Traceless linker and methods of use thereof

Abstract

The present disclosure provides reversible / traceless linkers that include functional groups for attaching a first and second molecule of interest, where the linker includes a reduction sensitive disulfide linker that undergoes cleavage under physiological reducing conditions, releasing the first molecule of interest. The release is fully reversible in that the first molecule is restored to its "pre-linker" state. The present disclosure provides compounds comprising a first and a second molecule of interest linked by a linker of the present disclosure. The present disclosure provides methods of using a compound of the present disclosure to deliver a molecule of interest. The present disclosure provides methods of modifying a target nucleic acid in a cell, which use a reversible / traceless linker to deliver a gene editing protein.

Description

TRACELESS LINKER AND METHODS OF USE THEREOF CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 608,771 filed December 11, 2023, which application is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Number NS115599 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE LISTING XML FILE

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 493PRV_SEQ_LIST.xml” created on December 6, 2023, and having a size of 69,866 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. INTRODUCTION

[0004] Reduction sensitive linkers conjoin two molecules of interest in a reversible manner. When the linker is exposed to a reducing environment, like the mammalian cell cytosol, tumor microenvironment, or by an externally imposed reducing reagent (such as GSH or BME), the linkage between the two molecules is split, and the molecules are no longer linked. These reversible linkers are ubiquitously used as prodrugs, in nanomaterial formulations and as components in drug delivery reagents. Reduction sensitive linkers have played an important role in the development of antibody drug conjugates, protein delivery vehicles, and nucleic acid delivery vehicles. Reduction sensitive linkers also have compelling synthetic and product development attributes because they are stable in aqueous environments, and can survive multi- step synthetic procedures without self-hydrolyzing.

[0005] However, there is a need for efficient reduction sensitive linkers suitable for linking bioactive molecules to the surface of therapeutically useful cargoes such as genome editing enzymes. There is a need for efficient reduction sensitive linkers that modify aliphatic amines and that provide for rapid release following disulfide reduction. Such are provided herein.SUMMARY

[0006] After reversibly tethering a protein (Cas9) with a subject linker (a para-nitro carbonate (PNC)- disulfide (DS)-PEG-TCO (PNC-DS-PEG-TCO) linker – described in further detail below), the inventors observed unexpectedly rapid and efficient conjugation of functional groups (e.g., peptide functional groups) bearing a tetrazine moiety (tetrazine performs the copper-free click reaction to covalently bond to the TCO moiety). This provided an unexpectedly convenient way to conjugate functional groups such as small molecules, proteins such as antibodies and functional fragments thereof, targeting and delivery moieties such as targeting and / or delivery peptides, lipids, nucleic acids, polysaccharides, or any combination thereof to other molecules of interest (e.g., proteins such as CRISPR-Cas effector proteins, especially those that may have poor solubility and may not conjugate efficiently due to their hydrophobic properties). The inventors also discovered that a reduction sensitive linker functionalized with disulfide carbamate (DCB) provided for unexpectedly efficient delivery of proteins to cells (e.g., ribonucleoprotein complexes such as CRISPR complexes) (e.g., when functionalized with delivery and targeting moieties).

[0007] An ideal linker for attaching a bifunctional compound to an active enzyme would include 1) an amine reactive group (for attaching a first molecule of interest, e.g., an enzyme), 2) a reduction sensitive linker, 3) an antifouling component (e.g., antifouling polymer such as PEG), and 4) a chemical handle for attaching a bifunctional compound (e.g., a targeting moiety, a delivery moiety, etc.), and such a linker would allow for regeneration of the first molecule of interest (e.g., enzyme) after cleavage of the reduction sensitive linker. Such linkers are provided herein.

[0008] The present disclosure provides an amine reactive linker capable of reversibly and covalently being conjugated to any molecule of interest, e.g., a protein such as a CRISPR-Cas RNP, or any other protein. Thus, a subject amine reactive linker includes an amine reactive portion, which is in some cases a para-nitro carbonate (PNC). The amine reactive linker also includes a disulfide cleavable linker portion (a reduction sensitive linker) that undergoes cleavage under physiological (e.g., intracellular) reducing conditions, releasing the conjugated protein cargo – and leaving no chemical trace on the cargo protein following reduction and release. The disulfide cleavable linker chemistry is reversible, allowing, e.g., stable surface decoration of the conjugated protein cargo until intracellular (cytosolic) delivery is successful, and then undergoing reversion (release) of the protein cargo to a pre-conjugation state due to the physiological reducing conditions. In some cases, the disulfide cleavable linker portion is a disulfide carbamate (DCB). The amine reactive linker also includes a reactive moiety functional group (e.g., a click reaction moiety) for attaching molecules of interest (e.g., a small molecule, a protein such as an antibody and / or a functional fragment thereof, a targeting and / or deliverymoiety such as a targeting peptide or protein transduction peptide such as Tet1 (T1) (HLNILSTLWKYR) (SEQ ID NO: 62), F4 (SEQ ID NO: 63), P55 (KLFEAIEGFIENGWEGMIDLWNGYGRKKRRQRR) (SEQ ID NO: 61), Angiopep2 (TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 3), TAT (e.g., YGRKKRRQRR (SEQ ID NO:19)), lipids, nucleic acids, polysaccharides, and any combination thereof). In some cases, the reactive moiety functional group is trans-cyclooctene (TCO), which facilitates copper-free click conjugation. In some cases, the amine reactive linker also includes an inert linker portion (e.g., polyethylene glycol (PEG)) between the disulfide cleavable linker portion and the reactive moiety functional group. Thus, a subject amine reactive linker includes an amine reactive portion (e.g., PNC), a disulfide cleavable linker portion (e.g., a DCB), and a reactive moiety functional group (e.g., TCO) – and optionally includes an inert linker portion between the disulfide cleavable linker portion and the reactive moiety functional group.

[0009] The present disclosure provides compounds comprising a first molecule of interest (e.g., a gene editing protein such as Cre, a CRISPR-Cas effector protein, and the like) and a second molecule of interest (e.g., small molecules, proteins such as antibodies and functional fragments thereof, targeting and delivery moieties such as targeting and / or delivery peptides such as T1, P55, Angiopep2, TAT, and the like (e.g., AP22, P2, F17, nPF4, PT551), lipids, nucleic acids, polysaccharides, or any combination thereof) linked by a linker of the present disclosure. In some cases, the first molecule of interest (e.g., a CRISPR-Cas effector protein) is conjugated to two or more different linkers of the present disclosure, e.g., where the two or more different linkers provide different ‘second’ molecules of interest (e.g., different targeting peptides and / or protein transduction peptides, e.g., one provides T1 and another provides P55). The present disclosure provides methods of using a compound of the present disclosure to deliver a molecule of interest. The present disclosure provides methods of modifying a target nucleic acid.

[0010] As discussed above, the present disclosure provides a linker with the capacity to couple two elements (two molecules of interest): one bearing primary amines (via the amine reactive portion, e.g., a PNC moiety) and the other bearing a conjugation compatible moiety (e.g., click- compatible moiety such as a copper free click-compatible moiety). The linker provides for a fully reversible / scarless conjugation (covalent bond) to the material being delivered (e.g. the first molecule of interest such as a protein “cargo”), which bears the primary amine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG.1A-1B Data demonstrating conjugation of a subject reversible / traceless linker to a ribonucleoprotein (RNP).

[0012] FIG.2A-2C Data demonstrating gene editing in neurons of mice via Cas9 / peptide conjugates using a subject reversible / traceless linker.

[0013] FIG.3 Data demonstrating conjugation, using a subject linker, of an RNP to a peptide, e.g., for blood-brain barrier (BBB) transit.

[0014] FIG.4A-4B Data demonstrating transit of an RNP, conjugated to AG2 (SEQ ID NO: 3) or KLPGWSG (SEQ ID NO. 60) sequence using a subject linker, across an artificial blood brain barrier (BBB) grown using BEND.3 cells on a trans-well insert.

[0015] FIG.5 Data demonstrating BBB transit in mice of RNPs conjugated to a transit peptide using a subject linker.

[0016] FIG. 6 NMR data demonstrating generation of a subject reversible / traceless linker. 1H-NMRspectrum of compound 6 (TCO-PEG23-DEC-lite) (CDCl3, 400 MHz).

[0017] FIG.7 Screening of peptides for cellular uptake in SH-SY5Y cells. Peptides identified from the literature were synthesized on solid resin support using standard Fmoc-based peptide synthesis, with a tetrazine functional group attached via a terminal cysteine and Tetrazine-PEG- Maleimide. The resulting material was incubated with SH-SY5Y cells in a 96-well format for 24 hours. Flow cytometry analysis revealed dye uptake, indicating peptide-mediated cellular delivery particularly in the case of peptides P55, TET1, and AP22.

[0018] FIG.8 Cas9 RNPs either untreated, reacted with the PNC-DS-PEG-TCO linker (referred to as “RNP-TCO”), or the PNC-DS-PEG-TCO linker and the peptides P55 and T1 (referred to as “RNP-(P55,T1)”). A shift in size is observed following conjugation of material to the RNP: a shift following RNP conjugation to PNC-DS-PEG-TCO (“RNP-TCO”), and another shift upon additional conjugation of the Tet1 and P55 peptide (“RNP-(P55,T1)”). As observed to the right of the latter, reduction with BME causes complete release of the conjugated materials.

[0019] FIG.9 Transmission electron microscopy images of uranyl acetate (UA) stained samples of RNP and RNP-(P55, T1) demonstrate minimal perturbation of RNP size after peptidilaytion.

[0020] FIG.10 Characterization of RNP Conjugates: The hydrodynamic size and polydispersity of pegylated and peptidylated RNP conjugates were measured via Dynamic Light Scattering (DLS). Samples were diluted in RNP buffer to achieve optimal concentrations. Measurements show a small increase in hydrodynamic diameter, with the addition of linker and again peptidylation, indicative of coupling of the material.

[0021] FIG.11 Flow cytometry analysis of tdTomato activation in Ai9 neural progenitor cells (NPCs) treated with pegylated and peptidylated RNP conjugates. Ai9 NPCs were treated with RNP conjugates targeting the tdTomato locus in a 96-well plate format and incubated under standard conditions for 72 hours. Post-treatment, cells were harvested, washed with PBS, and resuspended in FACS buffer (PBS with 1% FBS and EDTA). Flow cytometry revealed tdTomato expression,with untreated cells serving as a negative control. The percentage of tdTomato-positive cells indicates successful editing, and improved editing with both the conjugation of P55, (P55, T1) and P2.

[0022] FIG.12A-12B. Enhanced neuronal targeting and editing efficiency of peptide-conjugated RNPs in Ai9 mice. Ai9 mice were treated Via Convection enhanced delivery to the striatum, either unilaterally [RNP, RNP-TCO, RNP-P55, RNP-(P55,T1), RNP-F17, RNP-(F17,T1)] or bilaterally [RNP-(P55, T1, PEG5K), RNP-(P2)] with 5µL of peptide-conjugated RNPs, at a concentration of 40µM, where successful excision of the loxP-flanked repressor activates tdTomato expression. Whole-brain imaging revealed robust tdTomato fluorescence in mice treated with Tet1-tethered RNPs (compare FIG.12A to FIG.12B), significantly outperforming untethered RNPs (“RNP”), PEG-conjugated RNPs without targeting peptides (“RNP-TCO”), and RNPs tethered to P55 alone (“RNP-P55”). Additionally, RNPs conjugated to F17-P55 dimer constructs demonstrate greater editing efficiency compared to P55 alone. The inclusion of 5kPEG further enhanced delivery and editing efficiency. These results highlight the potential of Tet1 and peptide-based constructs to improve neuronal targeting and delivery of gene-editing therapeutics.

[0023] FIG.13 Stacked brain slice images were processed in MatLab by thresholding for the edited signal, aligning slices along the midline in 3D space, and segmenting the edited regions. The program calculated the edited volume from voxel data and generated 3D reconstructions of both the whole brain and the edited regions, enabling spatial analysis and quantification of editing patterns. Tet1-tethered RNPs, significantly outperforming untethered RNPs, PEG-conjugated RNPs without targeting peptides, and RNPs tethered to P55 alone. Additionally, RNPs conjugated to F17-P55 dimer constructs demonstrate greater editing efficiency compared to P55 alone. The inclusion of 5kPEG further enhanced delivery and editing efficiency. These results highlight the potential of Tet1 and peptide-based constructs to improve neuronal targeting and delivery of gene-editing therapeutics.

[0024] FIG.14 Improved motor performance in R6 / 2 mice following treatment with RNP- (P55,T1,PEG). R6 / 2 mice, a model of Huntington’s disease, were treated via CED (5uL of concentrated RNP material to the murine striatum) with the lead compound RNP-(P55,T1,PEG), designed for enhanced neuronal targeting. Motor function was assessed using the Rotorod test before and after treatment. Mice treated with RNP-(P55,T1,PEG) showed significant improvement in latency to fall, while those treated with control RNPs displayed no improvement. These findings highlight the potential of RNP-(P55,T1,PEG) to enhance therapeutic outcomes in neurodegenerative disease models.

[0025] FIG.15 Enhanced tdTomato expression in Ai9 murine thalamus following convection- enhanced delivery (CED) of RNP-(P55,T1). Ai9 mice were treated via CED with 2µL of compound RNP-(P55,T1) (40µM), designed to target neuronal cells. Robust tdTomato fluorescence was observed in the thalamus of mice treated with RNP-(P55,T1), indicating effective gene editing, while minimal expression was seen in mice treated with control RNPs lacking targeting moieties. These results demonstrate the superior neuronal targeting and editing efficiency of RNP-(P55,T1) in vivo.

[0026] FIG.1663X images of the same tissue as FIG.15, these images demonstrate enhanced neuronal editing with the addition of the T1 peptide.

[0027] FIG.17 Comparable tdTomato activation in Ai9 NPCs treated with RNPs conjugated to P55, S10, and S315 peptides. Ai9 NPCs were treated with 40pMol of RNPs conjugated to P55, S10, or S315 peptides, targeting the loxP-flanked tdTomato locus. Flow cytometry analysis revealed similar levels of tdTomato expression across all three peptide conjugates, indicating comparable editing efficiencies. RNPs without peptide conjugation showed significantly reduced tdTomato activation, confirming the importance of peptide targeting for efficient editing in Ai9 NPCs.

[0028] FIG.18 Optic nerve editing in Ai9 mice following retro-orbital administration of RNP- (P55,TET1,Angiopep2). Ai9 mice were treated via retro-orbital injection with RNP- (P55,TET1,Angiopep2), 2nMol dose, designed to target neural tissues and cross the blood brain barrier. tdTomato fluorescence, indicative of successful gene editing at the loxP-flanked locus, was observed in the optic nerve, demonstrating the ability of this conjugate to mediate precise editing in ocular and neural tissues. Untreated mice and those receiving non-targeted RNPs showed no detectable fluorescence, highlighting the specificity and efficiency of RNP- (P55,TET1,Angiopep2) for editing in vivo.

[0029] FIG.19 Striatal neuron editing in Ai9 mice following retro-orbital administration of RNP- (nPF4,TET1,Angiopep2). Ai9 mice were treated via retro-orbital injection with RNP- (nPF4,TET1,Angiopep2), 2nMol dose, a conjugate designed to target neural tissues and cross the blood brain barrier. tdTomato fluorescence (shown), indicating successful gene editing at the loxP-flanked locus, was observed in a subset of striatal neurons. Untreated mice and those receiving non-targeted RNPs showed no detectable fluorescence. These findings demonstrate the ability of RNP-(nPF4,TET1,Angiopep2) to achieve limited editing in deep-brain neurons through systemic administration.

[0030] FIG.20 Comparable editing efficiency of RNP-(PT551) in Ai9 mice following CED administration. Convection-enhanced delivery (CED) of RNP-(PT551), a P55-T1 fusion peptide conjugate, was performed in Ai9 mice to target the loxP-flanked tdTomato locus.5µL of40µM RNP constructs were dosed to the murine Striatum. tdTomato fluorescence was observed in the treated region, with editing efficiency comparable to other peptide-RNP constructs tested. Untreated controls showed no fluorescence, confirming the specificity of RNP-(PT551) for gene editing. These results demonstrate that the P55-T1 fusion peptide maintains editing performance similar to individual targeting peptides while offering potential advantages for streamlined material production.

[0031] FIG.21 Data demonstrating that the combination of P55 and targeting peptides K9_04, K9_12, & K9_16 enhance editing in Ai9 NPCs (40pMol dose). Where “A” and “B” indicate statistically significant differences between groups (p < 0.05, ANOVA with Tukey’s post hoc test). Bars represent mean ± SEM (n=3).

[0032] FIG.22 These data demonstrate that RNPs modified with Cholesterol-Tz targeting ligands are able to edit Ai9 NPCs.

[0033] FIG.23 spCas9-A22P (RNPk) self delivering fusion protein tethered with either PEG5k-Tz or TET1 and PEG5K and dosed via CED into the striatum of Ai9 Mice (5uL bilateral, 40µM). Immediately after the completion of CED, mice were dosed intraperitoneally with N-Acetyl cystine ethyl ester (NACET) a BBB permeable reducing reagent. This triggers the release of PEG or targeting peptide, promoting self-delivery of the RNP. This demonstrates the ability of two step administration to edit the murine striatum in vivo. DEFINITIONS

[0034] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0035] The terms “linked” and “linkage” in their various forms in the context of linking molecules to one another are generally used to refer to covalent binding, e.g., covalently bound / covalently tethered / covalently connected / connected via a covalent bond.

[0036] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0037] The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C1 carbon atom) have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, - SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NRaRb, wherein Raand Rbmay be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0038] “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -O-, -NR10-, -NR10C(O)-, - C(O)NR10- and the like. This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like. R10may be chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0039] “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below.

[0040] The term “alkane” refers to alkyl group and alkylene group, as defined herein.

[0041] The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl” refers to the groups R’NHR”- where R’is alkyl group as defined herein and R”is alkylene, alkenylene or alkynylene group as defined herein.

[0042] The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.

[0043] “Alkoxy” refers to the group –O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n- pentoxy, and the like. The term “alkoxy” also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0044] The term “substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0045] The term “alkoxyamino” refers to the group –NH-alkoxy, wherein alkoxy is defined herein.

[0046] The term “haloalkoxy” refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.

[0047] The term “haloalkyl” refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group. Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like.

[0048] The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0049] The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0050] “Alkenyl” refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.

[0051] The term “substituted alkenyl” refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0052] “Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH2C≡CH).

[0053] The term “substituted alkynyl” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol,thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0054] “Alkynyloxy” refers to the group –O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0055] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)-.

[0056] “Acylamino” refers to the groups –NR20C(O)alkyl, -NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, -NR20C(O)substituted cycloalkyl, - NR20C(O)cycloalkenyl, -NR20C(O)substituted cycloalkenyl, -NR20C(O)alkenyl, - NR20C(O)substituted alkenyl, -NR20C(O)alkynyl, -NR20C(O)substituted alkynyl, -NR20C(O)aryl, -NR20C(O)substituted aryl, -NR20C(O)heteroaryl, -NR20C(O)substituted heteroaryl, -NR20C(O)heterocyclic, and -NR20C(O)substituted heterocyclic, wherein R20is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0057] “Aminocarbonyl” or the term “aminoacyl” refers to the group -C(O)NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0058] “Aminocarbonylamino” refers to the group –NR21C(O)NR22R23where R21, R22, and R23are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.

