodiments, the scFv or VHH domain following step (i) would have the structure scFv-linker-cysteine residue or VHH domain- linker-cysteine. In some embodiments, the linker between the scFv (or VHH domain) and the cysteine residue is an amino acid linker. In some such embodiments, the amino acid linker is a flexible amino acid linker. In some embodiments, the linker is a rigid amino acid linker. In some embodiments, the linker can comprise a glycine (G) and a serine residue (S). In some embodiments, the linker can comprise GS residues. In some embodiments, the linker can comprise GGGS or GGS residues. In some embodiments, the linker can comprise repeats of GGGS, GGCS, GGS, or GS residues.

[0120] In other embodiments, a portion of a hinge region of an antibody can be introduced (e.g. recombinantly added) at the C-terminus between the scFv or VHH domain and the engineered cysteine residue. In such embodiments, the scFv or VHH domain following step (i) would have the structure scFv-hinge portion-cysteine residue or VHH domain-hinge portion-cysteine residue. In some such embodiments, DKTHT can be recombinantly added at the C-terminus of the scFv / VHH, followed by a cysteine. In some embodiments, both a linker and a hinge portion can be added to the C-terminus of the scFv or VHH domain prior to the engineered cysteine residue.

[0121] It will be understood that introducing a cysteine residue into a scFv or VHH domain, particularly at the C-terminus, can potentially result in dimerization of the scFv or VHH domainsvia formation of an intermolecular disulfide bond. Reduction of the disulfide bond in step (ii) through methods disclosed herein regenerates the free cysteine residues, which can then react with a thiol-reactive group in step (iii), hence generating a targeted LNP.

[0122] In some aspects, the disclosure provides a conjugate produced by a method comprising:(i) contacting a scFv or VHH domain that comprises an engineered cysteine at the C- terminus of the scFv or VHH domain with a reducing reagent; 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.D. Targeted LNP production using intrachain disulfide reduction followed by conjugation (VHH domains)

[0123] In another aspect, the disclosure provides a method of making a targeted LNP, said method comprising:(i) contacting a VHH domain with a reducing reagent, wherein the VHH domain comprises an intramolecular disulfide bond, whereby the reducing reagent reduces the intramolecular disulfide bond of the VHH domain 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.

[0124] In some aspects, the disclosure provides a conjugate produced by a method comprising:(i) contacting a VHH domain with a reducing reagent, wherein the VHH domain comprises an intramolecular disulfide bond, whereby the reducing reagent reduces the intramolecular disulfide bond of the VHH domain 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.

[0125] In some embodiments, the intramolecular disulfide bond in the VHH domain that is reduced is between Cys 22 and Cys 92 of the VHH A3 domain defined in the Kabat numbering scheme. In some embodiments, the free Cys 22 residue, following the reduction reaction in step (i), covalently binds to the precursor LNP in step (ii), thereby forming the targeted LNP. In someembodiments, the free Cys 92 residue, following the reduction reaction in step (i), covalently binds to the precursor LNP in step (ii), thereby forming the targeted LNP. In some embodiments, the free Cys 22 and the free Cys 92 residue, following the reduction reaction in step (i), covalently bind to the precursor LNP in step (ii), thereby forming the targeted LNP.

[0126] In some aspects, the disclosure provides a conjugate comprising an LNP and a VHH domain, wherein the LNP is covalently bonded to either or both of a first cysteine residue in the VHH domain and / or a second cysteine residue in the VHH domain, wherein the first cysteine is Cys 22 of the VHH domain defined by Kabat numbering and the second cysteine is Cys 92 of the VHH domain defined by Kabat numbering.

[0127] 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 VHH domain. The reaction between the thiol-reactive group and the at least one free cysteine residue of the VHH domain can be completed prior to or after formation of the LNP with the modified lipid.

[0128] Any suitable reducing reagent can be used to reduce the intrachain disulfide bond of the VHH domain. 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 intrachain disulfide bond in step (i). In some embodiments, the reduction reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reduction reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reduction reaction is performed at physiological temperature (e.g., about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reduction reaction is performed at ambient temperature (e.g. , about 23 to about 25° C). In some embodiments, the reduction reaction is performed at about 0° C to about 4° C.

[0129] In some embodiments, the thiol-reactive group is maleimide. In some embodiments, maleimide reacts with one of the two free cysteine residues of the VHH to form a thiosuccinimidemoiety. In some embodiments, maleimide reacts with a cysteine residue Cys 22 on the VHH domain. In some embodiments, maleimide reacts with a cysteine residue Cys 92 on the VHH domain. In some embodiments, two maleimide groups each react with the VHH domain, wherein one maleimide reacts with residue Cys 22 and the other maleimide reacts with Cys92 on the VHH domain.

[0130] 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 VHH domain. In some embodiments, the reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reaction is performed at physiological temperature (e.g., about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reaction is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the reaction is performed at about 0° C to about 4° C.

[0131] It will be understood that FIGs. 3-7, which relate to forming conjugates with Fab fragments, also apply to VHH domains.

[0132] The processes described herein also enable the ability to conjugate two or more different VHH domains to the surface of an LNP. Additionally, the process described herein enable the ability to conjugate a VHH domain and a Fab fragment (or F(ab')2 fragments or Fab' fragments) to the surface of an LNP.E. Conjugate synthesis using a sortase mediated enzymatic ligation process

[0133] In another aspect, the disclosure provides a targeted LNP made by a process comprising contacting an antibody or antigen binding fragment (e.g., Fab fragment, F(ab')2 fragment, Fab' fragment or VHH domain) that has been functionalized with a sortase tag with a precursor LNP comprising a plurality of polyglycine molecules covalently bonded to the surface of the LNP in the presence of sortase, whereby the antigen-binding fragment is conjugated to the surface of the precursor LNP through a sortase mediated ligation of the sortase tag and one or more polyglycine molecules, thereby forming a targeted LNP. A schematic of the process is depicted in FIG. 8.

[0134] In some embodiments, the antigen-binding fragment is a Fab fragment. In some embodiments, the antigen-binding fragment is a VHH domain.

[0135] In another aspect, the disclosure provides a method of making a targeted LNP comprising contacting a Fab fragment that has been functionalized with a sortase tag with a precursor LNP comprising a plurality of polyglycine molecules covalently bonded to the surface of the LNP in the presence of sortase, whereby the Fab fragment is conjugated to the surface of the precursor LNP through a sortase mediated ligation of the sortase tag and one or more polyglycine molecules, thereby forming a targeted LNP. A schematic of the process is depicted in FIG. 8.

[0136] In some embodiments, the sortase tag of the antigen-binding fragment (e.g., Fab fragment) is located at the C-terminus of the heavy chain of the Fab fragment. In some embodiments, the sortase tag comprises the amino acid sequence of LPXTG, wherein X is any amino acid (SEQ ID: 62). In some embodiments, the sortase tag is LPETG (SEQ ID: 63). In some embodiments, the sortase tag is linked to the antigen-binding fragment (e.g., Fab fragment) through an amino acid linker.

[0137] In some embodiments, a poly glycine molecule of the plurality of the precursor LNP comprises between 3 and 10 glycine residues. In some embodiments, the polyglycine molecule comprises 3, 4, 5, or 6 glycine residues. In some embodiments, the poly glycine molecule is linked to the Fab fragment through an amino acid linker.

[0138] Any suitable sortase enzyme can be used to catalyze the ligation reaction. In some embodiments, sortase is sortase A5. In some embodiments, sortase is Streptococcus pyogenes sortase A (SpSrtA WT). Any suitable conditions can be used for the sortase mediated ligation. In some embodiments, the ligation can occur in water, aqueous buffer, or cell culture media. In some embodiments, the ligation is performed at physiological pH (e.g., about 7.4). In some embodiments, the ligation is performed at physiological temperature (e.g., about 37° C). In some embodiments, the ligation is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the ligation is performed at about 0° C to about 4° C.

[0139] In some embodiments, the sortase mediated ligation forms a covalent bond. In some embodiments, the sortase mediated ligation is reversible (e.g., the Fab fragment can be released from the LNP surface). In some embodiments, the sortase mediated ligation is irreversible (e.g., the sortase mediate ligation is performed as described in Liu et al., The Journal of Organic Chemistry 2014 79 (2), 487-492). In some embodiments, the ligation efficiency between the sortase tag of the Fab fragment and the poly glycine molecule of the LNP is greater than 5%, greater than 10%, greater than 25%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the ligation efficiency between the sortase tagof the Fab fragment and the poly glycine molecule of the LNP is from about 5% to about 30%, about 10% to about 20%, about 25% to about 50%, about 30% to about 40%, about 50% to about 80%, about 60% to about 70%, about 70% to about 95%, or about 80% to about 90%. In some embodiments, the conjugate product of the disclosed method can be purified from remaining intermediate product (e.g., the antibody or functional fragment thereof) using any suitable technique such as, but not limited to, ultrafiltration and diafiltration.

[0140] 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.

[0141] In some embodiments, the conjugates prepared by the method or process disclosed herein comprise more than one Fab fragment (e.g., second, third, fourth, fifth, etc.) that bind to different antigens. In some embodiments, the conjugates prepared by the method or process disclosed herein comprise more than one Fab fragment (e.g., second, third, fourth, fifth, etc.) that bind to different epitopes on the same antigen.

[0142] In some embodiments, the polyglycine molecule conjugated to the surface of the LNP can be chemically reacted with a lipid molecule to afford a modified lipid capable of reacting with a sortase tag of a Fab fragment through a sortase mediated ligation. The reaction between the polyglycine molecule and the sortase tag of the Fab fragment can be completed prior to or after formation of the LNP. For instance, as shown in FIG. 2, the various components (e.g., lipids) comprising the LNP can be mixed with lipid molecules that each comprise a poly glycine molecule, thus generating an LNP that comprises a plurality of poly glycine molecules. A poly glycine molecule of the plurality can then be reacted with the sortase tag of the antibody or antigen-bindingfragment thereof, hence generating a conjugate. Alternatively, a lipid that has been modified with the polyglycine molecule can be directly reacted with the sortase tag of the antibody or antigenbinding fragment thereof. The resultant modified lipid can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.

[0143] The polyglycine molecule can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, the poly glycine molecule is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid- PEGx-poly glycine molecule, wherein x is 2-120 ethylene glycol units. In such embodiments, the sortase tag of the Fab fragment reacts with the polyglycine molecule bonded the one or more of the pegylated lipids comprising the LNP. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the polyglycine molecule. In some embodiments, the PEG spacer between the lipid and the polyglycine molecule comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the polyglycine molecule is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the poly glycine molecule is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the polyglycine molecule is bonded to at least one of the non-pegylated phospholipids comprising the LNP. In some embodiments, the polyglycine molecule is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the polyglycine molecule is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, the lipid component of the pegylated lipid bonded to the polyglycine molecule is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid. [

[0144] In some embodiments, the LNP comprises a sortase tag conjugated to the surface of the LNP. In such embodiments, an Fab fragment comprises a polyglycine molecule, wherein the Fab fragment is conjugated to the surface of the LNP through a sortase mediated ligation of the sortase tag of the LNP and the polyglycine molecule of the antibody or antigen-binding fragment thereof. In some embodiments, the sortase tag of the LNP comprises the amino acid sequence of LPXTG, wherein X is any amino acid (SEQ ID: 62). In some embodiments, the sortase tag is LPETG (SEQ ID: 63).

[0145] In some embodiments, the sortase tag conjugated to the surface of the LNP can be chemically reacted with a lipid molecule to afford a modified lipid capable of reacting with a polyglycine molecule of an Fab fragment through a sortase mediated ligation. The reaction between the sortase tag and the polyglycine molecule of the Fab fragment can be completed prior to or after formation of the LNP. For instance, as shown in FIG. 2, the various components (e.g., lipids) comprising the LNP can be mixed with lipid molecules that each comprise a sortase tag, thus generating an LNP that comprises a plurality of sortase tags. A sortase tag of the plurality can then be reacted with the polyglycine molecule of the antibody or antigen-binding fragment thereof, hence generating a conjugate. Alternatively, a lipid that has been modified with the sortase tag can be directly reacted with the polyglycine molecule of the antibody or antigen-binding fragment thereof. The resultant modified lipid can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.

[0146] The sortase tag can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, the sortase tag is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid-PEGx-sortase tag, wherein x is 2-120 ethylene glycol units. In such embodiments, a polyglycine molecule of an Fab fragment reacts with the sortase tag bonded the one or more of the pegylated lipids comprising the LNP. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the sortase tag. In some embodiments, the PEG spacer between the lipid and the sortase tag comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the sortase tag is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the sortase tag is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the sortase tag is bonded to at least one of the non- pegylated phospholipids comprising the LNP. In some embodiments, the sortase tag is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the sortase tag is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, the lipid component of the pegylated lipid bonded to the sortase tag is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.III. Lipid nanoparticles

[0147] 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.

[0148] 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 WO20 19217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers descried in Table 5 of WO2019217941, incorporated by reference.

[0149] 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.

[0150] 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.

[0151] 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 % ofthe 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 po ypeptide, 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.

[0152] In some embodiments, the average LNP diameter of the targeted LNP formulation may be between 10 nm and 150 nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 70 nm to about 150 nm, from about 80 nm to about 120 nm, from about 80 nm to about 110 nm, from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the targeted LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation ranges from about 1 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.

[0153] An LNP described herein, e.g., a targeted LNP, may, in some instances, be relatively homogenous. A poly dispersity 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.