[0059] The term “alkoxycarbonylamino” refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0060] The term “acyloxy” refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl- C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl- C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0061] “Aminosulfonyl” refers to the group –SO2NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

[0062] “Sulfonylamino” refers to the group –NR21SO2R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0063] “Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents,selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0064] “Aryloxy” refers to the group –O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.

[0065] “Amino” refers to the group –NH2.

[0066] The term “substituted amino” refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.

[0067] “Carboxyl,” “carboxy” or “carboxylate” refers to –CO2H or salts thereof.

[0068] “Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0069] “(Carboxyl ester)oxy” or “carbonate” refers to the groups –O-C(O)O- alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O- C(O)O-alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O- C(O)O-cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O- substituted cycloalkenyl, -O-C(O)O-heteroaryl, -O-C(O)O-substituted heteroaryl, -O-C(O)O- heterocyclic, and -O-C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0070] “Cyano” or “nitrile” refers to the group –CN.

[0071] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

[0072] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, - SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0073] “Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.

[0074] The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0075] “Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.

[0076] “Cycloalkoxy” refers to –O-cycloalkyl.

[0077] “Cycloalkenyloxy” refers to –O-cycloalkenyl.

[0078] “Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

[0079] “Hydroxy” or “hydroxyl” refers to the group –OH.

[0080] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic. To satisfy valence requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent group, e.g., an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, - SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0081] The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0082] “Heteroaryloxy” refers to –O-heteroaryl.

[0083] “Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, where, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or –SO2- moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein.

[0084] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1- dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0085] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, - SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0086] “Heterocyclyloxy” refers to the group –O-heterocyclyl.

[0087] The term “heterocyclylthio” refers to the group heterocyclic-S-.

[0088] The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein.

[0089] The term “hydroxyamino” refers to the group -NHOH.

[0090] “Nitro” refers to the group –NO2.

[0091] “Oxo” refers to the atom (=O).

[0092] “Sulfonyl” refers to the group SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cylcoalkyl, SO2-cycloalkenyl, SO2-substituted cylcoalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2- heterocyclic, and SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0093] “Sulfonyloxy” refers to the group –OSO2-alkyl, OSO2-substituted alkyl, OSO2-alkenyl, OSO2- substituted alkenyl, OSO2-cycloalkyl, OSO2-substituted cylcoalkyl, OSO2-cycloalkenyl, OSO2-substituted cylcoalkenyl, OSO2-aryl, OSO2-substituted aryl, OSO2-heteroaryl, OSO2-substituted heteroaryl, OSO2-heterocyclic, and OSO2substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0094] The term “aminocarbonyloxy” refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0095] “Thiol” refers to the group -SH.

[0096] “Thioxo” or the term “thioketo” refers to the atom (=S).

[0097] “Alkylthio” or the term “thioalkoxy” refers to the group -S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.

[0098] The term “substituted thioalkoxy” or “substituted alkylthio” refers to the group -S-substituted alkyl.

[0099] The term “thioaryloxy” refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.

[0100] The term “thioheteroaryloxy” refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.

[0101] The term “thioheterocyclooxy” refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.

[0102] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[0103] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O–M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, -P(O)(O–)2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O–M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)O- M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5(“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N- morpholinyl.

[0104] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S–M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3–M+, -SO3R70, -OSO2R70, -OSO3–M+, -OSO3R70, -PO3-2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2–M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2–M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+.

[0105] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS(O)2O-M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined.

[0106] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0107] It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl- (substituted aryl)-substituted aryl.

[0108] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.

[0109] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0110] An “inert linker” is a moiety that covalently attaches, and optionally spaces, one moiety in a compound from another and which no substantial negative effect on the activity of the overall compound, e.g., in context of the present disclosure, the ability of the reactive groups, such as a tetrazine group, a succidimidyl group, or a dicarboxylic acid anhydride groups, such as maleic anhydride to react with their intended targets, and form and maintain a bond according to the methods described herein. Aside from serving to covalently-link two moieties, a linker may have a beneficial effect, such as in the physical separation of moieties to which it is attached, e.g., to optimize spacing to avoid steric effects. A linker also may serve some additional function, such as altering the hydrophobicity / hydrophilicity of the overall molecule, to provide an additional site, e.g., an amine protected by a protective group for linking additional moieties to the compound, or to rigidize the overall molecule. A linker may be attached to a subject compound by any suitable linkage moiety (“linkage”), e.g., by a carbon-carbon bond, an ester, a thioester, an amine, an ether, an amide, a carbonate, or a carbamate linkage to the additional moieties of the compound. The linker may be hydrocarbyl, that is including only carbons and hydrogens, or optionally comprising one or more hetero-atom, such as N, O, and / or S. In the context of the present disclosure, in some embodiments, one suitable linker is a divalent moiety comprising apolyethylene glycol (PEG) group (O-CH2-CH2)n, where n can have a range of values. A PEG linker may comprise one or more methylene groups at either end in addition to suitable linkages attaching the PEG group to the other moieties of a subject compound.

[0111] A “reduction sensitive disulfide linker “ is a self-immolative disulfide linker that, under reducing conditions, provides for dissociation of the covalent bond between a subject linker compound and the first molecule of interest (Z1) – thus releasing Z1from the linker. A reduction sensitive disulfide linker of the disclosure renders a subject linker reversible / traceless because under reducing conditions, the released Z1is indistinguishable from the Z1molecule prior to conjugation. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. Thus, in some cases, a reduction sensitive disulfide linker undergoes cleavage under physiological (e.g., intracellular) reducing conditions – thereby releasing the conjugated Z1molecule of interest from the subject reversible / traceless linker. The disulfide cleavable linker chemistry is reversible, allowing, e.g., stable surface decoration of the conjugated protein cargo until intracellular (cytosolic) delivery is successful, and then undergoing reversion (release) of the protein cargo to a pre-conjugation state due to the physiological reducing conditions.

[0112] The term “pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0113] The term “salt thereof” means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0114] “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or amixture of both. Some examples of solvents include, but are not limited to, methanol, N,N- dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0115] “Stereoisomer” and “stereoisomers” refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0116] “Tautomer” refers to alternate forms of a molecule that differ only in electronic bonding of atoms and / or in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a -N=C(H)-NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.

[0117] It will be appreciated that the term “or a salt or solvate or stereoisomer thereof” is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound.

[0118] “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and / or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[0119] The terms "polypeptide," "peptide," and "protein", are used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0120] “Heterologous,” as used herein, refers to a nucleotide or amino acid sequence that is not found in the native nucleic acid or protein, respectively. For example, relative to a CRISPR-Cas effector polypeptide, a heterologous polypeptide comprises an amino acid sequence from a protein other than the CRISPR-Cas effector polypeptide. Thus, for example, a polymerase polypeptide is heterologous to a CRISPR-Cas effector polypeptide.

[0121] The terms “antibodies” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to antigen, including, but not limited to, Fab, Fv, single-chain Fv (scFv), and Fd fragments, chimeric antibodies, humanizedantibodies, single-chain antibodies (scAb), single domain antibodies (dAb), single domain heavy chain antibodies, a single domain light chain antibodies, nanobodies, bi-specific antibodies, multi-specific antibodies, nanobodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen binding) portion of an antibody and a non-antibody protein. The antibodies can be detectably labeled, e.g., with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. The antibodies can be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin- avidin specific binding pair), and the like. Also encompassed by the term are Fab’, Fv, F(ab’)2, and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which were produced from a single cell by repetitive cellular replication. That is, the clone of cells only produces a single antibody species. While a monoclonal antibody can be produced using hybridoma production technology, other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). An antibody can be monovalent or bivalent. An antibody can be an Ig monomer, which is a “Y-shaped” molecule that consists of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0122] The term “nanobody” (Nb), as used herein, refers to the smallest antigen binding fragment or single variable domain (VHH) derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996). In the family of “camelids” immunoglobulins devoid of light polypeptide chains are found. “Camelids” comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Llama paccos, Llama glama, Llama guanicoe and Llama vicugna). A single variable domain heavy chain antibody is referred to herein as a nanobody or a VHH antibody.

[0123] “Antibody fragments” comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; scFv; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol.21:484); single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab’)2fragment that has two antigen combining sites and is still capable of cross-linking antigen. “Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise the VHand VLdomains of antibody, wherein thesedomains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VHand VLdomains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994). The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0124] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0125] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, non-human primates, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.

[0126] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, 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, since the scope of the present invention will be limited only by the appended claims.

[0127] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0128] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0129] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a linker” includes a plurality of such linkers and reference to “the biomolecule” includes reference to one or more biomolecules and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0130] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0131] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION

[0132] The present disclosure provides a linker that includes functional groups for attaching molecules of interest, where the linker includes a reduction sensitive disulfide group and a reactive moiety that undergoes cleavage under physiological reducing conditions, releasing the molecules of interest. The present disclosure provides compounds comprising a first and a second molecule ofinterest linked by a linker of the present disclosure. The present disclosure provides methods of using a compound of the present disclosure to deliver a molecule of interest. LINKER COMPOUNDS REVERSIBLE (I.E., TRACELESS) LINKER OF FORMULA (I)

[0133] The present disclosure provides a compound (e.g., a reversible / traceless linker conjugate) comprising molecules of interest linked to a reversible / traceless linker. For example, in some cases, a reversible linker conjugate disclosed herein comprises a first molecule of interest linked by a linker to a second molecule of interest.

[0134] In some cases, a subject compound is a reversible / traceless linker (e.g., one that is not yet conjugated to a first or second molecule of interest). For example, a subject compound can comprise a reversible / traceless linker of the formula (I): O 2 H R R1N Oan L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X1is an amine-reactive moiety; a salt thereof, or a stereoisomer thereof.

[0135] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, PEG has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an averagemolecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0136] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-10 kDa. In some cases, the PEG (of L1) has an average molecular weight in a range of from 0.5-2 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0137] In some cases, the PEG is (C2H4O)n: O n where n is in a range of from 1-435 (e.g., 5-435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 10-350, 15-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22. In some cases, n is 21. In some cases, n is 23.

[0138] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0139] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, whereinn is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0140] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0141] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0142] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0143] In some cases, X1comprises an amine-reactive moiety. An “amine-reactive moiety” is a reactive functional group that provides for attachment (i.e., covalent attachment) between the compound and an amine-containing moiety of interest (e.g., a first molecule of interest as described herein, e.g., a protein). An amine-reactive moiety can react with an amine (e.g., primary or secondaryamine) of the amine-containing moiety of interest to form a covalent bond between the compound at X1and the amine-containing moiety of interest. Any convenient amine-reactive moiety can be used in X1, such as, but not limited to, carbonate ester, acyl halide, acid anhydride, alkyl halide, alcohol, and the like. In some cases, X1is an amine-reactive moiety, such as carbonate ester. In some cases, X1is a carbonate ester amine-reactive moiety that reacts with an amine of the amine-containing moiety of interest to form a carbamate linkage between the compound at X1and the amine-containing moiety of interest. In some cases, X1is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. In some cases, X1is a carbonate. In some cases, X1is a para-nitrophenolcarbonate.

[0144] In some cases, the attachment between the compound and the amine-containing moiety of interest (e.g., a protein such as a gene editing protein) is reversible, such that under appropriate conditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine-containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine-containing moiety of interest that was present before attachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker (i.e., a reversible / traceless linker). In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self-immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. For example, in some cases, L2is a reduction sensitive disulfide linker. In some cases, a self-immolative linker does not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker. Z1- LINKER INTERMEDIATE OF CONJUGATE OF FORMULA (I)

[0145] The present disclosure provides a compound comprising a first molecule of interest (Z1) and a reversible (i.e., traceless) linker of the present disclosure. For example, in some instances, a firstmolecule of interest (Z1) is attached (e.g., covalently attached) to the reversible (i.e., traceless) linker, thus producing a conjugate comprising the first molecule of interest (Z1) and the reversible (i.e., traceless) linker; i.e., an Z1-linker conjugate. In some cases, the Z1-linker can be used as an intermediate in a process for producing a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2). For instance, after attaching Z1to the reversible (i.e., traceless) linker, in a subsequent reaction the Z1-linker conjugate can be reacted with a second molecule of interest to produce a compound comprising a first molecule (Z1) linked, via a reversible (i.e., traceless) linker of the present disclosure, to a second molecule (Z2).

[0146] In some cases, the Z1-linker conjugate is a compound of the formula (Ia): O 2 H R R1N Oan L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X2is a connecting group; Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof.

[0147] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having anaverage molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0148] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0149] In some cases, the PEG is (C2H4O)n: O n where n is in a range of from 1-435 (e.g., 5-435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 10-350, 15-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0150] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0151] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0152] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0153] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0154] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0155] In some cases, the attachment between the compound and the amine-containing moiety of interest (e.g., a protein such as a gene editing protein) is reversible, such that under appropriate conditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine-containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine-containing moiety of interest that was present beforeattachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker (i.e., a reversible / traceless linker). In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self-immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. For example, in some cases, L2is a reduction sensitive disulfide linker. In some cases, a self-immolative linker does not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker.

[0156] X2is a connecting group, e.g., resulting from the conjugation of Z1(a first molecule of interest) to the X1of formula (I) above. In some cases, X2is -NH-, -NHCOO-, -NH(CO)NH-, -NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof. LINKER-Z2INTERMEDIATE OF CONJUGATE OF FORMULA (I)

[0157] The present disclosure provides a compound comprising a second molecule of interest (Z2) and a reversible (i.e., traceless) linker of the present disclosure. For example, in some instances, a second molecule of interest (Z2) is attached (e.g., covalently attached) to the reversible (i.e., traceless) linker, thus producing a conjugate comprising the second molecule of interest (Z2) and the reversible (i.e., traceless) linker; i.e., a linker-Z2conjugate. The linker-Z2conjugate can therefore be the result of conjugating Z2to the compound of formula (I) via click chemistry – e.g., a tetrazine-bearing Z2(e.g. tetrazine-bearing peptide) can react with the trans-cyclooctene (TCO) group of the compound of formula (I) to generate the compound of formula (Ib). In some cases, the linker-Z2conjugate comprises a reactive functional group as described herein and can be used as an intermediate in a process for producing a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2). For instance, after attaching Z2to the reversible (i.e., traceless) linker, in a subsequent reaction the linker-Z2conjugate (which comprises an amine-reactive moiety) can be reacted with a first molecule of interest to produce a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2).

[0158] In some cases, the linker-Z2conjugate is a compound of the formula (Ib): R6NHL2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; R6is H, alkyl or a substituted version thereof; X1is an amine-reactive moiety; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

[0159] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0160] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0161] In some cases, the PEG is (C2H4O)n: O n where n is in a range of from 1-435 (e.g., 5- 435, 20-435, 22-435, 1-400, 5-400,10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0162] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0163] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0164] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In somecases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0165] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0166] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. Forinstance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0167] In some cases, X1comprises an amine-reactive moiety. An “amine-reactive moiety” is a reactive functional group that provides for attachment (i.e., covalent attachment) between the compound and an amine-containing moiety of interest (e.g., a first molecule of interest as described herein). An amine-reactive moiety can react with an amine (e.g., primary or secondary amine) of the amine-containing moiety of interest to form a covalent bond between the compound at X1and the amine-containing moiety of interest. Any convenient amine-reactive moiety can be used in X1, such as, but not limited to, carbonate ester, acyl halide, acid anhydride, alkyl halide, alcohol, and the like. In some cases, X1is an amine-reactive moiety, such as carbonate ester. In some cases, X1is a carbonate ester amine-reactive moiety that reacts with an amine of the amine- containing moiety of interest to form a carbamate linkage between the compound at X1and the amine-containing moiety of interest. In some cases, X1is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. In some cases, X1is a carbonate. In some cases, X1is a para-nitrophenol carbonate.

[0168] In some cases, the attachment between the compound and the amine-containing moiety of interest (e.g., a protein such as a gene editing protein) is reversible, such that under appropriateconditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine-containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine-containing moiety of interest that was present before attachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker (i.e., a reversible / traceless linker). In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In somecases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self-immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. For example, in some cases, L2is a reduction sensitive disulfide linker. In some cases, a self-immolative linker does not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker.

[0169] In some cases, R6is H, alkyl or a substituted version thereof. In some cases, R6is H. In some cases, R6is alkyl or a substituted version thereof. For instance, R6is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R6is isopropyl, sec-butyl, tert- butyl, or so on. CONJUGATES OF FORMULA (I)

[0170] As noted above, the present disclosure provides a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2); i.e., an Z1-linker-Z2conjugate.

[0171] In some cases, the Z1-linker-Z2conjugate is a compound of the formula (Ic): R6L1is an inert linker;L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; R6is H, alkyl or a substituted version thereof; X2is a connecting group; Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

[0172] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k Daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0173] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0174] In some cases, the PEG is (C2H4O)n:O n where n is in a range of from 1-435 (e.g., 5- 435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0175] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or(iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0176] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0177] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0178] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, whereinn is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0179] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0180] In some cases, R6is H, alkyl or a substituted version thereof. In some cases, R6is H. In some cases, R6is alkyl or a substituted version thereof. For instance, R6is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R6is isopropyl, sec-butyl, tert- butyl, or so on.

[0181] X2is a connecting group, e.g., resulting from the conjugation of Z1(a first molecule of interest) to the X1of formula (I) or (Ib) above. In some cases, X2is -NH-, -NHCOO-, -NH(CO)NH-, - NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof.

[0182] In some cases, Z1is a first molecule of interest as described herein. In some cases, the first molecule of interest (Z1) comprises an amine functional group. In some cases, the amine functional group of the first molecule of interest (Z1) can react with an amine-reactive functional group of the linker as described herein to produce the Z1-linker-Z2conjugate as described herein. As such, Z1can be attached to the compound through the amine functional group of the first molecule of interest (Z1).

[0183] In some cases, the first molecule of interest (Z1) comprises an amine functional group. In some cases, the first molecule of interest (Z1) comprises one amine functional group. In some cases, the first molecule of interest (Z1) comprises more than one amine functional group. In some cases, the first molecule of interest (Z1) comprises 1 to 50 amine functional groups, such as 1 to 40, or 1 to 30, or 1 to 20, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2 amine functional groups.

[0184] In embodiments where the first molecule of interest (Z1) comprises more than one amine functional group, one or more of the amine functional groups can be attached to a linker or a Z2- linker conjugate as described herein. In some cases, each amine functional group of the first molecule of interest (Z1) is attached to a linker or an Z2-linker conjugate as described herein. For example, conjugates of the present disclosure include compounds where Z2and Z1are present in the compound in a ratio of 1:1. In some cases, where the first molecule of interest (Z1) includes more than one amine functional group, two or more of the amine functional groups of the first molecule of interest (Z1) can be attached to a linker or an Z2-linker conjugate as described herein. For example, conjugates of the present disclosure include compounds where Z2and Z1are present in the compound in a ratio of from 2:1 to 50:1, such as from 2:1 to 40:1, or 2:1 to 30:1, or 2:1 to 20:1, or 2:1 to 10:1, or 2:1 to 9:1, or 2:1 to 8:1, or 2:1 to 7:1, or 2:1 to 6:1, or 2:1 to 5:1, or 2:1 to 4:1, or 2:1 to 3:1.

[0185] In some cases, a compound (i.e., a reversible linker conjugate) of present disclosure is: O H OOS ON OREVERSIBLE (I.E., TRACELESS) LINKER OF FORMULA (II)

[0186] The present disclosure provides a compound (i.e., a reversible linker conjugate) comprising molecules of interest linked to a linker. For example, the compound disclosed herein comprises a first molecule of interest linked by a linker to a second molecule of interest. In some cases, the compound comprises a linker. For example, the compound can comprise a compound (a reversible / traceless linker) of the formula (II): O OX1is an amine-reactive moiety; Y1is a reactive moiety; L1is an inert linker; a salt thereof, or a stereoisomer thereof.

[0187] In some cases, X1comprises an amine-reactive moiety. An “amine-reactive moiety” is a reactive functional group that provides for attachment (i.e., covalent attachment) between the compound and an amine-containing moiety of interest (e.g., a first molecule of interest as described herein).An amine-reactive moiety can react with an amine (e.g., primary or secondary amine) of the amine-containing moiety of interest to form a covalent bond between the compound at X1and the amine-containing moiety of interest. Any convenient amine-reactive moiety can be used in X1, such as, but not limited to, carbonate ester, acyl halide, acid anhydride, alkyl halide, alcohol, and the like. In some cases, X1is an amine-reactive moiety, such as carbonate ester. In some cases, X1is a carbonate ester amine-reactive moiety that reacts with an amine of the amine- containing moiety of interest to form a carbamate linkage between the compound at X1and the amine-containing moiety of interest. In some cases, X1is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. In some cases, X1is acarbonate. In some cases, X1is a para-nitrophenol carbonate.

[0188] In some cases, the attachment between the compound and the amine-containing moiety of interest is reversible, such that under appropriate conditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine- containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine- containing moiety of interest that was present before attachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker. In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self- immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. In some cases, a self-immolative linker does not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker.

[0189] In some cases, Y1comprises a reactive moiety. A “reactive moiety” refers to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases, after optional activation of one of the functional groups. Reactive moieties of interest include, but are not limited to, thiols and maleimide or iodoacetamide, amines and carboxylic acids or active esters thereof, as well as click chemistry reactive moieties, i.e., groupsthat can react with other groups via click chemistry, e.g., azide and alkyne groups (e.g., cyclooctyne groups), tetrazine, transcyclooctene, dienes and dienophiles, and azide, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, as well as hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne, phosphine, epoxide, succinimide, pentafluorophenyl (PFP) ester, and carboxylic acid (e.g. on a lysine residue).

[0190] In some cases, Y1comprises a click chemistry reactive moiety. In some cases, Y1is trans- cyclooctene (TCO), tetrazine, alkyne, thiol, maleimide, iodoacetamide, amine, carboxyl, ester, triazole, diene, dienophile, sulfonyl fluoride, hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, phosphine, epoxide, succinimide, and pentafluorophenyl (PFP) ester, or a substituted version thereof.

[0191] Click-chemistry reactions that may be employed include (i) nucleophilic substitutions; (ii) additions to C–C multiple bonds (e.g., Michael addition, epoxidation, dihydroxylation, aziridination); (iii) nonaldol like chemistry (e.g., N-hydroxysuccinimide active ester couplings); and (iv) cycloadditions (e.g., Diels–Adler reaction, Huisgen’s cycloaddition). Huisgen’s cycloaddition has been applied in various branches of chemistry. It consists of the condensation of organic azides with alkyne groups to form 1,2,3-triazole linkages. Azide and alkyne functionalities can be easily introduced in the scaffold of large organic constructs of biological relevance. The reaction may be catalyzed by introducing copper(I). The Cu(I) core has a dual effect in that it activates the slow-reacting alkyne group thus accelerating the azide–alkyne condensation kinetics by ∼107–108-fold, and it organizes the reacting groups by “templation” so that only a regiospecific 1,4-disubstituted adduct is formed. This reaction is known as the copper-catalyzed azide alkyne cycloaddition (CuAAC), and its compatibility with a wide range of biological substrates and synthetic conditions makes CuAAC the flagship among click conjugations. Since its discovery, Cu(I)-catalyzed azide alkyne cycloaddition has been widely used within the fields of biology, biochemistry, and biotechnology. Click-chemistry reactions that may be employed to attach the nucleotide or polynucleotide to the particle include, but are not limited to, Huisgen Azide-Alkyne 1,3-Dipolar Cycloaddition, Copper-Catalyzed Azide- Alkyne Cycloaddition (CuAAC), Ruthenium-Catalyzed Azide-Alkyne Cycloaddition (RuAAC), and the like. Details regarding click-chemistry with nucleic acids are found, e.g., in Fantoni et al. (2021) Chem. Rev.121(12):7122–7154.

[0192] In some cases, Y1comprises a copper free click chemistry reactive moiety. In some cases, Y1comprises a copper free click chemistry reactive moiety that is not an azide (i.e., it can be a reactive moiety that can react with an azide but is not itself an azide). Copper-free click chemistry is an alternative approach to click chemistry that proceeds at a lower activation barrier and is free of cytotoxic transition metal catalysts. The copper-free reaction can use ring strain inplace of the copper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction. For example, 8-carbon closed ring structure of a cyclooctyne comprising an internal alkyne bond induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole. Thus, cyclooctyne derivatives may be used for copper- free click reactions, without the toxic copper catalyst. Another type of copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving strain-promoted alkyne-nitron cycloaddition. To address the slow rate of the original cyclooctyne reaction, electron-withdrawing groups can be attached adjacent to the triple bond. Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne. An alternative copper-free reaction involved strain-promoted alkyne-nitrone cycloaddition to give N-alkylated isoxazolines.

[0193] Accordingly, in some embodiments the copper-free click chemistry useful in forming compounds of the present disclosure can be selected from: (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels- Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbonene click chemistry; (d) Diels-Alder maleimide / furan click-chemistry; (e) Staudinger ligation; and (f) nitrile-oxide / norbonene cycloaddition click chemistry. Thus, in some cases, Y1comprises an appropriate copper free click chemistry reactive moiety, e.g., TCO. Likewise, in some cases, Y1comprises DBCO.

[0194] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0195] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0196] In some cases, the PEG is (C2H4O)n: O n

[0197] where n is in a range of from 1-435 (e.g., 5- 435, 20-435, 22-435, 1-400, 5-400,10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22. Z1- LINKER INTERMEDIATE OF CONJUGATE OF FORMULA (II)

[0198] As noted above, the present disclosure provides a compound comprising a first molecule of interest (Z1) and a reversible (i.e., traceless) linker of the present disclosure. For example, in some instances, a first molecule of interest (Z1) is attached (e.g., covalently attached) to the reversible (i.e., traceless) linker, thus producing a conjugate comprising the first molecule of interest (Z1) and the reversible (i.e., traceless) linker; i.e., an Z1-linker conjugate. In some cases, the Z1-linker conjugate comprises a reactive moiety (Y1, e.g., a click reactive moiety) and can be used as an intermediate in a process for producing a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2). For instance, after attaching Z1to the reversible (i.e., traceless) linker, in a subsequent reaction the Z1-linker conjugate (which comprises the reactive moiety) can be reacted with a second molecule of interest to produce a compound comprising a first molecule (Z1) linked, via a reversible (i.e., traceless) linker of the present disclosure, to a second molecule (Z2).

[0199] In some cases, the Z1-linker conjugate is a compound of the formula (IIa):O O Z1X2S L1Y1NL1is an inert linker; X2is a connecting group; Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof.

[0200] In some cases, Y1comprises a reactive moiety. A “reactive moiety” refers to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases, after optional activation of one of the functional groups. Reactive moieties of interest include, but are not limited to, thiols and maleimide or iodoacetamide, amines and carboxylic acids or active esters thereof, as well as click chemistry reactive moieties, i.e., groups that can react with other groups via click chemistry, e.g., azide and alkyne groups (e.g., cyclooctyne groups), tetrazine, transcyclooctene, dienes and dienophiles, and azide, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, as well as hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne, phosphine, epoxide, succinimide, pentafluorophenyl (PFP) ester, and carboxylic acid (e.g. on a lysine residue).

[0201] In some cases, Y1comprises a click chemistry reactive moiety. In some cases, Y1is trans- cyclooctene (TCO), tetrazine, alkyne, thiol, maleimide, iodoacetamide, amine, carboxyl, ester, triazole, diene, dienophile, sulfonyl fluoride, hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, phosphine, epoxide, succinimide, and pentafluorophenyl (PFP) ester, or a substituted version thereof.

[0202] Click-chemistry reactions that may be employed include (i) nucleophilic substitutions; (ii) additions to C–C multiple bonds (e.g., Michael addition, epoxidation, dihydroxylation, aziridination); (iii) nonaldol like chemistry (e.g., N-hydroxysuccinimide active ester couplings); and (iv) cycloadditions (e.g., Diels–Adler reaction, Huisgen’s cycloaddition). Huisgen’s cycloaddition has been applied in various branches of chemistry. It consists of the condensation of organic azides with alkyne groups to form 1,2,3-triazole linkages. Azide and alkyne functionalities can be easily introduced in the scaffold of large organic constructs of biological relevance. The reaction may be catalyzed by introducing copper(I). The Cu(I) core has a dual effect in that it activates the slow-reacting alkyne group thus accelerating the azide–alkyne condensation kinetics by ∼107–108-fold, and it organizes the reacting groups by “templation” so that only a regiospecific 1,4-disubstituted adduct is formed. This reaction is known as thecopper-catalyzed azide alkyne cycloaddition (CuAAC), and its compatibility with a wide range of biological substrates and synthetic conditions makes CuAAC the flagship among click conjugations. Since its discovery, Cu(I)-catalyzed azide alkyne cycloaddition has been widely used within the fields of biology, biochemistry, and biotechnology. Click-chemistry reactions that may be employed to attach the nucleotide or polynucleotide to the particle include, but are not limited to, Huisgen Azide-Alkyne 1,3-Dipolar Cycloaddition, Copper-Catalyzed Azide- Alkyne Cycloaddition (CuAAC), Ruthenium-Catalyzed Azide-Alkyne Cycloaddition (RuAAC), and the like. Details regarding click-chemistry with nucleic acids are found, e.g., in Fantoni et al. (2021) Chem. Rev.121(12):7122–7154.

[0203] In some cases, Y1comprises a copper free click chemistry reactive moiety. In some cases, Y1comprises a copper free click chemistry reactive moiety that is not an azide (i.e., it can be a reactive moiety that can react with an azide but is not itself an azide). Copper-free click chemistry is an alternative approach to click chemistry that proceeds at a lower activation barrier and is free of cytotoxic transition metal catalysts. The copper-free reaction can use ring strain in place of the copper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction. For example, 8-carbon closed ring structure of a cyclooctyne comprising an internal alkyne bond induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole. Thus, cyclooctyne derivatives may be used for copper- free click reactions, without the toxic copper catalyst. Another type of copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving strain-promoted alkyne-nitron cycloaddition. To address the slow rate of the original cyclooctyne reaction, electron-withdrawing groups can be attached adjacent to the triple bond. Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne. An alternative copper-free reaction involved strain-promoted alkyne-nitrone cycloaddition to give N-alkylated isoxazolines.

[0204] Accordingly, in some embodiments the copper-free click chemistry useful in forming compounds of the present disclosure can be selected from: (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels- Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbonene click chemistry; (d) Diels-Alder maleimide / furan click-chemistry; (e) Staudinger ligation; and (f) nitrile-oxide / norbonene cycloaddition click chemistry. Thus, in some cases, Y1comprises an appropriate copper free click chemistry reactive moiety, e.g., TCO. Likewise, in some cases, Y1comprises DBCO.

[0205] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG isunsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0206] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0207] In some cases, the PEG is (C2H4O)n: n

[0208] where n is in a range of from 1-435 (e.g., 5-435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 10-350, 15-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0209] In some cases, X2is a connecting group, e.g., resulting from the conjugation of Z1(a first molecule of interest) to the X1of formula (II) above. In some cases, X2is -NH-, -NHCOO-, -NH(CO)NH-, -NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof. LINKER-Z2INTERMEDIATE OF CONJUGATE OF FORMULA (II)

[0210] The present disclosure provides a compound comprising a second molecule of interest (Z2) and a reversible (i.e., traceless) linker of the present disclosure. For example, in some instances, a second molecule of interest (Z2) is attached (e.g., covalently attached) to the reversible (i.e., traceless) linker, thus producing a conjugate comprising the second molecule of interest (Z2) and the reversible (i.e., traceless) linker; i.e., a linker-Z2conjugate. In some cases, the linker-Z2conjugate comprises an amine-reactive moiety (X1) and can be used as an intermediate in a process for producing a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2). For instance, after attaching Z2to the reversible (i.e., traceless) linker, in a subsequent reaction the linker-Z2conjugate (which comprises the amine-reactive moiety) can be reacted with a first molecule of interest as to produce a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2).

[0211] In some cases, the linker-Z2conjugate is a compound of the formula (IIb): O OY2is a post-reaction chemical group; L1is an inert linker; X1is an amine-reactive moiety; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

[0212] In some cases, Y2is a post-reaction chemical group or a connecting group, e.g., resulting from the conjugation of Z2(a second molecule of interest) to the Y1of formula (II) above, e.g., in some cases via click chemistry. In some cases, Y2is TCO-tetrazine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, thioether, succinimidyl-thioether, carbamate acetal, disulfide or a substituted version thereof. In some cases, Y2is TCO-tetrazine.

[0213] In some cases, X1comprises an amine-reactive moiety. An “amine-reactive moiety” is a reactive functional group that provides for attachment (i.e., covalent attachment) between the compound and an amine-containing moiety of interest (e.g., a first molecule of interest as described herein).An amine-reactive moiety can react with an amine (e.g., primary or secondary amine) of the amine-containing moiety of interest to form a covalent bond between the compound at X1and the amine-containing moiety of interest. Any convenient amine-reactive moiety can be used in X1, such as, but not limited to, carbonate ester, acyl halide, acid anhydride, alkyl halide, alcohol, and the like. In some cases, X1is an amine-reactive moiety, such as carbonate ester. In some cases, X1is a carbonate ester amine-reactive moiety that reacts with an amine of the amine- containing moiety of interest to form a carbamate linkage between the compound at X1and the amine-containing moiety of interest. In some cases, X1is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. In some cases, X1is acarbonate. In some cases, X1is a para-nitrophenol carbonate.

[0214] In some cases, the attachment between the compound and the amine-containing moiety of interest is reversible, such that under appropriate conditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine- containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine- containing moiety of interest that was present before attachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker. In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self- immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. In some cases, a self-immolative linker does not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker.

[0215] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecularweight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0216] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0217] In some cases, the PEG is (C2H4O)n: O n

[0218] where n is in a range of from 1-435 (e.g., 5-435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 10-350, 15-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22. CONJUGATES OF FORMULA (II)

[0219] As noted above, the present disclosure provides a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2); i.e., an Z1-linker-Z2conjugate.

[0220] In some cases, the Z1-linker-Z2conjugate is a compound of the formula (IIc): O O Z1X2S L1Y2Z2L1is a linker; X2is a connecting group; Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

[0221] In some cases, Y2is a post-reaction chemical group or a connecting group, e.g., resulting from the conjugation of Z2(a second molecule of interest) to the Y1of formula (IIa) above, e.g., in some cases via click chemistry. In some cases, Y2is TCO-tetrazine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, thioether, succinimidyl-thioether, carbamate acetal, disulfide or a substituted version thereof. In some cases, Y2is TCO-tetrazine.

[0222] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0223] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0224] In some cases, the PEG is (C2H4O)n: O n

[0225] where n is in a range of from 1-435 (e.g., 5- 435, 20-435, 22-435, 1-400, 5-400,10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0226] X2is a connecting group, e.g., resulting from the conjugation of Z1(a first molecule of interest) to the X1of formula (II) or (IIb) above. In some cases, X2is -NH-, -NHCOO-, -NH(CO)NH-, - NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof.

[0227] In some cases, Z1is a first molecule of interest as described herein. In some cases, the first molecule of interest (Z1) comprises an amine functional group. In some cases, the amine functional group of the first molecule of interest (Z1) can react with an amine-reactive functional group of the linker as described herein to produce the Z1-linker-Z2conjugate as described herein. As such, Z1can be attached to the compound through the amine functional group of the first molecule of interest (Z1).

[0228] In some cases, the first molecule of interest (Z1) comprises an amine functional group. In some cases, the first molecule of interest (Z1) comprises one amine functional group. In some cases, the first molecule of interest (Z1) comprises more than one amine functional group. In some cases, the first molecule of interest (Z1) comprises 1 to 50 amine functional groups, such as 1 to 40, or 1 to 30, or 1 to 20, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2 amine functional groups.

[0229] In embodiments where the first molecule of interest (Z1) comprises more than one amine functional group, one or more of the amine functional groups can be attached to a linker or a Z2- linker conjugate as described herein. In some cases, each amine functional group of the firstmolecule of interest (Z1) is attached to a linker or an Z2-linker conjugate as described herein. For example, conjugates of the present disclosure include compounds where Z2and Z1are present in the compound in a ratio of 1:1. In some cases, where the first molecule of interest (Z1) includes more than one amine functional group, two or more of the amine functional groups of the first molecule of interest (Z1) can be attached to a linker or an Z2-linker conjugate as described herein. For example, conjugates of the present disclosure include compounds where Z2and Z1are present in the compound in a ratio of from 2:1 to 50:1, such as from 2:1 to 40:1, or 2:1 to 30:1, or 2:1 to 20:1, or 2:1 to 10:1, or 2:1 to 9:1, or 2:1 to 8:1, or 2:1 to 7:1, or 2:1 to 6:1, or 2:1 to 5:1, or 2:1 to 4:1, or 2:1 to 3:1. REVERSIBLE (I.E., TRACELESS) LINKER OF FORMULA (III)

[0230] The present disclosure provides a compound (e.g., a reversible / traceless linker conjugate) comprising molecules of interest linked to a reversible / traceless linker. For example, in some cases, a reversible linker conjugate disclosed herein comprises a first molecule of interest linked by a linker to a second molecule of interest.

[0231] In some cases, a subject compound is a reversible / traceless linker (e.g., one that is not yet conjugated to a first or second molecule of interest). For example, a subject compound can comprise a reversible / traceless linker of the formula (III): O HL1is an inert linker; L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X1is an amine-reactive moiety; Y1is a reactive moiety (e.g., a copper free click chemistry reactive moiety); a salt thereof, or a stereoisomer thereof.