[0154] 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.

[0155] 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%.

[0156] 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.

[0157] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by W02020061457, which is incorporated herein by reference in its entirety.

[0158] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Minis 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-[l,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.

[0159] 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

[0160] The LNPs of the disclosure (e.g., targeted LNPs) comprise one or more ionizable lipids. In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine- containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s), which bear the positive charge. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid, and a therapeutic agent as described herein (e.g., one or more nucleic acids (e.g., RNA) comprising a gene modifying system) encapsulated within or associated with the lipid nanoparticle. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) is coformulated with the cationic lipid. The therapeutic agent (e.g., one or more nucleic acids) may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments,the therapeutic agent (e.g., one or more nucleic acids) may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described 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).

[0161] 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.

[0162] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid V003, depicted below. V003 is described in U.S. Patent No. 10,059,655.Lipid V003

[0163] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid shown in Table 1. In some cases, an LNP containing an ionizable lipid of Table 1 exhibits higher levels of transduction in immune cells (e.g., T cells) and / or higher expression of a payload protein in immune cells (e.g., T cells) relative to an LNP that contains V003 as the ionizable lipid. In other embodiments, Lipid 092 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 093 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 153 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipidl54is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 155 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 162 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipidl63 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 169 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 176 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 178 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 183 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).

[0164] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid shown in Table 1. In some cases, an LNP containing an ionizable lipid of Table 1 exhibits higher levels of transduction in HSCs (e.g., HSPCs or LT-HSCs) and / or higher expression of a pay load protein in HSCs (e.g., HSPCs or LT-HSCs) relative to an LNP that contains V003 as the ionizable lipid. In other embodiments, Lipid 092 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 093 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 153 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipidl54 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 155 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 162 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipidl63 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 169 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 176 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 178 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs). In other embodiments, Lipid 183 is used as an ionizable lipid to generate LNPs for delivery to HSCs (e.g., HSPCs or LT-HSCs).Table 1: Exemplary Ionizable Lipids

[0165] 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-ioalkyl;R2is Ci-4alkyl;R3and R4are independently Ci-4 alkyl; andY is a lipophilic tail, branched or unbranched.

[0166] In some embodiments of the formula (I), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. In some embodiments, R2is C4 lkyl. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y isIn 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.

[0167] In some embodiments, compounds of formula (I) are compounds of formula (I- A):formula (I- A), or a pharmaceutically acceptable salt thereof, wherein:X is O or CH2; m is 1, 2, or 3;R1is C4-ioalkyl;R2is Ci-4alkyl;R3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle; andY is a lipophilic tail, branched or unbranched.

[0168] In some embodiments of the formula (I- A), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. In some embodiments, R2is C4 lkyl. In some embodiments, Y isin some embodiments, Y isIn 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 / / -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.

[0169] 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-ioalkyl;R2is Ci-4alkyl; andR3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0170] In some embodiments of the formula (I-B), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. 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 7 - ropyl. 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.

[0171] 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-ioalkyl;R2is Ci-4alkyl; andR3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0172] In some embodiments of the formula (I-C), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. In some embodiments, R2is C4 lkyl. 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 7 - ropyl. 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.

[0173] 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-ioalkyl;R2is Ci-4alkyl; andR3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0174] In some embodiments of the formula (I-D), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. In some embodiments, R2is C4 lkyl. 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 7 - ropyl. 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.

[0175] 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-ioalkyl;R2is Ci-4alkyl; andR3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0176] In some embodiments of the formula (I-E), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. 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 7 - ropyl. 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.

[0177] 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-ioalkyl;R2is Ci-4alkyl; andR3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0178] In some embodiments of the formula (I-F), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. In some embodiments, R2is C4 lkyl. 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 7 - ropyl. 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.

[0179] 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-ioalkyl;R2is Ci-4alkyl; andR3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0180] In some embodiments of the formula (I-G), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. 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 7 - ropyl. 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.

[0181] 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-ioalkyl;R2is Ci-4alkyl; andR3and R4are independently Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0182] In some embodiments of the formula (I-H), R1is Ce-salkyl. In some embodiments, R1is Cealkyl. In some embodiments, R2is Caalkyl. 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 7 - ropyl. 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.

[0183] 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; andY is a lipophilic tail, branched or unbranched.In some embodiments of the formula (II), Y is. In some embodiments, Y isIn 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.

[0184] 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.

[0185] In some embodiments of the formula (II-A), Y is

[0186] 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 Ci-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle; andY is a lipophilic tail, branched or unbranched.

[0187] In some embodiments of the formula (III), Y is. In some embodiments, Y is. In some embodiments, Y is, is,. In some embodiments, Y is o . In some embodiments, Y is. In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0188] In some embodiments, compounds of formula (III) are compounds of formula (III- A):formula (III-A), or a pharmaceutically acceptable salt thereof, wherein:Y is a lipophilic tail, branched or unbranched.

[0189] In some embodiments of the formula (III- A), Y is. In some embodiments, Y is. In some embodiments, Y is

[0190] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 2.Table 2: Exemplary Ionizable Lipids

[0191] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (IV):or a pharmaceutically acceptable salt thereof, wherein:X is -0- or -CH2-; m is 0, 1, 2, or 3;R1is Ci-4alkyl;R2is Ci-4alkyl; n is 1, 2, 3, or 4;R3is C4-ioalkyl;R4is C4-ioalkyl; p is 2, 3, 4, 5, or 6;R5is C4-ioalkyl; andR6is C4-ioalkyl.

[0192] In some embodiments, compounds of formula (IV) are compounds of formula (IV-A):or a pharmaceutically acceptable salt thereof, wherein: p is 2, 3, 4, 5, or 6;R5is C4-ioalkyl; andR6is C4-ioalkyl.

[0193] In some embodiments, compounds of formula (IV) are compounds of formula (IV-B):or a pharmaceutically acceptable salt thereof, wherein:R5is C4-6alkyl; andR6is C4-6alkyl.

[0194] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 3.Table 3: Exemplary Ionizable Lipids

[0195] In some embodiments, the ionizable lipid has one of the structures depicted below:Lipid 092Lipid 154Lipid 163Lipid 178Lipid 232

[0196] Other exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016 / 0311759; I of US20150376115 or in US2016 / 0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013 / 0178541; I of US2013 / 0303587 or US2013 / 0123338; I of US2015 / 0141678; II, III, IV, or V of US2015 / 0239926; I of US2017 / 0119904; I or II of WO2017 / 117528; A of US2012 / 0149894; A of US2015 / 0057373; A of WO2013 / 116126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of W02013 / 016058; A of W02012 / 162210; I of US2008 / 042973; I, II, III, or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131; A of US2012 / 01011478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of US2013 / 0323269; I of US2011 / 0117125; I, II, or III of US2011 / 0256175; I,II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242; XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 0116307; I, II, or III of US2013 / 0116307; I or II of US2010 / 0062967; I-X of US2013 / 0189351; I of US2014 / 0039032; V of US2018 / 0028664; I of US2016 / 0317458; I of US2013 / 0195920; 5, 6, or 10 of US10,221,127; III-3 of W02018 / 081480; 1-5 or 1-8 of W02020 / 081938; 18 or 25 of US9,867,888; A of US2019 / 0136231; II of WO2020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013; cKK-E12 / A6 of Miao et al (2020); C12-200 of W02010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of US9,708,628; I of W02020 / 106946; I of W02020 / 106946.

[0197] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3- nonyldocosa-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US9,867,888 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of W02015 / 095340(incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-l-yl) 9-((4- dimethylamino)butanoyl)oxy)heptadecanedioate (L319), , e.g. as synthesized in Example 7, 8, or 9 of US2012 / 0027803(incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is l,l'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxy dodecyl) amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of W02010 / 053572(incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is; Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)- 10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4-yl)propanoate, e.g., Structure (I) from W02020 / 106946 (incorporated by reference herein in its entirety).

[0198] 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%.

[0199] The compounds disclosed herein (e.g., lipids in Table 1, Table 2, and Table 3), or their pharmaceutically acceptable salts, may include an asymmetric center and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another and “diastereomers,” which refers to stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (), or (7?) 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

[0200] 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 16-0-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, l-stearoyl-2- oleoyl- phosphatidy ethanolamine (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.

[0201] Other examples of non-cationic lipids suitable for use in the lipid nanoparticles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017 / 099823 or US patent publication US2018 / 0028664, the contents of which is incorporated herein by reference in their entirety. In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety.

[0202] 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.

[0203] 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 about8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0204] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 21% to about 23%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%. As set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs (e.g., targeted LNPs) with these percentages of helper lipids provides enhanced transduction and expression of the payloads relative to targeted LNPs with smaller or larger quantities of helper lipid.

[0205] 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.

[0206] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table 1, Table 2 or Table 3 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table 1, Table 2 or Table 3 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. Insome 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%.

[0207] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid of Formula I and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid of Formula I and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0208] 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%.

[0209] n some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0210] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 153 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 153 and a sphingomyelin. Insome 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%.

[0211] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 154 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 154 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0212] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 155 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 155 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.=

[0213] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 162 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 163 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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, isabout 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0214] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 169 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 169 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0215] 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%.

[0216] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 178 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 178 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0217] 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, isfrom 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%.

[0218] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid V003 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid V003 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.Sterols

[0219] 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)-butyl 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.

[0220] In some embodiments, the component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30- 40% (mol) of the total lipid content of the lipid nanoparticle.

[0221] 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 someembodiments, 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

[0222] 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.

[0223] 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 phosphatidy lethanoloamine (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 US5,885,613, US6,287,591,

[0224] 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, thePEG-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:

[0225] 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-lipidconjugates (such as ATTA-lipid conjugates), and cationic -polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.

[0226] 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 W02020106946A1, the contents of all of which are incorporated herein by reference in their entirety.

[0227] In some embodiments, the pegylated lipid has at least one C16 (palmitoyl) PEG lipid anchor. In some embodiments, the pegylated lipid has two C16 PEG lipid anchors (i.e., dialkyl chains of 16 carbons long). In some embodiments, the pegylated lipid is 1,2-dipalmitoyl sn- g]ycero-3-phosphoethanolamine-N-[methox (polyethylene glycol)-2000] (DPPE-PEG2000.). In some embodiments, the pegylated lipid is l,2-Dipalmitoyl-rac-glycero-3 -methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is C16 PEG ceramide. In some embodiments, the targeted LNPs comprising the C16 pegylated lipids show reduced liver uptake than otherwise identical LNPs comprising C14 pegylated lipids.

[0228] In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0229] In some embodiments, the pegylated lipid has at least one C18 PEG lipid anchor. In some embodiments, the pegylated lipid has two C18 PEG lipid anchors (i.e., dialkyl chains of 18 carbons long). In some embodiments, the C18 pegylated lipid is l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the C18 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG-PEG2000).

[0230] 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

[0231] 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 therapeuticagent, 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, wherein the 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.

[0232] 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.

[0233] In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation is produced using a plurality of lipid reagents, and each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph.

[0234] 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.

[0235] 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.IV. Therapeutic payloads

[0236] The targeted LNPs (conjugates) described herein can be used to deliver payloads (e.g., comprising therapeutic agents) to cells, such as, but not limited to, immune cells (e.g., T cells) or HSCs (e.g., LT-HSCs) or HSC progenitors.

[0237] 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 pay load is an RNA template (for example, an RNA template for reverse transcription, e.g., Target Primed Reverse Transcription (TPRT)). In other embodiments the pay load is a siRNA or a microRNA (miRNA). In other embodiments, the payload comprises a guide RNA for a CRISPR-Cas system. In other embodiments, the payload is a tRNA. In other embodiments, the pay load is an antisense oligonucleotide (ASO). In otherembodiments, 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. In other embodiments, the payload is a small molecule. In some embodiments, the payload is a peptide or protein. In some embodiments, the conjugates disclosed herein can include two or more payloads, for example, selected from RNA (such as mRNA, ncRNA, guide RNA, siRNA, miRNA), an ASO, DNA vector, small molecule, peptide, and protein.

[0238] The conjugates (targeted LNPs) described herein can be used to deliver a therapeutic of interest to a cell, such as, but not limited to an immune cell (e.g., a T cell), HSC or HSC progenitor. In some embodiments, the conjugate (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 such as, but not limited to, an immune cell, such as a T cell, 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.

[0239] 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 or comprise 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, such as, but not limited to, an immune cell, such as 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 such as, but not limited to, an immune cell such as 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 nucleicacids (e.g., RNA and / or DNA), or combinations thereof. In some embodiments, the therapeutic agent can be a fusion protein, e.g., a fusion protein comprising a nuclease (e.g., an endonuclease such as Cas9 or a functional portion thereof) and a protein domain comprising recombinase, integrase, transposase, retrotransposase, helicase, transcriptase, polymerase, reverse transcriptase, deaminase, methylase, demethylase, or ligase activity.

[0240] 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 such as, but not limited to, an immune cell, such as 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 or RNA comprising 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, transcriptase, polymerase, reverse transcriptase, deaminase, methylase, demethylase, or ligase, or combinations thereof.