[0232] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, PEG has a molecular weight in the range of1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0233] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0234] In some cases, the PEG is (C2H4O)n: O n where n is in a range of from 1-435 (e.g., 5-435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 10-350, 15-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0235] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0236] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0237] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0238] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0239] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0240] In some cases, X1comprises an amine-reactive moiety. An “amine-reactive moiety” is a reactive functional group that provides for attachment (i.e., covalent attachment) between the compound and an amine-containing moiety of interest (e.g., a first molecule of interest as described herein, e.g., a protein). An amine-reactive moiety can react with an amine (e.g., primary or secondary amine) of the amine-containing moiety of interest to form a covalent bond between the compound at X1and the amine-containing moiety of interest. Any convenient amine-reactive moiety can be used in X1, such as, but not limited to, carbonate ester, acyl halide, acid anhydride, alkyl halide, alcohol, and the like. In some cases, X1is an amine-reactive moiety, such as carbonate ester. In some cases, X1is a carbonate ester amine-reactive moiety that reacts with an amine of the amine-containing moiety of interest to form a carbamate linkage between the compound at X1and the amine-containing moiety of interest. In some cases, X1is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. In some cases, X1is a carbonate. In some cases, X1is a para-nitrophenol carbonate.

[0241] In some cases, the attachment between the compound and the amine-containing moiety of interest (e.g., a protein such as a gene editing protein) is reversible, such that under appropriate conditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine-containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine-containing moiety of interest that was present before attachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker (i.e., a reversible / traceless linker). In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self-immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. For example, in some cases, L2is a reduction sensitive disulfide linker. In some cases, a self-immolative linker does not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker.

[0242] In some cases, Y1comprises a copper free click chemistry reactive moiety. In some cases, Y1comprises a copper free click chemistry reactive moiety that is not an azide (i.e., it can be a reactive moiety that can react with an azide but is not itself an azide). Copper-free click chemistry is an alternative approach to click chemistry that proceeds at a lower activation barrier and is free of cytotoxic transition metal catalysts. The copper-free reaction can use ring strain in place of the copper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction. For example, 8-carbon closed ring structure of a cyclooctyne comprising an internal alkyne bond induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole. Thus, cyclooctyne derivatives may be used for copper- free click reactions, without the toxic copper catalyst. Another type of copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving strain-promoted alkyne-nitron cycloaddition. To address the slow rate of the original cyclooctyne reaction, electron-withdrawing groups can be attached adjacent to the triple bond. Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne. An alternative copper-free reaction involved strain-promoted alkyne-nitrone cycloaddition to give N-alkylated isoxazolines.

[0243] Accordingly, in some embodiments the copper-free click chemistry useful in forming compounds of the present disclosure can be selected from: (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels- Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbonene click chemistry; (d) Diels-Alder maleimide / furan click-chemistry; (e) Staudinger ligation; and (f) nitrile-oxide / norbonene cycloaddition click chemistry. Thus, in some cases, Y1comprises an appropriate copper free click chemistry reactive moiety, e.g., TCO. Likewise, in some cases, Y1comprises DBCO. Z1- LINKER INTERMEDIATE OF CONJUGATE OF FORMULA (III)

[0244] The present disclosure provides a compound comprising a first molecule of interest (Z1) and a reversible (i.e., traceless) linker of the present disclosure. For example, in some instances, a first molecule of interest (Z1) is attached (e.g., covalently attached) to the reversible (i.e., traceless) linker, thus producing a conjugate comprising the first molecule of interest (Z1) and the reversible (i.e., traceless) linker; i.e., an Z1-linker conjugate. In some cases, the Z1-linker conjugate comprises a reactive moiety (e.g., a copper free click chemistry reactive moiety) and can be used as an intermediate in a process for producing a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2). For instance, after attaching Z1to the reversible (i.e., traceless) linker, in a subsequent reaction theZ1-linker conjugate (which comprises the reactive moiety, e.g., copper free click chemistry reactive moiety), can be reacted with a second molecule of interest to produce a compound comprising a first molecule (Z1) linked, via a reversible (i.e., traceless) linker of the present disclosure, to a second molecule (Z2).

[0245] In some cases, the Z1-linker conjugate is a compound of the formula (IIIa): O R2R1H NOL2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; Y1is a reactive moiety (e.g., a copper free click chemistry reactive moiety); X2is a connecting group; Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof.

[0246] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0247] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0248] In some cases, the PEG is (C2H4O)n: O n where n is in a range of from 1-435 (e.g., 5- 435, 20-435, 22-435, 1-400, 5-400,10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0249] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0250] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0251] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4islinear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0252] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0253] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0254] In some cases, the attachment between the compound and the amine-containing moiety of interest (e.g., a protein such as a gene editing protein) is reversible, such that under appropriate conditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine-containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine-containing moiety of interest that was present before attachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker (i.e., a reversible / traceless linker). In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditionsfound in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self-immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. For example, in some cases, L2is a reduction sensitive disulfide linker. In some cases, a self-immolative linker does not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker.

[0255] In some cases, Y1comprises a copper free click chemistry reactive moiety. In some cases, Y1comprises a copper free click chemistry reactive moiety that is not an azide (i.e., it can be a reactive moiety that can react with an azide but is not itself an azide). Copper-free click chemistry is an alternative approach to click chemistry that proceeds at a lower activation barrier and is free of cytotoxic transition metal catalysts. The copper-free reaction can use ring strain in place of the copper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction. For example, 8-carbon closed ring structure of a cyclooctyne comprising an internal alkyne bond induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole. Thus, cyclooctyne derivatives may be used for copper- free click reactions, without the toxic copper catalyst. Another type of copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving strain-promoted alkyne-nitron cycloaddition. To address the slow rate of the original cyclooctyne reaction, electron-withdrawing groups can be attached adjacent to the triple bond. Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne. An alternative copper-free reaction involved strain-promoted alkyne-nitrone cycloaddition to give N-alkylated isoxazolines.

[0256] Accordingly, in some embodiments the copper-free click chemistry useful in forming compounds of the present disclosure can be selected from: (a) strain-promoted azide / dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels- Alder (IED-DA) tetrazine / trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine / norbonene click chemistry; (d) Diels-Alder maleimide / furan click-chemistry; (e) Staudinger ligation; and (f) nitrile-oxide / norbonene cycloaddition click chemistry. Thus, in some cases, Y1comprises an appropriate copper free click chemistry reactive moiety, e.g., TCO. Likewise, in some cases, Y1comprises DBCO.

[0257] In some cases, X2is a connecting group, e.g., resulting from the conjugation of Z1(a first molecule of interest) to the X1of formula (I) above. In some cases, X2is -NH-, -NHCOO-, - NH(CO)NH-, -NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino,alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof. LINKER-Z2INTERMEDIATE OF CONJUGATE OF FORMULA (III)

[0258] The present disclosure provides a compound comprising a second molecule of interest (Z2) and a reversible (i.e., traceless) linker of the present disclosure. For example, in some instances, a second molecule of interest (Z2) is attached (e.g., covalently attached) to the reversible (i.e., traceless) linker, thus producing a conjugate comprising the second molecule of interest (Z2) and the reversible (i.e., traceless) linker; i.e., a linker-Z2conjugate. The linker-Z2conjugate can therefore be the result of conjugating Z2to the compound of formula (III) via click chemistry – e.g., a azide-bearing Z2(e.g. azide-bearing peptide) can react with the reactive moiety, e.g., copper free click chemistry reactive moiety (e.g., dibenzocylcooctyne (DBCO) group, TCO, and the like) of the compound of formula (III) to generate the compound of formula (IIIb). In some cases, the linker-Z2conjugate comprises an amine-reactive moiety (X1) and can be used as an intermediate in a process for producing a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2). For instance, after attaching Z2to the reversible (i.e., traceless) linker, in a subsequent reaction the linker-Z2conjugate (which comprises the amine-reactive moiety) can be reacted with a first molecule of interest to produce a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2).

[0259] In some cases, the linker-Z2conjugate is a compound of the formula (IIIb): O HL1is an inert linker; L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X1is an amine-reactive moiety; Y2is a post-reaction chemical group (e.g., a copper free click chemistry post-reaction group); Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

[0260] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0261] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0262] In some cases, the PEG is (C2H4O)n: n where n is in a range of from 1-435 (e.g., 5-435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 10-350, 15-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0263] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0264] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0265] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0266] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0267] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl orsubstituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0268] In some cases, X1comprises an amine-reactive moiety. An “amine-reactive moiety” is a reactive functional group that provides for attachment (i.e., covalent attachment) between the compound and an amine-containing moiety of interest (e.g., a first molecule of interest as described herein). An amine-reactive moiety can react with an amine (e.g., primary or secondary amine) of the amine-containing moiety of interest to form a covalent bond between the compound at X1and the amine-containing moiety of interest. Any convenient amine-reactive moiety can be used in X1, such as, but not limited to, carbonate ester, acyl halide, acid anhydride, alkyl halide, alcohol, and the like. In some cases, X1is an amine-reactive moiety, such as carbonate ester. In some cases, X1is a carbonate ester amine-reactive moiety that reacts with an amine of the amine- containing moiety of interest to form a carbamate linkage between the compound at X1and the amine-containing moiety of interest. In some cases, X1is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. In some cases, X1is a carbonate. In some cases, X1is a para-nitrophenol carbonate.

[0269] In some cases, the attachment between the compound and the amine-containing moiety of interest (e.g., a protein such as a gene editing protein) is reversible, such that under appropriate conditions the covalent bond is broken between the compound at X1and the amine-containing moiety of interest, thus releasing the amine-containing moiety of interest from the compound. In some cases, release of the amine-containing moiety of interest from the compound provides the same amine functional group on the amine-containing moiety of interest that was present before attachment of the amine-containing moiety of interest to the compound. In some cases, this type of linker, which provides for reversible attachment of the amine-containing moiety of interest and regeneration of the same amine functional group upon release of the amine-containing moiety of interest, is referred to as a “traceless” linker (i.e., a reversible / traceless linker). In some cases, the conditions that provide for dissociation of the covalent bond between the compound at X1and the amine-containing moiety of interest are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. A “self-immolative” linker is a linker where one or more covalent bonds in the linker undergo dissociation when contacted with appropriate conditions, such as physiological reducing conditions described above. For example, in some cases, L2is a reduction sensitive disulfide linker. In some cases, a self-immolative linkerdoes not require the application of an external reagent or stimulus to cause dissociation of one or more covalent bonds in the linker.

[0270] In some cases, Y2is a post-reaction chemical group or a connecting group, e.g., resulting from the conjugation of Z2(a second molecule of interest) to the Y1of formula (III) above, e.g., in some cases via copper free click chemistry. For example, in some cases, Y2is dibenzocyclooctyne (DBCO)-azide, Likewise, in some cases Y2is a TCO-tetrazine. CONJUGATES OF FORMULA (III)

[0271] As noted above, the present disclosure provides a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2); i.e., an Z1-linker-Z2conjugate.

[0272] In some cases, the Z1-linker-Z2conjugate is a compound of the formula (IIIc): O R2R1H NO Y2Z2an L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X2is a connecting group; Y2is post-reaction chemical group (e.g., a copper free click chemistry post-reaction group); Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

[0273] In some cases, L1is polyethylene glycol (PEG). In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 200k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 1k to 180k daltons. In some embodiments, polyethylene glycol has a molecular weight in the range of 10k to 160k daltons. In some embodiments, polyethylene glycolhas a molecular weight of about 1k daltons. In some embodiments, polyethylene glycol has a molecular weight of about 20k Daltons. The PEG reagent used to produce a subject linker can be any of a wide variety of molecular weights. For example, a PEG200 reagent is PEG having an average molecular weight of 200 daltons (Da). Likewise PEG1000 has an average molecular weight of 1 kilodalton (kDa) and PEG20000 has an average molecular weight of 20 kDa.

[0274] In some cases, the PEG (of L1) has an average molecular weight in a range of from 200 daltons (Da) to 160 kilodaltons (kDa) (e.g., 200 Da to 20 kDa, 200 Da to 15 kDa, 200 Da to 10 kDa, 200 Da to 6 kDa, 200 Da to 4 kDa, 200 Da to 2 kDa, 200 Da to 1 kDa, 400 Da to 160 kDa, 400 Da to 20 kDa, 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 160 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 400 daltons (Da) to 20 kilodaltons (kDa) (e.g., 400 Da to 15 kDa, 400 Da to 10 kDa, 400 Da to 6 kDa, 400 Da to 4 kDa, 400 Da to 2 kDa, 400 Da to 1 kDa, 600 Da to 20 kDa, 600 Da to 15 kDa, 600 Da to 10 kDa, 600 Da to 6 kDa, 600 Da to 4 kDa, 600 Da to 2 kDa, or 600 Da to 1 kDa). In some cases, the PEG (of L1) has an average molecular weight in a range of from 1-20 kDa. In some cases, the PEG (of L1) has an average molecular weight of about 1 kDa.

[0275] In some cases, the PEG is (C2H4O)n: O n where n is in a range of from 1-435 (e.g., 5-435, 20-435, 22-435, 1-400, 5-400, 10-400, 15-400, 20-400, 22-400, 1-350, 5-350, 10-350, 15-350, 20-350, 22-350, 1-300, 5-300, 10-300, 15-300, 20-300, 22-300, 1-250, 5-250, 10-250, 15-250, 20-250, 22-250, 1-200, 5-200, 10-200, 15-200, 20-200, 22-200, 1-150, 5-150, 10-150, 15-150, 20-150, 22-150, 1-100, 5-100, 10-100, 15-100, 20-100, 22-100, 1-50, 5-50, 10-50, 15-50, 20-50, or 22-50). In some cases, n is in a range of from 15-30 (e.g., 15-25, 15-23, 18-30, 18-25, 18-23, 20-30, 20-25, or 21-23). In some cases, n is about 22.

[0276] In some cases, L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. In some cases, L2is a disulfide. In some cases, L2is a disulfide carbamate. In some cases, L2is a dithiobenzyl carbamate. In some cases, L2is a dithiol-ethyl carbonate plus a benzyl carbamate.

[0277] In some cases, R1is absent. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R1is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R1is -(CH2)n-, whereinn is an integer ranging from 2 to 5. For instance, in some cases, R1is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0278] In some cases, R1is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0279] In some cases, R2is absent. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 1 to 10. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 8. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 6. In some cases, R2is -(CH2)n-, wherein n is an integer ranging from 2 to 5. For instance, in some cases, R2is -(CH2)1-, -(CH2)2-, -(CH2)3- , -(CH2)4-, -(CH2)5-¸-(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2)10-.

[0280] In some cases, R2is -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof. In some cases, R3is H. In some cases, R3is linear alkyl or substituted versions thereof. For instance, R3is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R3is branched alkyl or substituted versions thereof. For instance, R3is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R3is amine or substituted versions thereof. In some cases, R3is azide or substituted versions thereof. In some cases, R4is H. In some cases, R4is linear alkyl or substituted versions thereof. For instance, R4is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl or so on. In some cases, R4is branched alkyl or substituted versions thereof. For instance, R4is isopropyl, sec-butyl, tert-butyl, or so on. In some cases, R4is amine or substituted versions thereof. In some cases, R4is azide or substituted versions thereof.

[0281] In some cases, X2is a connecting group, e.g., resulting from the conjugation of Z1(a first molecule of interest) to the X1of formula (I) or (Ib) above. In some cases, X2is -NH-, -NHCOO- , -NH(CO)NH-, -NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino,alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof.

[0282] In some cases, Y2is a post-reaction chemical group or a connecting group, e.g., resulting from the conjugation of Z2(a second molecule of interest) to the Y1of formula (IIIa) above, e.g., in some cases via copper free click chemistry. For example, in some cases, Y2is dibenzocyclooctyne (DBCO)-azide, Likewise, in some cases Y2is a TCO-tetrazine.

[0283] In some cases, Z1is a first molecule of interest as described herein. In some cases, the first molecule of interest (Z1) comprises an amine functional group. In some cases, the amine functional group of the first molecule of interest (Z1) can react with an amine-reactive functional group of the linker as described herein to produce the Z1-linker-Z2conjugate as described herein. As such, Z1can be attached to the compound through the amine functional group of the first molecule of interest (Z1).

[0284] In some cases, the first molecule of interest (Z1) comprises an amine functional group. In some cases, the first molecule of interest (Z1) comprises one amine functional group. In some cases, the first molecule of interest (Z1) comprises more than one amine functional group. In some cases, the first molecule of interest (Z1) comprises 1 to 50 amine functional groups, such as 1 to 40, or 1 to 30, or 1 to 20, or 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2 amine functional groups.

[0285] In embodiments where the first molecule of interest (Z1) comprises more than one amine functional group, one or more of the amine functional groups can be attached to a linker or a Z2- linker conjugate as described herein. In some cases, each amine functional group of the first molecule of interest (Z1) is attached to a linker or an Z2-linker conjugate as described herein. For example, conjugates of the present disclosure include compounds where Z2and Z1are present in the compound in a ratio of 1:1. In some cases, where the first molecule of interest (Z1) includes more than one amine functional group, two or more of the amine functional groups of the first molecule of interest (Z1) can be attached to a linker or an Z2-linker conjugate as described herein. For example, conjugates of the present disclosure include compounds where Z2and Z1are present in the compound in a ratio of from 2:1 to 50:1, such as from 2:1 to 40:1, or 2:1 to 30:1, or 2:1 to 20:1, or 2:1 to 10:1, or 2:1 to 9:1, or 2:1 to 8:1, or 2:1 to 7:1, or 2:1 to 6:1, or 2:1 to 5:1, or 2:1 to 4:1, or 2:1 to 3:1. Z1AND Z2

[0286] As noted above, the present disclosure provides: a) a compound comprising a first molecule of interest (Z1) and a linker of the present disclosure; b) a compound comprising a second molecule of interest (Z2) and a reversible (i.e., traceless) linker of the present disclosure; and c) acompound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2). Z2is a molecule or compound that contains, or is modified to contain, one or more aliphatic amines (an amine functional group) (e.g., a lysine). Z1and Z2are molecules of interest. In some cases, Z1and Z2are independently any one of the following: a polypeptide, a polynucleotide, a carbohydrate, a lipid, a fatty acid, a steroid, a synthetic polymer, and combinations thereof. In some cases, Z1and / or Z2is a biomolecule. In some cases, Z1and Z2are independently selected from: an active agent (e.g., a small molecule drug); an affinity moiety; a moiety that enhances in vivo half-life; a targeting moiety, e.g., a moiety that targets a particular cell, tissue, or organ; an antibody; an enzyme; a targeting peptide; a protein transduction peptide; a protein (e.g., a gene editing protein); a ligand for a receptor; and a receptor. In some cases, Z1is an active agent. In some cases, Z2is an active agent. In some cases, Z1and / or Z2is a small molecule drug.

[0287] In some cases, Z1and / or Z2is a polynucleotide or comprises a polynucleotide. Suitable polynucleotides include single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, and the like. Suitable polynucleotides include aptamers, silencing RNA, microRNA, long non-coding RNA, ribozymes, and the like. Suitable polynucleotides include guide RNAs.