[0241] In certain embodiments, the therapeutic agent comprises a nuclease (e.g., an endonuclease) or a nucleic acid encoding the nuclease (e.g., an endonuclease). In some embodiments, the nuclease cleaves DNA to create a double stranded break, leading to the introduction of 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 some embodiments, the nuclease is a CRISPR-Cas protein. In some embodiments, the nuclease is a recombinant nuclease. In some embodiments, the nuclease is a restriction endonuclease, meganuclease, homing endonuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[0242] In some embodiments, the conjugates (targeted LNPs) of the disclosure can be used to deliver gene editing components into cells. In some embodiments, the conjugates described herein can be used to deliver a therapeutic agent comprising a CRISPR-Cas system or a component thereof into a cell. In some embodiments, the therapeutic agent comprises a Class 1 (type I, type III, or type IV) CRISPR-Cas protein or a nucleic acid encoding the CRISPR-Cas protein. In some embodiments, the therapeutic agent comprises a Class 2 (type II, type V, or type VI) CRISPR - Cas protein or a nucleic acid encoding the CRISPR-Cas protein. In some embodiments, the therapeutic agent comprises a CRISPR-Cas9 system, or a nucleic acid encoding one or more components of the CRISPR-Cas9 system. In some embodiments, the therapeutic agent comprisesa CRISPR-Casl2 system (e.g., a Casl2a system), or a nucleic acid encoding one or more components of the CRISPR-Casl2 system. In some such embodiments, the conjugates described herein can comprise two RNA molecules, such as an RNA comprising a guide RNA (gRNA) and an mRNA encoding a CRISPR-Cas protein. In some embodiments, the Cas is Cas9 or Casl2a. In some embodiments, the Cas is Cas9. In some embodiments, the Cas is Cas 12a. 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.

[0243] In some embodiments, the therapeutic agent comprises one of the following CRISPR- Cas proteins or a nucleic acid (e.g., mRNA) encoding one of the following CRISPR-Cas proteins: Cas9 (e.g., dCas9 and nCas9), Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Cas 12e / CasX, Cas 12g, Cas 12h, or Cas 12i. In some embodiments, the therapeutic agent comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof, or a nucleic acid encoding the Cas9 or functional fragment thereof. In some embodiments, the therapeutic agent comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference. In embodiments, the therapeutic agent comprises one of the following CRISPR-Cas proteins or a nucleic acid (e.g., mRNA) encoding one of the following CRISPR-Cas proteins: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csxl2), CaslO, CaslOd, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2b / C2cl, Casl2c / C2c3, SpCas9(K855A), eSpCas9(l.l), SpCas9-HFl, hyper accurate Cas9 variant (HypaCas9), SpRYCas9, homologues thereof, modified or engineered versions thereof, and / or functional fragments thereof. In embodiments, the Cas9 comprises one or more substitutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, D1125A, W 1126A, and DI 127A. In embodiments, the Cas9 comprises one or more mutations at positions selected from: DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, e.g., one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. In some embodiments, the therapeutic agent comprises a Cas (e.g., Cas9) or a nucleic acid encoding a Cas from Corynebacterium ulcerans,Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or a fragment or variant thereof.

[0244] In some embodiments, the therapeutic agent comprises a Cpfl domain, e.g., comprising one or more substitutions, e.g., at position D917, E1006A, D1255 or any combination thereof, e.g., selected from D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, and D917A / E1006A / D1255A, or a nucleic acid encoding the same.

[0245] In some embodiments, the therapeutic agent comprises an spCas9, spCas9-VRQR, spCas9- VRER, xCas9, saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9- LRVSQL, or a nucleic acid encoding the same.

[0246] In some embodiments, the therapeutic agent comprises a Cas9 nickase (nCas9), such as an S. pyogenes nCas9, e.g., wherein the Cas9 comprises an amino acid substitution at position DIO or H840, e.g., D10A or H840A, or a nucleic acid (e.g., mRNA) encoding the Cas9 nickase (nCas9). In some embodiments, the therapeutic agent comprises a catalytically inactive or “dead” Cas9 (dCas9), such as an S. pyogenes Cas9, e.g., wherein the Cas9 comprises an amino acid substitution at positions DIO and H840, e.g., D10A and H840A, or a nucleic acid (e.g., an mRNA) encoding the catalytically inactive Cas9 (dCas9).

[0247] In certain embodiments, the therapeutic agent comprises a deaminase, such as a cytidine deaminase or an adenine deaminase, or a nucleic acid (e.g., mRNA) encoding a deaminase. In some embodiments, conjugates (targeted LNPs) described herein deliver the deaminase to a target cell to generate a substitution mutation in the DNA, e.g., genomic DNA, of the cell. In some embodiments, the therapeutic agent is a base editor, as described in the art, e.g., a cytidine base editor (CBE) or an adenine nucleobase editor (ABE). Examples of therapeutic agents comprising a deaminase or nucleic acids encoding deaminases can be found in PCT Application Nos. PCT / US2014 / 038359, PCT / US2017 / 045381, PCT / US2018 / 024208, PCT / US2018 / 056146, and PCT / US2019 / 050112 incorporated herein by reference in their entirety, including the sequence listing and sequences therein.

[0248] In certain embodiments, the therapeutic agent can be used for epigenome editing. In some embodiments, the therapeutic agent comprises a methylase and / or a demethylase, or one or more nucleic acids (e.g., one or more mRNA) encoding a demethylase and / or a methylase. In some embodiments, the therapeutic agent demethylates DNA (e.g., genomic DNA) and / orhistones. In some embodiments, the therapeutic agent methylates DNA (e.g., genomic DNA) and / or histones. In some embodiments, the therapeutic agent can be useful in altering the transcription of a gene (e.g., via gene silencing or gene activation using CRISPRoff and / or CRISPRon). In some embodiments, the therapeutic agent can comprise a DNA methyltransferase domain or a nucleic acid encoding a DNA methyltransferase domain. In some embodiments, the therapeutic agent can comprise a KRAB domain, DNMT3A domain, DNMT3B domain, DNMT1 domain, DNMMT3L domain, or SETDB1 domain, or can comprise a nucleic acid encoding a KRAB domain, DNMT3A domain, DNMT3B domain, DNMT1 domain, DNMMT3L domain, SETDB1 domain, VP64 domain, p65 domain, TET1 domain, TET2 domain, or TET3 domain. Examples of therapeutic agents comprising a methylase and / / or demethylase or nucleic acids encoding methylases and / or demethylases can be found in PCT Application Nos. PCT / IB 2015 / 058202, PCT / US2021 / 035244, and PCT / US2021 / 035937 incorporated herein by reference in their entirety, including the sequence listing and sequences therein.

[0249] In certain embodiments, the therapeutic agent can be used to alter or modify a nucleic acid sequence, e.g., to introduce an indel or a substitution into DNA (e.g., genomic DNA) by inducing target-primed reverse transcription (TPRT) to insert a heterologous sequence into the DNA. In certain embodiments, the therapeutic agent (i.e., payload) can be a gene modifying protein, a nucleic acid encoding a gene modifying protein, or a gene modifying system, as described herein. In some embodiments, the therapeutic agent can be a gene modifying polypeptide or nucleic acid encoding a gene modifying polypeptide. In some embodiments, the therapeutic agent can be a template RNA for use with the gene modifying polypeptide. In some embodiments, the therapeutic agent delivered by a conjugate (targeted LNP) described herein is one or more components of a gene modifying system, e.g., a gene modifying polypeptide (or nucleic acid encoding the gene modifying polypeptide) and / or a template RNA for use with the gene modifying polypeptide. In some embodiments, the therapeutic agent comprises or is derived from a retrotransposon or mobile genetic element (MGE). In some embodiments, the therapeutic agent can be a heterologous gene modifying polypeptide or nucleic acid encoding a heterologous gene modifying polypeptide, as described herein. In some embodiments, the therapeutic agent can be an RNA for use with the heterologous gene modifying polypeptide. In some embodiments, the therapeutic agent delivered by a conjugate (targeted LNP) described herein is one or more components of a heterologous gene modifying system, e.g., a heterologous gene modifying polypeptide (or nucleic acid encoding the heterologous gene modifying polypeptide) and / or an RNA for use with the heterologous gene modifying polypeptide. In some embodiments, the therapeutic agent is a fusion protein, e.g., an endonuclease protein fused to a reverse transcriptase,e.g., an endonuclease nickase fused to a reverse transcriptase. In some embodiments, the therapeutic agent is a fusion protein, e.g., an endonuclease protein fused to a polymerase, e.g., an endonuclease nickase fused to a polymerase.

[0250] Other examples of therapeutic agents can be found in PCT Application Nos. PCT / US2020 / 023730, PCT / US2021 / 031439, PCT / US2020 / 055156, PCT / US2021 / 052097, PCT / US2022 / 012054, PCTUS2022 / 023175, PCT / US2022 / 074628, and PCT / US2023 / 065947 incorporated herein by reference in their entirety, including the sequence listing and sequences therein.

[0251] 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 CRISPR-Cas nuclease (such as a nickase), restriction endonuclease, meganuclease, homing endonuclease, zinc finger nuclease (ZFN), or a transcription activator- like effector nuclease (TALEN).

[0252] In certain embodiments, the therapeutic agent can be a small molecule. In certain embodiments, the therapeutic agent can be an siRNA or miRNA.A. Gene Modifying Systems

[0253] The conjugates (targeted LNPs) described herein can be formulated to comprise one or more components of a gene modifying system or one or more nucleic acids encoding said components. Accordingly, in some embodiments, the pay load comprises a gene modifying system, or one or more nucleic acids encoding the components of the gene modifying system. For instance, in some embodiments, the payload comprises a template RNA and an mRNA encoding the gene modifying polypeptide. In some embodiments, conjugates prepared in accordance with this disclosure are used to deliver to target cells systems that are capable of inserting a heterologous object sequence (e.g., a sequence encoding a CAR) into the genome of the cell, e.g., an immune cell, such as a T cell. In some embodiments, the system comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises: (i) an endonuclease and / or DNA binding domain; and (ii) a reverse transcriptase (RT) domain, where (i) and (ii) are both derived from a retrotransposon (e.g., from the same retrotransposon or different retrotransposons); and (B) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence. A gene modifying polypeptide, in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule inthe host cell), substantially without relying on host machinery. The heterologous object sequence may include, e.g., a coding sequence, a regulatory sequence, a gene expression unit.

[0254] In some embodiments, systems described herein can have a number of advantages relative to various earlier systems. For instance, the disclosure describes retrotransposases capable of inserting long sequences of heterologous nucleic acid into a genome. In addition, retrotransposases described herein can insert heterologous nucleic acid in an endogenous site in the genome, such as the rDNA locus. This is in contrast to Cre / loxP systems, which require a first step of inserting an exogenous loxP site before a second step of inserting a sequence of interest into the loxP site.(i) Gene modifying polypeptides

[0255] Non-long terminal repeat (LTR) retrotransposons are a type of mobile genetic elements that are widespread in eukaryotic genomes. They include, for example, the apurinic / apyrimidinic endonuclease (APE)-type, the restriction enzyme-like endonuclease (RLE)-type, and the Penelope-like element (PLE)-type.

[0256] The APE class retrotransposons are comprised of two functional domains: an endonuclease / DNA binding domain, and a reverse transcriptase domain. Examples of APE-class retrotransposons can be found, for example, in Table 1 of PCT Application No. PCT / US2019 / 048607, US 2023 / 0235358, US 2023 / 0242899, and US 2020 / 0109398, the disclosures of which are incorporated herein by reference in their entireties, including the sequence listing and sequences referred to in Table 1 in PCT / US2019 / 048607 and US 2020 / 0109398.

[0257] The RLE class are comprised of three functional domains: a DNA binding domain, a reverse transcription domain, and an endonuclease domain. Examples of RLE-class retrotransposons can be found, for example, in Table 2 of PCT Application No. PCT / US2019 / 048607, US 2023 / 0235358, US 2023 / 0242899, and US 2020 / 0109398, the disclosures of which are incorporated herein by reference in their entireties, including the sequence listing and sequences referred to in Table 2 in in PCT / US2019 / 048607 and US 2020 / 0109398.

[0258] The reverse transcriptase domain of non-LTR retrotransposon functions by binding an RNA sequence template and reverse transcribing it into the host genome’s target DNA. The RNA sequence template has a 3’ untranslated region which is specifically bound to the retrotransposase, and a variable 5’ region generally having Open Reading Frame(s) (“ORF”) encodingretrotransposase proteins. The RNA sequence template may also comprise a 5’ untranslated region which specifically binds the retrotransposase.

[0259] Penelope-like elements (PLEs) are distinct from both LTR and non-LTR retrotransposons. PLEs generally comprise a reverse transcriptase domain distinct from that of APE and RLE elements, but similar to that of telomerases and Group II introns, and an optional GIY-YIG endonuclease domain.

[0260] Other exemplary classes of retrotransposon include, without limitation, RTE (e.g., RTE- 1_MD, RTE-3_BF, and RTE-25_LMi), CR1 (e.g., CR1-1_PH), Crack (e.g., Crack-28_RF), L2 (e.g., L2-2_Dre and L2-5_GA), and Vingi (e.g., Vingi-l_Acar) retrotransposons.

[0261] As described herein, the elements of such retrotransposons can be functionally modularized and / or modified to target, edit, modify or manipulate a target DNA sequence, e.g., to insert an object (e.g., heterologous) nucleic acid sequence into a target genome, e.g., a mammalian genome, by reverse transcription. In some embodiments, a gene modifying system comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a retrotransposase reverse transcriptase domain, and (ii) a retrotransposase endonuclease domain that contains DNA binding functionality; and (B) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence. The RNA template element of a gene modifying system is typically heterologous to the polypeptide element and provides an object sequence to be inserted (reverse transcribed) into the host genome.

[0262] In some embodiments, the gene modifying system comprises a retrotransposase sequence of an element listed in any one of Table 10, Table 11, Table X, Table Z1 Table 3A, or 3B of PCT Pub. No.: WO / 2021 / 178717, US 2023 / 0235358, and US 2023 / 0242899, which are incorporated herein by reference as they relate to domains from retrotransposons.

[0263] In some embodiments, an amino acid sequence encoded by an element of Table 4 is an amino acid sequence encoded by the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the full-length sequence of an element listed in Table 4 may comprise one or more (e.g., all of) of a 5’ UTR, polypeptide-encoding sequence, or 3’ UTR of a retrotransposon as described herein. In some embodiments, an amino acid sequence of Table 4 is an amino acid sequence encoded by the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identitythereto. In some embodiments, a 5’ UTR of an element of Table 4 comprises a 5’ UTR of the full length sequence of an element listed in Table 7, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, a 3’ UTR of an element of Table 4 comprises a 3’ UTR of the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0264] Also indicated in Table 4 are the host organisms from which the nucleic acid sequences were obtained and a listing of domains present within the polypeptide encoded by the open reading frame of the nucleic acid sequence.

[0265] In certain embodiments, the gene modifying polypeptide further comprises a heterologous protein domain.

[0266] Table 4 provides gene modifying polypeptides comprising retrotransposon elements, altered for improved efficiency of integration into the human genome. Retrotransposase polypeptides were improved through consensus mapping to re-derive the optimal amino acid sequence. Template molecules for use with cognate retrotransposase enzymes were mapped back to their host genomes and flanking genomic DNA used to elucidate target site motifs. When detectable, conserved sequence motifs from the flanking genomic DNA of endogenous occurrences of an element were aligned to the human genome, and new sequences were derived from the human genome as 5’ or 3’ “Human Homology Arms.” In some embodiments, a template RNA described herein comprises one or both of a first homology domain comprising a sequence of a 5' Human Homology Arm of Table 4 (or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto) and a second homology domain comprising a sequence of a 3' Human Homology Arm of Table 4 (or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto).Table 4: Retrotransposase systems with improved integration activityRetrotransposon discovery tools

[0267] As the result of repeated mobilization over time, transposable elements in genomic DNA often exist as tandem or interspersed repeats (Jurka Curr Opin Struct Biol 8, 333-337 (1998)). Tools capable of recognizing such repeats can be used to identify new elements from genomic DNA and for populating databases, e.g., Repbase (Jurka et al Cytogenet Genome Res 110, 462- 467 (2005)). One such tool for identifying repeats that may comprise transposable elements is RepeatFinder (Volfovsky et al Genome Biol 2 (2001)), which analyzes the repetitive structure of genomic sequences. Repeats can further be collected and analyzed using additional tools, e.g., Censor (Kohany et al BMC Bioinformatics 7, 474 (2006)). The Censor package takes genomic repeats and annotates them using various BLAST approaches against known transposable elements. An all-frames translation can be used to generate the ORF(s) for comparison.

[0268] Other exemplary methods for identification of transposable elements include RepeatModeler2, which automates the discovery and annotation of transposable elements in genome sequences (Flynn et al bioRxiv (2019)). In addition to accomplishing this via available packages like Censor, one can perform an all-frames translation of a given genome or sequence and annotate with a protein domain tool like InterProScan, which tags the domains of a given amino acid sequence using the InterPro database (Mitchell et al. Nucleic Acids Res 47, D351-360 (2019)), allowing the identification of potential proteins comprising domains associated with known transposable elements.

[0269] Retrotransposons can be further classified according to the reverse transcriptase domain using a tool such as RTclassl (Kapitonov et al Gene 448, 207-213 (2009)).(ii) Polypeptide component of gene modifying system a) RT domain

[0270] In certain aspects, the reverse transcriptase domain of the gene modifying system is based on a reverse transcriptase domain of an APE-type or RLE-type non-LTR retrotransposon, or of a PLE-type retrotransposon. A wild-type reverse transcriptase domain of an APE-type, RLE-type, or PLE-type retrotransposon can be used in a gene modifying system or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) to alter the reverse transcriptase activity for target DNA sequences. In some embodiments, the reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the reverse transcriptase domain is a heterologous reverse transcriptase from a different LTR-retrotransposon, non-LTR retrotransposon, or other source. In certainembodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a RTE (e.g., RTE-1_MD, RTE-3_BF, and RTE-25_LMi), CR1 (e.g., CR1- 1_PH), Crack (e.g., Crack-28_RF), L2 (e.g., L2-2_Dre and L2-5_GA), and Vingi (e.g., Vingi- l_Acar) retrotransposon.

[0271] In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a retrotransposon listed in Table 10, Table 11, Table X, Table Zl, Table Z2, or Table 3A or 3B of PCT Pub. No.: WO / 2021 / 178717.

[0272] In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a retrotransposon listed in Table 4. In some embodiments, the amino acid sequence of the reverse transcriptase domain of a gene modifying system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of a reverse transcriptase domain of a retrotransposon whose DNA sequence is referenced in Table 4. Reverse transcriptase domains can be identified, for example, based upon homology to other known reverse transcription domains using routine tools as Basic Local Alignment Search Tool (BLAST). In some embodiments, reverse transcriptase domains are modified, for example by site-specific mutation. In some embodiments, the reverse transcriptase domain is engineered to bind a heterologous template RNA.

[0273] In some embodiments, a polypeptide (e.g., RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence. In some embodiments, a template RNA comprises an RNA sequence that is specifically bound by the RNA-binding domain.

[0274] In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, the RT domain is monomeric. In some embodiments, an RT domain naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer. Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, the RT function of the system is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, the multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.

[0275] In some embodiment, a gene modifying polypeptide described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of the RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, mutation of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16(l):265-277 (1988), e.g., lower by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is abolished.

[0276] In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. For instance, in some embodiments, a YADD (SEQ ID NO: 39) or YMDD (SEQ ID NO: 40) motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD (SEQ ID NO: 41). In embodiments, replacement of the YADD (SEQ ID NO: 39) or YMDD (SEQ ID NO: 40) or YVDD (SEQ ID NO: 41) results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011.) b) Endonuclease domain

[0277] In some embodiments, the polypeptide comprises an endonuclease domain (e.g., a heterologous endonuclease domain). In certain embodiments, the endonuclease / DNA binding domain of an APE-type retrotransposon, the endonuclease domain of an RLE-type retrotransposon, or the endonuclease domain of a PLE-type retrotransposon can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein. In some embodiments, the endonuclease domain or endonuclease / DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the endonuclease element is a heterologous endonuclease element. The amino acid sequence of an endonuclease domain of a gene modifying system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table X, Zl, Z2, 3A, or 3B of PCT Pub. No: WO / 2021 / 178717.

[0278] In certain embodiments, a gene modifying system includes a polypeptide that comprises an endonuclease domain of a retrotransposon listed in Table 4. In some embodiments, the amino acid sequence of the endonuclease domain of a gene modifying system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of a endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table 4. Endonuclease domains can be identified, for example, based upon homology to other known endonuclease domains using tools as Basic Local Alignment Search Tool (BLAST).

[0279] In some embodiments, a gene modifying polypeptide possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA) binding domain. In certain embodiments, the endonuclease / DNA binding domain of an APE-type retrotransposon or the endonuclease domain of an RLE-type retrotransposon can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein. c. Template nucleic acid binding domain

[0280] A gene modifying polypeptide typically contains regions capable of associating with the template nucleic acid (e.g., template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs, e.g., secondary structures present in the 3’ UTR in non-LTR retrotransposons. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) RNA binding domain is contained within the reverse transcription domain, e.g., the reverse transcriptase-derived component has a known signature for RNA preference, e.g., secondary structures present in the 3’ UTR in non-LTR retrotransposons. d) DNA binding domain

[0281] In certain aspects, the DNA-binding domain of a gene modifying polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence. In certain embodiments, the DNA-binding domain of the engineered retrotransposon is a heterologous DNA-binding protein or domain relative to a native retrotransposon sequence. In certain embodiments, the heterologous DNA-binding domain is a DNA binding domain of aretrotransposon described in Table 4 herein or in Table X, Table Zl, Table Z2, or Table 3A or 3B of PCT Pub. No.: WO / 2021 / 178717. In some embodiments, DNA binding domains can be identified based upon homology to other known DNA binding domains using tools as Basic Local Alignment Search Tool (BLAST). In still other embodiments, DNA-binding domains are modified, for example by site-specific mutation. In some embodiments, the DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells.

[0282] In embodiments, the DNA binding domain comprises one or more modifications relative to a wild-type DNA binding domain, e.g., a modification via directed evolution, e.g., phage- assisted continuous evolution (PACE).

[0283] In certain aspects of the present invention, the host DNA-binding site integrated into by the gene modifying system can be in a gene, in an intron, in an exon, an ORF, outside of a coding region of any gene, in a regulatory region of a gene, or outside of a regulatory region of a gene. In other aspects, the engineered retrotransposon may bind to one or more than one host DNA sequence. In other aspects, the engineered retrotransposon may have low sequence specificity, e.g., bind to multiple sequences or lack sequence preference.

[0284] In some embodiments, a gene modifying system is used to edit a target locus in multiple alleles. In some embodiments, a gene modifying system is designed to edit a specific allele. For example, a gene modifying polypeptide may be directed to a specific sequence that is only present on one allele, e.g., comprises a template RNA with homology to a target allele, e.g., an annealing domain, but not to a second cognate allele. In some embodiments, a gene modifying system can alter a haplotype- specific allele. In some embodiments, a gene modifying system that targets a specific allele preferentially targets that allele, e.g., has at least a 2, 4, 6, 8, or 10-fold preference for a target allele. e) Localization sequences for gene modifying systems

[0285] In certain embodiments, a gene modifying system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence.

[0286] The nuclear localization sequence may be an RNA sequence that promotes the import of the RNA into the nucleus. In certain embodiments, the nuclear localization signal is located on the template RNA. In certain embodiments, the retrotransposase polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nuclear localization signal is located on the template RNA and not on an RNA encoding the retrotransposase polypeptide.While not wishing to be bound by theory, in some embodiments, the RNA encoding the retrotransposase is targeted primarily to the cytoplasm to promote its translation, while the template RNA is targeted primarily to the nucleus to promote its retrotransposition into the genome. In some embodiments, the nuclear localization signal is at the 3’ end, 5’ end, or in an internal region of the template RNA. In some embodiments the nuclear localization signal is 3’ of the heterologous sequence (e.g., is directly 3’ of the heterologous sequence) or is 5’ of the heterologous sequence (e.g., is directly 5’ of the heterologous sequence). In some embodiments, the nuclear localization signal is placed outside of the 5’ UTR or outside of the 3’ UTR of the template RNA. In some embodiments the nuclear localization signal is placed between the 5’ UTR and the 3’ UTR, wherein optionally the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is an anti-sense orientation or is downstream of a transcriptional termination signal or poly adenylation signal). In some embodiments, the nuclear localization sequence is situated inside of an intron. In some embodiments a plurality of the same or different nuclear localization signals are in the RNA, e.g., in the template RNA. In some embodiments, the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bp in length. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences, which drive RNA localization into the nucleus. In some embodiments, the nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments, the nuclear localization signal binds a nuclear-enriched protein. In some embodiments, the nuclear localization signal binds the HNRNPK protein. In some embodiments the nuclear localization signal is rich in pyrimidines, e.g., is a C / T rich, C / U rich, C rich, T rich, or U rich region. In some embodiments, the nuclear localization signal is derived from a long noncoding RNA. In some embodiments, the nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments, the nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments, the nuclear localization sequence is described in Shukla et al., The EMBO Journal e98452 (2018). In some embodiments, the nuclear localization signal is derived from a non-LTR retrotransposon, an LTR retrotransposon, retrovirus, or an endogenous retrovirus.

[0287] In some embodiments, a polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example, a nuclear localization sequence (NLS), e.g., as described herein. In some embodiments, the NLS is a bipartite NLS. In some embodiments, anNLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, the NLS is fused to the N-terminus of a gene modifying polypeptide described herein. In some embodiments, the NLS is fused to the C-terminus of the gene modifying polypeptide. In some embodiments, a linker sequence is disposed between the NLS and the neighboring domain of the gene modifying polypeptide. In some embodiments, an NLS comprises the amino acid sequence of an NLS described herein.

[0288] In some embodiments, a nucleic acid described herein (e.g., an RNA encoding a gene modifying polypeptide, or a DNA encoding the RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a gene modifying system. For instance, the microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the RNA encoding the gene modifying polypeptide is present in a non-target cell, it would be bound by the miRNA, and when the RNA encoding the gene modifying polypeptide is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the RNA encoding the gene modifying polypeptide may reduce production of the gene modifying polypeptide, e.g., by degrading the mRNA encoding the polypeptide or by interfering with translation. Accordingly, the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells. A system having a microRNA binding site in the RNA encoding the gene modifying polypeptide (or encoded in the DNA encoding the RNA) may also be used in combination with a template RNA that is regulated by a second microRNA binding site.