[0288] In some cases, Z1and / or Z2is a lipid or comprises a lipid. Suitable lipids include, e.g., 3-N- [(methoxypoly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C- DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w- methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, 1,2- dilinoleyloxy-3-N,Ndimethylaminopropane, 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG- cDMA, 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), and the like.

[0289] In some cases, Z2is an affinity moiety. Suitable affinity moieties include His5 (HHHHH) (SEQ ID NO:12); HisX6 (HHHHHH) (SEQ ID NO:13); c-myc (EQKLISEEDL) (SEQ ID NO:14); Flag (DYKDDDDK) (SEQ ID NO:15); StrepTag (WSHPQFEK) (SEQ ID NO:7) hemagglutinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:16); glutathione-S-transferase (GST); thioredoxin; cellulose binding domain, RYIRS (SEQ ID NO:17); Phe-His-His-Thr (SEQ ID NO:10); chitin binding domain; S-peptide; T7 peptide; SH2 domain; C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:18); metal binding domains, e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large-subunit.S100 proteins. parvalbumin,calbindin D9K, calbindin D28K, and calretinin; biotin; streptavidin; MyoD; leucine zipper polypeptides; and maltose binding protein.

[0290] In some cases, Z2is a synthetic polymer. Suitable synthetic polymers include, but are not limited to, polyalkylenes such as polyethylene and polypropylene and polyethyleneglycol (PEG); polychloroprene; polyvinyl ethers such as poly(vinyl acetate); polyvinyl halides such as poly(vinyl chloride); polysiloxanes; polystyrenes; polyurethanes; polyacrylates such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl (meth)acrylate), poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyacrylamides such as poly(acrylamide), poly(methacrylamide), poly(ethyl acrylamide), poly(ethyl methacrylamide), poly(N-isopropyl acrylamide), poly(n, iso, and tert- butyl acrylamide); and copolymers and mixtures thereof. In some cases, Z2is poly(ethylene) glycol. The term “PEG” as used herein means any polyethylene glycol or other polyalkylene ether polymer. In some cases, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some cases, PEG is unsubstituted. In one embodiment, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some cases, “PEG” includes PEG copolymers such as PEG-polyurethane or PEG-polypropylene. In some cases, “PEG” does not include PEG copolymers. In some cases, the PEG has a molecular weight (in Da) of from about 130 to about 50,000, from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, from about 150 to about 5,000, from about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3,000, from about 1,000 to about 3,000, or from about 1,500 to about 2,500.

[0291] In some cases, Z2is a polypeptide. In some cases, Z1is a polypeptide. In some cases, Z2and Z1are both polypeptides. Suitable polypeptides include, e.g., fluorescent proteins; antibodies; receptors; polypeptide ligands for a receptor; targeting peptides; protein transduction peptides; enzymes (e.g., a gene editing protein); structural proteins; affinity tags; and the like.

[0292] Examples of suitable molecules that can be used as Z2include, but are not necessarily limited to: small molecules, proteins such as antibodies and functional fragments thereof, targeting and delivery moieties such as targeting and / or delivery peptides, lipids, nucleic acids, polysaccharides, or any combination thereof.Fluorescent proteins

[0293] Suitable fluorescent proteins (e.g., to be used as a Z2) include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t- HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol.17:969-973, is suitable for use. Antibodies

[0294] In some cases, Z2is an antibody. In some cases, Z1is an antibody. In some cases, both Z2and Z1are antibodies. In some cases, Z2and Z1are antibodies, where Z2specifically binds a first epitope; and where Z1specifically binds a second epitope. In some cases, Z1is a small molecule drug; and Z2is an antibody (and vice versa). In some cases, Z2is a moiety that enhances in vivo half-life; and Z1is an antibody. In some cases, Z2is a targeting moiety (e.g., one that provides for targeting to a particular cell type, tissue, or organ); and Z1is an antibody.

[0295] The antibody can be any antigen-binding antibody-based polypeptide, a wide variety of which are known in the art. In some instances, the antibody is a single chain Fv (scFv). Other antibody- based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable domains are suitable for use. In some instances, T-cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain two-domain TCR containing VαVβ) are also suitable for use. In some instances, NK-cell receptor (NKR) based recognition domains are suitable for use.

[0296] An antibody can be specific for an antigen such as CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecularweight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. In some cases, the antibody is specific for a cytokine. In some cases, the antibody is specific for a cytokine receptor. In some cases, the antibody is specific for a growth factor. In some cases, the antibody is specific for a growth factor receptor. In some cases, the antibody is specific for a cell-surface receptor. In some cases, the antibody is an anti-CD3 antibody.

[0297] An antibody can be specific for an antigen selected from: carbonic anhydrase IX, alpha- fetoprotein (AFP), α-actinin-4, A3, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD11B, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD34, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD49f / ITGA6, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD71 / TFRC, CD74, CD79a, CD80, CD83, CD90 / THY1, CD95, CD117 / KIT / c-Kit, CD123 / IL3RA, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEACAM5, CEACAM6, c-Met, DAM, epidermal growth factor receptor (EGFR), EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep- CAM, EPOR, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, histone H2B, histone H3, histone H4, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, insulin-like growth factor-1 receptor (IGF-1R), IFN-γ IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL- 23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART- 2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, PD-1, PD-L1, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, and C5.

[0298] In some cases, the antibody is selected from: 806, 9E10, 3F8, 81C6, 8H9, Abagovomab, Abatacept, Abciximab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alefacept, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, AMG 102, Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atacicept, Atezolizumab, Atinumab, Atlizumab / tocilizumab,Atorolimumab, AVE1642, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, BMS-936559, Bococizumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, cBR96- doxorubicin immunoconjugate, CC49, CDP791, Cedelizumab, Certolizumab pegol, Cetuximab, cG250, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab, Concizumab, CP 751871, CR6261, Crenezumab, CS-1008, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etanercept, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, F19, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, HGS-ETR2, hu3S193, huA33, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, IGN101, IgN311, Igovomab, IIIA4, IM-2C6, IMAB362, Imalumab, IMC-A12, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab, Inolimomab, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, J591, KB004, Keliximab, KW-2871, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab, MEDI4736, Mepolizumab, Metelimumab, METMAB, Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab, MK-0646, MK-3475, MM-121, Mogamulizumab, MORAb-003, Morolimumab, Motavizumab, MOv18, Moxetumomab pasudotox, MPDL33280A, Muromonab- CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab,Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, R1507, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Rinucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sarilumab, Satumomab pendetide, SCH 900105, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, TGN1412, Ticilimumab / tremelimumab, Tigatuzumab, Tildrakizumab, TNX-650, Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urt oxazumab, Ustekinumab, Vandortuzumab vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab and Zolimomab aritox. Therapeutic agents

[0299] In some cases, Z1and / or Z2is a therapeutic agent. In some cases, Z1is a small molecule drug, and Z2is a moiety that increases in vivo half-life of Z1. Suitable therapeutic agents include, but are not limited to, cancer chemotherapeutic agents, steroids, immunotherapeutic agents, anti- inflammatory agents, immunosuppressive agents, anti-angiogenic agents, antibiotics, anti-fungal agents, and the like.

[0300] Non-limiting examples of anti-angiogenic agents include a vascular endothelial growth factor (VEGF) inhibitor, bevacizumab, thalidomide, itraconazole, carboxyamidotriazole, TNP-470,IFN-α, IL-12, platelet factor-4, suramin, thrombospondin, angiostatin, endostatin, 2- methoxyestradiol, tecogalan, prolactin, linomide, ranibizumab, sorafenib, sunitinib, pazopanib, and everolimus.

[0301] Polypeptide therapeutic agents include, but are not limited to, a blood factor, betatrophin, exendin, enzyme, asparaginase, glutamase, arginase, arginine deaminase, adenosine deaminase (ADA), ADA-2, ribonuclease, cytosine deaminase, trypsin, chymotrypsin, papain, growth factor, epidermal growth factor (EGF), insulin, insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), bone morphogenic protein (BMP), fibroblast growth factor (FGF), somatostatin, somatotropin, somatropin, somatrem, calcitonin, parathyroid hormone, colony stimulating factors (CSF), clotting factors, tumor necrosis factors (TNF), gastrointestinal peptides, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), gastrin, secretin, erythropoietins, growth hormone, GRF, vasopressins, octreotide, pancreatic enzymes, superoxide dismutase, thyrotropin releasing hormone (TRH), thyroid stimulating hormone, luteinizing hormone, luteinizing hormone- releasing hormone (LHRH), growth hormone releasing hormone (GHRH), tissue plasminogen activators, interleukins, interleukin-1, interleukin-15, interleukin-2, interleukin-10, colony stimulating factor, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor antagonist (IL-1RA), glucagon-like peptide-1 (GLP-1), exenatide, GLP-1 R multi- agonist, GLP-1 R antagonist, GLP-2, TNF-related apoptosis-inducing ligand (TRAIL), leptin, ghrelin, granulocyte monocyte colony stimulating factor (GM-CSF), interferons, interferon-α, interferon-γ, human growth hormone (hGH), macrophage activator, chorionic gonadotropin, heparin, atrial natriuretic peptide, hemoglobin, relaxin, cyclosporine, oxytocin, ankyrin repeat proteins, affibodies, activin receptor 2A extracellular domain, alpha-2 macroglobulin, alpha- melanocyte, apelin, bradykinin B2 receptor antagonist, cytotoxic T-lymphocyte-associated protein (CTLA-4), elafin, Factor IX, Factor VIII, hepcidin, infestin-4, kallikrein inhibitor, L4F peptide, lacritin, parathyroid hormone (PTH), peptide YY (PYY), thioredoxin, thymosin B4, urate oxidase, and urodilatin.

[0302] Non-limiting examples of immunosuppressants include, e.g., azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab, infliximab, methotrexate, and tacrolimus.

[0303] Examples of steroids include, but are not limited to, corticosteroids such as cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.

[0304] Suitable cancer chemotherapeutic agents include, e.g., chemotherapeutics include, but are not limited to, an alkylating agent (e.g., busulfan, carmustine), an anti-metabolite (e.g., 5-fluoro uracil, gemcitabine, methotrexate), an anti-tumor antibiotic (e.g. dactinomycin, doxorubicin, epirubicin), a topoisomerase inhibitor (e.g. topotecan, irinotecan), a mitotic inhibitor (e.g.,paclitaxel, ixabepilone, vinblastine, estramustine), a plant alkaloid or a microtubule inhibitor (e.g. docetaxel, irinotecan, etoposide), a DNA linking agent (e.g., carboplatin, cisplatin, oxaliplatin), an immunotherapeutic agent (e.g., rituximab, alemtuzumab, lenalidomide), and a differentiating agent (e.g., tretinoin, bexarotene), cisplatin, doxorubicin, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, gefitinib, erlotinib hydrochloride, imatinib mesylate, cytarabine, gemcitabine, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, 6- mercaptopurine, 6-thioguanine, fludarabine phosphate, oxaliplatin, pentostatin, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, epirubicin, idarubicin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide, testolactone, estramustine, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, vinorelbine, anastrazole, letrozole, capecitabine, raloxifene, xeloda, vinorelbine, cetuximab, N,N’N’- triethylenethiophosphoramide, altretamine, trastuzumab, fulvestrant, exemestane, and any combination thereof.

[0305] Suitable antibiotics include, e.g., rifampicin, rifabutin, rifalazil, rifapentine, rifaximin, oxacillin, methicillin, ampicillin, cloxacillin, carbenicillin, piperacillin, tricarcillin, flucloxacillin, nafcillin, azithromycin, clarithromycin, erythromycin, telithromycin, cethromycin, solithromycin, aztreonam, BAL30072, meropenem, doripenem, imipenem, ertapenem, biapenem, tomopenem, panipenem, tigecycline, omadacycline, eravacycline, doxycycline, minocycline, ciprofloxacin, levofloxacin, moxifloxacin, delafloxacin, fusidic acid, novobiocin, teichoplanin, telavancin, dalbavancin, and oritavancin.

[0306] Suitable antibiotics include aminoglycoside antibiotics, e.g., streptomycin, neomycin, kanamycin, amikacin, gentamycin, tobramycin, sisomicin, arbekacin, netilmicin, paromomycin, spectinomycin, amikacin chloride, tobramycin sulfate, gentamycin sulfate, or gentamycin chloride. Suitable antibiotics include quinolone antibiotics, e.g., ciprofloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, sparfloxacin, trvafloxacin, gatifloxacin, gemifloxacin, cinoxacin, or nalidixic acid. Suitable antibiotics include dibasic macrolide antibiotics, e.g., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, or spectinomycin.

[0307] Suitable anti-fungal agents include, e.g., Polyene antifungals (such as Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin and Rimocidin), Imidazoles (such as Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole and Tioconazole), Triazoles (such as Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole,Ravuconazole, Terconazole and Voriconazole), Thiazoles (such as Abafungin), Allylamines (such as Amorolfin, Butenafine, Naftifine and Terbinafine), Echinocandins (such as Anidulafungin, Caspofungin and Micafungin) and others such as Benzoic acid, Ciclopirox, Flucytosine or 5-fluorocytosine, Griseofulvin, Haloprogin, Polygodial, Tolnaftate, Undecylenic acid, and Crystal violet. Moieties that increase in vivo half-life

[0308] In some cases, Z2is a moiety that increases in vivo half-life of a molecule Z1. For example, in some cases, Z2is a moiety that, when present in a conjugate of the present disclosure, where the conjugate includes a molecule of interest Z1, increases the in vivo half-life of Z1, compared to the in vivo half-life of free Z1, i.e., Z1not conjugated to Z2. In some cases, Z2is a moiety that, when present in a conjugate of the present disclosure, where the conjugate includes a molecule of interest Z1, increases the in vivo half-life of Z1by at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or more than 50-fold, compared to the in vivo half-life of free Z1, i.e., Z1not conjugated to Z2. The increase in in vivo half-life can be an increase in circulation half-life, e.g., an increase in the half-life in blood.

[0309] Moieties that increase in vivo half-life of a molecule include, but are not limited to, polyethylene glycol; a polysialyl group; an XTEN polypeptide (see, e.g., Schellenberger et al. (2009) Nature Biotechnol.27:1186; and Haeckel et al. (2016) PLoSOne 11:e0157193); HEMA- phosphorylcholine; a biocompatible fatty acid; an anti-serum albumin antibody (e.g., a domain antibody (dAb) that binds serum albumin; see, e.g., Holt et al. (2008) Protein Eng. Des. Sel. 21:283); a hydroxy alkyl starch (HAS), e.g. hydroxy ethyl starch (HES); Poly (Glyx-Sery) (HAP); hyaluronic acid (HA); a heparosan polymer (HEP); a fleximer; dextran; a poly-sialic acid (PSA); an Fc polypeptide; a transferrin polypeptide; an albumin polypeptide; an elastin like (ELP) peptide; a proline-alanine-serine (PAS) polymer; a PA polymer; an albumin binding peptide; a carboxyl-terminal peptide (CTP); a gelatin-like polypeptide; an FcRn binding peptide; and the like.

[0310] Examples of polymer half-life extending moieties include, e.g., polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), starch, hydroxylethyl starch (HES), hydroxyalkyl starch (HAS), pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co- maleic acid anyhydride, polystyrene-co-maleic acid anhydride, poly(1-hydroxymethyethylene hydroxymethylformal) (PHF), a zwitterionic polymer, a phosphorylcholine containing polymerand a polymer comprising MPC, Poly (Glyx-Sery), hyaluronic acid (HA), heparosan polymers (HEP), Fleximers, Dextran, and Poly-sialic acids (PSA). Targeting moieties

[0311] In some cases, Z2is a targeting moiety (e.g., a targeting peptide). A targeting moiety is a moiety that targets a particular cell type, tissue, or organ. For example, in some cases, Z2is a moiety that, when present in a conjugate of the present disclosure, where the conjugate includes a molecule of interest Z1, targets the conjugate to a particular cell type, tissue, or organ. Suitable targeting moieties include, e.g., an antibody specific for a cell surface protein, see, e.g., antibodies as described above. Targeting moieties can be any convenient molecule and many different targeting moietites will be known to one of ordinary skill in the art, including, e.g., but not necessariliy limited to: small molecules, proteins such as antibodies and functional fragments thereof, targeting peptides, lipids, nucleic acids, polysaccharides, or any combination thereof.

[0312] In some cases, the targeting moiety targets a conjugate of the present disclosure to a cancer cell. In some cases, the targeting moiety is an antibody specific for a cancer-associated antigen. Examples of cancer-associated antigens include, but are not limited to, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, VEGFR2, HMW-MAA, MAGE-A1, IL-13R-a2, GD2, and the like.

[0313] A targeting moiety can be an antibody specific for an antigen selected from: carbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4, A3, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEACAM5, CEACAM6, c-Met, DAM, epidermal growth factor receptor (EGFR), EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep- CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, histone H2B, histone H3, histone H4, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, insulin-like growth factor-1 receptor (IGF-1R), IFN-γ IFN-α, IFN-β, IFN-λ, IL-4R, IL- 6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR / FUT,macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY- ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, PD-1, PD-L1, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, and C5.

[0314] In some cases, a targeting moiety is a peptide and can be referred to as a targeting peptide. Examples include, but are not limited, to those presented in Table 2. In some cases, a targeting moiety target a lung cell (e.g., a lung epithelial cell).

[0315] In some cases, a targeting moiety (e.g., a targeting peptide) is a transcytosis peptide. For example, in some cases, Z2is a transcytosis peptide. In some cases, a transcytosis peptide comprises blood-brain barrier (BBB) transit polypeptide. In some cases, the BBB transit polypeptide is an Angiopep2 (AG2) peptide (TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 3)) (see, e.g., Demeule et al., J. Pharmacol. Exp. Ther.2008;324:1064–1072). In some cases, the BBB transit polypeptide is rabies virus glycoprotein (RVG) protein YTIWMPENPRPGTPCDIFTNSRGKRASN (SEQ ID NO: 4).

[0316] In some cases, a targeting moiety promotes transferrin receptor (TfR)-mediated transcytosis, e.g., that targeting moiety can include Tfr (also known as CD71) or a functional fragment thereof. Likewise, in some cases, the targeting moiety can include an anti-transferrin antibody (e.g. anti- CD71) or a functional fragment thereof.

[0317] In some cases, a transcytosis peptide is a blood-cerebrospinal fluid barrier (BCSFB) transit polypeptide. For example, in some cases a BCSFB transit polypeptide includes PMKSHTN (SEQ ID NO: 5) (see, e.g., Yang et al., Amino Acids.2021 Aug;53(8):1181-1186). Protein Transduction Domain

[0318] In some cases, Z2is a Protein Transduction Domain (PTD). For example, in some cases, Z2includes a PTD; and Z1is a gene editing protein such as CRISPR-Cas effector polypeptide (e.g., Cas9).

[0319] “Protein Transduction Domain” or PTD or PTD peptide (and also known as a CPP – a cell penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and / or a nanoparticle, facilitates the moleculetraversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some cases, Z2is a PTD; and Z1is a CRISPR-Cas effector polypeptide. PTDs are also referred to herein as delivery moieties, e.g., delivery peptides.

[0320] In some cases, a PTD includes a nuclear localization signal (NLS). Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRR; SEQ ID NO:19); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther.9(6):489- 96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:20); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:21); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:22); and RQIKIWFQNRRMKWKK (SEQ ID NO:23). Exemplary PTDs include but are not limited to, YGRKKRRQRR (SEQ ID NO:19), YGRKKRRQRRR (SEQ ID NO:24), RKKRRQRRR (SEQ ID NO:25); and an arginine homopolymer of from 3 arginine residues to 50 arginine residues. For example, in some cases, a PTD is Argn, where n is an integer from 3 to 50 (e.g., n is an integer from 3 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 40, or from 40 to 50). In some cases, a PTD is Argn, where n is from 5 to 15, from 5 to 10, or from 10 to 15. a PTD is Argn, where n is 10. Exemplary PTD amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRR (SEQ ID NO:19); RKKRRQRR (SEQ ID NO:27); YARAAARQARA (SEQ ID NO:28); THRLPRRRRRR (SEQ ID NO:29); and GGRRARRRRRR (SEQ ID NO:30). In some cases, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane.

[0321] In some cases, a protein transduction domain is an endosomolytic peptide. Thus, in some cases, Z2comprises an endosomolytic peptide (also referred to as an endosomal escape peptide). For example, in some cases, Z2is an endosomal escape peptide (and in some such cases, Z1is a gene editing protein, e.g., a CRISPR / Cas effector protein such a Cas9 protein). In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO:32), wherein each X is independently selectedfrom lysine, histidine, and arginine. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO:33).

[0322] In some cases, an endosomal escape polypeptide comprises an HA2-TAT (CGLFEAIAGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 1)) or HA2-TAT- derived fusion peptide. In some cases, an endosomal escape polypeptide comprises an INF7- TAT (CGLFEAIEGFIENGWEGMIDGWYGYGRKKRRQRR (SEQ ID NO: 2)) or INF7-TAT- derived fusion peptide (see, e.g., Algayer et al., Molecules.2019 May 31;24(11):2079).

[0323] For example, CRISPR cargo (e.g., a CRISPR-Cas effector polypeptide, an RNP, etc.) can in some cases accumulate in endosomes and / or lysosomes (intracellular compartments), but that accumulation is not necessarily functionally meaningful because such cargo does not efficiently reach the cytosol and thus is not efficiently transported into the nucleus. When a subject delivery peptide (via a subject linker) is used, however, endosomal escape can be promoted, which allows the cargo to reach the cytosol, which is reducing; the endosomal / lysosomal compartment is not. Therefore, reduction (and linker release) is synchronized with “successful” delivery into the cytosol.

[0324] Tables 1 and 2 provide non-limiting examples of protein transduction domain peptides and targeting peptides, respectively. For all listed peptides that do not include a tetrazine (Tz), a Tz can be added as would be understood by one of ordinary skill in the art (e.g., to allow for click chemistry reactions with a subject linker that has a TCO), e.g., to the C-terminal or N-terminal end. For those peptides that do include a Tz, the Tz could instead be added elsewhere, e.g., to the C-terminal or N-terminal end. For Table 1 and Table 2, the following descriptions of modified amino acids and / or non-amide bond linkages apply: i){Ac} is an acetyl group; ii) {β-Ala} is beta alanine; iii) {Ctz} refers to a cysteine that has been chemically modified using methyltetrazine- PEG4-maleimide; iv) {Cpy} refers to Cys(3-nitro-2-pyridinesulfenyl) incorporated into the peptide backbone; v) –PEG2– refers to incorporation of 2 copies (monomeric units) of polyethylene glycol into the peptide backbone; vi) –PEG4– refers to incorporation of 4 copies (monomeric units) of polyethylene glycol into the peptide backbone; vii) –PEG6– refers to incorporation of 6 copies (monomeric units) of polyethylene glycol into the peptide backbone;viii) –PEG8– refers to incorporation of 8 copies (monomeric units) of polyethylene glycol into the peptide backbone; ix){Kpi} refers to Lys(3-Nitro-pyridine-2-carboxylic acid); x){Kp46} refers to Lys(PEG23)2, a branched PEG moiety extending off of the peptide backbone; xi) {Kp46py} refers to Lys(PEG23)2)-(3-Nitro-pyridine-2-carboxylic acid) extending off of the peptide backbone; xii) {Kfam} refers to a Lys that is chemically modified via conjugation to a succinimidyl ester form of 5-FAM (5-carboxyfluorescein); xiii) {Cd} refers to Cys that is chemically modified post-synthesis with 1,4- bis(bromomethyl)-benzene, creating a peptide dimer; and xiv) {Ktz} refers to Lys that is chemically modified via conjugation to methyltetrazine- PEG4-NHS ester. Alpha (α): α-aminoadipic acid; Abu: Aminobutyric Acid; Mtz: methyltetrazine; Mal: maleimide, site of conjugation to Cys residue at C-term; PEG6: polyethlyene glycol (6x ethylene glycol repeats); PEG4: polyethlyene glycol (4x ethylene glycol repeats); cyclo: cyclized via Glu / Lys lactam bond; Tz: tetrazine; Dap: 2,3-diaminopropionic acid; *: cyclized between the marked positions (e.g., cyclized between Dap and D for MiniAP4- tz, cyclized between A and T for cR11A-Tz); underscore (_) is a blank for visual purposes when comparing different sequences, but can be ignored (i.e., is not part of the sequence).

[0325] In some cases, the peptides listed in Table 1 and / or Table 2 can be used in their “retro” and / or “inverso” peptides. “Retro” refers to reversing the order of the amino acids in terms of their N- to-C sequence. “Inverso” refers to changing the chirality of the peptide backbone (“L” chirality is typical in human cells; “D” is the inverso form), which causes a “mirror image” flip of some peptide features (e.g. an alpha helix will be left-handed instead of right-handed). In many cases, the peptide sequence (the amino acids adjacent to one another) will retain its biological function following the “retro” and / or “inverso” modification.

[0326] Table 1. Examples of protein transduction domain peptides # Amino Acid Sequence Short Name SEQ ID NO.# Amino Acid Sequence Short Name SEQ ID NO. 5 GLFEAIAEFIENGWEGLIEGWYGYGRKKRRQRRRR E5- 26# Amino Acid Sequence Short Name SEQ ID NO. 43 RLFEKIEGFIENGWEGMIDGWYGYGRKKRRQRR G1R, A5K 98# Amino Acid Sequence Short Name SEQ ID NO. 82 GLFEAIEGFIENGWEGMIDGWYGYGHKKHHQHH R(26,29,30,32,33)H 135# Amino Acid Sequence Short Name SEQ ID NO. f6 KLFEAIEGFIENGWEGMIDLWNG-PEG2-{Kp46}-PEG2- f691-PEG2K- 170

[0327] a e . a pes o age g pep es. Peptide Name Sequence Alternative Names SEQ ID NO.Peptide Name Sequence Alternative Names SEQ ID NO. ANP-Tz K(Tz)-PEG4- 195

[0328] Additional examples of peptides that can be used for Z2are provided in international patent application publication No. WO2023097222, which disclosure is hereby incorporated by reference in its entirety, e.g., see disclosure therein of peptides that can be used as cargo delivery fusion polypeptides. Gene Editing Proteins

[0329] In some cases, Z1is a gene editing protein. Gene editing, or genome editing, is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using a nuclease. The gene editing protein may be artificially engineered or may be found in nature. In some embodiments, the gene editing protein is a nuclease that creates a specific break (in some cases one or more single-stranded breaks, i.e., nicks, and in some cases one or more double-stranded breaks (DSBs)) at desired locations in the genome. The cell's endogenous repair mechanisms subsequently repairs the induced break(s) by natural processes, such as homologous recombination (HR) and non-homologous end-joining (NHEJ). Examples of such gene editing proteins include, but are necessarily limited to: Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), CRISPR-Cas effector proteins, (e.g., the CRISPR / Cas system), and meganucleases (homing endonucleases).

[0330] Examples of gene editing proteins include, but are necessarily limited to: a ZFN, a TALEN, a CRISPR-Cas effector protein (which functions in combination with a guide RNA), a meganuclease, a site-specific recombinase, a resolvase / integrase, a transposase, a transposon, and the like. In some cases, the gene editing protein is a site-specific recombinase (e.g., Cre recombinase, Dre recombinase, Flp recombinase, KD recombinase, B2 recombinase, B3 recombinase, R recombinase, Hin recombinase, Tre recombinase, PhiC31 integrase, Bxb1 integrase, R4 integrase, lambda integrase, HK022 integrase, HP1 integrase, and the like). In some cases, the gene editing protein is a meganulease (homing endonuclease) (e.g., I-SceI, I- SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I- CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I- DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I- NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I- SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I- TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII, and the like). In some cases, the gene editing protein is a resolvase and / or invertase (e.g., Gin, Hin, γδ3, Tn3, Sin, Beta, and the like). In some cases, the gene editing protein is a transposon (e.g., bacterial transposons such as Tn3, Tn5, Tn7, Tn9, Tn10, Tn903, Tn1681, and the like; eukaryotic transposons such as Tc1 / mariner super family transposons, PiggyBac superfamily transposons, hAT superfamily transposons, PiggyBac, Sleeping Beauty, Frog Prince, Minos, Himar1, and the like). In some cases, a gene editing protein is a zinc finger nuclease (ZFN) or a transcription activator like effector nuclease (TALEN). CRISPR-Cas effector polypeptides

[0331] In some cases, Z1is a CRISPR-Cas effector polypeptide. A suitable CRISPR-Cas effector polypeptide is a class 2 CRISPR-Cas effector polypeptide such as a type II, type V, or type VI CRISPR-Cas effector polypeptide. In some cases, a suitable RNA-guided endonuclease is a class 2 CRISPR-Cas effector polypeptide. In some cases, a suitable RNA-guided endonuclease is a class 2 type II CRISPR-Cas effector polypeptide (e.g., a Cas9 protein). In some cases, a CRISPR-Cas effector polypeptide is a class 2 type V CRISPR-Cas effector polypeptide (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein). In some cases, a suitable CRISPR-Cas effector polypeptide is a class 2 type VI CRISPR-Cas effector polypeptide (e.g., a C2c2 protein; also referred to as a “Cas13a” protein). Also suitable is a CasX protein. Also suitable is a CasY protein. The term “class 2 CRISPR / Cas protein” or “CRISPR-Cas effector polypeptide” (and the like) is used herein to mean the effector protein from class 2 CRISPR systems – for example, type II CRISPR / Cas proteins (e.g., Cas9), type V CRISPR / Cas proteins (e.g., Cpf1 / Cas12a, C2c1 / Cas12b, C2C3 / Cas12c, Cas12d / CasY, Cas12e / CasX), and type VI CRISPR / Cas proteins (e.g., C2c2 / Cas13a, C2C7 / Cas13c, C2c6 / Cas13b). Class 2 CRISPR / Cas effector proteins include type II, type V, and type VI CRISPR / Cas proteins, but the term is also meant to encompass any class 2 CRISPR / Cas protein suitable for binding to a corresponding guide RNA and forming a ribonucleoprotein (RNP) complex.

[0332] In some cases, the CRISPR / Cas effector polypeptide is a Type II CRISPR / Cas effector polypeptide. In some cases, the CRISPR / Cas effector polypeptide is a Cas9 polypeptide. In some cases, a Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99%, amino acid sequence identity to the Streptococcus pyogenes Cas9 of SEQ ID NO: 50. In some cases, a Cas9 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 50-55.

[0333] In some cases, the Cas9 polypeptide is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the saCas9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the saCas9 amino acid sequence of SEQ ID NO: 56.

[0334] In some cases, a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide. Kleinstiver et al. (2016) Nature 529:490. For example, amino acids N497, R661, Q695, and Q926 of the amino acid sequence of SEQ ID: 50 are substituted, e.g., with alanine. For example, an HF Cas9 polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence of SEQ ID NO: 50, where amino acids N497, R661, Q695, and Q926 are substituted, e.g., with alanine. In some cases, a suitable Cas9 polypeptide exhibits altered PAM specificity. See, e.g., Kleinstiver et al. (2015) Nature 523:481.

[0335] In some cases, a suitable CRISPR / Cas effector polypeptide is a type V CRISPR / Cas effector polypeptide. In some cases, a type V CRISPR / Cas effector polypeptide is a Cpf1 protein. In some cases, a Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100%, amino acid sequence identity to the Cpf1 amino acid sequence of SEQ ID NO: 57, 58, or 59.

[0336] In some cases, a suitable CRISPR / Cas effector polypeptide is a fusion protein comprising a CRISPR / Cas effector polypeptide that is fused to a heterologous polypeptide (also referred to as a “fusion partner”). In some cases, a CRISPR / Cas effector polypeptide is fused to an amino acid sequence (a fusion partner) that provides for subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).

[0337] In some cases, the CRISPR / Cas effector polypeptide is a class 2 CRISPR / Cas effector polypeptide. In class 2 CRISPR systems, the functions of the effector complex (e.g., the cleavage of target DNA) are carried out by a single endonuclease (e.g., see Zetsche et al., Cell.2015 Oct 22;163(3):759-71; Makarova et al., Nat Rev Microbiol.2015 Nov;13(11):722-36; Shmakov et al., Mol Cell.2015 Nov 5;60(3):385-97); and Shmakov et al. (2017) Nature Reviews Microbiology 15:169. As such, the term “class 2 CRISPR / Cas protein” is used herein to encompass the CRISPR / Cas effector polypeptide (e.g., the target nucleic acid cleaving protein) from class 2 CRISPR systems. Thus, the term “class 2 CRISPR / Cas effector polypeptide” as used herein encompasses type II CRISPR / Cas effector polypeptides (e.g., Cas9); type V-A CRISPR / Cas effector polypeptides (e.g., Cpf1 (also referred to a “Cas12a”)); type V-B CRISPR / Cas effector polypeptides (e.g., C2c1 (also referred to as “Cas12b”)); type V-CCRISPR / Cas effector polypeptides (e.g., C2c3 (also referred to as “Cas12c”)); type V-U1 CRISPR / Cas effector polypeptides (e.g., C2c4); type V-U2 CRISPR / Cas effector polypeptides (e.g., C2c8); type V-U5 CRISPR / Cas effector polypeptides (e.g., C2c5); type V-U4 CRISPR / Cas proteins (e.g., C2c9); type V-U3 CRISPR / Cas effector polypeptides (e.g., C2c10); type VI-A CRISPR / Cas effector polypeptides (e.g., C2c2 (also known as “Cas13a”)); type VI-B CRISPR / Cas effector polypeptides (e.g., Cas13b (also known as C2c4)); and type VI-C CRISPR / Cas effector polypeptides (e.g., Cas13c (also known as C2c7)). To date, class 2 CRISPR / Cas effector polypeptides encompass type II, type V, and type VI CRISPR / Cas effector polypeptides, but the term is also meant to encompass any class 2 CRISPR / Cas effector polypeptide suitable for binding to a corresponding guide RNA and forming an RNP complex.

[0338] In some cases, the CRISPR / Cas effector polypeptide is a Type II CRISPR / Cas effector polypeptide, such as a Cas9 polypeptide. In some cases, the fusion protein comprising a Cas9 polypeptide that is fused to a heterologous protein (referred to as a fusion partner), where the heterologous protein provides an activity (e.g., one that is not provided by the Cas9 protein). The fusion partner can provide an activity, e.g., enzymatic activity (e.g., nuclease activity, activity for DNA and / or RNA methylation, activity for DNA and / or RNA cleavage, activity for histone acetylation, activity for histone methylation, activity for RNA modification, activity for RNA- binding, activity for RNA splicing etc.). In some cases, a portion of the Cas9 protein (e.g., the RuvC domain and / or the HNH domain) exhibits reduced nuclease activity relative to the corresponding portion of a wild type Cas9 protein (e.g., in some cases the Cas9 protein is a nickase). In some cases, the Cas9 protein is enzymatically inactive, or has reduced enzymatic activity relative to a wild-type Cas9 protein (e.g., relative to Streptococcus pyogenes Cas9).

[0339] In some cases, a Cas9 protein is a variant Cas9 protein. A variant Cas9 protein has an amino acid sequence that is different by at least one amino acid (e.g., has a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of a corresponding wild type Cas9 protein. In some instances, the variant Cas9 protein has an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 protein. For example, in some instances, the variant Cas9 protein has 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein has no substantial nuclease activity. When a Cas9 protein is a variant Cas9 protein that has no substantial nuclease activity, it can be referred to as a nuclease defective Cas9 protein or “dCas9” for “dead” Cas9. A protein (e.g., a class 2 CRISPR / Cas protein, e.g., a Cas9 protein) that cleaves one strand but not the other of a double stranded target nucleic acid is referred to herein as a “nickase” (e.g., a “nickase Cas9”).

[0340] In some cases, a variant Cas9 protein has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target nucleic acid. As a non-limiting example, in some cases, the variant Cas9 protein harbors mutations at amino acid positions corresponding to residues D10 and H840 (e.g., D10A and H840A) of a Cas9 protein comprising the amino acid sequence of SEQ ID NO: 50 (or the corresponding residues of another Cas9 protein) such that the polypeptide has a reduced ability to cleave (e.g., does not cleave) both the complementary and the non-complementary strands of a target nucleic acid. Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded or double stranded target nucleic acid) but retains the ability to bind a target nucleic acid. A Cas9 protein that cannot cleave target nucleic acid (e.g., due to one or more mutations, e.g., in the catalytic domains of the RuvC and HNH domains) is referred to as a “dead” Cas9 or simply “dCas9.”

[0341] In some cases, a suitable CRISPR / Cas effector polypeptide is a type V or type VI CRISPR / Cas endonuclease (i.e., the CRISPR / Cas effector polypeptide is a type V or type VI CRISPR / Cas endonuclease) (e.g., Cpf1, C2c1, C2c2, C2c3). Type V and type VI CRISPR / Cas endonucleases are a type of class 2 CRISPR / Cas endonuclease. Examples of type V CRISPR / Cas endonucleases include but are not limited to: Cpf1, C2c1, and C2c3. An example of a type VI CRISPR / Cas effector polypeptide is C2c2. In some cases, a suitable CRISPR / Cas effector polypeptide is a type V CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c3). In some cases, a Type V CRISPR / Cas effector polypeptide is a Cpf1 protein. In some cases, a suitable CRISPR / Cas effector polypeptide is a type VI CRISPR / Cas endonuclease (e.g., Cas13a).

[0342] Examples and guidance related to type V and type VI CRISPR / Cas proteins (e.g., Cpf1, C2c1, C2c2, and C2c3 guide RNAs) can be found in the art, for example, see Zetsche et al., Cell.2015 Oct 22;163(3):759-71; Makarova et al., Nat Rev Microbiol.2015 Nov;13(11):722-36; Shmakov et al., Mol Cell.2015 Nov 5;60(3):385-97; and Shmakov et al. (2017) Nature Reviews Microbiology 15:169.

[0343] In some cases, the Type V or type VI CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3) is enzymatically active, e.g., the Type V or type VI CRISPR / Cas polypeptide, when bound to a guide RNA, cleaves a target nucleic acid. In some cases, the Type V or type VI CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3) exhibits reduced enzymatic activity relative to a corresponding wild-type a Type V or type VI CRISPR / Cas endonuclease (e.g., Cpf1, C2c1, C2c2, C2c3), and retains DNA binding activity.

[0344] Suitable CRISPR / Cas effector polypeptides include CasX and CasY polypeptides. See, e.g., Burstein et al. (2017) Nature 542:237. Suitable CasX polypeptides include those described in WO 2018 / 064371. Suitable CasY polypeptides include those described in WO 2018 / 064352.

[0345] In some cases, a CRISRP / Cas effector polypeptide is a CRISRP / Cas fusion protein comprising: a) a CRISRP / Cas effector polypeptide; and b) a heterologous polypeptide (also referred to as a “heterologous fusion partner”). In some cases, a CRISRP / Cas effector polypeptide is a prime editor (e.g. Anzalone et al (2019) Nature 576: 149–157 https: / / doi.org / 10.1038 / s41586-019- 1711-4). As such, in some cases, a CRISRP / Cas effector polypeptide (e.g., a nickase version such as nickase Cas9 that cuts the non-complementary strand of the DNA) is fused to a reverse transcriptase (RT) [e.g., M-MLV RT]. As would be known to one of ordinary skill in the art, the guide RNA for prime editing can be fused to an RNA sequence that serves as a donor template for the RT.

[0346] In some cases, a CRISRP / Cas effector polypeptide is a base editor. As such, in some cases, a CRISRP / Cas effector polypeptide (e.g., a dead or nickase version such as a dCas9 or nickase Cas9) is fused to a base editing enzyme such as a deaminase enzyme such as APOBEC. Examples of base editing enzymes include, but are not necessarily limited to: cytosine base editors (CBEs), adenine base editors (ABEs), cytosine-to-guanine base editors (CGBEs), and simultaneous adenine and cytosine base editors (ACBEs). The deaminase fused to the CRISRP / Cas effector polypeptide (e.g., nickase) – be it a cytosine deaminase (e.g. APOBEC) or an adenosine deaminase (e.g. engineered TadA) – will dictate whether the base editor is a CBE or an ABE. CBEs contain cytosine deaminases, while ABEs typically contain adenine deaminases (though several groups have recently engineered CBEs from the conventional TadA adenosine deaminase domain.

[0347] In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones, and the like). In some cases the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones, and the like).

[0348] In some cases, a CRISPR / Cas effector fusion polypeptide includes a heterologous polypeptide that has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity such as FokI nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity,DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).

[0349] In some cases, a CRISPR / Cas effector fusion polypeptide includes a heterologous polypeptide that has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity).

[0350] Examples of proteins (or fragments thereof) that can be used in increase transcription, and that are suitable as heterologous fusion partners, include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.

[0351] Examples of proteins (or fragments thereof) that can be used in decrease transcription, and that are suitable as heterologous fusion partners, include but are not limited to: transcriptional repressors such as the Krüppel associated box (KRAB or SKD); KOZ2 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr- SET7 / 8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0352] In some cases, the fusion partner has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like) , DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).

[0353] In some cases, the fusion partner has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the fusion partner include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetyl transferase (e.g., catalytic core / fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[0354] For all of the proteins listed above as possible fusion partners, in some cases, these proteins need not be fused to the CRISPR-Cas effector protein, but can instead be recruited inside the cell non- covalently. For example, the CRISPR-Cas effector protein (or a CRISPR-Cas guide RNA in some cases) can be modified to contain a domain that can bind (non-covalently) specifically to an endogenous functional protein with any of the functions described above.

[0355] In some cases, Z1(e.g., a CRISPR-Cas effector protein) is complexed with an RNA (e.g., a guide RNA) and is therefore a ribonucleoprotein (RNP). As will be known to one of ordinary skill in the art, in some embodiments, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.” In some embodiments, the guide RNA is one molecule (e.g., for some class 2 CRISPR / Cas proteins, the corresponding natural guide RNA is a single molecule; and in some cases, an activator and targeter are covalently linked to one another, e.g., via intervening nucleotides using genetic engineering), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.” Anti-CRISPR polypeptides

[0356] In some cases, Z2is an anti-CRISPR polypeptide; and Z1is a CRISPR-Cas effector polypeptide.

[0357] Suitable ACR polypeptides include, e.g., AcrIIC1, AcrIIA1, AcrIIA2, AcrIIA3, AcrIIA4, AcrIIC2, AcrIIC3, AcrE1, AcrID1, Acrf10, anti-CRISPR protein 30, Acrf2, and Acrf1. See, e.g., WO 2017 / 160689; and Nakamura et al. (2019) Nature Communications 10:194; Harrington et al. (2017) Cell 170:1224; Shin et al. (2017) Sci. Adv.3:e1701620; Zhu et al. (2019) Mol. Cell 74:296.

[0358] As an example, an AcrIIA4 polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: NDLIREIKNKDYTVKLSGTDSNSITQLIIRVNNDGNEYVISESENESIVEKFISAFKNGWNQ EYEDEEEFYNDMQTITLKSELN (SEQ ID NO:31).Nuclear localization signal

[0359] In some cases, Z1includes a nuclear localization signal (NLS) (e.g., in some cases, Z1is a CRSIPR-Cas effector polypeptide that is fused to one or more NLSs). In some cases, Z2is a nuclear localization signal (NLS).

[0360] Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 34); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 35)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 36) or RQRRNELKRSP (SEQ ID NO: 37); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 38); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 39) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 40) and PPKKARED (SEQ ID NO: 41) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 42) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 43) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 44) and PKQKKRK (SEQ ID NO: 45) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 46) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 47); the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 48) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 49) of the steroid hormone receptors (human) glucocorticoid. METHODS OF MAKING A CONJUGATE

[0361] The present disclosure provides a method of making a conjugate as described herein, such as a method of making a compound comprising a first molecule (Z1) linked, via a linker of the present disclosure, to a second molecule (Z2); i.e., an Z1-linker- Z2conjugate.

[0362] In some cases, the method of making the conjugate includes modifying a molecule Z1with a linker compound of the present disclosure (i.e., a compound of formula (I) or formula (II) or formula (III) as described herein), thus generating a Z1-linker intermediate; i.e. a compound of formula (Ia) or (IIa) or (IIIa) as described herein. In some cases, a method includes contacting the Z1-linker intermediate with a molecule Z2, thereby reversibly linking Z1to Z2to form a conjugate.

[0363] Likewise, in some cases, the method of making the conjugate includes modifying a molecule Z2with a linker compound of the present disclosure (i.e., a compound of formula (I) or formula (II) or formula (III) as described herein), thus generating a linker-Z2intermediate; i.e. a compound of formula (Ib) or (IIb) or (IIIb) as described herein. In some cases, a method includes contactingthe linker-Z2intermediate with a molecule Z1, thereby reversibly linking Z1to Z2to form a conjugate.

[0364] For example, a subject method of making a conjugate can include modifying a molecule Z1with a compound of formula (I) or (II) or (III) as described herein, thus generating a linker intermediate (a Z1-linker conjugate) of the formula (Ia) or (IIa) or (IIIa), respectively: O H R2 12R 1N Owhere the are as

[0365] A subject method of making a conjugate can include modifying a compound of formula (Ia) or (IIa) or (IIIa) as described herein with a molecule Z2, thus generating a conjugate of the formula (Ic) or (IIc) or (IIIc), respectively: R6where the variables are each as described above for the respective compounds.

[0366] A subject method of making a conjugate can include modifying a molecule Z2with a compound of formula (I) or (II) or (III) as described herein, thus generating a linker intermediate (a linker- Z2conjugate) of the formula (Ib) or (IIb) or (IIIb), respectively: R6NH compounds.

[0367] A subject method of making a conjugate can include modifying a compound of formula (Ib) or (IIb) or (IIIb) as described herein with a molecule Z1, thus generating a conjugate of the formula (Ic) or (IIc) or (IIIc), respectively: R6O 21H N221R R 1O YZ Z X2L2HN L where compounds.COMPOSITIONS

[0368] The present disclosure provides compositions, including pharmaceutical compositions, comprising a compound of the present disclosure.

[0369] The present disclosure provides a composition comprising: a) a compound of the present disclosure; and b) at least one additional compound. Suitable additional compounds include, but are not limited to: a salt, such as a magnesium salt, a sodium salt, etc., e.g., NaCl, MgCl, KCl, MgSO4, etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a protease inhibitor; and the like.

[0370] The present disclosure provides a composition comprising: a) a compound of the present disclosure; and b) a pharmaceutically acceptable excipient. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20thedition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7thed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rded. Amer. Pharmaceutical Assoc. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride, sulfate salts, solvates (e.g., mixed ionic salts, water, organics), hydrates (e.g., water), and the like.METHODS OF DELIVERING A MOLECULE OF INTEREST

[0371] The present disclosure provides a method of delivering a molecule of interest to an individual. In some cases, the method includes administering a compound of the present disclosure to the individual. In some cases, the method includes administering a compound of the present disclosure to the individual, where the compound is a Z1-linker- Z2conjugate as described herein. In some cases, Z1is a molecule of interest as described herein, where Z1is released from the conjugate under physiological reducing conditions.

[0372] For example, the attachment (i.e., covalent bond) between the amine group of the first molecule of interest (Z1) and the compound can be labile, such that under appropriate conditions the covalent bond dissociates and thus releases the first molecule of interest (Z1) from the compound (and as noted elsewhere herein, the “release” is a complete reversal of the conjugation - in other words, Z1will return to the state it had before conjugation with the linker). In some cases, the conditions that provide for dissociation of the covalent bond between the compound and the first molecule of interest (Z1) are reducing conditions. In some cases, the reducing conditions are physiological reducing conditions, such as reducing conditions found in a target area in a subject in vivo. In some cases, the compound comprises a linker as described above, where the linker is a self-immolative linker. For example, in some cases, the linker is a reduction sensitive disulfide linker.

[0373] Compounds of the present disclosure can be administered to a subject in any suitable form, e.g., in the form of a pharmaceutically acceptable composition, and can be formulated for any suitable route of administration, e.g., oral, topical or parenteral administration. Suitable routes of administration include oral, intravenous, subcutaneous, intramuscular, intraperitoneal, intratumoral, peritumoral, intracranial (e.g., convection enhanced delivery), intraocular, topical, retro-orbital (e.g., retro-orbital injection), etc. In some cases, administering the compound to a subject is via local administration (e.g., injection such as intratumoral injection). In some cases, administering the compound to a subject is via systemic administration. Where the compound is provided as a liquid injectable (such as in those embodiments where the compound is administered intravenously or directly into a tissue), the compound can be provided as a ready- to-use dosage form, or as a reconstitutable storage-stable powder or liquid composed of pharmaceutically acceptable carriers and excipients.

[0374] Methods for formulating compounds can be adapted from those readily available. For example, compounds can be provided in a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition may optionally include other additives (e.g., buffers,stabilizers, preservatives, and the like). In some embodiments, the formulations are suitable for administration to a mammal, such as those that are suitable for administration to a human.

[0375] In some cases, the method includes administering to a subject an effective amount of any of the compounds of the present disclosure. In certain aspects, provided are methods of delivering a molecule of interest (e.g., an active agent) to a target site in a subject, the method including administering to the subject a pharmaceutical composition including any of the compounds of the present disclosure, where the administering is effective to release a therapeutically effective amount of the molecule of interest from the compound at the target site in the subject.

[0376] In some cases, one or multiple doses of a compound are administered to the subject. The frequency of administration of a compound can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc. For example, in some embodiments, a compound is administered once per month, twice per month, three times per month, every other week, once per week (qwk), twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily (qd / od), twice a day (bds / bid), or three times a day (tds / tid), etc.

[0377] The present disclosure provides a method of delivering a molecule of interest to a cell (e.g., a “target cell”), the method comprising contacting the cell with a conjugate of the present disclosure, where the conjugate enters the cell, and where the molecule of interest Z1is released from the conjugate under reducing conditions within the cell. In some cases, the cell is in vitro. In some cases, the cell is in vivo. In some cases, the cell is a eukaryotic cell, and the cell is in vitro. In some cases, the cell is a eukaryotic cell, and the cell is in vivo.

[0378] Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0379] A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual – in some cases a primary cell, e.g., a primary human cell). A cell can be an in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.

[0380] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a lung cell (e.g., a lung epithelial cell), etc.

[0381] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells.

[0382] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0383] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[0384] Adult stem cells are resident in differentiated tissue, but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[0385] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[0386] Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

[0387] In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm- derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+and CD3-. HSCs can repopulate the erythroid, neutrophil- macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0388] In other embodiments, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[0389] In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

[0390] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon.

[0391] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a sub-order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola , Hymenoptera , Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera , Mecoptera , Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[0392] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.

[0393] The present disclosure provides a method of delivering a molecule of interest to an individual, the method comprising administering a conjugate of the present disclosure to the individual, wherein Z1is a molecule of interest, and wherein Z1is released from the conjugate under physiological reducing conditions, e.g., reducing conditions within a cell of the individual.

[0394] A conjugate of the present disclosure can be administered to an individual in need thereof via any of a variety of routes of administration and via any of a variety of modes of administration. For example, a conjugate of the present disclosure can be administered intramuscularly, intravenously, subcutaneously, intracranially, intratumorally, peritumorally, intraocularly, etc. A conjugate of the present disclosure can be administered by injection, via a catheter, or any other convenient mode of administration. METHODS OF MODIFYING A TARGET NUCLEIC ACID

[0395] Provided is a method of modifying a target nucleic acid in a cell, the method comprising contacting the cell with a composition comprising a conjugate of the formula (Ic) or (IIc) or (IIIc): R6O 21H NO 221R 2 R 1YZ whereCas effector polypeptide, a Cre recombinase, and the like); Z2is a small molecule, a protein such as an antibody or functional fragment thereof, a targeting and / or delivery moiety such as a targeting and / or delivery peptides such as T1, P55, Angiopep2, TAT, and the like (e.g., AP22, P2, F17, nPF4, PT551), a lipid, a nucleic acid, a polysaccharide, or any combination thereof [In some cases Z2is a targeting moiety (e.g., a targeting peptide) or a protein transduction peptide (e.g., a polypeptide that facilitates entry of Z1into a cell); a polypeptide that inhibits enzymatic activity of Z1(e.g., an ACR polypeptide); a moiety that targets a particular cell type, organ, and / or tissue; an endosomal escape polypeptide; a transcytosis polypeptide; a blood-brain barrier (BBB) transit polypeptide; a blood-cerebrospinal fluid barrier (BCSFB) transit polypeptide; and the like]; and the other variable are each as described above for the respective compounds. In some embodiments, e.g., in some cases when Z1is a CRISPR-Cas effector polypeptide (e.g., Cas9), the composition further comprises a guide RNA or a nucleic acid encoding the guide RNA. In some such cases the CRISPR-Cas effector polypeptide (e.g., Cas9) is complexed with a guide RNA thus forming an RNP. In some cases, the guide RNA is a single-molecule guide RNA.

[0396] In some cases, the method includes contacting the cell with a composition comprising a conjugate of the formula (Ic). In some cases, the method includes contacting the cell with a composition comprising a conjugate of the formula (IIc). In some cases, the method includes contacting the cell with a composition comprising a conjugate of the formula (IIIc).

[0397] In some cases, Z1is a CRISPR-Cas effector polypeptide (e.g., Cas9).

[0398] In some cases, Z2is a polypeptide that facilitates entry of Z1into a cell. In some cases, Z2is a polypeptide that inhibits enzymatic activity of Z1(e.g., an ACR polypeptide). In some cases, Z2is a moiety that targets a particular cell type, organ, and / or tissue. In some cases, Z2is an endosomal escape polypeptide. In some cases, Z2is a transcytosis polypeptide. In some cases, Z2is a blood-brain barrier (BBB) transit polypeptide. In some cases, Z2is a blood-cerebrospinal fluid barrier (BCSFB) transit polypeptide.

[0399] In some cases, upon entry of the conjugate into a cell (e.g., a eukaryotic cell, such as a mammalian cell), the linker is cleaved and Z1is released from the conjugate. In some cases, Z1(e.g., a CRISPR / Cas effector polypeptide such as Cas9) is fused to a nuclear localization signal (NLS). In some cases, the composition comprises a DNA donor template. In some cases, Z1is a gene editing peptide (e.g., CRISPR / Cas effector polypeptide such as Cas9) that is a fused to a heterologous polypeptide. In some such cases, the CRISPR / Cas effector polypeptide (e.g., Cas9) has reduced nuclease activity (e.g., can be a nickase or catalytically inactive, e.g., a dCas9).

[0400] In some cases, the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, viral DNA, and extrachromosomal DNA. In some cases, the contacting takes place inside of a cell in vivo. In some cases, the contacting takes place inside of a cell in vitro. In some cases, the contacting takes place inside of a cell ex vivo.

[0401] In some cases, the cell is a eukaryotic cell. In some cases, the cell is a non-human primate cell. In some cases, the cell is a human cell. In some cases, the cell is a mammalian cell. In some cases, the cell is a plant cell. In some cases, the cell is an arthropod cell. In some cases, the cell is an insect cell. In some cases, the cell is an arachnid cell. In some cases, the cell is an avian cell. In some cases, the cell is fish cell. In some cases, the cell is an invertebrate cell.

[0402] A subject method can be used, e.g., for gene editing in vivo (e.g., to edit neurons in an individual, e.g., using a BBB transit polypeptide as Z2; to edit cells of the lung, e.g., lung epithelium; to target T cells, B cells, and / or hematopoietic stem cells, e.g., in humans, in humanized mice, etc.; to generate humanized mice; etc.).

[0403] For example, the present disclosure provides a method of modifying a target nucleic acid in a cell, the method comprising contacting the cell with a composition comprising a conjugate of the present disclosure, where Z1is a CRISPR-Cas effector polypeptide, and Z2is a targeting moiety (e.g., a targeting peptide) or a protein transduction peptide (e.g., a polypeptide that facilitates entry of Z1into a cell); a polypeptide that inhibits enzymatic activity of Z1(e.g., an ACR polypeptide); a moiety that targets a particular cell type, organ, and / or tissue; an endosomal escape polypeptide; a transcytosis polypeptide; a blood-brain barrier (BBB) transit polypeptide; a blood-cerebrospinal fluid barrier (BCSFB) transit polypeptide; and the like). In some cases, the composition comprises one or more CRISPR-Cas guide RNAs, or a nucleic acid comprising a nucleotide sequence encoding the one or more CRISPR-Cas guide RNA (e.g., in some cases the CRISPR-Cas effector polypeptide is complexed with the guide RNA, forming an RNP) . Upon entry of the conjugate into a mammalian cell, the linker is cleaved and Z1(and in some cases a Z1-guide RNA RNP) is released from the conjugate. In some cases, the composition further comprises a donor template nucleic acid.

[0404] A target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial,chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the CRISPR-Cas guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted).

[0405] A target nucleic acid can be DNA or RNA. A target nucleic acid can be double stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is single stranded. In some cases, a target nucleic acid is a single stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and microRNA (miRNA). In some cases, a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some cases, a target nucleic acid is single stranded.

[0406] The target nucleic acid can be in a cell in vitro. The target nucleic acid can be in a cell in vivo. The target nucleic acid can be in a cell ex vivo. Suitable cells are as described above. In some cases, the cell is in an individual in vivo, e.g., the cell is in a mammal (e.g., a human; a non- human mammal; a non-human primate; a feline; a bovine; a canine; an ovine; an equine; an ungulate; and the like). In some cases, the individual is a reptile. In some cases, the individual is an amphibian. In some cases, the individual is an arthropod. In some cases, the individual is an arachnid. In some cases, the individual is an invertebrate.

[0407] In some cases, e.g., where Z2is a CPP, the conjugate can be introduced into a target cell without the need for a transfection reagent.

[0408] As noted above, the composition can comprise, in addition to a conjugate of the present disclosure, one or more CRISPR-Cas guide RNAs. In some cases, the composition comprises: i) a CRISPR / Cas effector polypeptide guide RNA (referred to herein as a “CRISPR / Cas guide RNA” or simply “guide RNA”); or ii) a nucleic acid comprising a nucleotide sequence encoding the CRISPR / Cas effector polypeptide guide RNA. A CRISPR / Cas guide RNA comprises a protein-binding segment that binds and activates a CRISPR / Cas effector polypeptide. A CRISPR / Cas guide RNA provides target specificity the CRISPR-Cas effector polypeptide by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid.

[0409] In some cases, a guide RNA includes two separate nucleic acid molecules: an “activator” (comprising the protein-binding segment) and a “targeter” (comprising the targeting segment) and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two- molecule guide RNA”, or a “dgRNA.” In some cases, the guide RNA is one molecule (e.g., for some class 2 CRISPR / Cas proteins, the corresponding guide RNA is a single molecule; and in some cases, an activator and targeter are covalently linked to one another, e.g., via interveningnucleotides, e.g., via artificial engineering), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.”

[0410] As noted above, in some cases, the composition comprises a donor template polynucleotide. A CRISPR-Cas effector polypeptide in some cases generates site-specific double strand breaks (DSBs) or single strand breaks (SSBs) (e.g., when the CRISPR-Cas effector polypeptide is a nickase variant) within double-stranded DNA (dsDNA) target nucleic acids, which are repaired either by non-homologous end joining (NHEJ) or homology-directed recombination (HDR).

[0411] In some cases, contacting a target nucleic acid (e.g., with a CRISPR-Cas effector polypeptide and a guide RNA) occurs under conditions that are permissive for nonhomologous end joining or homology-directed repair. Thus, in some cases, a subject method includes contacting the target DNA with a donor polynucleotide (e.g., by introducing the donor polynucleotide into a cell), wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. In some cases, the method does not comprise contacting a cell with a donor polynucleotide, and the target DNA is modified such that nucleotides within the target DNA are deleted.

[0412] In applications in which it is desirable to insert a polynucleotide sequence into the genome where a target sequence is cleaved, a donor polynucleotide (a nucleic acid comprising a donor sequence) can also be provided to the cell. By a “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site cleaved by the CRISPR-Cas effector polypeptide (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g.70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) can support homology-directed repair. Donor polynucleotides can be of any length, e.g.10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0413] In some cases, the donor sequence is not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long assufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease-causing base pair to a non-disease-causing base pair). In some cases, the donor sequence comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region. Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide. Examples of Non-Limiting Aspects of the Disclosure

[0414] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure, numbered 1-49, are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: 1. A reversible / traceless linker of formula (I): O HL1is an inert linker; L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X1is an amine-reactive moiety; a salt thereof, or a stereoisomer thereof.2. A reversible / traceless linker conjugate of formula (Ia): O 2 H R1N OZ1X2L2R O HN L1L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X2is a connecting group;Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof. 3. A reversible / traceless linker conjugate of formula (Ib): R6L1is an inert linker; L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; R6is H, alkyl or a substituted version thereof; Z2is an amine-reactive moiety; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof. 4. A reversible / traceless linker conjugate of formula (Ic):R6NHL2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; R6is H, alkyl or a substituted version thereof; X2is a connecting group; Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof. 5. The reversible / traceless linker of 1 or linker conjugate of 3, wherein Z2is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. 6. The reversible / traceless linker or linker conjugate of 5, wherein Z2is a carbonate. 7. The reversible / traceless linker or linker conjugate of 6, wherein Z2is a para-nitrophenol carbonate. 8. The reversible / traceless linker conjugate of any one of 2, 4, and 5-7, wherein X2is -NH-, -NHCOO-, -NH(CO)NH-, -NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof. 9. The reversible / traceless linker conjugate of any one of 2, 4, and 5-8, wherein Z1is a small molecule drug or a polypeptide. 10. The reversible / traceless linker conjugate of any one of 2, 4, and 5-8, wherein Z1is a gene editing protein. 11. The reversible / traceless linker conjugate of 10, wherein the gene editing protein is a Cre recombinase, a Flp recombinase, a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-Cas effector protein. 12. The reversible / traceless linker conjugate of 10, wherein the gene editing protein is Cas9.13. The reversible / traceless linker conjugate of any one of 3-12, wherein Z2is a cell penetrating polypeptide; an anti-CRISPR polypeptide; a moiety that targets a specific cell type, tissue, or organ; an endosomal escape polypeptide; a transcytosis polypeptide; a blood- brain barrier (BBB) transit polypeptide; or a blood-cerebrospinal fluid barrier (BCSFB) transit polypeptide. 14. The reversible / traceless linker conjugate of any one of 3-12, wherein Z2comprises: a Tet1 (T1) peptide, a peptide comprising the amino acid sequence of SEQ ID NO: 32, a peptide comprising the amino acid sequence of SEQ ID NO: 33, a P55 peptide, an F4 peptide, an angiopep2 peptide, a TAT peptide, or any combination thereof. 15. The reversible / traceless linker or linker conjugate of any one of 1-14, wherein L1ispolyethylene glycol (PEG). 16. The reversible / traceless linker or linker conjugate of 15, wherein the PEG has an average molecular weight in a range of from 200 daltons to 160 kilodaltons (kDa). 17. The reversible / traceless linker or linker conjugate of 15, wherein the PEG has an average molecular weight in a range of from 600 daltons to 2 kilodaltons (kDa). 18. The reversible / traceless linker or linker conjugate of any one of 1-17, wherein L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate. 19. The reversible / traceless linker of 1, wherein the reversible / traceless linker is: O H N20. A reversible / traceless linker of formula (II): O OZ2is an amine-reactive moiety;Y1is a reactive moiety; L1is an inert linker; a salt thereof, or a stereoisomer thereof. 21. A reversible / traceless linker conjugate of formula (IIa):O O Z1X2S L1Y1NL1is an inert linker; X2is a connecting group; Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof. 22. A reversible / traceless linker conjugate of formula (IIb): O O X1S L1Y2Z2L1is an inert linker; Z2is an amine-reactive moiety; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof. 23. A reversible / traceless linker conjugate of formula (IIc): O OY2is post-reaction chemical group; L1is a linker; X2is a connecting group; Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof. 24. The reversible / traceless linker of 20 or linker conjugate of 22, wherein Z2is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester. 25. The reversible / traceless linker or linker conjugate of 24, wherein Z2is a carbonate.26. The reversible / traceless linker or linker conjugate of 25, wherein Z2is a para-nitrophenol carbonate. 27. The reversible / traceless linker or linker conjugate of any one of 20-21 and 24-26, wherein Y1is trans-cyclooctene (TCO), tetrazine, alkyne, thiol, maleimide, iodoacetamide, amine, carboxyl, ester, triazole, diene, dienophile, sulfonyl fluoride, hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, phosphine, epoxide, succinimide, and pentafluorophenyl (PFP) ester, or a ...

Claims

Claims What is claimed is:

1. A reversible / traceless linker of formula (I): O 21H R 2 R 1N OL2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X1is an amine-reactive moiety; a salt thereof, or a stereoisomer thereof.

2. A reversible / traceless linker conjugate of formula (Ia): O R2R1H NOL1is an inert linker; L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X2is a connecting group; Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof.

3. A reversible / traceless linker conjugate of formula (Ib): R6NHL2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; R6is H, alkyl or a substituted version thereof; Z2is an amine-reactive moiety; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

4. A reversible / traceless linker conjugate of formula (Ic): R6L1is an inert linker; L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; R6is H, alkyl or a substituted version thereof; X2is a connecting group; Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

5. The reversible / traceless linker of claim 1 or linker conjugate of claim 3, wherein Z2is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester.

6. The reversible / traceless linker or linker conjugate of claim 5, wherein Z2is a carbonate.

7. The reversible / traceless linker or linker conjugate of claim 6, wherein Z2is a para-nitrophenol carbonate.

8. The reversible / traceless linker conjugate of any one of claims 2, 4, and 5-7, wherein X2is -NH-, - NHCOO-, -NH(CO)NH-, -NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof.

9. The reversible / traceless linker conjugate of any one of claims 2, 4, and 5-8, wherein Z1is a small molecule drug or a polypeptide.

10. The reversible / traceless linker conjugate of any one of claims 2, 4, and 5-8, wherein Z1is a gene editing protein.

11. The reversible / traceless linker conjugate of claim 10, wherein the gene editing protein is a Cre recombinase, a Flp recombinase, a meganuclease, a zinc finger nuclease (ZFN), a transcription activator- like effector nuclease (TALEN), or a CRISPR-Cas effector protein.

12. The reversible / traceless linker conjugate of claim 10, wherein the gene editing protein is Cas9.

13. The reversible / traceless linker conjugate of any one of claims 3-12, wherein Z2is a cell penetrating polypeptide; an anti-CRISPR polypeptide; a moiety that targets a specific cell type, tissue, or organ; an endosomal escape polypeptide; a transcytosis polypeptide; a blood-brain barrier (BBB) transit polypeptide; or a blood-cerebrospinal fluid barrier (BCSFB) transit polypeptide.

14. The reversible / traceless linker conjugate of any one of claims 3-12, wherein Z2comprises: a Tet1 (T1) peptide, a peptide comprising the amino acid sequence of SEQ ID NO: 32, a peptide comprising the amino acid sequence of SEQ ID NO: 33, a P55 peptide, an F4 peptide, an angiopep2 peptide, a TAT peptide, or any combination thereof.

15. The reversible / traceless linker or linker conjugate of any one of claims 1-14, wherein L1is polyethylene glycol (PEG).

16. The reversible / traceless linker or linker conjugate of claim 15, wherein the PEG has an average molecular weight in a range of from 200 daltons to 160 kilodaltons (kDa).

17. The reversible / traceless linker or linker conjugate of claim 15, wherein the PEG has an average molecular weight in a range of from 600 daltons to 2 kilodaltons (kDa).

18. The reversible / traceless linker or linker conjugate of any one of claims 1-17, wherein L2is (i) a disulfide, (ii) a disulfide carbamate, (iii) a dithiobenzyl carbamate, or (iv) a dithiol-ethyl carbonate plus a benzyl carbamate.

19. The reversible / traceless linker of claim 1, wherein the reversible / traceless linker is: O H OOS ON O20. A reversible / traceless linker of formula (II): O OZ2is an amine-reactive moiety;Y1is a reactive moiety; L1is an inert linker; a salt thereof, or a stereoisomer thereof.

21. A reversible / traceless linker conjugate of formula (IIa): O Owherein: Y1is a reactive moiety; L1is an inert linker; X2is a connecting group; Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof.

22. A reversible / traceless linker conjugate of formula (IIb): O O X1S L1Y2Z2L1is an inert linker; Z2is an amine-reactive moiety; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

23. A reversible / traceless linker conjugate of formula (IIc): O OY2is post-reaction chemical group; L1is a linker; X2is a connecting group; Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

24. The reversible / traceless linker of claim 20 or linker conjugate of claim 22, wherein Z2is isothiocyanate, isocyanate, acyl azide, N-Hydroxysuccinimide ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, or fluorophenyl ester.

25. The reversible / traceless linker or linker conjugate of claim 24, wherein Z2is a carbonate.

26. The reversible / traceless linker or linker conjugate of claim 25, wherein Z2is a para-nitrophenol carbonate.

27. The reversible / traceless linker or linker conjugate of any one of claims 20-21 and 24-26, wherein Y1is trans-cyclooctene (TCO), tetrazine, alkyne, thiol, maleimide, iodoacetamide, amine, carboxyl, ester, triazole, diene, dienophile, sulfonyl fluoride, hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, phosphine, epoxide, succinimide, and pentafluorophenyl (PFP) ester, or a substituted version thereof.

28. The reversible / traceless linker conjugate of any one of claims 22-26, wherein Y2is TCO- tetrazine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, thioether, succinimidyl-thioether, carbamate acetal, disulfide or a substituted version thereof.

29. The reversible / traceless linker conjugate of any one of claims 22-26, wherein Y2is TCO- tetrazine.

30. The reversible / traceless linker conjugate of any one of claims 21, 23, and 27-29, wherein X2is - NH-, -NHCOO-, -NH(CO)NH-, -NHSOO-, amide, imine, diimine, hydroxyamine, polyethylene glycol, amino, alkoxy, amide, triazole, ester, aryl, heteroaryl, succinimidyl-thioether, or a substituted version thereof.

31. The reversible / traceless linker conjugate of any one of claims 21, 23, and 27-30, wherein Z1is a small molecule drug or a polypeptide.

32. The reversible / traceless linker conjugate of any one of claims 21, 23, and 27-30, wherein Z1is a gene editing protein.

33. The reversible / traceless linker conjugate of claim 32, wherein the gene editing protein is a Cre recombinase, a Flp recombinase, a meganuclease, a zinc finger nuclease (ZFN), a transcription activator- like effector nuclease (TALEN), and a CRISPR-Cas effector protein.

34. The reversible / traceless linker conjugate of claim 32, wherein the gene editing protein is Cas9.

35. The reversible / traceless linker conjugate of any one of claims 22-26, and 28-34, wherein Z2is a cell penetrating polypeptide; an anti-CRISPR polypeptide; a moiety that targets a specific cell type, tissue, or organ; or an endosomal escape polypeptide.

36. The reversible / traceless linker conjugate of claim 35, wherein Z2comprises: a Tet1 (T1) peptide, a peptide comprising the amino acid sequence of SEQ ID NO: 32, a peptide comprising the amino acid sequence of SEQ ID NO: 33, a P55 peptide, an F4 peptide, an angiopep2 peptide, a TAT peptide, or any combination thereof.

37. The reversible / traceless linker or linker conjugate of any one of claims 20-36, wherein L1ispolyethylene glycol (PEG).

38. The reversible / traceless linker or linker conjugate of claim 37, wherein the PEG has an average molecular weight in a range of from 200 daltons to 160 kilodaltons (kDa).

39. The reversible / traceless linker or linker conjugate of claim 37, wherein the PEG has an average molecular weight in a range of from 600 daltons to 2 kilodaltons (kDa).

40. A pharmaceutical composition comprising: the reversible / traceless linker conjugate of any one of claims 4, 8-19, 23, and 29-39; and a pharmaceutical excipient.

41. A method of producing a reversible / traceless linker conjugate, the method comprising: (a) covalently attaching the reversible / traceless linker of claim 1 or claim 20 to a first molecule of interest and / or a second molecule of interest, or (b) covalently attaching the reversible / traceless linker conjugate of claim 2 or claim 21 to a second molecule of interest, or (c) covalently attaching the reversible / traceless linker conjugate of claim 3 or claim 22 to a first molecule of interest; thereby producing a reversible / traceless linker conjugate.

42. A method of delivering a molecule of interest to an individual, the method comprising administering the reversible / traceless linker conjugate of any one of claims 4, 8, 15-19, 23, 28-30 and 37- 39 to an individual, wherein: (a) Z1is a small molecule drug or a polypeptide,(b) Z2comprises a targeting moiety, a moiety that increases in vivo half-life of Z1, or a protein transduction domain, and (c) Z1is released from the conjugate under physiological reducing conditions.

43. The method of claim 42, wherein Z2comprises a Tet1 (T1) peptide, an F4 peptide, a P55 peptide, or both a T1 peptide and a P55 peptide.

44. The method of claim 42 or claim 43, wherein the individual has a neurodegenerative disease or disorder.

45. The method of any one of claims 42-44, wherein said administering comprises systemic administration.

46. A method of modifying a target nucleic acid in a cell, the method comprising contacting a cell with a composition comprising: (1) the reversible / traceless linker conjugate of any one of claims 4, 8, 15-17, 22, 27-29 and 36, wherein: (a) Z1is a CRISPR-Cas effector protein, and (b) Z2comprises a targeting moiety, a moiety that increases in vivo half-life of Z1, or a protein transduction domain, and (2) a guide RNA, or a nucleic acid encoding the guide RNA, wherein said contacting results in modification of the target nucleic acid.

47. The method of claim 46, wherein, upon entry of the reversible / traceless linker conjugate into the cell, the linker is cleaved and Z1is released from the conjugate.

48. The method of claim 46 or claim 47, wherein the CRISPR-Cas effector protein is fused to a nuclear localization signal.

49. The method of any one of claims 46-48, wherein the composition comprises a DNA donor template.

50. The method of any one of claims 46-49, wherein the CRISPR-Cas effector protein is a fused to a heterologous polypeptide.

51. The method of any one of claims 46-50, wherein the guide RNA is a single-molecule guide RNA.

52. The method of any one of claims 46-51, wherein the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, viral DNA, and extrachromosomal DNA.

53. The method of any one of claims 46-52, wherein the cell is in vivo.

54. The method of any one of claims 46-53, wherein the cell is a eukaryotic cell.

55. A reversible / traceless linker of formula (III): O R2R1H NOL1is an inert linker; L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X1is an amine-reactive moiety; Y1is a reactive moiety (e.g., a copper free click chemistry reactive moiety); a salt thereof, or a stereoisomer thereof 56. A reversible / traceless linker conjugate of formula (IIIa): O HL1is an inert linker; L2is a reduction sensitive disulfide linker;R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; Y1is a reactive moiety (e.g., a copper free click chemistry reactive moiety); X2is a connecting group; Z1is a first molecule of interest; a salt thereof, or a stereoisomer thereof.

57. A reversible / traceless linker conjugate of formula (IIIb): O H R2R1N O Y2Z2L2is a reduction sensitive disulfide linker; R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X1is an amine-reactive moiety; Y2is a post-reaction chemical group (e.g., a copper free click chemistry post-reaction group); Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

58. A reversible / traceless linker conjugate of formula (IIIc): O HL1is an inert linker; L2is a reduction sensitive disulfide linker;R1and R2are independently absent, -(CH2)n-, or -CR3R4-, wherein R3and R4are each independently selected from the group consisting of H, linear alkyl, branched alkyl, amine, azide or substituted versions thereof, and n is independently an integer ranging from 1 to 10; X2is a connecting group; Y2is post-reaction chemical group (e.g., a copper free click chemistry post-reaction group); Z1is a first molecule of interest; Z2is a second molecule of interest; a salt thereof, or a stereoisomer thereof.

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