[0289] In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide sequence of multiple retrotransposons. In some embodiments, a 5’ or 3’ untranslated region for use in any of the systems described herein can be a molecular reconstruction based upon the aligned 5’ or 3’ untranslated region of multiple retrotransposons. Based on the Accession numbers, polypeptides or nucleic acid sequences can be aligned, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD-Search for conserved domain analysis. Molecular reconstructions can be created based upon sequence consensus, e.g. using approaches described in Ivies et al., Cell 1997, 501 - 510 ; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99. In some embodiments, the retrotransposon from which the 5’ or 3’ untranslated region or polypeptide is derived is a young or a recently active mobile element, asassessed via phylogenetic methods such as those described in Boissinot et al., Molecular Biology and Evolution 2000, 915-928. f) Inteins

[0290] In some embodiments, the gene modifying system comprises an intein. Generally, an intein comprises a polypeptide that has the capacity to join two polypeptides or polypepide fragments together via a peptide bond. In some embodiments, the intein is a trans-splicing intein that can join two polypeptide fragments, e.g., to form the polypeptide component of a system as described herein.(iii) Promoters

[0291] In some embodiments, one or more promoter or enhancer elements are operably linked to a nucleic acid encoding a gene modifying protein or a template nucleic acid, e.g., that controls expression of the heterologous object sequence. In certain embodiments, the one or more promoter or enhancer elements comprise cell-type or tissue specific elements. In some embodiments, the promoter or enhancer is the same or derived from the promoter or enhancer that naturally controls expression of the heterologous object sequence.

[0292] In some embodiments, a gene modifying system is capable of producing a substitution into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides. In some embodiments, the substitution is a transition mutation. In some embodiments, the substitution is a transversion mutation. In some embodiments, the substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine.

[0293] In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capableof producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases).

[0294] In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g. transcription or translation) of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g. transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g. sequences that bind transcription factors. In some embodiments, an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g. alters an amino acid sequence), inserts or deletes a start or stop codon, alters or fixes the translation frame of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g. by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g. from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g. adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g. improves) protein folding (e.g. to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g. a protein encoded by the gene.

[0295] In some embodiments, a system or method described herein results in “scarless” insertion of the heterologous object sequence, while in some embodiments, the target site can show deletions or duplications of endogenous DNA as a result of insertion of the heterologous sequence. The mechanisms of different retrotransposons could result in different patterns of duplications or deletions in the host genome occurring during retrotransposition at the target site. In someembodiments, the system results in a scarless insertion, with no duplications or deletions in the surrounding genomic DNA. In some embodiments, the system results in a deletion of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA upstream of the insertion. In some embodiments, the system results in a deletion of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA downstream of the insertion. In some embodiments, the system results in a duplication of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA upstream of the insertion. In some embodiments, the system results in a duplication of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA downstream of the insertion.

[0296] In some embodiments, a gene modifying system described herein, or a DNA-binding domain thereof, binds to its target site specifically, e.g., as measured using an assay of Example 21 of PCT Application No. PCT / US2019 / 048607. In some embodiments, the gene modifying polypeptide or DNA-binding domain thereof binds to its target site more strongly than to any other binding site in the human genome. For example, in some embodiments, in an assay of Example 21 of PCT Application No. PCT / US2019 / 048607, the target site represents more than 50%, 60%, 70%, 80%, 90%, or 95% of binding events of the gene modifying polypeptide or DNA-binding domain thereof to human genomic DNA. In some embodiments, the DNA binding domain of the gene modifying polypeptide is heterologous to the remainder of the gene modifying polypeptide, e.g., such that the gene modifying polypeptide targets a different target site that the endogenous DNA binding domain associated with the remainder of the gene modifying polypeptide.(\\}Genetically engineered, e.g., dimerized gene modifying polypeptides

[0297] Some non-LTR retrotransposons utilize two subunits to complete retrotransposition (Christensen et al PNAS 2006). In some embodiments, a retrotransposase described herein comprises two connected subunits as a single polypeptide. For instance, two wild-type retrotransposases could be joined with a linker to form a covalently “dimerized” protein. In some embodiments, the nucleic acid coding for the retrotransposase codes for two retrotransposase subunits to be expressed as a single polypeptide. In some embodiments, the subunits are connected by a peptide linker. Based on mechanism, not all functions are required from both retrotransposase subunits. In some embodiments, the fusion protein may consist of a fully functional subunit and a second subunit lacking one or more functional domains. In some embodiments, one subunit may lack reverse transcriptase functionality. In some embodiments, one subunit may lack the reverse transcriptase domain. In some embodiments, one subunit may possess only endonuclease activity. In some embodiments, one subunit may possess only an endonuclease domain. In someembodiments, the two subunits comprising the single polypeptide may provide complimentary functions.

[0298] In some embodiments, one subunit may lack endonuclease functionality. In some embodiments, one subunit may lack the endonuclease domain. In some embodiments, one subunit may possess only reverse transcriptase activity. In some embodiments, one subunit may possess only a reverse transcriptase domain. In some embodiments, one subunit may possess only DNA- dependent DNA synthesis functionality.(v) Evolved Variants of Gene Modifying Polypeptides

[0299] In some embodiments, the invention provides evolved variants of gene modifying polypeptides. Evolved variants are described, e.g., at p. 1179-1182 of PCT application WO / 2021 / 178720.(vi) Template RNA component of gene modifying system

[0300] The gene modifying systems described herein can transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription. By writing DNA sequence(s) via reverse transcription of the RNA sequence template directly into the host genome, the gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step. Therefore, the gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and / or function information.

[0301] In some embodiments, the template RNA encodes a gene modifying protein in cis with a heterologous object sequence. Various cis constructs were described, for example, in Kuroki- Kami et al (2019) Mobile DNA 10:23 (incorporated by reference herein in its entirety), and can be used in combination with any of the embodiments described herein. For instance, in some embodiments, the template RNA comprises a heterologous object sequence, a sequence encoding a gene modifying protein (e.g., a protein comprising (i) a reverse transcriptase domain and (ii) an endonuclease domain, e.g., as described herein), a 5’ untranslated region, and a 3’ untranslated region. The components may be included in various orders. In some embodiments, the gene modifying protein and heterologous object sequence are encoded in different directions (sense vs. anti-sense), e.g., using an arrangement shown in Figure 3A of Kuroki-Kami et al, Id. In some embodiments, the gene modifying protein and heterologous object sequence are encoded in the same direction. In some embodiments, the nucleic acid encoding the polypeptide and the templateRNA or the nucleic acid encoding the template RNA are covalently linked, e.g., are part of a fusion nucleic acid, and / or are part of the same transcript. In some embodiments, the fusion nucleic acid comprises RNA or DNA.

[0302] The nucleic acid encoding the gene modifying polypeptide may, in some instances, be 5’ of the heterologous object sequence. For example, in some embodiments, the template RNA comprises, from 5’ to 3’, a 5’ untranslated region, a sense-encoded gene modifying polypeptide, a sense-encoded heterologous object sequence, and 3’ untranslated region. In some embodiments, the template RNA comprises, from 5’ to 3’, a 5’ untranslated region, a sense-encoded gene modifying polypeptide, anti-sense-encoded heterologous object sequence, and 3’ untranslated region.

[0303] It is understood that, when a template RNA is described as comprising an open reading frame or the reverse complement thereof, in some embodiments the template RNA must be converted into double stranded DNA (e.g., through reverse transcription) before the open reading frame can be transcribed and translated.

[0304] In certain embodiments, customized RNA sequence template can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure / altemative splicing; causing disruption of an endogenous gene; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up- or down-regulation of operably liked genes, etc. In certain embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and / or transgenes, provide for binding sites to transcription factor activators, repressors, enhancers, etc., and combinations of thereof. In other embodiments, the coding sequence can be further customized with splice acceptor sites, poly-A tails. In certain embodiments the RNA sequence can contain sequences coding for an RNA sequence template homologous to the retrotransposase, be engineered to contain heterologous coding sequences, or combinations thereof.

[0305] The template RNA may have some homology to the target DNA. In some embodiments the template RNA has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of exact homology to the target DNA at the 3’ end of the RNA. In some embodiments the template RNA has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, 180, or 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% homology to the target DNA, e.g., at the 5’ end of the template RNA. In some embodiments, the template RNA has a 3’ untranslated region derived from a retrotransposon, e.g. a retrotransposons described herein. In some embodiments the template RNA has a 3’ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the 3’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g. a retrotransposon in Table 4. In some embodiments, the template RNA has a 5’ untranslated region derived from a retrotransposon, e.g. a retrotransposons described herein. In some embodiments the template RNA has a 5’ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, or 200 or more bases of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater homology to the 5’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g. a retrotransposon described in Table 4.

[0306] The template RNA component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template RNA has a 3’ region that is capable of binding a gene modifying genome editing protein. The binding region, e.g., 3’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system.

[0307] The template RNA component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template RNA has a 5’ region that is capable of binding a gene modifying protein. The binding region, e.g., 5’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system. In some embodiments, the 5’ untranslated region comprises a pseudoknot, e.g., a pseudoknot that is capable of binding to the gene modifying protein.

[0308] In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a stem-loop sequence. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a hairpin. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a helix. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a psuedoknot. In some embodiments, the template RNA comprises a ribozyme. In some embodiments the ribozyme is similar to a hepatitis delta virus (HDV) ribozyme, e.g., has a secondary structure like that of the HDV ribozyme and / or has one or more activities of the HDVribozyme, e.g., a self-cleavage activity. See, e.g., Eickbush et al., Molecular and Cellular Biology, 2010, 3142-3150.

[0309] In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 3’ untranslated region) comprises one or more stem-loops or helices. Exemplary structures of R2 3’ UTRs are shown, for example, in Ruschak et al. “Secondary structure models of the 3' untranslated regions of diverse R2 RNAs” RNA. 2004 Jun; 10(6): 978-987, e.g., at Figure 3, therein, and in Eikbush and Eikbush, “R2 and R2 / R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements” Mobile DNA (2012) 3:10; e.g., at Figure 3 therein, which articles are hereby incorporated by reference in their entirety.

[0310] In some embodiments, a template RNA described herein comprises a sequence that is capable of binding to a gene modifying protein described herein. For instance, in some embodiments, the template RNA comprises an MS2 RNA sequence capable of binding to an MS2 coat protein sequence in the gene modifying protein. In some embodiments, the template RNA comprises an RNA sequence capable of binding to a B-box sequence. In some embodiments, in addition to or in place of a UTR, the template RNA is linked (e.g., covalently) to a non-RNA UTR, e.g., a protein or small molecule.

[0311] In some embodiments, the template RNA has a poly- A tail at the 3’ end. In some embodiments, the template RNA does not have a poly- A tail at the 3’ end.

[0312] In some embodiments the template RNA has a 5’ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 or more bases of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater homology to the 5’ sequence of a retrotransposon, e.g., a retrotransposon described herein.

[0313] The template RNA of the system typically comprises an object sequence for insertion into a target DNA. The object sequence may be coding or non-coding.

[0314] In some embodiments, a system or method described herein comprises a single template RNA. In some embodiments, a system or method described herein comprises a plurality of template RNAs.

[0315] In some embodiments, the object sequence may contain an open reading frame. In some embodiments, the template RNA has a Kozak sequence. In some embodiments, the template RNA has an internal ribosome entry site. In some embodiments, the template RNA has a self-cleavingpeptide such as a T2A or P2A site. In some embodiments, the template RNA has a start codon. In some embodiments, the template RNA has a splice acceptor site. In some embodiments, the template RNA has a splice donor site. Exemplary splice acceptor and splice donor sites are described in US 10435677, incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those of skill in the art and include, by way of example only, CTGACCCTTCTCTCTCTCCCCCAGAG (SEQ ID NO: 42) (from human HBB gene) and TTTCTCTCCCACAAG (SEQ ID NO: 43) (from human immunoglobulin-gamma gene). In some embodiments the template RNA, has a microRNA binding site downstream of the stop codon. In some embodiments, the template RNA has a polyA tail downstream of the stop codon of an open reading frame. In some embodiments, the template RNA comprises one or more exons. In some embodiments, the template RNA comprises one or more introns. In some embodiments, the template RNA comprises a eukaryotic transcriptional terminator. In some embodiments, the template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments, the RNA comprises the human T-cell leukemia virus (HTLV-1) R region. In some embodiments, the RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WPRE). In some embodiments, in the template RNA, the heterologous object sequence encodes a polypeptide and is coded in an antisense direction with respect to the 5’ and 3’ UTR. In some embodiments, in the template RNA, the heterologous object sequence encodes a polypeptide and is coded in a sense direction with respect to the 5’ and 3’ UTR.

[0316] In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a gene modifying system. For instance, the microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the template RNA is present in a non-target cell, it would be bound by the miRNA, and when the template RNA is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the template RNA may interfere with insertion of the heterologous object sequence into the genome. Accordingly, the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells. A system having a microRNA binding site in the template RNA (or DNA encoding it) may also be used in combination with a nucleic acid encoding a gene modifying polypeptide, wherein expression of the gene modifyingpolypeptide is regulated by a second microRNA binding site, e.g., as described herein, e.g., in the section entitled “Polypeptide component of gene modifying system.”

[0317] In some embodiments, the object sequence may contain a non-coding sequence. For example, the template RNA may comprise a promoter or enhancer sequence. In some embodiments, the template RNA comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional. In some embodiments, the promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments, the promoter comprises a TATA element. In some embodiments, the promoter comprises a B recognition element. In some embodiments, the promoter has one or more binding sites for transcription factors. In some embodiments, the non-coding sequence is transcribed in an antisense-direction with respect to the 5’ and 3’ UTR. In some embodiments, the non-coding sequence is transcribed in a sense direction with respect to the 5’ and 3’ UTR.

[0318] In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a promoter sequence, e.g., a tissue specific promoter sequence. In some embodiments, the tissue- specific promoter is used to increase the target-cell specificity of a gene modifying system. For instance, the promoter can be chosen on the basis that it is active in a target cell type but not active in (or active at a lower level in) a non-target cell type. Thus, even if the promoter integrated into the genome of a non-target cell, it would not drive expression (or only drive low-level expression) of an integrated gene. A system having a tissuespecific promoter sequence in the template RNA may also be used in combination with a microRNA binding site, e.g., in the template RNA or a nucleic acid encoding a gene modifying protein, e.g., as described herein. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a DNA encoding a gene modifying polypeptide, driven by a tissue-specific promoter, e.g., to achieve higher levels of gene modifying protein in target cells than in non-target cells.

[0319] In some embodiments, a heterologous object sequence comprised by a template RNA (or DNA encoding the template RNA) is operably linked to at least one regulatory sequence. In some embodiments, the heterologous object sequence is operably linked to a tissue-specific promoter, such that expression of the heterologous object sequence, e.g., a therapeutic protein, is upregulated in target cells, as above. In some embodiments, the heterologous object sequence is operably linked to a miRNA binding site, such that expression of the heterologous object sequence, e.g., a therapeutic protein, is downregulated in cells with higher levels of the corresponding miRNA, e.g., non-target cells, as above.

[0320] In some embodiments, the template RNA comprises a microRNA sequence, a siRNA sequence, a guide RNA sequence, a piwi RNA sequence.

[0321] In some embodiments, the template RNA comprises a non-coding heterologous object sequence, e.g., a regulatory sequence. In some embodiments, integration of the heterologous object sequence thus alters the expression of an endogenous gene. In some embodiments, integration of the heterologous object sequence upregulates expression of an endogenous gene. In some embodiments, integration of the heterologous object sequence downregulated expression of an endogenous gene.

[0322] In some embodiments, the template RNA comprises a site that coordinates epigenetic modification. In some embodiments, the template RNA comprises an element that inhibits, e.g., prevents, epigenetic silencing. In some embodiments, the template RNA comprises a chromatin insulator. For example, the template RNA comprises a CTCF site or a site targeted for DNA methylation.

[0323] In order to promote higher level or more stable gene expression, the template RNA may include features that prevent or inhibit gene silencing. In some embodiments, these features prevent or inhibit DNA methylation. In some embodiments, these features promote DNA demethylation. In some embodiments, these features prevent or inhibit histone deacetylation. In some embodiments, these features prevent or inhibit histone methylation. In some embodiments, these features promote histone acetylation. In some embodiments, these features promote histone demethylation. In some embodiments, multiple features may be incorporated into the template RNA to promote one or more of these modifications. CpG dinucleotides are subject to methylation by host methyl transferases. In some embodiments, the template RNA is depleted of CpG dinucleotides, e.g., does not comprise CpG nucleotides or comprises a reduced number of CpG dinucleotides compared to a corresponding unaltered sequence. In some embodiments, the promoter driving transgene expression from integrated DNA is depleted of CpG dinucleotides.

[0324] In some embodiments, the template RNA comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. The effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA).

[0325] In some embodiments, the object sequence of the template RNA is inserted into a target genome in an endogenous intron. In some embodiments, the object sequence of the template RNA is inserted into a target genome and thereby acts as a new exon. In some embodiments, the insertionof the object sequence into the target genome results in replacement of a natural exon or the skipping of a natural exon.

[0326] In some embodiments, the object sequence of the template RNA is inserted into the target genome in a genomic safe harbor site, such as AAVS1, CCR5, or ROSA26. In some embodiments, the object sequence of the template RNA is inserted into the albumin locus. In some embodiments, the object sequence of the template RNA is inserted into the TRAC locus. In some embodiments, the object sequence of the template RNA is added to the genome in an intergenic or intragenic region. In some embodiments, the object sequence of the template RNA is added to the genome 5’ or 3’ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous active gene. In some embodiments, the object sequence of the template RNA is added to the genome 5’ or 3’ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous promoter or enhancer. In some embodiments, the object sequence of the template RNA can be, e.g., 50-50,000 base pairs (e.g., between 50-40,000 bp, between 500-30,000 bp between 500-20,000 bp, between 100-15,000 bp, between 500-10,000 bp, between 50-10,000 bp, between 50-5,000 bp. In some embodiments, the heterologous object sequence is less than 1,000, 1,300, 1500, 2,000, 3,000, 4,000, 5,000, or 7,500 nucleotides in length.

[0327] The template nucleic acid (e.g., template RNA) component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template nucleic acid (e.g., template RNA) has a 3’ region that is capable of binding a gene modifying protein. The binding region, e.g., 3’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system. The binding region may associate the template nucleic acid (e.g., template RNA) with any of the polypeptide modules. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with an RNA-binding domain in the polypeptide. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with the reverse transcription domain of the polypeptide (e.g., specifically bind to the RT domain). For example, where the reverse transcription domain is derived from a non-LTR retrotransposon, the template nucleic acid (e.g., template RNA) may contain a binding region derived from a non-LTR retrotransposon, e.g., a 3’ UTR from a non-LTR retrotransposon. In some embodiments a system or method described herein comprises a single template nucleic acid (e.g., template RNA). In some embodiments a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs). In some embodiments, when the systemcomprises a plurality of nucleic acids, each nucleic acid comprises a conjugating domain. In some embodiments, a conjugating domain enables association of nucleic acid molecules, e.g., by hybridization of complementary sequences.

[0328] In some embodiments, the template nucleic acid may comprise one or more UTRs (e.g., a 5’ UTR or a 3’ UTR, e.g., from an R2-type retrotransposon). In some embodiments, the UTR facilitates interaction of the template with the reverse transcriptase domain of the polypeptide. In some embodiments, the template possesses one or more sequences aiding in association of the template with the gene modifying polypeptide. In some embodiments, these sequences may be derived from retrotransposon UTRs. In some embodiments, the UTRs may be located flanking the desired insertion sequence. In some embodiments, a sequence with target site homology may be located outside of one or both UTRs. In some embodiments, the sequence with target site homology can anneal to the target sequence to prime reverse transcription. In some embodiments, the 5’ and / or 3’ UTR may be located terminal to the target site homology sequence. In some embodiments, the gene modifying system may result in the insertion of a desired payload without any additional sequence (e.g., a gene expression unit without UTRs used to bind the gene modifying protein).

[0329] The template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein the reverse transcription will result in insertion of the heterologous sequence into the target DNA. In other embodiments, the RNA template may be designed to write a deletion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In other embodiments, the template nucleic acid (e.g., template RNA) may be designed to write an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.

[0330] In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionallyno more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases).(vii) Methods and Compositions for Modified RNA (e.g., template RNA)

[0331] In some embodiments, an RNA component of the system (e.g., a template RNA, as described herein) comprises one or more nucleotide modifications. In some embodiments, the modification pattern of the template RNA can significantly affect in vivo activity compared to unmodified or end-modified guides. Without wishing to be bound by theory, this process may be due, at least in part, to a stabilization of the RNA conferred by the modifications. Non-limiting examples of such modifications may include 2'-O-methyl (2'-0-Me), 2'-O-(2-methoxyethyl) (2'- O-MOE), 2'- fluoro (2'-F), phosphorothioate (PS) bond between nucleotides, G-C substitutions, and inverted basic linkages between nucleotides and equivalents thereof.

[0332] In some embodiments, the template RNA (e.g., at the portion thereof that binds a target site) comprises a 5' terminus region. In some embodiments, the template RNA does not comprise a 5' terminus region. In some embodiments, the 5' terminus region comprises a 5' end modification. In some embodiments, the template RNA comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the template RNA comprises a 2'-O-(2-methoxy ethyl) (2'-O-moe) modified nucleotide. In some embodiments, the template RNA comprises a 2'-fluoro (2'- F)modified nucleotide. In some embodiments, the template RNA comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the template RNA comprises a 5' end modification, a 3' end modification, or 5' and 3' end modifications. In some embodiments, the 5' end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the 5' end modification comprises a 2'-O-methyl (2'-0-Me), 2'-O-(2-methoxy ethyl) (2'-0-M0E), and / or 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the 5' end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2'-O-methyl (2'-O- Me), 2'-O-(2-methoxyethyl) (2'-0-M0E), and / or 2'-fluoro (2'-F) modified nucleotide. The end modification may comprise a phosphorothioate (PS), 2'-O-methyl (2'-0-Me) , 2'-O-(2- methoxyethyl) (2'-0-M0E), and / or 2'-fluoro (2'-F) modification. Equivalent end modifications are also encompassed by embodiments described herein. In some embodiments, the template RNA comprises an end modification in combination with a modification of one or more regions of the template RNA. In some embodiments, structure-guided and systematic approaches are used to introduce modifications (e.g., 2'-0Me-RNA, 2'-F-RNA, and PS modifications) to a template RNA, for example, as described in Mir et al. Nat Commun 9:2641 (2018) (incorporated by reference herein in its entirety). In some embodiments, the incorporation of 2'-F-RNAs increases thermal and nuclease stability of RNA:RNA or RNA:DNA duplexes, e.g., while minimally interfering with C3'-endo sugar puckering. In some embodiments, 2'-F may be better tolerated than 2'-OMe at positions where the 2'-OH is important for RNA:DNA duplex stability. In some embodiments, structure-guided and systematic approaches (e.g., as described in Mir et al. Nat Commun 9:2641 (2018); incorporated herein by reference in its entirety) are employed to find modifications for the template RNA. In some embodiments, a structure of polypeptide bound to template RNA is used to determine non-protein-contacted nucleotides of the RNA that may then be selected for modifications, e.g., with lower risk of disrupting the association of the RNA with the polypeptide. Secondary structures in a template RNA can also be predicted in silico by software tools, e.g., the RNAstructure tool available at ma.urmc.rochester.edu / RNAstructureWeb (Bellaousov et al. Nucleic Acids Res 4EW471-W474 (2013); incorporated by reference herein in its entirety), e.g., to determine secondary structures for selecting modifications, e.g., hairpins, stems, and / or bulges.

[0333] It is contemplated that it may be useful to employ circular and / or linear RNA states during the formulation, delivery, or gene modifying reaction within the target cell. Thus, in some embodiments of any of the aspects described herein, a gene modifying system comprises one or more circular RNAs (circRNAs). In some embodiments of any of the aspects described herein, a gene modifying system comprises one or more linear RNAs. In some embodiments, a nucleic acid as described herein (e.g., a template nucleic acid, a nucleic acid molecule encoding a genemodifying polypeptide, or both) is a circRNA. In some embodiments, a circular RNA molecule encodes the gene modifying polypeptide. In some embodiments, the circRNA molecule encoding the gene modifying polypeptide is delivered to a host cell. In some embodiments, the circRNA molecule encoding the gene modifying polypeptide is linearized (e.g., in the host cell, e.g., in the nucleus of the host cell) prior to translation. Circular RNAs are described, e.g., at p. 1215-1218 of PCT Pub. No. WO / 2021 / 178720.

[0334] Further included here are compositions and methods for the assembly of full or partial template RNA molecules. Methods of making template RNAs are described, e.g., at p. 1150-1155 of PCT Pub. No. WO / 2021 / 178720.(viii) Additional Template Features

[0335] In some embodiments, the template (e.g., template RNA) comprises certain structural features, e.g., determined in silico. In embodiments, the template RNA is predicted to have minimal energy structures between -280 and -480 kcal / mol (e.g., between -280 to -300, -300 to - 350, -350 to -400, -400 to -450, or -450 to -480 kcal / mol), e.g., as measured by RNAstructure, e.g., as described in Turner and Mathews Nucleic Acids Res 38:D280-282 (2009) (incorporated herein by reference in its entirety).

[0336] In some embodiments, the template (e.g., template RNA) comprises certain structural features, e.g., determined in vitro. In embodiments, the template RNA is sequence optimized, e.g., to reduce secondary structure as determined in vitro, for example, by SHAPE-MaP (e.g., as described in Siegfried et al. Nat Methods 11:959-965 (2014); incorporated herein by reference in its entirety). In some embodiments, the template (e.g., template RNA) comprises certain structural features, e.g., determined in cells. In embodiments, the template RNA is sequence optimized, e.g., to reduce secondary structure as measured in cells, for example, by DMS-MaPseq (e.g., as described in Zubradt et al. Nat Methods 14:75-82 (2017); incorporated by reference herein in its entirety).(ix) Additional Functional Characteristics and Features of gene modifying systems

[0337] A gene modifying system as described herein may, in some instances, be characterized by one or more functional measurements or characteristics. In some embodiments, the DNA binding domain has one or more of the functional characteristics described below. In some embodiments, the RNA binding domain has one or more of the functional characteristics described below. In some embodiments, the endonuclease domain has one or more of the functional characteristics described below. In some embodiments, the reverse transcriptase domain has oneor more of the functional characteristics described below. In some embodiments, the template (e.g., template RNA) has one or more of the functional characteristics described below. In some embodiments, the target site bound by the gene modifying polypeptide has one or more of the functional characteristics described below.(x) Gene modifying Polypeptide a) DNA Binding Domain

[0338] In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with greater affinity than a reference DNA binding domain. In some embodiments, the reference DNA binding domain is a DNA binding domain from R2_BM of B. mori. In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM).

[0339] In some embodiments, the affinity of a DNA binding domain for its target sequence (e.g., dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety).

[0340] In embodiments, the DNA binding domain is capable of binding to its target sequence (e.g., dsDNA target sequence), e.g, with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM) in the presence of a molar excess of scrambled sequence competitor dsDNA, e.g., of about 100-fold molar excess.

[0341] In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) more frequently than any other sequence in the genome of a target cell, e.g., human target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated herein by reference in its entirety). In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) at least about 5-fold or 10-fold, more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.

[0342] In some embodiments, a gene modifying polypeptide comprises a modification to a DNA-binding domain, e.g., relative to the wild-type polypeptide. In some embodiments, the DNA-binding domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original DNA-binding domain. In some embodiments, the DNA-bindingdomain is modified to include a heterologous functional domain that binds specifically to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain replaces at least a portion (e.g., the entirety of) the prior DNA-binding domain of the polypeptide. In some embodiments, a gene modifying polypeptide comprises a modification to an endonuclease domain, e.g., relative to the wild-type polypeptide. In some embodiments, the endonuclease domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original endonuclease domain. In some embodiments, the endonuclease domain is modified to include a heterologous functional domain that binds specifically to and / or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest. b) RNA Binding Domain

[0343] In some embodiments, the RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, the reference RNA binding domain is an RNA binding domain from R2_BM of B . mori. In some embodiments, the RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in cells (e.g., by FRET or CLIP-Seq).

[0344] In some embodiments, the RNA binding domain is associated with the template RNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(l l):5490-5501 (incorporated by reference herein in its entirety). In some embodiments, the RNA binding domain is associated with the template RNA in cells (e.g., in HEK293T cells) at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra. c) Endonuclease Domain

[0345] In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA. In someembodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA, e.g., in a cell (e.g., a HEK293T cell). In some embodiments, the frequency of association between the endonuclease domain and the target DNA or scrambled DNA is measured by ChlP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated by reference herein in its entirety).

[0346] In some embodiments, the endonuclease domain can catalyze the formation of a nick at a target sequence, e.g., to an increase of at least about 5-fold or 10-fold relative to a non-target sequence (e.g., relative to any other genomic sequence in the genome of the target cell). In some embodiments, the level of nick formation is determined using NickSeq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org / 10.1101 / 867937 (incorporated herein by reference in its entirety).

[0347] In some embodiments, the endonuclease domain is capable of nicking DNA in vitro. In embodiments, the nick results in an exposed base. In embodiments, the exposed base can be detected using a nuclease sensitivity assay, e.g., as described in Chaudhry and Weinfeld (1995) Nucleic Acids Res 23(19):3805-3809 (incorporated by reference herein in its entirety). In embodiments, the level of exposed bases (e.g., detected by the nuclease sensitivity assay) is increased by at least 10%, 50%, or more relative to a reference endonuclease domain. In some embodiments, the reference endonuclease domain is an endonuclease domain from R2_BM of B. mori.

[0348] In some embodiments, the endonuclease domain is capable of nicking DNA in a cell. In embodiments, the endonuclease domain is capable of nicking DNA in a HEK293T cell. In embodiments, an unrepaired nick that undergoes replication in the absence of Rad51 results in increased NHEJ rates at the site of the nick, which can be detected, e.g., by using a Rad51 inhibition assay, e.g., as described in Bothmer et al. (2017) Nat Commun 8:13905 (incorporated by reference herein in its entirety). In embodiments, NHEJ rates are increased above 0-5%. In embodiments, NHEJ rates are increased to 20-70% (e.g., between 30%-60% or 40-50%), e.g., upon Rad51 inhibition.

[0349] In some embodiments, the endonuclease domain releases the target after cleavage. In some embodiments, release of the target is indicated indirectly by assessing for multiple turnovers by the enzyme, e.g., as described in Yourik at al. RNA 25(l):35-44 (2019) (incorporated herein by reference in its entirety) and shown in Figure 2. In some embodiments, the kexpof an endonuclease domain is 1 x 10'3- 1 x 10'5 min-1 as measured by such methods.

[0350] In some embodiments, the endonuclease domain has a catalytic efficiency (kcat / Km) greater than about 1 x 108s'1M'1in vitro. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1 x 105, 1 x 106, 1 x 107, or 1 x 108, s'1M'1in vitro. In embodiments, catalytic efficiency is determined as described in Chen et al. (2018) Science 360(6387):436-439 (incorporated herein by reference in its entirety). In some embodiments, the endonuclease domain has a catalytic efficiency (fcCat / Km) greater than about 1 x 108s'1M'1in cells. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1 x 105, 1 x 106, 1 x 107, or 1 x 108s'1M'1in cells.(xi) Reverse Transcriptase Domain a) Target Site and Integration

[0351] In some embodiments, after gene editing, the target site surrounding the integrated sequence contains a limited number of insertions or deletions, for example, in less than about 50% orlO% of integration events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. (2020) bioRxiv doi.org / 10.1101 / 645903 (incorporated by reference herein in its entirety). In some embodiments, the target site does not show multiple insertion events, e.g., head-to-tail or head-to-head duplications, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In some embodiments, the target site does not contain insertions resulting from endogenous RNA in more than about 1% or 10% of events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains the integrated sequence corresponding to the template RNA.

[0352] In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In embodiments, the target site does not comprise sequence outside of the template, e.g., as determined by long-read amplicon sequencing of the target site (for example, as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020); incorporated herein by reference in its entirety).(xii) DNA Damage Response

[0353] In some embodiments, modifying a genome of a cell (e.g., a primary cell, e.g., a T cell) using a gene modifying system does not result in activation of the endogenous DNA damageresponse (DDR) pathway. In some embodiments, modifying a genome of a cell (e.g., a primary cell) using a gene modifying system results in activation of the cell’s endogenous DDR pathway less than in an otherwise similar cell treated with Cas9.

[0354] In some embodiments, modifying a genome of a cell (e.g., a primary cell, e.g., a T cell) using a gene modifying system does not result in activation of the endogenous interferon response. In some embodiments, modifying a genome of a cell using a gene modifying system results in activation of the cell’s interferon response less than in an otherwise similar cell treated with a gene modifying system comprising elements from a LINE-1 retrotransposase.

[0355] In some embodiments, the gene modifying polypeptide systems described herein includes a self-inactivating module. The self-inactivating module leads to a decrease of expression of the gene modifying polypeptide, the gene modifying template, or both. Self-inactivating modules are described, e.g., at p. 1200-1201 of PCT Pub. No. WO / 2021 / 178720.

[0356] In some embodiments a polypeptide described herein (e.g., a gene modifying polypeptide) is controllable via a small molecule. In some embodiments, the polypeptide is dimerized via a small molecule. Polypeptides of this type are described, e.g., at p. 1201-1203 of WO / 2021 / 178720.B. Heterologous Gene Modifying Systems

[0357] The conjugates (targeted LNPs) described herein can be formulated to comprise one or more components of a heterologous gene modifying system, or one or more nucleic acids encoding said components. Accordingly, in some embodiments, the pay load comprises a heterologous gene modifying system, or one or more nucleic acids encoding the components of the heterologous gene modifying polypeptide. For instance, in some embodiments, the payload comprises a template RNA and an mRNA encoding the heterologous gene modifying polypeptide.

[0358] A heterologous gene modifying system may comprise a heterologous gene modifying polypeptide and a template RNA. The heterologous gene modifying polypeptide may comprise an endonuclease domain, a DNA binding domain, a linker, and a reverse transcriptase domain derived from a retrovirus. The heterologous gene modifying polypeptide may comprise a Cas domain, a linker, and a reverse transcriptase domain derived from a retrovirus. The template RNA compatible with the heterologous gene modifying polypeptide may comprise (e.g., from 5' to 3') (i) a gRNA spacer that binds a target site, (ii) a gRNA scaffold that binds the heterologous gene modifying polypeptide, e.g., the Cas domain of the heterologous gene modifying polypeptide, (iii)a heterologous object sequence, and (iv) a primer binding site (PBS) sequence. These components are now described in more detail.

[0359] In some aspects, a heterologous gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide that comprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain, wherein the RT domain is C-terminal of the Cas domain; and a linker disposed between the RT domain and the Cas domain.

[0360] In some embodiments, a heterologous gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 59 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 59 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the N-terminal end of the heterologous gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 59 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N- terminal end of the heterologous gene modifying polypeptide.Exemplary N-terminal NLS-Cas9 domainMPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHERKKEVDSTDKADEREIYEAEAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDGG (SEQ ID NO: 59)

[0361] In some embodiments, a heterologous gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 60 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 60 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the C-terminal end of the heterologous gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 60 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C- terminal end of the heterologous gene modifying polypeptide.Exemplary C-terminal sequence comprising an NLSAGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 60)

[0362] In some embodiments, the heterologous gene modifying polypeptide comprises an amino acid sequence according to SEQ ID NO: 61, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.Exemplary benchmarking sequenceMPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDGGSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGTLNIEDEYRLHETSK EPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIK PHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNL LSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYR ASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCR LFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVD EKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLP LPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG WLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAI TETPDTSTLLIENSSPSGGSKRTADGSEFEAGKRTADGSEFEKRTADGSEFESPKKKAKV E (SEQ ID NO: 61)Exemplary N-terminal NLS-Cas9 domainMPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRK SEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDGG (SEQ ID NO: 44)Exemplary C-terminal sequence comprising an NLSAGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 45)

[0363] In some embodiments, a heterologous gene modifying polypeptide described herein comprises an RT domain having an amino acid sequence according to Table 6 of International Application WO / 2023 / 039440 (which Table is incorporated herein by reference in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0364] In some embodiments, a heterologous gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a heterologous gene modifying polypeptide comprises an amino acid sequence according to Table 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.Table 5: Selection of exemplary gene modifying polypeptidesTable 6: Full length amino acid sequence corresponding to Table

[0365] In some embodiments, the heterologous gene modifying polypeptide has a sequence disclosed in International Application WO / 2023 / 039424 (which is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. For instance, in some embodiments, the heterologous gene modifying polypeptide has an RT domain as described in Table 6 of International Application WO / 2023 / 039424 (which table is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, a heterologous gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 10 of International Application WO / 2023 / 039424 (which Table is incorporated herein by reference in its entirety). In some embodiments, the heterologous gene modifying polypeptide has a sequence according to Table T2 of International Application WO / 2023 / 039424 (which table is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, the heterologous gene modifying polypeptide has a sequence according to Table Al of International Application WO / 2023 / 039424 (which table is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0366] In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., an M-MLV RT, e.g., comprising the following sequence:TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQ GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK TPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTA PALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLR MVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR YAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEA RGNRMADQAARKAAITETPDTSTLL (SEQ ID NO: 55), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0367] In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, e.g., a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and W313F. In some embodiments, an M-MLV RT used herein comprises the mutations D200N, L603W, T330P, T306K and W313F. In embodiments, the mutant M-MLV RT comprises the following amino acid sequence:TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQ GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK TPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTA PALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLR MVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR YAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEA RGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO: 56)Exemplary gene modifying system comprises mutant M-MLV RT regionMPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDGGSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGTLNIEDEYRLHETSK EPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIK PHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNL LSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCR LFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVD EKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLT MGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLP LPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTE TEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRG WLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAI TETPDTSTLLIENSSPSGGSKRTADGSEFEAGKRTADGSEFEKRTADGSEFESPKKKAKV E (SEQ ID NO: 57)

[0368] In some embodiments, a template RNA molecule for use in the system comprises, from 5' to 3' (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence. In some embodiments:(1) Is a gRNA spacer of -18-22 nt, e.g., is 20 nt(2) Is a gRNA scaffold comprising one or more hairpin loops, e.g., 1, 2, of 3 loops for associating the template with a Cas domain, e.g., a nickase Cas9 domain. In some embodiments, the gRNA scaffold comprises the sequence, from 5' to 3', GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGGACCGAGTCGGTCC (SEQ ID NO: 58).(3) In some embodiments, the heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nt or, 80-90 nt in length.(4) In some embodiments, the PBS sequence that binds the target priming sequence after nicking occurs is e.g., 3-20 nt, e.g., 7-15 nt, e.g., 12-14 nt. In some embodiments, the PBS sequence has 40-60% GC content.V. Conjugates targeting immune cells

[0369] 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, naive 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 sequence encoding a T-cell receptor (TCR) in a T cell. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T-cells. For instance, in specific embodiments, the conjugates can be used to deliver an RNA encoding a CAR to T-cells. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a CAR to natural killer (NK) cells. In some embodiments, the conjugates be used to deliver at least one sequence encoding a CAR to natural killer T (NKT) cells. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a 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-T / CAR-NK / CAR-NKT 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(HERl), 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 (pro state- 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).

[0370] 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., RNA, such as 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.

[0371] hi 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) ,TCR-a, TCR- a / p, TCR-y / 5, PD1, CTLA4, TI 3, LAG3, CD18, IL-2 receptor, CDlla, TLR2, TLR4, TLR5, IL -7 receptor, or IL- 15 receptor.

[0372] hi 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, CDS, CD28, CD i 37, CD45, T-cell receptor (TCR)p,TCR-a, TCR- a / p, TCR-y / 5, PDF CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CDHa, TLR2, TLR4, TLR5, IL-7 receptor, and IL- 15 receptor. In some embodiments the CD80 targeting moiety is a CD80 extracellular domain (ECD).

[0373] In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that targets a receptor on the surface of the T cell selected from CD2, CD3, CD4, CD5, CD6, CD7, CD8, and CD28. In some embodiments, the targeting moiety targets a CD3 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD7 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD5 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD2 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD8 receptor on the surface ofthe T cell. In some embodiments, the targeting moiety targets a CD28 receptor on the surface of the T cell.

[0374] In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that targets CD3 on the surface of the T cell, wherein the targeting moiety is an antibody, Fab fragment or ScFv selected from SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab and visilizumab, or an antigen-binding portion thereof. In certain embodiments, the targeting moiety is SP34 or an antigen-binding portion thereof. In other embodiments, the targeting moiety is teclistamab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is mosunetuzumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is odronextamab or an antigen-binding portion thereof.In other embodiments, the targeting moiety is tebentafusp or an antigen-binding portion thereof.In other embodiments, the targeting moiety is muromonab or an antigen-binding portion thereof.In other embodiments, the targeting moiety is visilizumab or an antigen-binding portion thereof.In other embodiments, the targeting moiety is tepilizumab or an antigen-binding portion thereof.

[0375] In some embodiments, a targeted LNP (conjugate) for delivery of a therapeutic agent to immune cells (e.g., T cells) as described herein, comprises:

[0376] an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any or the lipids in Table 1, Table 2 and Table 3;

[0377] a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties bind to at least one targeting moiety on a T cell; and a therapeutic agent encapsulated within the LNP.

[0378] In some embodiments, the plurality of targeting moieties bind to two or more T-cell antigens selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR) ,TCR-a, TCR-a / p, TCR-y / 5, PD1, CTLA4, TIM3, LAG3, CD 18, IL-2 receptor, CD I la, TLR2, TLR4, TLR5, IL-7 receptor, and IL- 15 receptor, In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that targets a receptor on the surf...

Claims

CLAIMSWhat is claimed is:

1. A method of making a targeted LNP, said method comprising:(i) contacting an antigen-binding fragment comprising a light chain and a heavy chain (e.g., an antigen binding fragment of an antibody) with a reducing reagent, wherein the antigen-binding fragment comprises an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chain domain 1 (CHI), whereby the reducing reagent reduces the interchain disulfide bond of the antigen-binding 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.

2. The method of claim 1, wherein the antigen-binding fragment is a Fab fragment.

3. The method of claim 1, wherein the antigen-binding fragment is a F(ab')2 fragment.

4. The method of claim 1, wherein the antigen-binding fragment is a Fab' fragment.

5. A method of making a targeted LNP, said method comprising:(i) introducing a cysteine residue at the C-terminus of a VHH domain or scFv;(ii) (ii) contacting the scFv or VHH domain with a reducing reagent, whereby the reducing reagent reduces any intermolecular disulfide bonds between pairs of scFvs or pairs of VHH domains (e.g., in a solution or mixture comprising a plurality of the scFvs and / or VHH domains), thereby generating two free cysteine residues; and(iii) contacting the product of step (ii) 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.

6. The method of any one of claims 1-5, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP).

7. The method of claim 6, wherein the reducing agent is TCEP.

8. The method of any one of claims 1-7, wherein the thiol-reactive group is maleimide.

9. The method of any one of claims 1-7, wherein the thiol-reactive group is 2,3- dibromomaleimide (DBM).

10. The method of any one of claims 1-9, wherein the thiol-reactive group is covalently bonded to one or more of the lipids comprising the precursor LNP.

11. The method of claim 10, wherein the thiol-reactive group is covalently bonded to a pegylated lipid.

12. The method of any one of claims 1-4 or 6-11, wherein the antigen-binding fragment does not have a hinge region.

13. The method of any one of claims 1-4 or 6-11, where there are no additional disulfide linkages between the light chain and the heavy chain of the antigen-binding fragment.

14. A conjugate produced by a method of any one of claims 1-13.

15. A conjugate comprising an LNP and an antigen-binding 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 antigen-binding fragment and a second cysteine residue in the constant region of light chain of the antigen-binding fragment, wherein the antigen-binding fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the antigen-binding fragment and the constant region of the light chain of the antigen-binding fragment.

16. The conjugate of claim 15, wherein the antigen-bonding fragment is a Fab fragment.

17. The conjugate of claim 15, wherein the antigen-bonding fragment is a Fab' fragment.

18. The conjugate of claim 15, wherein the antigen-bonding fragment is a F(ab')2 fragment.

19. The conjugate of any one of claims 15-18, wherein both the constant region of the heavy chain and the constant region of the light chain of the antigen-binding fragment are covalently bonded to the LNP.

20. The conjugate of any one of claims 15-18, wherein only the constant region of the heavy chain of the antigen-binding fragment is covalently bonded to the LNP.

21. The conjugate of claim 20, wherein the light chain remains associated with the heavy chain that is covalently bonded to the surface of the LNP.

22. The conjugate of claim any one of claims 15-18, wherein only the constant region of the light chain of the antigen-binding fragment is covalently bonded to the LNP.

23. The conjugate of claim 22, wherein the heavy chain remains associated with the light chain that is covalently bonded to the surface of the LNP.

24. The conjugate of any one of claims 15-23, wherein the antigen-binding fragment is linked to the LNP through a thiosuccinimide moiety.

25. The conjugate of any one of claims 15-23, wherein the antigen-binding fragment is linked to the LNP through a dithiomalemide moiety.

26. The conjugate of any one of claims 15-23, wherein the antigen-binding fragment is linked to the LNP through a maleamic acid moiety.

27. A conjugate comprising an LNP and an IgGl Fab fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment is covalently bonded to the LNP.

28. A conjugate comprising an LNP and an IgGl Fab fragment, wherein the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab fragment is covalently bonded to the LNP.

29. A conjugate comprising an LNP and an IgGl Fab fragment, wherein the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment is covalently bonded to the LNP.

30. A conjugate comprising an LNP and an IgG2 Fab fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP.

31. A conjugate comprising an LNP and an IgG2 Fab fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently to the LNP.

32. A conjugate comprising an LNP and an IgG2 Fab fragment, wherein 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 is covalently bonded to the LNP.

33. A conjugate comprising an LNP and an IgG4 Fab fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently bonded to the LNP.

34. A conjugate comprising an LNP and an IgG4 Fab fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently bonded to the LNP.

35. A conjugate comprising an LNP and an IgG4 Fab fragment, wherein 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.

36. The conjugate of any one of claims 27-35, wherein the Fab fragment does not have a disulfide linkage between the heavy and light chains of the Fab fragment.

37. A conjugate comprising an LNP and an IgGl F(ab')2 fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl F(ab')2 fragment is covalently bonded to the LNP.

38. A conjugate comprising an LNP and an IgGl F(ab')2 fragment, wherein the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl F(ab')2 fragment is covalently bonded to the LNP.

39. A conjugate comprising an LNP and an IgGl F(ab')2 fragment, wherein the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl F(ab')2 fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl F(ab')2 fragment is covalently bonded to the LNP.

40. A conjugate comprising an LNP and an IgG2 F(ab')2 fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 F(ab')2 fragment is covalently bonded to the LNP.

41. A conjugate comprising an LNP and an IgG2 F(ab')2 fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 F(ab')2 fragment is covalently to the LNP.

42. A conjugate comprising an LNP and an IgG2 F(ab')2 fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 F(ab')2 fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 F(ab')2 fragment is covalently bonded to the LNP.

43. A conjugate comprising an LNP and an IgG4 F(ab')2 fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 F(ab')2 fragment is covalently bonded to the LNP.

44. A conjugate comprising an LNP and an IgG4 F(ab')2 fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 F(ab')2 fragment is covalently bonded to the LNP.

45. A conjugate comprising an LNP and an IgG4 F(ab')2 fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 F(ab')2 fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 F(ab')2 fragment is covalently to the LNP.

46. The conjugate of any one of claims 37-45, wherein the F(ab')2 fragment does not have a disulfide linkage between the heavy and light chains of the F(ab')2 fragment.

47. A conjugate comprising an LNP and an IgGl Fab' fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab' fragment is covalently bonded to the LNP.

48. A conjugate comprising an LNP and an IgGl Fab' fragment, wherein the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab' fragment is covalently bonded to the LNP.

49. A conjugate comprising an LNP and an IgGl Fab' fragment, wherein the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab' fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab' fragment is covalently bonded to the LNP.

50. A conjugate comprising an LNP and an IgG2 Fab' fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab' fragment is covalently bonded to the LNP.

51. A conjugate comprising an LNP and an IgG2 Fab' fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab' fragment is covalently to the LNP.

52. A conjugate comprising an LNP and an IgG2 Fab' fragment, wherein 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 is covalently bonded to the LNP.

53. A conjugate comprising an LNP and an IgG4 Fab' fragment, wherein the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab' fragment is covalently bonded to the LNP.

54. A conjugate comprising an LNP and an IgG4 Fab' fragment, wherein the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab' fragment is covalently bonded to the LNP.

55. A conjugate comprising an LNP and an IgG4 Fab' fragment, wherein 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.

56. The conjugate of any one of claims 47-55, wherein the Fab' fragment does not have a disulfide linkage between the heavy and light chains of the Fab' fragment.

57. The conjugate of any one of claims 37-56, wherein the Fab fragment, F(ab')2 fragment, orFab' fragment binds to a first antigen in the surface of a target cell.

58. The conjugate of claim 57, wherein the target cell is a T cell.

59. The conjugate of claim 57, wherein the target cell is a hematopoietic stem cell.

60. The conjugate of any one of claims 57-59, wherein the conjugate comprises at least one additional Fab fragment, F(ab')2 fragment or Fab' fragment bound to the surface of the LNP, wherein the one additional Fab fragment, F(ab')2 fragment or Fab' fragment binds to a second antigen on the surface of the target cell.

61. A conjugate comprising an LNP and a VHH domain, wherein the LNP is covalently bonded to either or both of a first cysteine residue in the VHH domain and / or a second cysteine residue in the VHH domain, wherein the first cysteine is Cys 22 of the VHH domain defined by Kabat numbering and the second cysteine is Cys 92 of the VHH domain defined by Kabat numbering.

62. The conjugate of claim 61, wherein the VHH domain binds to a first antigen on the surface of a target cell.

63. The conjugate of claim 62, wherein the target cell is a T cell.

64. The conjugate of claim 62, wherein the target cell is a hematopoietic stem cell.

65. The conjugate of any one of claims 62-64, wherein the conjugate comprises at least one additional VHH bound to the surface of the LNP, wherein the one additional VHH domain binds to a second antigen on the surface of the target cell.

66. The conjugate of any one of claims 62-64, wherein the conjugate further comprises a Fab fragment, F(ab')2 fragment or Fab' fragment bound to the surface of the LNP, wherein the Fab fragment, F(ab')2 fragment or Fab' fragment binds to a second antigen on the surface of the target cell.

67. The conjugate of claim 66, wherein the Fab fragment, F(ab')2 fragment or Fab' fragment is conjugated to the surface of the LNP by a method of any one of claims 1-13.

68. The conjugate of any one of claims 15-67, wherein the LNP comprises an ionizable lipid selected from Table 1, Table 2 or Table 3.

69. The conjugate of any one of claims 15-67, wherein the LNP comprises an ionizable lipid having one of the structures depicted below:Lipid 092Lipid 154Lipid 163Lipid 178Lipid 23270. A method of making a targeted LNP comprising contacting an antbody or antigenbinding fragment Fab fragment that has been functionalized with a sortase tag with a precursor LNP comprising a plurality of poly glycine molecules covalently bonded to the surface of the LNP in the presence of sortase, whereby the antibody or antigen-binding fragment is conjugated to the surface of the precursor LNP through a sortase mediated ligation of the sortase tag and one or more polyglycine molecules, thereby forming a targeted LNP.

71. The method of claim 70, wherein the antigen binding fragment is a Fab fragment.

72. The method of claim 70, wherein the antigen binding fragment is a VHH domain.

73. The method of any one of claims 70-72, wherein the sortase tag has the sequence LPXTG, wherein X is any amino acid (SEQ ID: 62).

74. The method of any one of claims 70-72, wherein the sortase tag has the sequence LPETG (SEQ ID: 63).

75. A conjugate comprising a plurality of antigen-binding fragments (e.g., the antigenbinding fragments of an antibody) attached to the surface of a lipid nanoparticle (LNP), whereineach antigen-binding fragment comprises a light chain and a heavy chain, the conjugate comprising: a plurality of thiol reactive groups conjugated to the surface of the LNP, wherein each antigen-binding fragment in the plurality of antigen-binding fragments comprises a reduced interchain disulfide bond such that a cysteine on the heavy chain of the antigen-binding fragment is no longer covalently linked to a cysteine on the light chain of the antigen-binding fragment, and wherein each antigen-binding fragment comprises: the heavy chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups, the light chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups, or the heavy chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups and the light chain cysteine covalently linked to a thiol reactive group in the plurality of thiol reactive groups.

76. The conjugate of claim 75, wherein the antigen binding fragment is a Fab fragment.

77. The conjugate of claim 75, wherein the antigen binding fragment is a F(ab')2 fragment.

78. The conjugate of claim 75, wherein the antigen binding fragment is a Fab' fragment.

79. The conjugate of any one of claims 75-78, comprising a second plurality of antigenbinding fragments conjugated to the LNP that that bind to a second antigen.

80. The conjugate of any one of claims 14-79, wherein the conjugate encapsulates a therapeutic agent.

81. The conjugate of claim 80, wherein the therapeutic agent is a gene modifying system.

82. The conjugate of claim 81, wherein the gene modifying system comprises:(i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and(ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence.