Engineered targeted protein degraders and methods of use thereof

Abstract

Described herein are methods and compositions relating to a chimeric molecule comprising: a degradation domain; and a targeting domain capable of specifically directing said degradation domain to a substrate, wherein said targeting domain is heterologous to said degradation domain.

Description

Attorney Docket No: 701586-000144WOPTENGINEERED TARGETED PROTEIN DEGRADERS AND METHODS OF USE THEREOFCROSS-REFERENCE TO REEATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 685,328 filed August 21, 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The technology described herein relates to methods and compositions for generation of protein-based targeted protein degraders using recombinant proteins and / or exogenous polynucleotides. Wherein said protein-based protein degrader specifically interacts with a target protein of interest and degrades the target protein using the proteasome without requiring covalent conjugation of ubiquitin(s) to the target protein.BACKGROUND

[0003] The current small molecule, inhibitor-based drug paradigm largely limits drug targets to enzymes or receptors with tight and well-defined pockets (“active sites”). Additionally, as these drugs act stoichiometrically, they typically require high dosing in order to achieve adequate concentrations for therapeutic efficacy (Bekes et al., 2022). To circumvent these issues, alternative therapeutic strategies have been employed to specifically knock down target proteins. While genetic techniques such as RNA interference (“RNAi”), and CRISPR / Cas9 can significantly reduce protein levels, the pharmacokinetic / pharmacodynamic properties (i.e., metabolic stability, tissue distribution, off-target knock-down) associated with these approaches have so far limited their development as clinical agents (Doudna, 2020; Guo et al., 2023; Setten et al., 2019). These genetic approaches have a limited capacity to achieve subpopulation specific knock-do wn / knock-out (e.g., mutant vs. wild-type, paralogs) due to significant sequence identity between protein subpopulations. Additionally, as these approaches target proteins at the pre-translational level (e.g., DNA or RNA), it is impossible to selectively knock-down post-translationally modified (e.g., phosphorylation, acetylation, etc.) subpopulations of protein, or eliminate / reduce target protein that has already accumulated before drug administration.

[0004] Direct proteome editing technology represents a shift in drug paradigm that operates at the post-translational level and has the potential to address complicated protein functions at higher resolution than methods targeting DNA or RNA and with post-translational precision. Two of the most notable methods in this emerging field of targeted protein degradation (“TPD”) are: (i) proteolysis targeting chimeras (“PROTACs”) (Bondeson et al., 2015; Chimomas et al., 2023; Deshaies, 2015; Gough et al., 2024; Hickey et al., 2024; Hines et al., 2013; Hines et al., 2019; Neklesa14926-6123-2472 2Attorney Docket No: 701586-000144WGPT et al., 2017; Schneekloth et al., 2008; Schneekloth et al., 2004) or (ii) monovalent / molecular glue degraders (“MGDs”) (Garber, 2024; Toriki et al., 2023; Zhao et al., 2022).

[0005] These small molecule PROTACs or MGDs hijack or re-target the machinery of the cellular ubiquitin-proteasome system (“UPS”) to specifically degrade proteins of interest. The canonical ubiquitination cascade requires the activities of three enzymes — ubiquitin activating enzyme (El), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) — which act sequentially to tag proteins for degradation through the covalent attachment of a poly-ubiquitin chain to exposed lysine residues in an energy-dependent manner.

[0006] PROTACs are heterobifimctional molecules containing one ligand for an E3 ubiquitin ligase, another ligand for the target protein to be degraded, and a linker connecting the two. These molecules form a ternary complex, binding to both the E3 ligase and the target protein, triggering target polyubiquitination followed by its proteasomal degradation. MGDs also bind to E3 ligase and target protein to form a ternary complex but are traditionally a “linker less” small molecule.

[0007] Due to their mechanism of action, PROTAC / MGD target proteins are limited to proteins which contain exposed lysine(s) (Zhao et al., 2022). In addition to this requirement, current TPD modalities face significant challenges developing small molecule binders which are specific for target proteins. For example, many PROTACs / MGDs rely on a small number of previously developed small molecule inhibitors as targeting moieties for target protein interactions (Mullard, 2023).

[0008] In addition to specific protein / polypeptide requirements, these small molecule TPD systems exhibit several undesirable pharmacokinetic / pharmacodynamic properties. At high concentrations, these systems exhibit a self-inhibitory effect (i.e., hook-effect), which poses challenges on effective dosing (Moreau et al., 2020). Recent evidence suggests that there are tissues where commonly targeted E3 ligase substrate receptors (e.g., von Hippel-Lindau (“VHL”) and Cereblon (“CRBN”)) are inactivated / suppressed, therefore preventing degradation of target protein in said tissue (Luo et al., 2022) Size / charge / hydrophobicity limitations of many PROTAC systems may also preclude oral delivery (Edmondson et al., 2019). Additionally, as these small molecules lack tissue targeting moieties, dosing is typically very high, often requiring concentrations up to 25 mM to induce sufficient degradation (Buckley & Crews, 2014), which may lead to a higher likelihood of off- target toxicities. Moreover, PROTAC / MGD small molecules may not subcellularly localize to compartments containing the target protein / polypeptide (e.g., the nucleus).

[0009] More recently, protein-based chimeras have been developed where native or engineered E3 ubiquitin ligases are genetically fused to a protein that natively binds the target of interest. Following ectopic expression in cells, the engineered protein chimera recruits the E3 ligase to the target protein, leading to its polyubiquitination and subsequent degradation by the proteasome (Hatakeyama et al., 2005; Kong et al., 2013; Ma et al., 2013; Zhang et al., 2003; Zhou et al., 2000). These approaches have been extended beyond naturally occurring binding pairs by fusing an E3 ligase to synthetic binding proteins / polypeptides such as a single-chain antibody fragment (“scFv”), a24926-6123-2472 2Attorney Docket No: 701586-000144WOPT designed ankyrin repeat protein (“DARPin”), or a fibronectin type III (“FN3”) monobody (Caussinus et al., 2011; Fulcher et al., 2017; Fulcher et al., 2016; Ju Shin et al., 2015; Kanner et al., 2017; Lim, Khoo, Juang, et al., 2020; Lim, Khoo, Peh, et al., 2020; Ludwicki et al., 2019; Portnoff et al., 2014). These bifimctional chimeras, commonly known as “ubiquibodies” (“uAbs”), offer several advantages over first generation small molecule TPD modalities. One of the greatest advantages is their highly modular architecture, where simply swapping synthetic targeting moieties can generate a new uAb that specifically targets a different substrate protein, while swapping E3 domains can alter the kinetics, mechanism, or tissue tropism of ubiquitin transfer. Moreover, by incorporating synthetic binding proteins that recognize particular protein states (e.g., active vs. inactive conformation, mutant vs. wild-type, post-translationally modified), it becomes possible to deplete certain protein subpopulations while sparing others (Baltz et al., 2018; Zhang et al., 2003). However, even the uAb system requires exposed lysine(s) residues on target proteins to achieve degradation through ubiquitination.

[0010] Recently, the first report of midnolin’s (Gene ID: MIDN) mechanism of action was published (Gu et al., 2023), wherein midnolin mediates the proteasomal degradation of immediate- early gene (“IEG”) transcription factors without requiring the conjugation of ubiquitin to the target proteins.

[0011] Inspired by this discovery, this disclosure outlines a mechanism for the production of synthetic protein-based targeted protein degraders which induce depletion of target proteins without requiring the formation of an isopeptide bond between ubiquitin and a lysine residue on the target protein. This potentially broadens the druggable proteome by allowing for targeted degradation of proteins which lack surface lysine residues, or where mutations cause said lysine residues to become cryptic. Said protein-based degrader may be purified recombinant protein or expressed in situ through the delivery of exogenous RNA or DNA. By delivering protein-based degraders to target cells, therapeutic effect can be achieved through targeted depletion of pathogenic protein(s) / polypeptide(s). This disclosure demonstrates the viability of polypeptide-based degraders in treating or preventing oncological, degenerative, fibrotic, cardiovascular, and immunological diseases.SUMMARY

[0012] Many diseases are caused, at least in part, by the expression of a mutated protein or the overexpression of a normal protein. Many attempts to treat such diseases by targeting these diseasecausing proteins have been made. One strategy is targeted protein degradation, in which the patient is administered a therapeutic protein, small molecule, or polynucleotide that binds to the disease-causing protein and causes the disease-causing protein to be degraded. The existing targeted protein degradation technologies are not readily adapted to new targets and rely upon ubiquitination of the disease-causing protein. They are also limited to disease-causing proteins which exhibit select34926-6123-2472 2Attorney Docket No: 701586-000144WOPT structural features, e.g., solvent-exposed lysine residues. Finally, these technologies suffer from the “hook effect.”

[0013] The novel chimeric molecules described herein provide strong binding, which is readily adjusted for a new target, coupled with degradation activity which does not require ubiquitination, e.g., of the target protein. The present chimeric molecules also do not suffer from the “hook effect” and can be carefully adjusted to provide proper dosages.

[0014] In one aspect of any of the embodiments, described herein is a chimeric molecule comprising: a degradation domain; and a targeting domain capable of specifically directing said degradation domain to a substrate, wherein said targeting domain is heterologous to said degradation domain.

[0015] In some embodiments of any of the aspects, the molecule is a polypeptide. In some embodiments of any of the aspects, the degradation domain is coupled to the targeting domain via a linker. In some embodiments of any of the aspects, the targeting domain is N-terminal to the degradation domain.

[0016] In some embodiments of any of the aspects, the degradation domain comprises a ubiquitin domain or ubiquitin-like domain. In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Midnolin (Gene ID: MIDN, SEQ ID NO: 1) including the ubiquitin-like domain (“Ubl”, residues 31 to 105), Catchl domain (residues 112 to 156), Catch2 domain (residues 266 to 332), nuclear localization sequence domain (“NLS”, residues 402 to 413), or C-terminal a helix domain (“aHelix-C”, residues 377 to 413). In some embodiments of any of the aspects, the degradation domain comprises a ubiquitin-like domain and a C-terminal a helix domain. In some embodiments of any of the aspects, the degradation domain comprises a midnolin ubiquitin-like domain and a midnolin C-terminal a helix domain. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal, a Catchl domain, or a Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal, a midnolin Catchl domain, or a midnolin Catch2 domain.

[0017] In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 80% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 80%44926-6123-2472 2Attorney Docket No: 701586-000144WGPT sequence identity to the entirety of each of SEQ ID NO: 100, 103, and 106 or b) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100, 104, and 106.

[0018] In some embodiments of any of the aspects, the sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 106 is the most C-terminal portion of the chimeric molecule. In some embodiments of any of the aspects, the sequence with the sequence of SEQ ID NO: 106 is the most C-terminal portion of the chimeric molecule.

[0019] In some embodiments of any of the aspects, the degradation domain does not comprise a sequence with at least 80% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises residues 31-105 and 377-413 of SEQ ID NO: 1.

[0020] In some embodiments of any of the aspects, the degradation domain comprises each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises a) each of SEQ ID NO: 100, 103, and 106 or b) each of SEQ ID NO: 100, 104, and 106.

[0021] In some embodiments of any of the aspects, the degradation domain does not comprise residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise the sequences of each of SEQ ID NO: 101, 102, and 105.

[0022] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: Midnolin; Ubiquitin-like protein 4A; Ubiquitin-like protein 4B; Ubiquitin-like protein 5; Ubiquitin-like protein 7; RAD23A; RAD23B; UBAC1; ISG15; DDI1; DDE; Ubiquitin-related modifier 1; ATG12; Ubiquitin D; Polyubiquitin-C; Ubiquilin-1; Ubiquilin-2; Ubiquilin-3; Ubiquilin-4; Ubiquilin-like protein; USP14, USP7; PARK2; BAG6; UBLCP1; and TMUB1. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: Midnolin; Ubiquitin-like protein 4A; Ubiquitin-like protein 4B; Ubiquitin-like protein 5; Ubiquitin-like protein 7; RAD23A; RAD23B; UBAC1; ISG15; DDI1; DDE; Ubiquitin- related modifier 1; ATG12; Ubiquitin D; Polyubiquitin-C; Ubiquilin-1; Ubiquilin-2; Ubiquilin-3; Ubiquilin-4; Ubiquilin-like protein; USP14, USP7; PARK2; BAG6; UBLCP1; and TMUB1.

[0023] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the54926-6123-2472 2Attorney Docket No: 701586-000144WOPT ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin- like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20.

[0024] In some embodiments of any of the aspects, the degradation domain contains the full- length, or a portion of the protein Ubiquitin-like protein 4 A (Gene ID: UBL4A, SEQ ID NO: 2). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 4B (Gene ID: UBL4B, SEQ ID NO: 3). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin- like protein 5 (Gene ID: UBL5, SEQ ID NO: 4). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 7 (Gene ID: UBL7, SEQ ID NO: 5). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog A (Gene ID: RAD23A, SEQ ID NO: 6). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog B (Gene ID: RAD23B, SEQ ID NO: 7). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-associated domain-containing protein 1 (Gene ID: UBAC1, SEQ ID NO: 8). In some embodiments of any ofthe aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ISG15 (Gene ID: ISG15, SEQ ID NO: 9). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 1 (Gene ID: DDI1, SEQ ID NO: 10). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 2 (Gene ID: DDI2. SEQ ID NO: 11). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-related modifier 1 (Gene ID: URM1, SEQ ID NO: 12). In some embodiments of any ofthe aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ATG12 (Gene ID: ATG12, SEQ ID NO: 13). In some embodiments of any of the aspects, the degradation domain contains the full- length, or a portion of the protein Ubiquitin D (Gene ID: UBD, SEQ ID NO: 14). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Polyubiquitin-C (Gene ID: UBC, SEQ ID NO: 15). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-1 (Gene ID: UBQLN1, SEQ ID NO: 16). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-2 (Gene ID: UBQLN2, SEQ ID NO: 17). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-3 (Gene ID: UBQLN3, SEQ ID NO: 18). In some embodiments of any of the64926-6123-2472 2Attorney Docket No: 701586-000144WGPT aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-4 (Gene ID: UBQLN4, SEQ ID NO: 19). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-like protein (Gene ID: UBQLNL, SEQ ID NO: 20).

[0025] In some embodiments of any of the aspects, the targeting domain binds specifically to the substrate. In some embodiments of any of the aspects, the targeting domain comprises an antibody reagent. In some embodiments of any of the aspects, the targeting domain or target-binding moiety comprises at least one monobody, fibronectin type III domain (FN3), antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab')2, Fv, scFv, tascFvs, bis- scFvs, sdAb, VH, VL, Vnar, scFvDIO, scFvl3R4, scFvDIO, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibody, minibody, non-antibody protein scaffold, Adnectin, Affibody and their two-helix variants, Anticalin, camelid antibody, VHH, knottin, DARPin, or Sso7d. In some embodiments of any of the aspects, targeting domain comprises a single-domain antibody. In some embodiments of any of the aspects, the targeting domain comprises a fibronectin type III monobody.

[0026] In some embodiments of any of the aspects, the targeting domain or target-binding moiety is a specific binder for an antigen selected from the group consisting of: RAS family (KRAS / HRAS / NRAS); RAF family (A / B / c-RAF); Tau (MAPT); MEK family (MEK1 / 2); ERK family (ERK1 / 2); PI3K complex (PIK3C3); AKT; (AKT1 / 2 / 3); RSK (RSK1 / 2 / 3 / 4); PIM1; PKA; PKCI; PKCE; PRKD1; PKC; p38; BIM; NOXA; c-myc; BAD; BAK; BOK; CDK5; AMPK; GSK3[3; CK1; MARKs; Dyrk-IA; FYN; ABL; SYK; Insulin Receptor (IR); IRS1; mTOR; FOXOs; JNK; c-JUN; IKKP; Nf-Kp (NFKB1 / 2); S0S1; Pyruvate Kinase (PKM); a-Synuclein (SNCA); STAT3; YAP; EGFR; PDK1; GYS1 / 2; HER2; Huntingtin (HTT); VHL; ITK; FGFR1 / 2 / 3 / 4; BRD4; MDM2; TBK1; Pyruvate Kinase PKLR; PIP (PIP2 / 3); Bruton’s tyrosine kinase (BTK); SMAD (SMAD1-7); P-catenin (CTNNB1); Programmed death-ligand 1 (PD-L1); Estrogen receptor (ER); Androgen receptor (AR); B-cell lymphoma 6 (BCL6); Hematopoietic progenitor kinase 1 (HPK1); Leucine-rich repeat kinase 2 (LRRK2); IRAK-4; STAT family; STAT6; BCL-XL; IKZF1, IKZF3, and BRPD9.

[0027] In some embodiments of any of the aspects, said targeting domain binds to a non-native substrate. In some embodiments of any of the aspects, said targeting domain binds to a substrate which is exogenous to a human cell. In some embodiments of any of the aspects, the substrate is a fluorescent protein selected from the group consisting of: green fluorescent protein, emerald fluorescent protein, venus fluorescent protein, cerulean fluorescent protein, red fluorescent protein, mCherry, and enhanced cyan fluorescent protein. In some embodiments of any of the aspects, the substrate is a Cas9 / gene editor, or modified variant of a Cas9 / gene editor which has a degron targetable by the chimeric molecule.74926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0028] In some embodiments of any of the aspects, the chimeric molecule achieves targeted protein catabolism which depletes target protein(s) without requiring covalent conjugation of ubiquitin(s) to the target protein.

[0029] In one aspect of any of the embodiments, described herein is a mRNA encoding the chimeric molecule described herein.

[0030] In one aspect of any of the embodiments, described herein is a nucleic acid molecule encoding the chimeric molecule described herein. In some embodiments of any of the aspects, the nucleic acid molecule is a mRNA or saRNA.

[0031] In one aspect of any of the embodiments, described herein is an expression vector comprising one or more polynucleotide(s) encoding the chimeric molecule described herein.

[0032] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, mRNA, or expression vector described herein and at least one of:A) a lipid nanoparticle encapsulating the chimeric molecule, mRNA, or expression vector,B) an AAV encoding the chimeric molecule or mRNA,C) a polymer based delivery vehicle, and / orD) an extracellular contractile injection system.

[0033] A cell comprising the chimeric molecule or expression vector described herein.

[0034] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule or expression vector described herein; and a pharmaceutically-acceptable carrier. In some embodiments of any of the aspects, the composition further comprises a second agent selected from the group consisting of: an anti-inflammatory agent, an antidiabetic agent, a hypolipidemic agent, a chemotherapeutic agent, an antiviral agent, an antibiotic, a metabolic agent, a small molecule inhibitor, a protein kinase inhibitor, adjuvants, apoptotic agents, a proliferative agent, organotropic targeting agents, and any combination thereof.

[0035] In one aspect of any of the embodiments, described herein is a method comprising expressing and localizing the chimeric molecule described herein to the cytoplasm of a cell, wherein the substrate or the target protein is localized in the cytoplasm or is associated with the cell membrane.

[0036] In one aspect of any of the embodiments, described herein is a method comprising expressing and localizing the chimeric molecule described herein to the nucleus of a cell, wherein the substrate or the target protein is localized in the nucleus or is associated with the nuclear membrane.

[0037] In one aspect of any of the embodiments, described herein is method comprising expressing and secreting extracellularly the chimeric molecule described herein, wherein the substrate or the target protein is localized extracellularly or is associated with a cell membrane.

[0038] In one aspect of any of the embodiments, described herein is a method comprising producing one or more chimeric molecules described herein from exogenously delivered84926-6123-2472 2Attorney Docket No: 701586-000144WGPT polynucleotides, whereby a specific protein target is degraded in a ubiquitin-independent fashion with a therapeutic effect.

[0039] In one aspect of any of the embodiments, described herein is a method of treating a disease in a subject in need thereof, the method comprising administering the chimeric molecule, expression vector, or composition described herein to the subject.

[0040] In one aspect of any of the embodiments, described herein is treating a disease comprising: selecting a subject having a disease and administering a chimeric molecule, expression vector, or composition of as described herein to the subject to deplete the expression of said target relative to a subject not afflicted with said disease. In some embodiments of any of the aspects, said disease is selected from the group consisting of cancer, metastatic cancer, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson’s disease, Alzheimer’s disease, cystic fibrosis diabetes, ALS, pathogenic diseases, idiopathic diseases, viral diseases, bacterial, diseases, prionic diseases, fungal diseases, parasitic diseases, arthritis, wound healing, immunodeficiency, inflammatory disease, aplastic anemia, anemia, genetic disorders, congenital disorders, type 1 diabetes, type 2 diabetes, gestational diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, and obesity.

[0041] In some embodiments of any of the aspects, the nucleotides are delivered intracellularly by a carrier; wherein that carrier is a lipid nanoparticle; wherein the carrier is a peptide conjugated to the nucleotides; wherein the carrier is a cationic polymer; wherein the carrier is of bacterial origin; wherein the carrier is of viral origin; or wherein the carrier is of phage origin.

[0042] In some embodiments of any of the aspects, the therapeutic effect is antifibrotic; wherein the therapeutic effect is vasodilatory; wherein the therapeutic effect is hemodynamic; wherein the therapeutic effect is angiogenic; wherein the therapeutic effect is apoptotic; wherein the therapeutic effect is antiviral; wherein the therapeutic effect is antifibrogenic; wherein the therapeutic effect is cytotoxic; wherein the therapeutic effect is proliferative; wherein the therapeutic effect is regenerative; wherein the therapeutic effect regulates cell cycle; or wherein the therapeutic results in epigenetic alterations.

[0043] In one aspect of any of the embodiments, described herein is method of degrading a target protein in a cell, comprising: introducing into the cell a chimeric molecule, expression vector, or composition as described herein. In some embodiments of any of the aspects, the substrate is selectively degraded in a ubiquitin-independent manner.

[0044] In some embodiments of any of the aspects, the chimeric molecule, expression vector, or composition is delivered as mRNA, saRNA, or a DNA transgene. In some embodiments of any of the aspects, the chimeric molecule is delivered as a purified protein. In some embodiments of any of the aspects, the chimeric molecule is delivered via a lipid nanoparticle or viral vector. In some94926-6123-2472 2Attorney Docket No: 701586-000144WGPT embodiments of any of the aspects, the substrate is a mutant isoform, a post-translationally modified protein, or a protein localized to a specific subcellular compartment. In some embodiments of any of the aspects, the cell is a mammalian cell, a plant cell, or a fungal cell. In some embodiments of any of the aspects, the chimeric molecule is engineered to avoid the “hook effect” and exhibits a linear doseresponse relationship with respect to target protein degradation. In some embodiments of any of the aspects, the chimeric molecule is engineered such that modification of the unstructured C-terminal region abrogates degradation activity. In some embodiments of any of the aspects, the chimeric molecule is used to treat a disease selected from the group consisting of cancer, genetic disorders, neurodegenerative diseases, and autoimmune diseases.

[0045] In one aspect of any of the embodiments, described herein is a library of chimeric molecules as described herein, wherein each chimeric molecule comprises a different binding moiety for targeting a different protein of interest.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 is a diagram of a synthetic midnolin (Syn-MIDN) degrader for degradation of intracellular protein targets. The diagram depicts a cell which expresses the Syn-MIDN degrader and a cytosolic target protein. The targeting domain on Syn-MIDN binds specifically to the target protein. Either previously, simultaneously, or following this binding, the C-terminal a helix and ubiquitin-like (Ubl) domain on Syn-MIDN facilitate interaction with the proteasome. Formation of a ternary complex leads to degradation of the cytosolic target protein in the absence of covalent conjugation of ubiquitin onto the target protein. (1) Syn-MIDN binding cytosolic target protein. (2) Syn-MIDN C- terminal a helix and Ubl domain facilitating ternary complex formation with the proteasome. (3) Due to induced proximity and Ubl domain, the cytosolic target protein is degraded by the proteasome. Figure was created with BioRender.com.

[0047] FIG. 2 is a diagram of a synthetic midnolin (Syn-MIDN) degrader for degradation of nuclear protein targets. The diagram depicts a cell which expresses the Syn-MIDN degrader and a nuclear localized target protein. The targeting domain on Syn-MIDN binds specifically to the target protein in the nucleus. Either previously, simultaneously, or following this binding, the C-terminal a helix and ubiquitin-like (Ubl) domain on Syn-MIDN facilitate interaction with nuclear localized proteasome. Formation of a ternary complex leads to degradation of the nuclear target protein in the absence of covalent conjugation of ubiquitin onto the target protein. (1) Syn-MIDN binding nuclear localized target protein. (2) Syn-MIDN C-terminal a helix and Ubl domain facilitating ternary complex formation with the proteasome. (3) Due to induced proximity and Ubl domain, the nuclear localized target protein is degraded by the proteasome. Figure was created with BioRender.com.

[0048] FIG. 3 is a diagram of a synthetic midnolin (Syn-MIDN) degrader for degradation of membrane bound protein targets. The diagram depicts a cell which expresses the Syn-MIDN degrader104926-6123-2472 2Attorney Docket No: 701586-000144WGPT and a membrane bound or transmembrane target protein. The targeting domain on Syn-MIDN binds specifically to an intracellular domain on the membrane bound or transmembrane target protein. Either previously, simultaneously, or following this binding, the C-terminal a helix and ubiquitin-like (Ubl) domain on Syn-MIDN facilitate interaction with the proteasome. Formation of a ternary complex leads to degradation of the membrane bound or transmembrane target protein in the absence of covalent conjugation of ubiquitin onto the target protein. (1) Syn-MIDN binding membrane bound or transmembrane target protein. (2) Syn-MIDN C-terminal a helix and Ubl domain facilitating ternary complex formation with the proteasome. (3) Due to induced proximity and Ubl domain, the membrane bound or transmembrane target protein is degraded by the proteasome. Figure was created with BioRender.com.

[0049] FIG. 4 shows Syn-MIDN mediated degradation of EGFP-fusion targets with sdAb replacing catch domain or appended on N-terminus of midnolin protein. HEK293T cells stably expressing (Left panel) Histone H2B-EGFP, (middle panel) PCNA-EGFP, or (right panel) KRASG12V- EGFP on bicistronic mRNA with mCherry internal control show reduced EGFP / mCherry ratio in flow cytometry panel when transiently transfected with degrader plasmid. Cells shown are positive for mCherry expression with counts normalized by unit area. GS2-IpaH9.8 and nonfunction IpaH9.8 mutant (C337A) used as comparison.

[0050] FIG. 5 shows Syn-MIDN mediated degradation of EGFP-fusion targets in co-transfection of degrader and EGFP-fusion target. HEK293T cells co-transfected with plasmid encoding syn-MIDN degrader and plasmid encoding either (left panel) Histone H2B-EGFP, (middle panel) PCNA-EGFP, or (right panel) KRASG12V-EGFP on bicistronic mRNA with mCherry internal control show reduced EGFP / mCherry ratio in flow cytometry panel. Cells shown are positive for mCherry expression with counts normalized by unit area. GS2-IpaH9.8 and nonfunction IpaH9.8 mutant (C337A) used as comparison.

[0051] FIG. 6 shows Syn-MIDN mediated degradation of EGFP-fusion targets with removal of catch domain from midnolin protein and transfer of HA-tag from N to C-terminus. HEK293T cells stably expressing (left panel) Histone H2B-EGFP, (middle panel) PCNA-EGFP, or (right panel) KRASG12V-EGFP on bicistronic mRNA with mCherry internal control show reduced EGFP / mCherry ratio in flow cytometry panel when transiently transfected with degrader plasmid. Cells shown are positive for mCherry expression with counts normalized by unit area. GS2-IpaH9.8 and vhhGFP4- SPOP and their corresponding nonfunctional mutants used as comparison.

[0052] FIG. 7 shows MIDN mediated degradation of EGFP-fusion targets in cells positive for degrader expression. HEK293T cells stably expressing (left panel) Histone H2B-EGFP, (middle panel) PCNA-EGFP, or (right panel) KRASG12V-EGFP on bicistronic mRNA with mCherry internal control show reduced EGFP / mCherry ratio in flow cytometry panel when transiently transfected with degrader plasmid. Cells shown are positive for mTagBFP2 expressed on bicistronic mRNA encoding114926-6123-2472 2Attorney Docket No: 701586-000144WGPT degrader gene with counts normalized by unit area. GS2-IpaH9.8 and vhhGFP4-SPOP and their corresponding nonfunctional mutants used as comparison.

[0053] FIG. 8 shows example library of syn-MIDN degraders mediating degradation of EGFP- fusion targets in cells positive for degrader expression. HEK293T cells stably expressing (left panel) Histone H2B-EGFP, (middle panel) PCNA-EGFP, or (right panel) KRASG12V-EGFP on bicistronic mRNA with mCherry internal control show reduced EGFP / mCherry ratio in flow cytometry panel when transiently transfected with degrader plasmid. Cells shown are positive for mTagBFP2 expressed on bicistronic mRNA encoding degrader gene with counts normalized by unit area. GS2- IpaH9.8 and vhhGFP4-SPOP and their corresponding nonfunctional mutants used as comparison.

[0054] FIG. 9 shows comparison between syn-MIDN degraders and sdAb linked with ubiquitin- like domain (Ubl) found in RAD23A or RAD23B. HEK293T cells stably expressing (left panel) Histone H2B-EGFP, (middle panel) PCNA-EGFP, or (right panel) KRASG12V-EGFP on bicistronic mRNA with mCherry internal control were transiently transfected with plasmid encoding syn-MIDN degrader or a sdAb linked to the RAD23A or RAD23B Ubl using a short linker (SL) or long linker (LL). Cells shown are positive for mTagBFP2 expressed on bicistronic mRNA encoding degrader gene with counts normalized by unit area. GS2-IpaH9.8 and vhhGFP4-SPOP and their corresponding nonfunctional mutants used as an additional comparison.

[0055] FIG 10 shows comparison between syn-MIDN degraders and sdAb linked with ubiquitin- like domain (Ubl) found in native MIDN. HEK293T cells stably expressing (left panel) Histone H2B- EGFP, (middle panel) PCNA-EGFP, or (right panel) KRASG12V-EGFP on bicistronic mRNA with mCherry internal control were transiently transfected with plasmid encoding syn-MIDN degrader or a sdAb linked to the MIDN Ubl using a short linker (SL) or long linker (LL). Cells shown are positive for mTagBFP2 expressed on bicistronic mRNA encoding degrader gene with counts normalized by unit area. GS2-IpaH9.8 and vhhGFP4-SPOP and their corresponding nonfunctional mutants used as an additional comparison.

[0056] Figs. 11A-11C: Midnolin structural prediction and Syn-MIDN design library. (Fig. 11A) Native midnolin structural prediction by AlphaFold (AF-Q504T8-Fl-v4). (Fig. 1 IB) Schematic of design library of Syn-MIDN for the degradation of EGFP-tagged proteins. sdAbs were placed at the N-terminus of midnolin or placed in the native position of the Catch domain. The Catch domain and / or the NLS were removed in some constructs to determine their necessity for degradation. In the “sdAb-Ubl” conformation, midnolin is truncated immediately following the Ubl at residue 105. Native midnolin, IpaH9.8 ubiquibody, and SPOP bioPROTACs are shown for comparison. (Fig. 11C) AlphaFold prediction of GS2 monobody fused to the N-terminus of midnolin with Catch and NLS deletion (GS2-ACatch-ANLS).

[0057] Figs. 12A-12D: GFP-tagged protein model system and validation of midnolin retargeting. (Fig. 12A) Schematic of protein stability assay used to screen functional Syn-MIDN degraders. Doxycycline (Dox)-induction promotes expression of bicistronic EGFP-fusion protein124926-6123-2472 2Attorney Docket No: 701586-000144WGPT target and mCherry. EGFP degradation is evaluated by EGFP / mCherry ratio. (Figs. 12B-12D) Representative flow cytometry results measuring EGFP / mCherry ratio in HEK 293T cells stably expressing (Fig. 12B) H2B-EGFP, (Fig. 12C) PCNA-EGFP, or (Fig. 12D) EGFP-KRASG12V 48 h following transient transfection of degrader plasmid and induction with 100 ng / mL dox (N=3). Histogram includes cells gated as positive for mCherry and TagBFP expression. Counts are normalized by unit area. Rows represent degrader construct and columns represent sdAb used.

[0058] Fig. 13: Biodegrader expression level informs substrate degradation mechanism. Flow cytometric analysis of HEK 293T Tet-On 3G cells with stable integration of EGFP-KRASG12V 48 h following transient transfection of degrader plasmid and induction with 100 ng / mL dox. Cells shown are gated as positive for mCherry and TagBFP expression. Cells in QI represent successful EGFP- KRASG12V depletion by the respective anti-EGFP biodegraders (N=3).

[0059] Figs. 14A-14C: Syn-MIDN targeting p-catenin as a therapeutic strategy for [3-catenin- driven tumorigenesis. (Fig. 14A) Dysregulation of Wnt / p-catenin signaling in cancer, adapted from 69. In the absence of Wnt ligands (left; Wnt “OFF”), P-catenin remains primarily associated with the cytosolic side of the cell membrane as a component of cell -cell junctions. Any cytosolic P-catenin is targeted by the destruction complex (CKla, APC, Axin, GSK-3P), phosphorylated, ubiquitinated by P-TrCP, and degraded by the proteasome. In the presence of Wnt ligands (right; Wnt “ON”), signaling through Frizzled protein (FZD), lipoprotein receptor-related protein 5 (LRP5) or LRP6 on the cell surface, and Disheveled (DVL) inhibits the destruction complex, allowing p-catenin to accumulate in the cytosol. Oncogenic mutations in this pathway similarly lead to cytosolic accumulation of P- catenin. Stabilized P-catenin translocates to the nucleus, where it activates TCF / LEF -dependent transcription of genes involved in proliferation, differentiation, and migration. (Fig. 14B) CRISPRi dependency scores (Chronos) for CTNNB1 in bowel (n=63), liver (n=24), and other cancer cell models (n=1091) in DepMap. The dotted black line at x = -1 indicates a cutoff used to define strong essentiality. (Fig. 14C) TOPflash / FOPflash luciferase assay measuring P-catenin activity in HEK 293T cells 48 h following co-transfection of degrader and reporter plasmids with or without stimulation with hWnt-3a. sdAb-ANLS Syn-MIDN architecture was used for all P-catenin binders (n=3, mean ± SD).

[0060] Fig. 15: Proposed mechanism of Syn-MIDN-mediated, ubiquitin-independent proteasomal substrate degradation. (1) In the Syn-MIDN-proteasome cycle, a Syn-MIDN-POI complex can bind to the substrate-free closed-core particle (CP) state. Simultaneous binding of the C- terminal a-Helix and the UBL domain of MIDN to RPN1 and RPN11, respectively, induces a conformational change positioning the POI substrate above the opening to the AAA ATPase channel, leading to substrate translocation. (2) Midnolin is then thought to disengage from the regulatory particle (RP) and possibly from the substrate prior to complete substrate translocation, suggesting a catalytic degradation mechanism. (3) Targeted protein of interest is subsequently degraded by the proteasome.134926-6123-2472 2Attorney Docket No: 701586-000144WOPT

[0061] Figs. 16A-16C: (Connected to Fig. 12). Characterization of GFP -tagged protein model system. Median GFP and mCherry fluorescence intensity in (Fig. 16A) H2B-EGFP, (Fig. 16B) PCNA-EGFP, or (Fig. 16C) EGFP-KRASG12V 48 h following induction with dose escalation of doxycycline (n = 6, mean ± SD).

[0062] Figs. 17A-17C: (Connected to Fig. 12). Median EGFP / mCherry ratio in HEK 293T cells stably expressing (Fig. 17A) H2B-EGFP, (Fig. 17B) PCNA-EGFP, or (Fig. 17C) EGFP-KRASG12V 48 h following transient transfection of degrader plasmid and induction with 100 ng / mL dox (N = 3, mean ± SD). Cells included are positive for mCherry and TagBFP expression. Statistical significance was determined by ANOVA, control for multiple comparisons via Dunnett’s method. * p < 0.05, ** p < 0.01, *** p < 0.001, ***** p < 0.0001, n.s. not significant (p > 0.05), relative to IpaH9.8 positive control.

[0063] Fig. 18: (Connected to Fig. 13). Flow cytometric analysis of HEK 293T Tet-On 3G cells with stable integration of H2B-EGFP 48 h following transient transfection of degrader plasmid and induction with 100 ng / mL dox. Cells shown are gated as positive for mCherry and TagBFP expression. Cells in QI represent successful H2B-EGFP depletion by the respective anti-EGFP biodegraders (N=3).

[0064] Fig. 19: (Connected to Fig. 13). Flow cytometric analysis of HEK 293T Tet-On 3G cells with stable integration of PCNA-EGFP 48 h following transient transfection of degrader plasmid and induction with 100 ng / mL dox. Cells shown are gated as positive for mCherry and TagBFP expression. Cells in QI represent successful PCNA-EGFP depletion by the respective anti-EGFP biodegraders (N=3).

[0065] Figs. 20A-20D: Modification of Syn-MIDN unstructured C-terminal domain abrogates bioactivity. (Fig. 20A) Representative flow cytometry results measuring EGFP / mCherry ratio in HEK 293T cells stably expressing H2B-EGFP, PCNA-EGFP, or EGFP-KRASG12V 48 h following transient transfection of degrader plasmids incorporating HA-tag (HA) and induction with 100 ng / mL dox (n=3). (Figs. 20B-20D) Representative flow cytometry results measuring EGFP / mCherry ratio in HEK 293T cells stably expressing H2B-EGFP, PCNA-EGFP, or EGFP-KRASG12V 24 h following transient transfection of degrader mRNA and induction with 100 ng / mL dox (n=3). Constructs in (Figs. 20B, 20C) contain a C-terminal self-cleaving peptide (T2A) with gating based on (Fig. 20B) mTagBFP+ cells or (Fig. 20C) all single cells. (Fig. 20D) Equivalent mRNA transfection with Syn- MIDN constructs lacking a C-terminal self-cleaving peptide and mTagBFP demonstrate more pronounced reduction in EGFP / mCherry ratio.

[0066] Fig. 21: (Connected to Fig. 14C). TOPflash (first series) and FOPflash (second series) FLuc luminescence signal normalized to RLuc luminescence (normalizing for cell count) following transient co-transfection of TOPflash / FOPflash plasmid and biodegrader plasmid (n=3, mean ± SD). Minimal differences in FOPflash signal reflect that differences in [3-catenin activity (i.e.,144926-6123-2472 2Attorney Docket No: 701586-000144WOPTTOPflash / FOPflash ratio) is specifically a result of differences in TCF / LEF driven transcription (i.e., TOPflash FLuc / RLuc ratio).

[0067] Figs. 22A-22B Syn-MIDN degradation following expression via self-amplifying RNA (saRNA) encapsulated in lipid nanoparticles (LNPs). HEK293T cells stably expressing EGFP- KRASG12V on a bicistronic mRNA with mCherry internal control show reduced (Fig. 22A) EGFP intensity and (Fig. 22B) EGFP / mCherry ratio in flow cytometry panel 24 hrs following treatment with saRNA-LNPs. Cells shown are positive for mTagBFP2 expressed in cis on a bicistronic saRNA via IRES sequence. Histogram counts are normalized by unit area. GS2-IpaH9.8 and its corresponding nonfunctional mutants used as comparison. Vertical line indicates GFP positive gate based on non- fluore scent control.

[0068] Figs. 23A-23B Syn-MIDN degradation validation via western blotting. HEK293T cells stably expressing a doxycycline (Dox) inducible (Fig. 23A) H2B-EGFP or (Fig. 23B) EGFP- KRASG12V show markedly reduced EGFP fusion protein levels 24 hrs following treatment with saRNA-LNPs and induction with 100 ng / mL doxycycline. Mock transfected groups represent cells treated with blank LNPs with (+) or without (-) Dox induction of EGFP fusion protein expression. GS2-IpaH9.8 and its corresponding nonfunctional mutant, GS2-IpaH9.8mut, was used as comparison.

[0069] FIG. 24 Syn-MIDN degradation is proteasome dependent. HEK293T cells stably expressing EGFP -KRASG 12V on a bicistronic mRNA with mCherry internal control show reduced degradation of EGFP -KRASG 12V protein in flow cytometry panel following treatment with saRNA- LNPs and the proteasome inhibitor MG132 for 16 hrs. Cells shown are positive for mTagBFP2 expressed in cis on a bicistronic saRNA via IRES sequence. Histogram counts are normalized by unit area. GS2-IpaH9.8 and its corresponding nonfunctional mutants used as comparison. Vertical line indicates GFP positive gate based on non-fluorescent control.

[0070] Fig. 25 Syn-MIDN degradation is ubiquitination-independent. HEK293T cells stably expressing EGFP -KRASG 12V on a bicistronic mRNA with mCherry internal control show reduced degradation of EGFP protein in flow cytometry panel 24 hrs following treatment with saRNA-LNPs with and without the ubiquitin activating enzyme inhibitor TAK-243. Cells shown are positive for mTagBFP2 expressed in cis on a bicistronic saRNA via IRES sequence. Histogram counts are normalized by unit area. GS2-IpaH9.8 and its corresponding nonfunctional mutants used as comparison. Vertical line indicates GFP positive gate based on non-fluorescent control.DETAILED DESCRIPTION OF THE INVENTION

[0071] The technology described herein relates to methods and compositions for generation of synthetic protein-based targeted protein degraders which induce depletion of target proteins without the requirement for target ubiquitination. Said proteins may be expressed intracellularly from exogenous polynucleotides and act on intracellular / membrane bound target proteins or be secreted to154926-6123-2472 2Attorney Docket No: 701586-000144WGPT act upon extracellular / membrane bound target proteins. Additionally, synthetic protein-based protein degraders may be expressed from one or more exogenous polynucleotides. Said synthetic proteins may also be expressed and purified for use as recombinant protein, and act upon intracellular / membrane bound target proteins or extracellular / membrane bound target proteins in a similar manner. One or more synthetic polypeptide protein degraders may be used simultaneously to deplete one or more target proteins.

[0072] Disclosed herein are methods and compositions for the creation of synthetic polypeptide- based protein degraders derived from the native human protein midnolin. This approach may be applied to additional native human proteins (e.g., ubiquitin-like protein 4A, ubiquitin-like protein 4B, UV excision repair protein RAD23 homolog A, UV excision repair protein RAD23 homolog B, ubiquitin-associated domain-containing protein 1, protein DDI1 homolog 2) for the creation of targeted protein degraders which deplete target protein without the requirement for target ubiquitination. These proteins are selected as they all contain a ubiquitin-like domain (Ubl) in the protein structure, which may serve as a degradation signal without the requirement for direct isopeptide bond formation between this protein subunit and the target protein to achieve target degradation.

[0073] The technology disclosed herein overcomes several limitations posed by small molecule targeted protein degradation technologies (e.g., PROTAC, MGD), such as the lack of available small molecule binders for target proteins and E3 ligases, the difficulty in directing small molecules to specific tissues or cells, self-inhibitory effect (i.e., hook effect), and the requirement for target proteins to have an exposed lysine residue for ubiquitination. Additionally, prior protein-based technologies, such as uAbs, still require an exposed lysine residue which limits the total druggable proteome. Advantageously, the technology described herein provides a highly modular approach to creating targeted protein degraders through a differentiated mechanism that does not require target protein ubiquitination.

[0074] Descriptions of the present invention are not intended to detail each disclosed embodiment or every implementation of the present invention. The description and examples that follow exemplify illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0075] In one aspect of any of the embodiments, described herein is a chimeric molecule comprising: a degradation domain; and a targeting domain capable of specifically directing said degradation domain to a substrate, wherein said targeting domain is heterologous to said degradation domain.

[0076] As used herein, “chimeric” refers to the property of being the product of the fusion of portions of at least two or more different biological molecules, e.g., two different naturally occurring polypeptides or two polypeptides of different origins / sources. In some embodiments of any of the164926-6123-2472 2Attorney Docket No: 701586-000144WGPT aspects, a chimeric polypeptide comprises at least a first domain and at least a second domain, wherein the first domain and second domain are not found in the same naturally occurring polypeptide. In some embodiments of any of the aspects, a chimeric polypeptide comprises at least a first domain and at least a second domain, wherein the first domain and second domain are not found in the same naturally occurring human polypeptide.

[0077] In some embodiments of any of the aspects, a chimeric polypeptide comprises at least a degradation domain and at least a targeting domain, wherein the degradation domain and targeting domain are not found in the same naturally occurring polypeptide. In some embodiments of any of the aspects, a chimeric polypeptide comprises at least a degradation domain and at least a targeting domain, wherein the degradation domain and targeting domain are not found in the same naturally occurring human polypeptide.

[0078] In some embodiments of any of the aspects, a chimeric polypeptide comprises at least a first domain and at least a second domain, wherein the first domain and second domain are heterologous with respect to each other. In some embodiments of any of the aspects, a chimeric polypeptide comprises at least a degradation domain and at least a targeting domain, wherein the degradation domain and targeting domain are heterologous with respect to each other. As used herein, “heterologous” refers to that which is not endogenous to, or naturally occurring in, a referenced sequence, molecule (including e.g., a protein), virus, cell, tissue, or organism. For example, a heterologous sequence of the present disclosure can be derived from a different naturally occurring polypeptide than the other domain, or be an engineered sequence not found in nature in the same polypeptide as the other domain. Also for example, a nucleic acid sequence that is not normally expressed in a cell or a virus is a heterologous nucleic acid sequence with regard to that cell or virus. The term “heterologous” can refer to DNA, RNA, or protein that does not occur naturally as part of the organism in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature.

[0079] In some embodiments of any of the aspects, the molecule is a polypeptide. In some embodiments of any of the aspects, the molecule comprises a polypeptide. In some embodiments of any of the aspects, the molecule consists of a polypeptide. In some embodiments of any of the aspects, the molecule consists essentially of a polypeptide.

[0080] In some embodiments of any of the aspects, the chimeric molecule is a polypeptide. In some embodiments of any of the aspects, the chimeric molecule comprises a polypeptide. In some embodiments of any of the aspects, the chimeric molecule consists of a polypeptide. In some embodiments of any of the aspects, the chimeric molecule consists essentially of a polypeptide.

[0081] As used herein, “domain” refers to a protein structure with a tertiary structure that distinguishes it from adjoining sections of a polypeptide. In some embodiments of any of the aspects, a domain can retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases can be added,174926-6123-2472 2Attorney Docket No: 701586-000144WGPT removed or transferred to other proteins without loss of function or properties of the remainder of the protein to which it is added or transferred and / or of the domain itself.

[0082] A domain described herein, e.g., a ubiquitin-like domain, can be identified in a given protein (e.g., a protein of Table 5 or Table 6) by aligning a domain reference sequence provided herein against the given protein to identify sequence homology and / or predicted structure homology. In some embodiments of any of the aspects, a domain described herein is the sequence in a protein which corresponds to the reference sequence for that domain provided herein. As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0083] The term “effector moiety”, “degradation domain”, “effector domain”, as used herein refers to a biological substance, compound, or molecule, which facilitates degradation of a target protein after said target protein is bound to the targeting moiety (e.g., the targeting domain). The effector moiety may achieve degradation of said target by engaging native cellular machinery involved in the ubiquitin-proteasome system (UPS), autophagy, or the endosomal-lysosomal system. In some embodiments of any of the aspects, the degradation domain is a polypeptide domain.

[0084] In some embodiments of any of the aspects, the degradation domain comprises a ubiquitin domain or ubiquitin-like domain.

[0085] In some embodiments of any of the aspects, the degradation domain comprises a ubiquitin domain. In some embodiments of any of the aspects, the degradation domain consists essentially of a ubiquitin domain. In some embodiments of any of the aspects, the degradation domain consists of a ubiquitin domain.

[0086] In some embodiments of any of the aspects, the degradation domain comprises a ubiquitin-like domain. In some embodiments of any of the aspects, the degradation domain consists essentially of a ubiquitin-like domain. In some embodiments of any of the aspects, the degradation domain consists of a ubiquitin-like domain.

[0087] As used herein, “ubiquitin-like domain” refers to a polypeptide sequence comprising five P-sheets surrounding an a helix to form a -grasp fold. The structure and function of the ubiquitin-like domain, as well as proteins comprising a ubiquitin-like domain, are discussed further at, e.g., Collins et al. PNAS 117:4664-4674 (2020); Elasser et al. Nat Cell Biol 4:725-730 (2002); and Hochstrasser. Nature 458:422 (2009); the contents of each of which are incorporated by reference herein in their entireties. As used herein a “ubiquitin domain” refers to a ubiquitin-like domain found in or having the sequence of a domain found in ubiquitin.

[0088] In the Examples, midnolin and engineered versions of midnolin are used as exemplary, non-limiting degradation domain. As used herein, “midnolin” or “MIDN” refers to a polypeptide that184926-6123-2472 2Attorney Docket No: 701586-000144WOPT can interact with substrates (e.g., targets) and induce their degradation by the proteasome without ubiquitination. The structure of midnolin is described herein and further discussion of midnolin’s structure and function can be found, e.g., in Gu et al. Science 381:eadh5021 (2023); which is incorporated by reference herein in its entirety. The term “midnolin” or gene ID “MIDN” as used herein, refers to a polypeptide or gene belonging to native midnolin (SEQ ID NO: 1) or any isoforms, a fragment (e.g., a bioactive fragment) or variant of any of the foregoing. Midnolin protein (SEQ ID NO: 1) domains are defined as follows: ubiquitin-like domain (Ubl), amino acid residues 31 to 105; Catchl, amino acid residues 112 to 156; Catch2, amino acid residues 266 to 332; nuclear localization sequence (NLS), amino acid residues 402 to 413; aHelix-C, amino acid residues 377 to 413.

[0089] Midnolin comprises a ubiquitin-like domain (‘Ubl”, e.g., residues 31 to 105 of SEQ ID NO: 1), a Catchl domain (e.g., residues 112 to 156 of SEQ ID NO: 1), a Catch2 domain (e.g., residues 266 to 332 of SEQ ID NO: 1), a nuclear localization domain (“NLS” e.g., residues 402 to 413 of SEQ ID NO: 1), and a C-terminal a helix domain (“aHelix-C”, e.g., residues 112 to 156 of SEQ ID NO: 1), and an unstructured C-terminal domain (e.g., residues 414-468 of SEQ ID NO: 1). The sequences of these domains of midnolin are shown in Table 4. The predicted structure of midnolin is also depicted in Fig. 11A.

[0090] In some embodiments of any of the aspects, the degradation domain contains the full- length, or a portion of the protein Midnolin (Gene ID: MIDN, SEQ ID NO: 1) including the ubiquitin- like domain (“Ubl”, residues 31 to 105), Catchl domain (residues 112 to 156), Catch2 domain (residues 266 to 332), nuclear localization sequence domain (“NLS”, residues 402 to 413), or C-terminal a helix domain (“aHelix-C”, residues 377 to 413). In some embodiments of any of the aspects, the degradation domain comprises the full-length, or a portion of the protein Midnolin (Gene ID: MIDN, SEQ ID NO: 1) including the ubiquitin-like domain (“Ubl”, residues 31 to 105), Catchl domain (residues 112 to 156), Catch2 domain (residues 266 to 332), nuclear localization sequence domain (“NLS”, residues 402 to 413), or C-terminal a helix domain (“aHelix-C”, residues 377 to 413). In some embodiments of any of the aspects, the degradation domain comprises the full-length midnolin. In some embodiments of any of the aspects, the degradation domain comprises a portion of the protein Midnolin (Gene ID: MIDN, SEQ ID NO: 1) including the ubiquitin-like domain (“Ubl”, residues 31 to 105), Catchl domain (residues 112 to 156), Catch2 domain (residues 266 to 332), nuclear localization sequence domain (“NLS”, residues 402 to 413), or C-terminal a helix domain (“aHelix-C”, residues 377 to 413).

[0091] In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 90% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the194926-6123-2472 2Attorney Docket No: 701586-000144WOPT degradation domain comprises a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with 100% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain consists essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain consists of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 1.

[0092] In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 80% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 85% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 90% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 95% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 98% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 99% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with 100% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain consists essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain consists of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1.

[0093] Table 4: Human Midnolin (SEQ ID NO: 1) domains204926-6123-2472 2Atorney Docket No: 701586-000144WGPT214926-6123-2472 2Attorney Docket No: 701586-000144WOPT

[0094] In some embodiments of any of the aspects, a ubiquitin-like domain is a domain comprising a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin-like domain is a domain comprising a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin-like domain is a domain comprising a sequence with at least 90% sequence identity to the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin- like domain is a domain comprising a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin-like domain is a domain comprising a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin-like domain is a domain comprising a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin-like domain is a domain comprising the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin-like domain is a domain consisting essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 100. In some embodiments of any of the aspects, a ubiquitin-like domain is a domain consisting of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 100.

[0095] As used herein, “Catch 1 domain” refers to a polypeptide domain comprising two antiparallel [3 strands and two a helices. The Catch 1 domain interacts with the Catch2 domain to form a Catch superdomain that interacts with substrates and which is not required for proteasome interaction. In some embodiments of any of the aspects, a Catch 1 domain is a domain comprising a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catch 1 domain is a domain comprising a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catch 1 domain is a domain comprising a sequence with at least 90% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catchl domain is a domain comprising a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catchl domain is a domain comprising a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catchl domain is a domain comprising a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catchl domain is a domain comprising the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catchl domain is a domain consisting essentially of a sequence with at least 80%, 85%,224926-6123-2472 2Attorney Docket No: 701586-000144WOPT90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, a Catch 1 domain is a domain consisting of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 101.

[0096] As used herein, “Catch2 domain” refers to a polypeptide domain comprising two antiparallel [3 strands and three a helices. The Catch2 domain interacts with the Catch 1 domain to form a Catch superdomain that interacts with substrates and which is not required for proteasome interaction. In some embodiments of any of the aspects, a Catch2 domain is a domain comprising a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain comprising a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain comprising a sequence with at least 90% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain comprising a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain comprising a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain comprising a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain comprising the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain consisting essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, a Catch2 domain is a domain consisting of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 102.

[0097] As used herein, “C-terminal alpha helix domain” or “C-terminal a helix domain” refers to an alpha helix which binds to the proteasome and which is found C-terminal of Catch 1 and Catch2 in naturally occurring ubiquitin-like proteins (e.g., midnolin). The naturally occurring C-terminal a helix domain comprises a nuclear localization signal, but embodiments of the C-terminal a helix domain described herein can omit the nuclear localization signal.

[0098] In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 104. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 104. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 90% sequence identity to the entirety of SEQ ID NO: 104. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 104. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 104. In some234926-6123-2472 2Attorney Docket No: 701586-000144WOPT embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 104. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising the entirety of SEQ ID NO: 104. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain consisting essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 104. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain consisting of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 104.

[0099] In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 90% sequence identity to the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain comprising the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain consisting essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 103. In some embodiments of any of the aspects, a C-terminal alpha helix domain is a domain consisting of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 103.

[0100] As used herein, “nuclear localization signal” or “NLS” refers to an amino acid sequence which directs a protein disposed in the cytoplasm of a cell across the nuclear membrane and into the nucleus of the cell. In some embodiments of any of the aspects, a NLS comprises amino acids in a range from about 4 to 25. Nuclear localization signal sequences are known in the art, e.g., for more discussion see Goswami et al. Chem Soc Rev 53:204-226 (2024); which is incorporated by reference herein it its entirety.

[0101] In some embodiments of any of the aspects, a nuclear localization signal is a domain comprising a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a nuclear localization signal is a domain comprising a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a Nuclear localization signal is a domain comprising a sequence244926-6123-2472 2Attorney Docket No: 701586-000144WGPT with at least 90% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a nuclear localization signal is a domain comprising a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a nuclear localization signal is a domain comprising a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a nuclear localization signal is a domain comprising a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a nuclear localization signal is a domain comprising the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a nuclear localization signal is a domain consisting essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, a nuclear localization signal is a domain consisting of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 105.

[0102] As used herein, “unstructured C-terminal domain” refers to a domain which does not exhibit alpha helix or beta strand tertiary structure and which is found C-terminal of the C-terminal a helix domain in naturally occurring ubiquitin-like proteins (e.g., midnolin). In some embodiments of any of the aspects, an unstructured C-terminal domain is a domain comprising a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, an unstructured C-terminal domain is a domain comprising a sequence with at least 85% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, an unstructured C-terminal domain is a domain comprising a sequence with at least 90% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, an unstructured C-terminal domain is a domain comprising a sequence with at least 95% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, an unstructured C-terminal domain is a domain comprising a sequence with at least 98% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, an unstructured C-terminal domain is a domain comprising a sequence with at least 99% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, a unstructured C-terminal domain is a domain comprising the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, an unstructured C-terminal domain is a domain consisting essentially of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, an unstructured C- terminal domain is a domain consisting of a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entirety of SEQ ID NO: 106.

[0103] As demonstrated in the examples herein, a ubiquitin-like domain and a C-terminal a helix domain can be sufficient to provide degradation activity. In some embodiments of any of the aspects, a degradation domain comprises a ubiquitin-like domain and a C-terminal a helix domain. In some embodiments of any of the aspects, a degradation domain consists essentially of a ubiquitin-like254926-6123-2472 2Attorney Docket No: 701586-000144WOPT domain and a C-terminal a helix domain. In some embodiments of any of the aspects, a degradation domain consists of a ubiquitin-like domain and a C-terminal a helix domain.

[0104] In some embodiments of any of the aspects, a degradation domain comprises a midnolin ubiquitin-like domain and a midnolin C-terminal a helix domain. In some embodiments of any of the aspects, a degradation domain consists essentially of a midnolin ubiquitin-like domain and a midnolin C-terminal a helix domain. In some embodiments of any of the aspects, a degradation domain consists of a midnolin ubiquitin-like domain and a midnolin C-terminal a helix domain.

[0105] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.

[0106] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 85% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 85% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 85% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 85% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.

[0107] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 90% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 90% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 90% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 90% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.

[0108] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 95% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 95% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 95% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103. In some embodiments of any of the aspects, the degradation domain comprises one264926-6123-2472 2Attorney Docket No: 701586-000144WGPT or more sequences with at least 95% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.

[0109] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 98% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 98% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 98% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 98% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.

[0110] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences comprising a) the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences comprising the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences comprising the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.

[0111] As demonstrated in the examples herein, a degradation domain can exhibit degradation activity when one or more of the nuclear localization domain, Catch 1 domain, and Catch2 domain are absent. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal or a Catch 1 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal or a Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a Catch 1 domain or a Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal, a Catch 1 domain, or a Catch2 domain.

[0112] In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal or a midnolin Catch 1 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal or a midnolin Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin Catch 1 domain or a midnolin Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal, a midnolin Catch 1 domain, or a midnolin Catch2 domain.

[0113] As demonstrated in the examples herein, a ubiquitin-like domain, a C-terminal a helix domain, and an unstructured C-terminal domain can provide high degradation activity. In some embodiments of any of the aspects, a degradation domain comprises a ubiquitin-like domain, a C-274926-6123-2472 2Attorney Docket No: 701586-000144WOPT terminal a helix domain, and an unstructured C-terminal domain. In some embodiments of any of the aspects, a degradation domain consists essentially of a ubiquitin-like domain, a C-terminal a helix domain, and an unstructured C-terminal domain. In some embodiments of any of the aspects, a degradation domain consists of a ubiquitin-like domain, a C-terminal a helix domain, and an unstructured C-terminal domain.

[0114] In some embodiments of any of the aspects, a degradation domain comprises a midnolin ubiquitin-like domain, a midnolin C-terminal a helix domain, and a midnolin unstructured C-terminal domain. In some embodiments of any of the aspects, a degradation domain consists essentially of a midnolin ubiquitin-like domain, a midnolin C-terminal a helix domain, and a midnolin unstructured C-terminal domain. In some embodiments of any of the aspects, a degradation domain consists of a midnolin ubiquitin-like domain, a midnolin C-terminal a helix domain, and a midnolin unstructured C-terminal domain.

[0115] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 80% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106, or b) at least 80% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 80% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 80% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106.

[0116] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 85% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106, or b) at least 85% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 85% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 85% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106.

[0117] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 90% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106, or b) at least 90% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 90% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 90% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106.284926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0118] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 95% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106, or b) at least 95% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 95% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 95% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106.

[0119] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with a) at least 98% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106, or b) at least 98% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 98% sequence identity to the entirety of each of SEQ ID NOs: 100, 103, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences with at least 98% sequence identity to the entirety of each of SEQ ID NOs: 100, 104, and 106.

[0120] In some embodiments of any of the aspects, the degradation domain comprises one or more sequences comprising a) the entirety of each of SEQ ID NOs: 100, 103, and 106, or b) entirety of each of SEQ ID NOs: 100, 104, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences comprising the entirety of each of SEQ ID NOs: 100, 103, and 106. In some embodiments of any of the aspects, the degradation domain comprises one or more sequences comprising the entirety of each of SEQ ID NOs: 100, 104, and 106.

[0121] In some embodiments of any of the aspects, the unstructured C-terminal domain is the most C-terminal domain or portion of the chimeric molecule. In some embodiments of any of the aspects, no domain or signal is located C-terminal of the unstructured C-terminal domain.

[0122] In some embodiments of any of the aspects, the sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 106 is the most C-terminal domain or portion of the chimeric molecule. In some embodiments of any of the aspects, no domain or signal is located C-terminal of the sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 106. In some embodiments of any of the aspects, the sequence of SEQ ID NO: 106 is the most C-terminal domain or portion of the chimeric molecule. In some embodiments of any of the aspects, no domain or signal is located C-terminal of the sequence of SEQ ID NO: 106.

[0123] As demonstrated in the examples herein, a degradation domain can exhibit degradation activity when one or more of the nuclear localization domain, Catch 1 domain, and Catch2 domain are absent. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal or a Catch 1 domain. In some embodiments of any of the294926-6123-2472 2Attorney Docket No: 701586-000144WOPT aspects, the degradation domain does not comprise a nuclear localization signal or a Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a Catch 1 domain or a Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a nuclear localization signal, a Catch 1 domain, or a Catch2 domain.

[0124] In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal or a midnolin Catch 1 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal or a midnolin Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin Catch 1 domain or a midnolin Catch2 domain. In some embodiments of any of the aspects, the degradation domain does not comprise a midnolin nuclear localization signal, a midnolin Catch 1 domain, or a midnolin Catch2 domain.

[0125] In some embodiments of any of the aspects, the degradation domain does not comprise a sequence with at least 80% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain does not comprise a sequence with at least 85% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain does not comprise a sequence with at least 90% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain does not comprise a sequence with at least 95% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain does not comprise a sequence with at least 98% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1. In some embodiments of any of the aspects, the degradation domain does not comprise the sequences of residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1.

[0126] In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 102, or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise : a sequence304926-6123-2472 2Attorney Docket No: 701586-000144WOPT with more than 80% sequence identity to the entirety of SEQ ID NO: 102 or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105.

[0127] In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 102, or a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise : a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 102 or a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 85% sequence identity to the entirety of SEQ ID NO: 105.

[0128] In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 102, or a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the314926-6123-2472 2Attorney Docket No: 701586-000144WOPT degradation domain does not comprise: a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise : a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 102 or a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 90% sequence identity to the entirety of SEQ ID NO: 105.

[0129] In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 102, or a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise : a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 102 or a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 95% sequence identity to the entirety of SEQ ID NO: 105.

[0130] In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 102, or a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 105.324926-6123-2472 2Attorney Docket No: 701586-000144WGPTIn some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 101 or a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise : a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 102 or a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence with more than 98% sequence identity to the entirety of SEQ ID NO: 105.

[0131] In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 101, a sequence comprising the entirety of SEQ ID NO: 102, and / or a sequence comprising the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 101, a sequence comprising the entirety of SEQ ID NO: 102, or a sequence comprising the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 101 or a sequence comprising the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 101 or a sequence comprising the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 102 or a sequence comprising the entirety of SEQ ID NO: 105. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 101. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 102. In some embodiments of any of the aspects, the degradation domain does not comprise: a sequence comprising the entirety of SEQ ID NO: 105.

[0132] Ubiquitin domains and / or ubiquitin-like domains from proteins other than midnolin are contemplated for use in the degradation domains described herein. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBL4B); Ubiquitin-like protein 5 (UBL5); Ubiquitin-like protein 7 (UBL7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2334926-6123-2472 2Attorney Docket No: 701586-000144WGPT(DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D (UBD); Polyubiquitin-C (ubiquitin C or UBC); Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); and Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D (UBD); Polyubiquitin-C (ubiquitin C or UBC); and Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D (UBD); Polyubiquitin-C (ubiquitin C or UBC); Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitinspecific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBUCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin- like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D (UBD); Polyubiquitin-C (ubiquitin C or UBC); Ubiquilin- 1 (UBQLN1); Ubiquilin-2 (UBQLN2); Ubiquilin-3 (UBQLN3); Ubiquilin-4 (UBQLN4); Ubiquilin- like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBLCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1).344926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0133] The sequences of the foregoing proteins are known in the art, e.g., are the sequences available in the NCBI database under the following Gene ID numbers.

[0134] Table 6

[0135] In some embodiments of any of the aspects, the sequences of the foregoing proteins are the sequences provided in Table 5 below.

[0136] Table 5354926-6123-2472 2Atorney Docket No: 701586-000144WGPT364926-6123-2472 2Atorney Docket No: 701586-000144WGPT374926-6123-2472 2Atorney Docket No: 701586-000144WGPT384926-6123-2472 2Atorney Docket No: 701586-000144WGPT394926-6123-2472 2Attorney Docket No: 701586-000144WOPT

[0137] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 85% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); and Ubiquilin-4 (UBQUN4); Ubiquilin- like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin- like domain comprises a sequence with at least 85% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin- like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin- related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; and Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain404926-6123-2472 2Attorney Docket No: 701586-000144WGPT or ubiquitin-like domain comprises a sequence with at least 85% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin- like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBUCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 85% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin- like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBLCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1).

[0138] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 90% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBL4B); Ubiquitin-like protein 5 (UBL5); Ubiquitin-like protein 7 (UBL7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQLN1); Ubiquilin-2 (UBQLN2); Ubiquilin-3 (UBQLN3); and Ubiquilin-4 (UBQLN4); Ubiquilin- like protein (UBQLNL). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin- like domain comprises a sequence with at least 90% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBL4B); Ubiquitin-like protein 5 (UBL5); Ubiquitin-414926-6123-2472 2Attorney Docket No: 701586-000144WGPT like protein 7 (UBL7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin- related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; and Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 90% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin- like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBUCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 90% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin- like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBLCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1).

[0139] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 95% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBL4B); Ubiquitin-like protein 5 (UBL5); Ubiquitin-like protein 7 (UBL7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1424926-6123-2472 2Attorney Docket No: 701586-000144WGPT(UBQLN1); Ubiquilin-2 (UBQLN2); Ubiquilin-3 (UBQLN3); and Ubiquilin-4 (UBQLN4); Ubiquilin- like protein (UBQLNL). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin- like domain comprises a sequence with at least 95% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin- like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin- related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; and Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 95% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin- like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBUCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 95% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin- like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBLCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1).

[0140] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 98% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A434926-6123-2472 2Attorney Docket No: 701586-000144WGPT(UBL4A); Ubiquitin-like protein 4B (UBL4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); and Ubiquilin-4 (UBQUN4); Ubiquilin- like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin- like domain comprises a sequence with at least 98% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin- like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin- related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; and Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 98% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin- like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBUCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 98% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin- like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone444926-6123-2472 2Attorney Docket No: 701586-000144WGPT2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBLCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1).

[0141] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBL4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); and Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; and Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); nucleotide excision repair protein A (RAD23A); nucleotide excision repair protein B (RAD23B); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-4 (UBQUN4); Ubiquilin-like protein (UBQUNU); Ubiquitin-specific peptidase 14 (USP14); Ubiquitin-specific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBUCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1). In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of: midnolin; Ubiquitin-like protein 4A (UBU4A); Ubiquitin-like protein 4B (UBU4B); Ubiquitin-like protein 5 (UBU5); Ubiquitin-like protein 7 (UBU7); UBA domain containing 1 (UBAC1); ISG15 ubiquitin like modifier (ISG15); DDI proteasomal shuttling factor 1 (DDI1); DDI proteasomal shuttling factor 2 (DDE); Ubiquitin-related modifier 1 (URM1); autophagy related 12 (ATG12); Ubiquitin D; Polyubiquitin-C; Ubiquilin-1 (UBQUN1); Ubiquilin-2 (UBQUN2); Ubiquilin-3 (UBQUN3); Ubiquilin-454926-6123-2472 2Attorney Docket No: 701586-000144WOPT4 (UBQLN4); Ubiquilin-like protein (UBQLNL); Ubiquitin-specific peptidase 14 (USP14); Ubiquitinspecific peptidase 7 (USP17); parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)); BAG cochaperone 2 (BAG6); ubiquitin like domain containing CTD phosphatase 1 (UBUCP1); and transmembrane and ubiquitin like domain containing 1 (TMUB1).

[0142] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 85% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 90% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 95% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 98% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with 100% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20.

[0143] In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 85% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 90% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin- like domain comprises a sequence with at least 95% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 98% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain comprises a sequence with 100% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205. In some embodiments of any of the aspects, the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205.464926-6123-2472 2Attorney Docket No: 701586-000144WOPT

[0144] In some embodiments of any of the aspects, the degradation domain contains the full- length, or a portion of the protein Ubiquitin-like protein 4 A (Gene ID: UBL4A, SEQ ID NO: 2). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 4B (Gene ID: UBE4B, SEQ ID NO: 3). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin- like protein 5 (Gene ID: UBL5, SEQ ID NO: 4). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 7 (Gene ID: UBL7, SEQ ID NO: 5). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog A (Gene ID: RAD23A, SEQ ID NO: 6). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog B (Gene ID: RAD23B, SEQ ID NO: 7). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-associated domain-containing protein 1 (Gene ID: UBAC1, SEQ ID NO: 8). In some embodiments of any ofthe aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ISG15 (Gene ID: ISG15, SEQ ID NO: 9). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 1 (Gene ID: DDI1, SEQ ID NO: 10). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 2 (Gene ID: DDI2. SEQ ID NO: 11). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-related modifier 1 (Gene ID: URM1, SEQ IDNO: 12). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ATG12 (Gene ID: ATG12, SEQ ID NO: 13). In some embodiments of any of the aspects, the degradation domain contains the full- length, or a portion of the protein Ubiquitin D (Gene ID: UBD, SEQ ID NO: 14). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Polyubiquitin-C (Gene ID: UBC, SEQ ID NO: 15). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-1 (Gene ID: UBQLN1, SEQ ID NO: 16). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-2 (Gene ID: UBQLN2, SEQ ID NO: 17). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-3 (Gene ID: UBQLN3, SEQ ID NO: 18). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-4 (Gene ID: UBQLN4, SEQ ID NO: 19). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquilin-like protein (Gene ID: UBQLNL, SEQ ID NO: 20). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein Ubiquitin-specific peptidase 14 (USP14) (SEQ ID NO: 200). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the474926-6123-2472 2Attorney Docket No: 701586-000144WOPT protein Ubiquitin-specific peptidase 7 (USP17)(SEQ ID NO: 201). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2))(SEQ ID NO: 202). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein BAG cochaperone 2 (BAG6) (SEQ ID NO: 203). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein ubiquitin like domain containing CTD phosphatase 1 (UBLCP1) (SEQ ID NO: 204). In some embodiments of any of the aspects, the degradation domain contains the full-length, or a portion of the protein transmembrane and ubiquitin like domain containing 1 (TMUB1) (SEQ ID NO: 205).

[0145] In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-like protein 4A (Gene ID: UBL4A, SEQ ID NO: 2). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-like protein 4B (Gene ID: UBL4B, SEQ ID NO: 3). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-like protein 5 (Gene ID: UBL5, SEQ ID NO: 4). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin- like protein 7 (Gene ID: UBL7, SEQ ID NO: 5). In some embodiments of any of the aspects, the degradation domain contains the full-length protein UV excision repair protein RAD23 homolog A (Gene ID: RAD23A, SEQ ID NO: 6). In some embodiments of any of the aspects, the degradation domain contains the full-length protein UV excision repair protein RAD23 homolog B (Gene ID: RAD23B, SEQ ID NO: 7). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-associated domain-containing protein 1 (Gene ID: UBAC1, SEQ ID NO: 8). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-like protein ISG15 (Gene ID: ISG15, SEQ ID NO: 9). In some embodiments of any of the aspects, the degradation domain contains the full-length protein DDI1 homolog 1 (Gene ID: DDI1, SEQ ID NO: 10). In some embodiments of any of the aspects, the degradation domain contains the full-length protein DDI1 homolog 2 (Gene ID: DDI2. SEQ ID NO: 11). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-related modifier 1 (Gene ID: URM1, SEQ IDNO: 12). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-like protein ATG12 (Gene ID: ATG12, SEQ ID NO: 13). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin D (Gene ID: UBD, SEQ ID NO: 14). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Polyubiquitin-C (Gene ID: UBC, SEQ ID NO: 15). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquilin-1 (Gene ID: UBQLN1, SEQ ID NO: 16). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquilin-2 (Gene ID: UBQLN2, SEQ ID NO: 17). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquilin-3 (Gene ID: UBQLN3, SEQ ID NO: 18). In some embodiments of any of the aspects, the484926-6123-2472 2Attorney Docket No: 701586-000144WOPT degradation domain contains the full-length protein Ubiquilin-4 (Gene ID: UBQLN4, SEQ ID NO: 19). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquilin-like protein (Gene ID: UBQLNL, SEQ ID NO: 20). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-specific peptidase 14 (USP14) (SEQ ID NO: 200). In some embodiments of any of the aspects, the degradation domain contains the full-length protein Ubiquitin-specific peptidase 7 (USP17)(SEQ ID NO: 201). In some embodiments of any of the aspects, the degradation domain contains the full-length protein parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)) (SEQ ID NO: 202). In some embodiments of any of the aspects, the degradation domain contains the full-length protein BAG cochaperone 2 (BAG6) (SEQ ID NO: 203). In some embodiments of any of the aspects, the degradation domain contains the full-length protein ubiquitin like domain containing CTD phosphatase 1 (UBLCP1) (SEQ ID NO: 204). In some embodiments of any of the aspects, the degradation domain contains the full-length protein transmembrane and ubiquitin like domain containing 1 (TMUB1) (SEQ ID NO: 205).

[0146] In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-like protein 4A (Gene ID: UBL4A, SEQ ID NO: 2). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-like protein 4B (Gene ID: UBL4B, SEQ ID NO: 3). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-like protein 5 (Gene ID: UBL5, SEQ ID NO: 4). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin- like protein 7 (Gene ID: UBL7, SEQ ID NO: 5). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein UV excision repair protein RAD23 homolog A (Gene ID: RAD23A, SEQ ID NO: 6). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein UV excision repair protein RAD23 homolog B (Gene ID: RAD23B, SEQ ID NO: 7). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-associated domain-containing protein 1 (Gene ID: UBAC1, SEQ ID NO: 8). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-like protein ISG15 (Gene ID: ISG15, SEQ ID NO: 9). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein DDI1 homolog 1 (Gene ID: DDI1, SEQ ID NO: 10). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein DDI1 homolog 2 (Gene ID: DDI2. SEQ ID NO: 11). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-related modifier 1 (Gene ID: URM1, SEQ ID NO: 12). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-like protein ATG12 (Gene ID: ATG12, SEQ ID NO: 13). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin D (Gene ID: UBD, SEQ ID NO: 14). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Polyubiquitin-C (Gene ID: UBC, SEQ ID NO:494926-6123-2472 2Attorney Docket No: 701586-000144WGPT15). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquilin-1 (Gene ID: UBQLN1, SEQ ID NO: 16). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquilin-2 (Gene ID: UBQLN2, SEQ ID NO: 17). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquilin-3 (Gene ID: UBQLN3, SEQ ID NO: 18). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquilin-4 (Gene ID: UBQLN4, SEQ ID NO: 19). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquilin-like protein (Gene ID: UBQLNL, SEQ ID NO: 20). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-specific peptidase 14 (USP14) (SEQ ID NO: 200). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein Ubiquitin-specific peptidase 7 (USP17)(SEQ ID NO: 201). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein parkin RBR E3 ubiquitin protein ligase (PRKN or Parkinsonism associated deglycase ((PARK2)) (SEQ ID NO: 202). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein BAG cochaperone 2 (BAG6) (SEQ ID NO: 203). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein ubiquitin like domain containing CTD phosphatase 1 (UBLCP1) (SEQ ID NO: 204). In some embodiments of any of the aspects, the degradation domain contains a portion of the protein transmembrane and ubiquitin like domain containing 1 (TMUB1) (SEQ ID NO: 205).

[0147] The term “targeting moiety”, “target-binding moiety”, “targeting domain”, as used herein refers to a biological substance, compound, or molecule, which is capable of binding specifically to a biological target. Targeting moieties may comprise at least one monobody, fibronectin type III domain (FN3), antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab')2, Fv, scFv, tascFvs, bis-scFvs, sdAb, VH, VL, Vnar, scFvDIO, scFvl3R4, scFvDIO, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibody, minibody, non-antibody protein scaffold, Adnectin, Affibody and their two-helix variants, Anticalin, camelid antibody, VHH, knottin, DARPin, or Sso7d. In some embodiments of any of the aspects, the substrate or biological target is a polypeptide.

[0148] The term “target”, “target protein”, or “biological target” as used herein refers to a biological substance, compound, or molecule, which after activation, inhibition, or degradation has a specific and intended downstream therapeutic effect. The term “target” encompasses cytosolic proteins (e.g., Bruton’s tyrosine kinase (BTK), Kirsten rat sarcoma viral oncogene homolog (KRAS)), nuclear proteins (e.g., c-myc, [3-catenin, MDM2), membrane bound proteins (e.g., G protein-coupled receptors, tyrosine kinase transmembrane receptors, hormone receptors, chemokine receptors, ligand gated ion channels, programmed death-ligand 1) and extracellular proteins (e.g., tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-J3)). The term “target” or “biological target”504926-6123-2472 2Attorney Docket No: 701586-000144WGPT may also encompass enzymes, voltage gated ion channels, structural proteins, nucleic acids, transporters, signaling proteins, or another ligand. The term “target” may refer to a single entity or a set of entities comprising multiple components of a shared signaling pathway. The “target” is also referred to herein interchangeably as a “substrate.”

[0149] In some embodiments of any of the aspects, the substrate is an individual polypeptide. In some embodiments of any of the aspects, the targeting domain binds specifically to the substrate. In some embodiments of any of the aspects, the targeting domain binds specifically to the substrate polypeptide as compared to the most closely related human polypeptide. In some embodiments of any of the aspects, the targeting domain binds specifically to the substrate polypeptide as compared to the most closely related human paralog. In some embodiments of any of the aspects, the targeting domain binds specifically to the substrate polypeptide as compared to the most closely related human protein family member.

[0150] In some embodiments of any of the aspects, the substrate is a mutant isoform, a post- translationally modified protein, or a protein localized to a specific subcellular compartment. In some embodiments of any of the aspects, the substrate is a mutant isoform. In some embodiments of any of the aspects, the substrate is a post-translationally modified protein. In some embodiments of any of the aspects, the substrate is a protein localized to a specific subcellular compartment.

[0151] In some embodiments of any of the aspects, the targeting domain (or target-binding moiety) comprises one or more of the following: monobody, fibronectin type III domain (FN3), antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab')2, Fv, scFv, tascFvs, bis-scFvs, sdAb, VH, VL, Vnar, scFvDIO, scFvl3R4, scFvDIO, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibody, minibody, non-antibody protein scaffold, Adnectin, Affibody and their two-helix variants, Anticalin, camelid antibody, VHH, knottin, DARPin, or Sso7d. In some embodiments of any of the aspects, the targeting domain (or target-binding moiety) comprises at least one: monobody, fibronectin type III domain (FN3), antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab')2, Fv, scFv, tascFvs, bis-scFvs, sdAb, VH, VL, Vnar, scFvDIO, scFvl3R4, scFvDIO, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibody, minibody, non-antibody protein scaffold, Adnectin, Affibody and their two-helix variants, Anticalin, camelid antibody, VHH, knottin, DARPin, or Sso7d. Targeting domains, e.g., antibodies, antibody reagents, nanobodies, and polypeptides specific for a selected target or substrate are readily available in the art. For example, antibodies for a selected target or substrate can be identified by one of skill in the art using The Antibody Registry (available on the world wide web at antibodyregistry.org); Antibodypedia (available on the world wide web at antibodypedia.com); the Validated Antibody Database (available on the world wide web at labome.com / index.html); or ABCD (available on the world wide web at514926-6123-2472 2Attorney Docket No: 701586-000144WOPT web.expasy.org / abcd / ); or commercial sources such as AbCam, Sigma, ThermoFisher, and BioLegend. One of skill in the art can readily select a targeting domain depending on the target or substrate to be bound.

[0152] In some embodiments of any of the aspects, the targeting domain comprises an antibody reagent. In some embodiments of any of the aspects, the targeting domain comprises a single-domain antibody. In some embodiments of any of the aspects, the targeting domain comprises a fibronectin type III monobody.

[0153] In some embodiments of any of the aspects, the sequence of the targeting domain is not a naturally occurring sequence. In some embodiments of any of the aspects, the sequence of the targeting domain is not a naturally occurring human sequence.

[0154] Suitable targets or substrates for therapeutic purposes include but are not limited to any oncological proteins with known gain-of-function mutations where degradation could suppress growth. Alternative suitable targets or substrates include BCL-XL, IKZF1, IKZF3, BRD9.

[0155] In some embodiments of any of the aspects, the targeting domain or target-binding moiety is a specific binder for an antigen selected from the group consisting of: RAS family (KRAS / HRAS / NRAS); RAF family (A / B / c-RAF); Tau (MAPT); MEK family (MEK1 / 2); ERK family (ERK1 / 2); PI3K complex (PIK3C3); AKT; (AKT1 / 2 / 3); RSK (RSK1 / 2 / 3 / 4); PIM1; PKA; PKCI; PKCE; PRKD1; PKC; p38; BIM; NOXA; c-myc; BAD; BAK; BOK; CDK5; AMPK; GSK3[3; CK1; MARKs; Dyrk-IA; FYN; ABL; SYK; Insulin Receptor (IR); IRS1; mTOR; FOXOs; JNK; c- JUN; IKKP; Nf-K|3 (NFKB1 / 2); S0S1; Pyruvate Kinase (PKM); a-Synuclein (SNCA); STAT3; YAP; EGFR; PDK1; GYS1 / 2; HER2; Huntingtin (HTT); VHL; ITK; FGFR1 / 2 / 3 / 4; BRD4; MDM2; TBK1; Pyruvate Kinase PKLR; PIP (PIP2 / 3); Bruton’s tyrosine kinase (BTK); SMAD (SMAD1-7); [3- catenin (CTNNB1); Programmed death-ligand 1 (PD-L1); Estrogen receptor (ER); Androgen receptor (AR); B-cell lymphoma 6 (BCL6); Hematopoietic progenitor kinase 1 (HPK1); Leucine-rich repeat kinase 2 (LRRK2); and IRAK-4. In some embodiments of any of the aspects, the targeting domain or target-binding moiety is a binds specifically to a protein selected from the group consisting of: RAS family (KRAS / HRAS / NRAS); RAF family (A / B / c-RAF); Tau (MAPT); MEK family (MEK1 / 2);ERK family (ERK1 / 2); PI3K complex (PIK3C3); AKT; (AKT1 / 2 / 3); RSK (RSK1 / 2 / 3 / 4); PIM1; PKA; PKCI; PKCE; PRKD1; PKC; p38; BIM; NOXA; c-myc; BAD; BAK; BOK; CDK5; AMPK; GSK3P; CK1; MARKs; Dyrk-IA; FYN; ABL; SYK; Insulin Receptor (IR); IRS1; mTOR; FOXOs; JNK; c-JUN; IKKP; Nf-Kp (NFKB1 / 2); S0S1; Pyruvate Kinase (PKM); a-Synuclein (SNCA); STAT3; YAP; EGFR; PDK1; GYS1 / 2; HER2; Huntingtin (HTT); VHL; ITK; FGFR1 / 2 / 3 / 4; BRD4; MDM2; TBK1; Pyruvate Kinase PKLR; PIP (PIP2 / 3); Bruton’s tyrosine kinase (BTK); SMAD (SMAD1-7); P-catenin (CTNNB1); Programmed death-ligand 1 (PD-L1); Estrogen receptor (ER); Androgen receptor (AR); B-cell lymphoma 6 (BCL6); Hematopoietic progenitor kinase 1 (HPK1); Leucine-rich repeat kinase 2 (LRRK2); and IRAK-4.524926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0156] In some embodiments of any of the aspects, the targeting domain or target-binding moiety is a binds specifically to a protein selected from the group consisting of: RAS family (KRAS / HRAS / NRAS); RAF family (A / B / c-RAF); Tan (MAPT); MEK family (MEK1 / 2); ERK family (ERK1 / 2); PI3K complex (PIK3C3); AKT; (AKT1 / 2 / 3); RSK (RSK1 / 2 / 3 / 4); PIM1; PKA; PKCI; PKCE; PRKD1; PKC; p38; BIM; NOXA; c-myc; BAD; BAK; BOK; CDK5; AMPK; GSK3[3; CK1; MARKs; Dyrk-IA; FYN; ABL; SYK; Insulin Receptor (IR); IRS1; mTOR; FOXOs; JNK; c-JUN; IKKJ3; Nf-Kp (NFKB1 / 2); S0S1; Pyruvate Kinase (PKM); a-Synuclein (SNCA); STAT3; YAP; EGFR; PDK1; GYS1 / 2; HER2; Huntingtin (HTT); VHL; ITK; FGFR1 / 2 / 3 / 4; BRD4; MDM2; TBK1; Pyruvate Kinase PKLR; PIP (PIP2 / 3); Bruton’s tyrosine kinase (BTK); SMAD (SMAD1-7); P-catenin (CTNNB1); Programmed death-ligand 1 (PD-L1); Estrogen receptor (ER); Androgen receptor (AR); B-cell lymphoma 6 (BCL6); Hematopoietic progenitor kinase 1 (HPK1); Leucine-rich repeat kinase 2 (LRRK2); and IRAK-4; BCL-XL; IKZF1; IKZF3; BRPD9; STAT family; and STAT6.

[0157] In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to a protein selected from the group consisting of: RAS family (KRAS / HRAS / NRAS); RAF family (A / B / c-RAF); a-Synuclein (SNCA); Huntingtin (HTT); P-catenin (CTNNB1); STAT family; and STAT6.

[0158] In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to a protein selected from the group consisting of: RAS family (KRAS / HRAS / NRAS). In some embodiments of any of the aspects, the targeting domain or targetbinding moiety binds specifically to at least one RAS family member. In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to a protein selected from the group consisting of: KRAS; HRAS; and NRAS. In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to KRAS. In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to HRAS. In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically NRAS.

[0159] In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically P-catenin (CTNNB1). In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to a-Synuclein (SNCA). In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to Huntingtin (HTT). In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to at least one STAT family member. In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to STAT6.

[0160] In some embodiments of any of the aspects, the targeting domain or target-binding moiety binds specifically to a protein selected from the group consisting of: RAS family (KRAS / HRAS / NRAS); a-Synuclein (SNCA); Huntingtin (HTT); P-catenin (CTNNB1); STAT family; and STAT6.534926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0161] Table 1. Exemplary target proteins544926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0162] In some embodiments of any of the aspects, the target or substate is a non -native substrate. A non-native substrate is a substrate which is not naturally occurring in the cell or environment in which554926-6123-2472 2Attorney Docket No: 701586-000144WGPT the chimeric molecule is present or expressed. In some embodiments of any of the aspects, the nonnative substrate is a substrate which is not naturally occurring in the cell in which the chimeric molecule is present or expressed. In some embodiments of any of the aspects, the non-native substrate is a substrate which is not naturally occurring in a human or human cell. In some embodiments of any of the aspects, the targeting domain binds to a substrate which is exogenous to a human cell.

[0163] In some embodiments of any of the aspects, the targeting domain specifically binds to a non-native substrate. In some embodiments of any of the aspects, the targeting domain binds to a nonnative substrate.

[0164] In some embodiments of any of the aspects, the non-native substrate is a fluorescent protein. Exemplary, non-limiting non-native substrates include fluorescent protein selected from the group consisting of: green fluorescent protein, emerald fluorescent protein, venus fluorescent protein, cerulean fluorescent protein, red fluorescent protein, mCherry, and enhanced cyan fluorescent protein.

[0165] In some embodiments of any of the aspects, the substrate is a Cas9 / gene editor, or modified variant of a Cas9 / gene editor which has a degron targetable by the chimeric molecule. In some embodiments of any of the aspects, the substrate is a Cas9 / gene editor.

[0166] In some embodiments of any of the aspects, the targeting domain is N-terminal to the degradation domain. In some embodiments of any of the aspects, the chimeric polypeptide molecule comprises, from N-terminus to C-terminus, the targeting domain and the degradation domain. In some embodiments of any of the aspects, the chimeric polypeptide molecule consists of, from N-terminus to C-terminus, the targeting domain and the degradation domain. In some embodiments of any of the aspects, the chimeric polypeptide molecule consists essentially of, from N-terminus to C-terminus, the targeting domain and the degradation domain.

[0167] In some embodiments of any of the aspects, the chimeric molecule further comprises a linker between the targeting domain and the degradation domain. In some embodiments of any of the aspects, the degradation domain is coupled to the targeting domain via a linker.

[0168] In some embodiments of any of the aspects, the chimeric molecule further comprises a linker between each domain, e.g., between each of any of the targeting domain, ubiquitin-like domain, Catch 1, Catch2, alpha-helix domain, and / or unstructured c-terminal domain. In some embodiments of any of the aspects, the chimeric molecule comprises, from N-terminus to C-terminus: a targeting domain, a first linker, a ubiquitin-like domain, a second linker, a C-terminal a helix domain, a third linker, and an unstructured C-terminal domain. In some embodiments of any of the aspects, the chimeric molecule comprises, from N-terminus to C-terminus: a targeting domain, a first linker, a ubiquitin-like domain, a second linker, a Catch 1 domain, a third linker, a Catch2 domain, a fourth linker, a C-terminal a helix domain, a fifth linker, and an unstructured C-terminal domain. In some embodiments of any of the aspects, the chimeric molecule comprises, from N-terminus to C-terminus: a targeting domain, a first linker, a ubiquitin-like domain, a second linker, and a C-terminal a helix domain. In some embodiments of any of the aspects, the chimeric molecule comprises, from N-564926-6123-2472 2Attorney Docket No: 701586-000144WGPT terminus to C-terminus: a targeting domain, a first linker, a ubiquitin-like domain, a second linker, a Catch 1 domain, a third linker, a Catch2 domain, a fourth linker, and a C-terminal a helix domain. In some embodiments of any of the aspects, the chimeric molecule comprises, from N-terminus to C- terminus: a targeting domain, a first linker, a ubiquitin-like domain, a second linker, a Catch 1 domain, a third linker, a Catch2 domain, a fourth linker, a C-terminal a helix domain, and an unstructured C- terminal domain. In some embodiments of any of the aspects, the chimeric molecule comprises, from N-terminus to C-terminus: a targeting domain, a first linker, a ubiquitin-like domain, a second linker, a C-terminal a helix domain, and an unstructured C-terminal domain.

[0169] As used herein, “linker” refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the other domains or sequences of the polypeptides as described herein. In some embodiments of any of the aspects, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable.

[0170] In some embodiments of any of the aspects, the linker comprises glycine and serine residues. In some embodiments of any of the aspects, the linker consists of glycine and serine residues. In some embodiments of any of the aspects, the linker comprises (GGGGS)n, wherein n= 1-10. In some embodiments of any of the aspects, the linker consists of (GGGGS)n, wherein n= 1-10.

[0171] In some embodiments of any of the aspects, the linker is a rigid linker. In some embodiments of any of the aspects, the linker comprises (EAAAK)nwherein n = 1-10. In some embodiments of any of the aspects, the linker consists of (EAAAK)nwherein n = 1-10. In some embodiments of any of the aspects, the linker comprises A(EAAAK)nALEA(EAAAK)nA wherein n = 1-10. In some embodiments of any of the aspects, the linker consists of A(EAAAK)nALEA(EAAAK)nA wherein n = 1-10.

[0172] In some embodiments of any of the aspects, the linker is at least 2 amino acids in length. In some embodiments of any of the aspects, the linker is 2-200 amino acids in length. In some embodiments of any of the aspects, the linker is 2-100 amino acids in length. In some embodiments of any of the aspects, the linker is 2-50 amino acids in length. In some embodiments of any of the aspects, the linker is 2-20 amino acids in length. In some embodiments of any of the aspects, the linker is 2-10 amino acids in length. In some embodiments of any of the aspects, the linker is at least 50 amino acids in length. In some embodiments of any of the aspects, the linker is 50-300 amino acids in length. In some embodiments of any of the aspects, the linker is 30-300 amino acids in length. In some embodiments of any of the aspects, the linker is 20-300 amino acids in length. In some embodiments of any of the aspects, the linker is 20-290 amino acids in length. In some embodiments of any of the aspects, the linker is 30-290 amino acids in length. In some embodiments of any of the aspects, the linker is 50-290 amino acids in length.574926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0173] In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is at least 20 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is at least 30 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is at least 40 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is at least 50 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is at least 51 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is 20-300 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is 50-300 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is 20-290 amino acids in length. In some embodiments of any of the aspects, a linker between the ubiquitin-like domain and the alpha-helix domain is 50-290 amino acids in length.

[0174] In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is at least 15 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is at least 20 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is at least 25 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is at least 30 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 15-100 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 15-150 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 15-200 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 15-250 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 15-300 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 20-100 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 20-150 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 20-200 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 20-250 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 20-300 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 25-100 amino acids in length. In some embodiments of any of the aspects, a linker between584926-6123-2472 2Attorney Docket No: 701586-000144WGPT the targeting domain and the ubiquitin-like domain is 25-150 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 25-200 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 25-250 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 25-300 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 30-100 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 30-150 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 30-200 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 30-250 amino acids in length. In some embodiments of any of the aspects, a linker between the targeting domain and the ubiquitin-like domain is 30-300 amino acids in length.

[0175] In some embodiments of any of the aspects, a linker between the Catch 1 domain and the Catch2 domain is at least 10 amino acids in length. In some embodiments of any of the aspects, a linker between the Catch 1 domain and the Catch2 domain is at least 11 amino acids in length. In some embodiments of any of the aspects, a linker between the Catch 1 domain and the Catch2 domain is at least 15 amino acids in length. In some embodiments of any of the aspects, a linker between the Catch 1 domain and the Catch2 domain is at least 20 amino acids in length. In some embodiments of any of the aspects, a linker between the Catch 1 domain and the Catch2 domain is 10-100 amino acids in length. In some embodiments of any of the aspects, a linker between the Catchl domain and the Catch2 domain is 11-100 amino acids in length. In some embodiments of any of the aspects, a linker between the Catchl domain and the Catch2 domain is 15-100 amino acids in length. In some embodiments of any of the aspects, a linker between the Catchl domain and the Catch2 domain is 20- 100 amino acids in length.

[0176] The chimeric molecules described herein can achieve targeted protein catabolism which depletes target protein(s) (i.e. the substrate). The chimeric molecules described herein can achieve targeted protein catabolism which depletes target protein(s) (i.e. the substrate) without requiring covalent conjugation of ubiquitin(s) to the target protein.

[0177] In some embodiments of any of the aspects, the chimeric molecule is engineered to avoid the “hook effect” and exhibits a linear dose-response relationship with respect to substrate (e.g., target protein) degradation. In some embodiments of any of the aspects, the chimeric molecule is engineered to exhibit a linear dose-response relationship with respect to substrate (e.g., target protein) degradation. In some embodiments of any of the aspects, the chimeric molecule exhibits a linear doseresponse relationship with respect to substrate (e.g., target protein) degradation. In some embodiments of any of the aspects, the chimeric molecule does not exhibit a “hook effect.”594926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0178] In some embodiments of any of the aspects, the chimeric molecule is engineered such that modification of the unstructured C-terminal region abrogates degradation activity. In some embodiments of any of the aspects, modification of the unstructured C-terminal region abrogates degradation activity. In some embodiments of any of the aspects, the unstructured C-terminal region of the chimeric molecule is not modified. In some embodiments of any of the aspects, the unstructured C-terminal region of the chimeric molecule is not mutated or engineered or substituted as compared to a naturally occurring unstructured C-terminal region sequence. In some embodiments of any of the aspects, the unstructured C-terminal region of the chimeric molecule is not mutated or engineered or substituted as compared to a naturally occurring unstructured C-terminal region reference sequence.

[0179] In one aspect of any of the embodiments, described herein is a nucleic acid molecule encoding a chimeric molecule described herein. In some embodiments of any of the aspects, the nucleic acid molecule is a mRNA molecule. In some embodiments of any of the aspects the nucleic acid molecule is a DNA molecule. In some embodiments of any of the aspects, the nucleic acid molecule is a saRNA molecule. In some embodiments of any of the aspects, the nucleic acid molecule is a mRNA molecule or saRNA molecule.

[0180] In one aspect of any of the embodiments, described herein is a polynucleotide encoding a chimeric molecule described herein. In some embodiments of any of the aspects, the polynucleotide is a mRNA molecule. In some embodiments of any of the aspects the polynucleotide is a DNA molecule. In some embodiments of any of the aspects, the polynucleotide is a saRNA molecule. In some embodiments of any of the aspects, the polynucleotide is a mRNA molecule or saRNA molecule.

[0181] In some embodiments, a nucleic acid encoding a polypeptide as described herein is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a polypeptide is operably linked to a vector.

[0182] A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory sequences and the sequence sought to be expressed are connected in such a way as to permit gene expression as polypeptides in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the art.

[0183] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or604926-6123-2472 2Attorney Docket No: 701586-000144WGPT antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0184] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0185] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0186] In one aspect of any of the embodiments, described herein is an expression vector comprising a nucleic acid encoding a chimeric molecule described herein. In one aspect of any of the embodiments, described herein is an expression vector comprising a polynucleotide encoding a chimeric molecule described herein.

[0187] In some embodiments, a nucleic acid encoding a polypeptide as described herein is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0188] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).

[0189] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered614926-6123-2472 2Attorney Docket No: 701586-000144WGPT or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0190] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0191] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0192] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0193] Methods of making polypeptides are well known in the art, and numerous commercial sources offer pre-made polypeptides / antibody reagents or production services suitable for providing elements according to the methods and compositions described herein, e.g. the antibody sources described elsewhere herein. Methods of making oligonucleotides are well known in the art, and numerous commercial sources offer oligonucleotide synthesis services suitable for providing elements according to the methods and compositions described herein, e.g. INVITROGEN™ Custom DNA Oligos; Life Technologies; Grand Island, NY or custom DNA Oligos from IDT; Coralville, IA).624926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0194] In one aspect of any of the embodiments, described herein is a cell comprising a chimeric molecule, polypeptide as described herein, or a nucleic acid encoding such a polypeptide. In one aspect of any of the embodiments, described herein is a cell comprising a chimeric molecule as described herein. In one aspect of any of the embodiments, described herein is a cell comprising an expression vector comprising a nucleic acid encoding a polypeptide as described herein. The cell can be either a prokaryotic or eukaryotic cell. In some embodiments, the nucleotide sequence is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient cell. Any of a wide variety of vectors can be employed for this purpose and are known and available to those or ordinary skill in the art. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.

[0195] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, nucleic acid molecule, or expression vector described herein and at least one of:1. a lipid nanoparticle encapsulating the chimeric molecule, nucleic acid molecule, or expression vector,2. an AAV encoding the chimeric molecule or comprising or encoding the nucleic acid molecule,3. a polymer based delivery vehicle, and / or4. an extracellular contractile injection system.

[0196] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, nucleic acid molecule, or expression vector described herein and a lipid nanoparticle encapsulating the chimeric molecule, nucleic acid molecule, or expression vector. In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, nucleic acid molecule, or expression vector described herein and an AAV encoding the chimeric molecule or comprising or encoding the nucleic acid molecule. In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, nucleic acid molecule, or expression vector described herein and polymer based delivery vehicle. In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, nucleic acid molecule, or expression vector described herein and an extracellular contractile injection system.

[0197] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, mRNA, or expression vector as described herein and at least one of:1. a lipid nanoparticle encapsulating the chimeric molecule, mRNA, or expression vector,2. an AAV encoding the chimeric molecule or mRNA,3. a polymer based delivery vehicle, and / or634926-6123-2472 2Attorney Docket No: 701586-000144WGPT4. an extracellular contractile injection system.

[0198] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, mRNA, or expression vector as described herein and a lipid nanoparticle encapsulating the chimeric molecule, mRNA, or expression vector. In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, mRNA, or expression vector as described herein and an AAV encoding the chimeric molecule or comprising or encoding the mRNA. In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, mRNA, or expression vector as described herein and a polymer based delivery vehicle. In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule, mRNA, or expression vector as described herein and an extracellular contractile injection system.

[0199] As used herein, the term “nanoparticle” refers to particles that are on the order of about 1 to 1,000 nanometers in diameter or width. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. Exemplary nanoparticles include lipid nanoparticles or ferritin nanoparticles. Lipid nanoparticles can comprise multiple components, including, e.g., ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.

[0200] Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.

[0201] In some embodiments, a viral vector such as an adeno-associated virus (AAV) vector is used. AAVs, which normally infect mammals, including humans, but are non-pathogenic, have been developed and employed as gene therapy vectors in clinical trials in the United States and Europe (Daya and Berns, Clinical Microbiology Reviews 2008, 21, 583-593). AAV vectors may be prepared using any one of a number of methods available to those of ordinary skill in the art. Exemplary AAV vectors are disclosed in Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146 which is incorporated herein by reference; Gao et al., Gene Therapy 2005, 5, 285-297; Vandenberghe et al., Gene Therapy 2009, 16, 311-319; Gao et al., PNAS 2002, 99, 11854-11859; Gao et al., PNAS 2003, 100, 6081-6086; Gao et al., J. of Virology 2004, 78, 6381-6388.

[0202] In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.HR, AAVrh.10, AAVMYO, or AAV2.5.

[0203] Polymers can be combined with nucleic acids, e.g., in a nanocomplex or polyplex, can reduce degradation of the nucleic acid and extend the half-life of the nucleic acid, while facilitating644926-6123-2472 2Attorney Docket No: 701586-000144WGPT intracellular transport. As used herein, the term “polymer” refers to oligomers, co-oligomers, polymers and co-polymers, e.g., random block, multiblock, star, grafted, gradient copolymers and combination thereof. The average molecular weight of the polymer, as determined by gel permeation chromatography, can range from 500 to about 500,000, e.g., from 20,000 to about 500,000. A polymer formulation that can reduce degradation of the nucleic acid, extend the half-life of the nucleic acid, and / or facilitate intracellular transport of a nucleic acid is referred to herein as a “polymer based delivery vehicle.” Exemplary polymer based delivery vehicles include linear polycations (e.g., diethylaminomethyl , polylysines, polyamines), proton sponges (e.g., polyamidoamine, polyethylenimine, poly(2-dimethylaminoethyl methacrylate)), amphiphilic polymers (e.g., poly-alkylcarboxylic acids, polyvinyl ethers, pyridyl disulfide), chitosan, cyclodextrins, PLA, PGA, PLGA, and PBAEs. Polymer based delivery vehicles are known in the art and described further in e.g., Piotrowski-Daspit et la. Adv Drug Deliv Rev 156: 119-132 (2020); the contents of each of which are incorporated by reference herein in its entirety.

[0204] As used herein, “extracellular contractile injection system” refers to proteins or a group of proteins that, when present in the extracellular environment can deliver one or more effectors or nucleic acids into the cytoplasm using a contractile element to form a syringe like structure. Extracellular contractile injection systems are known in the art, see, e.g., Kreitz et al. Nature 616:357- 364 (2023); Wang et al. Frontiers in Microbiology 11:366 (2020); and Vlisidou et al. eLife 8:e46259 (2019); the contents of each of which are incorporated by reference herein in its entirety.

[0205] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule or expression vector described herein; and a carrier. The term “carrier” as used herein refers to a formulation which allows for the intracellular delivery of polypeptides or nucleotides. The term “carrier” may refer to a formulation of lipids, polymers, peptides or any other compound capable of intracellular nucleic acid delivery. The term “carrier” may also refer to an excipient, diluent, cream, lotion, gel, or solution. In some embodiments of any of the aspects, the carrier is a lipid nanoparticle. In some embodiments of any of the aspects, the carrier is a peptide conjugated to the nucleotides. In some embodiments of any of the aspects, the carrier is a cationic polymer. In some embodiments of any of the aspects, the carrier is of bacterial origin. In some embodiments of any of the aspects, the carrier is of viral origin. In some embodiments of any of the aspects, the carrier is of phage origin.

[0206] In one aspect of any of the embodiments, described herein is a composition comprising the chimeric molecule or expression vector described herein; and a pharmaceutically-acceptable carrier.

[0207] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a chimeric molecule, nucleic acid molecule, or expression vector as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise a chimeric molecule, nucleic acid654926-6123-2472 2Attorney Docket No: 701586-000144WGPT molecule, or expression vector as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of a chimeric molecule, nucleic acid molecule, or expression vector as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of a chimeric molecule, nucleic acid molecule, or expression vector as described herein.

[0208] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other nontoxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a chimeric molecule, nucleic acid molecule, or expression vector as described herein.

[0209] In some embodiments, the pharmaceutical composition comprising a chimeric molecule, nucleic acid molecule, or expression vector as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.

[0210] Suitable vehicles that can be used to provide parenteral dosage forms of a chimeric molecule, nucleic acid molecule, or expression vector as disclosed within are well known to those664926-6123-2472 2Attorney Docket No: 701586-000144WGPT skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of an agent as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0211] Pharmaceutical compositions comprising a chimeric molecule, nucleic acid molecule, or expression vector can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0212] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the chimeric molecule, nucleic acid molecule, or expression vector can be administered in a sustained release formulation.

[0213] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or674926-6123-2472 2Attorney Docket No: 701586-000144WGPT systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0214] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0215] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.

[0216] In some embodiments of any of the aspects, the chimeric molecule, nucleic acid molecule, or expression vector described herein is administered as a monotherapy, e.g., another treatment for the disease is not administered to the subject.

[0217] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy.

[0218] In some embodiments of any of the aspects, a composition described herein further comprises a second agent selected from the group consisting of: an anti-inflammatory agent, an antidiabetic agent, a hypolipidemic agent, a chemotherapeutic agent, an antiviral agent, an antibiotic, a metabolic agent, a small molecule inhibitor, a protein kinase inhibitor, adjuvants, apoptotic agents, a proliferative agent, organotropic targeting agents, and any combination thereof. One of skill in the art can readily identify the foregoing therapeutic agents, e.g., see Eippincott, Williams, and Wilkins, 2025-2026 Drug handbook published by EWW, 2024 (ISBN 19752171 IX); Skidmore -Roth, Mosby’s 2026 Nursing Drug Reference published by Mosby, 2025 (ISBN 0443282781); Collins, Elsevier’s 2026 Intravenous Medications published by Elsevier, 2025 (ISBN 0443269599); Davis’s Drug Guide; the contents of which are all incorporated by reference herein in their entireties. For example, one of684926-6123-2472 2Attorney Docket No: 701586-000144WGPT skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Edward Chu et al., Physicians' Cancer Chemotherapy Drug Manual, published by Jones & Bartlett Learning, 2024 (ISBN 1284000001, 978-1284000009); Joseph Loscalzo et al., Harrison's Principles of Internal Medicine, 21st edition, published by McGraw Hill / Medical, 2022 (ISBN 1264268505, 978-1264268504); and John E. Neiderhuber et al, AbelofE s Clinical Oncology, 6th Edition, published by Elsevier, 2019 (ISBN 0323476740, 978-0323476744), the contents of which are all incorporated by reference herein in their entireties.

[0219] Non-limiting examples of a second agent and / or treatment can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1- TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone,694926-6123-2472 2Attorney Docket No: 701586-000144WGPT dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor- free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0220] In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.

[0221] By way of non-limiting example, if a subject is to be treated for pain or inflammation according to the methods described herein, the subject can also be administered a second agent and / or treatment known to be beneficial for subjects suffering from pain or inflammation. Examples of such agents and / or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone); methotrexate; sulfasalazine; leflunomide; anti-TNF medications; cyclophosphamide; pro-resolving drugs; my cophenolate; or opiates (e.g. endorphins, enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.704926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0222] In certain embodiments, an effective dose of a composition comprising a chimeric molecule, nucleic acid molecule, expression vector, or composition as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a chimeric molecule, nucleic acid molecule, expression vector, or composition as described herein can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising a chimeric molecule, nucleic acid molecule, expression vector, or composition as described herein, such as, e.g. 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0223] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. substrate levels by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.

[0224] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active ingredient. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a chimeric molecule, nucleic acid molecule, expression vector, or composition as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0225] The dosage ranges for the administration of a chimeric molecule, nucleic acid molecule, expression vector, or composition as described herein, according to the methods described herein depend upon, for example, the form of the active ingredient, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for substrate levels are desired to be induced. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age,714926-6123-2472 2Attorney Docket No: 701586-000144WGPT condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0226] The efficacy of a chimeric molecule, nucleic acid molecule, expression vector, or composition as described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. reduction of substrate levels) can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. substrate levels. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, forthat disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. symptoms and / or substrate levels). It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of a murine model of cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. tumor size, tumor growth, or survival.

[0227] The chimeric molecules used herein, when in combination with the substrate (e.g., target), will bind specifically to and then cause the degradation of the substrate. Thus, the chimeric molecules described herein can be used to inhibit or reduce a given substrate.

[0228] In one aspect of any of the embodiments, described herein is a method of degrading a target protein in a cell, comprising: introducing into the cell a chimeric molecule, expression vector, or composition described herein. In one aspect of any of the embodiments, described herein is a method of degrading a substrate in a cell, comprising: introducing into the cell a chimeric molecule, expression vector, or composition described herein. In some embodiments of tiny of the aspects, the substrate (e.g., target) is selectively degraded in a ubiquitin-independent manner.724926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0229] The term “ubiquitin-independent”, “ubiquitin-independent process” or “ubiquitin- independent fashion” refers to a degradation mechanism that does not rely on the covalent attachment of a poly-ubiquitin chain to exposed lysine residues on a target protein to achieve degradation of said target protein. Ubiquitin-independent degradation may still rely on ubiquitin-like peptides, compounds, or molecules to achieve target protein degradation; however, it is understood that these ubiquitin-like peptides, compounds, or molecules do not covalently interact with the target protein. This process differs from the canonical ubiquitination cascade which relies on E3 ligases to tag proteins for degradation through the covalent attachment of a poly-ubiquitin chain to exposed lysine residues in an energy-dependent manner.

[0230] In some embodiments of any of the aspects, the chimeric molecule, expression vector, or composition is delivered as mRNA, saRNA, or a DNA transgene. In some embodiments of any of the aspects, the chimeric molecule is delivered as a protein. In some embodiments of any of the aspects, the chimeric molecule is delivered as a purified protein. In some embodiments of any of the aspects, the chimeric molecule, expression vector, or composition is delivered via a lipid nanoparticle or viral vector.

[0231] In some embodiments of any of the aspects, the cell is a mammalian cell, a plant cell, or a fungal cell. In some embodiments of any of the aspects, the cell is a mammalian cell. In some embodiments of any of the aspects, the cell is a human cell.

[0232] The activity of the chimeric molecule can be further directed and / or limited by providing the chimeric molecule to certain biological locations, e.g., the cytoplasm or nucleus.

[0233] In one aspect of any of the embodiments, described herein is a method comprising expressing and localizing the chimeric molecule described herein to the cytoplasm of a cell, wherein the target protein is localized in the cytoplasm or is associated with the cell membrane. In some embodiments of any of the aspects, localizing the chimeric molecule to the cytoplasm comprises providing a chimeric molecule which does not comprise a NLS.

[0234] In one aspect of any of the embodiments, described herein is a method comprising expressing and localizing the chimeric molecule described herein to the nucleus of a cell, wherein the target protein is localized in the nucleus or is associated with the nuclear membrane. In some embodiments of any of the aspects, localizing the chimeric molecule to the nucleus comprises providing a chimeric molecule which does comprise a NLS.

[0235] In one aspect of any of the embodiments, described herein is a method comprising expressing and secreting extracellularly the chimeric molecule described herein, wherein the target protein is localized extracellularly or is associated with a cell membrane. In some embodiments of any of the aspects, localizing the chimeric molecule to the extracellular space or cell membrane comprises providing a chimeric molecule with a signal sequence that traffics the chimeric molecule to these compartments. Such signal sequences are well known in the art.734926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0236] In one aspect of any of the embodiments, described herein is a method comprising producing one or more chimeric molecules described herein from exogenously delivered polynucleotides, whereby a specific protein target is degraded in a ubiquitin-independent fashion with a therapeutic effect. In one aspect of any of the embodiments, described herein is a method of treating a disease in a subject in need thereof, the method comprising administering the chimeric molecule, expression vector, or composition described herein to the subject. In one aspect of any of the embodiments, described herein is a method of treating a disease comprising: selecting a subject having a disease and administering a chimeric molecule, expression vector, or composition described herein to the subject to deplete the expression of a substrate (e.g., target) relative to a subject not afflicted with said disease.

[0237] In one aspect of any of the embodiments, described herein is a method of inhibiting or reducing a substrate (e.g. target) in a cell or organism, the method comprising administering a chimeric molecule, expression vector, or composition described herein to the cell or organism.

[0238] In one aspect of any of the embodiments, described herein is a chimeric molecule, expression vector, or composition described herein for use in a method of treating a disease.

[0239] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having, e.g., cancer with a chimeric molecule, nucleic acid molecule, expression vector, or composition described herein. Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and / or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, fevers, weight loss, bumps or tumors. Tests that may aid in a diagnosis of, e.g. cancer include, but are not limited to, biopsy and imaging exams. A family history of cancer, or exposure to risk factors for cancer can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.

[0240] The compositions and methods described herein can be administered to a subject having or diagnosed as having a disease described herein, e.g., cancer. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. to a subject in order to alleviate a symptom of a disease. As used herein, "alleviating a symptom " is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration.Administration can be local or systemic.

[0241] The term “effective amount" as used herein refers to the amount of the active ingredient needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient744926-6123-2472 2Attorney Docket No: 701586-000144WGPT amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the active ingredient that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0242] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the active ingredient which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for substrate levels among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0243] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the minimal effective dose and / or maximal tolerated dose. The dosage can vary depending upon the dosage form employed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a dosage range between the minimal effective dose and the maximal tolerated dose. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor growth and / or size among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0244] In some embodiments of any of the aspects, the disease is selected from the group consisting of cancer, metastatic cancer, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson’s disease, Alzheimer’s disease, cystic fibrosis diabetes, ALS, pathogenic diseases, idiopathic diseases, viral diseases, bacterial, diseases, prionic diseases, fungal diseases, parasitic diseases, arthritis, wound healing, immunodeficiency, inflammatory disease,754926-6123-2472 2Attorney Docket No: 701586-000144WGPT aplastic anemia, anemia, genetic disorders, congenital disorders, type 1 diabetes, type 2 diabetes, gestational diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, and obesity. Suitable substrates (e.g., targets) for these diseases are known in the art. Exemplary targets are provided elsewhere herein.

[0245] In some embodiments of any of the aspects, a chimeric molecule described herein is used to treat a disease selected from the group consisting of cancer, genetic disorders, neurodegenerative diseases, and autoimmune diseases. In some embodiments of any of the aspects, a chimeric molecule, nucleic acid molecule, or composition described herein is used to treat a disease selected from the group consisting of cancer, genetic disorders, neurodegenerative diseases, and autoimmune diseases.

[0246] In some embodiments of any of the aspects, the disease is cancer. In some embodiments of any of the aspects, the disease is a genetic disorder. In some embodiments of any of the aspects, the disease is a neurodegenerative disease. In some embodiments of any of the aspects, the disease is an autoimmune disease.

[0247] Administration of a chimeric molecule, expression vector, or composition described herein can provide a therapeutic effect, e.g., by inhibiting or reducing the substrate (e.g., target). In some embodiments of any of the aspects, the therapeutic effect is antifibrotic; wherein the therapeutic effect is vasodilatory; wherein the therapeutic effect is hemodynamic; wherein the therapeutic effect is angiogenic; wherein the therapeutic effect is apoptotic; wherein the therapeutic effect is antiviral; wherein the therapeutic effect increases cell proliferation; wherein the therapeutic effect is antifibrogenic; wherein the therapeutic effect is cytotoxic; wherein the therapeutic effect is proliferative; wherein the therapeutic effect is regenerative; wherein the therapeutic effect regulates cell cycle; or wherein the therapeutic results in epigenetic alterations. In some embodiments of any of the aspects, the therapeutic effect is antifibrotic; wherein the therapeutic effect is vasodilatory; wherein the therapeutic effect is hemodynamic; wherein the therapeutic effect is angiogenic; wherein the therapeutic effect is apoptotic; wherein the therapeutic effect is antiviral; wherein the therapeutic effect is antifibrogenic; wherein the therapeutic effect is cytotoxic; wherein the therapeutic effect is proliferative; wherein the therapeutic effect is regenerative; wherein the therapeutic effect regulates cell cycle; or wherein the therapeutic results in epigenetic alterations.

[0248] In one aspect of any of the embodiments, described herein is a library of chimeric as described herein, wherein each chimeric molecule comprises a different binding moiety for targeting a different protein of interest. In one aspect of any of the embodiments, described herein is a library of chimeric as described herein, wherein each chimeric molecule comprises a different targeting domain for targeting a different substrate (e.g., target). In one aspect of any of the embodiments, described herein is a library of chimeric as described herein, wherein each chimeric molecule comprises a different targeting domain.764926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0249] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0250] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, 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. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0251] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0252] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference774926-6123-2472 2Attorney Docket No: 701586-000144WGPT level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0253] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0254] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease. A subject can be male or female.

[0255] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0256] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0257] The inventions illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations which are not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present inventions have been specifically disclosed by preferred methods, embodiments and optional features, modification and variation of the concepts herein784926-6123-2472 2Attorney Docket No: 701586-000144WGPT disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions as defined by the embodiments and elsewhere in the invention. In the case of conflict, the specification, including definitions, will control.

[0258] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

[0259] Certain aspects and embodiments of the invention and inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic invention also form part of some aspects and embodiments of inventions contemplated herein. This includes the generic description of inventions with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0260] The term “about” as used herein means in quantitative terms plus or minus 10%. For example, “About 3%” would encompass 2.7-3.3% and “About 10%” would encompass 9-11%”. Moreover, where “about” is used herein in conjunction with a quantitative term it is understood that in addition to the value plus or minus 10%, the exact value of the quantitative term is also contemplated and described — for example, the term “about 3%” expressly contemplates, describes, and includes exactly 3%.

[0261] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA, genomic DMA), RNA molecules (e.g., mRNA, saRNA, siRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products.

[0262] These terms also encompass a gene. The term "gene" or “gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences.

[0263] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds. As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including794926-6123-2472 2Attorney Docket No: 701586-000144WGPT modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, e.g. degradation activity or binding activity, of the full length polypeptide. Conservative substitution variants that maintain the activity of wildtype will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species. Amino acids that are identical between homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of disease or substrate targeting as described herein.

[0264] In some embodiments, a polypeptide can be a variant of a sequence described herein. In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., at least 50% of the degradation or binding activity as the wildtype. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of wildtype, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.804926-6123-2472 2Attorney Docket No: 701586-000144WOPT

[0265] One method of identifying amino acid residues which can be substituted is to align, for example, the human protein to a homolog from one or more non-human species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence, or a nucleic acid encoding one of those amino acid sequences. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp or BLASTn (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set.

[0266] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0267] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired814926-6123-2472 2Attorney Docket No: 701586-000144WGPT activity, e.g. degradation or binding activity, and specificity of a native or reference polypeptide is retained.

[0268] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0269] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu. Typically conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0270] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0271] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative824926-6123-2472 2Attorney Docket No: 701586-000144WGPT substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0272] In some embodiments, a polypeptide can comprise one or more amino acid substitutions or modifications. In some embodiments, the substitutions and / or modifications can prevent or reduce proteolytic degradation and / or prolong half-life of the polypeptide in a subject. In some embodiments, a polypeptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example, transferrin (WO06096515A2), albumin (Y eh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0273] In some embodiments, a polypeptide as described herein can comprise at least one peptide bond replacement. A polypeptide as described herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g. 2 types, 3 types, 4 types, 5 types, or more types of peptide bond replacements. Non-limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0274] In some embodiments, a polypeptide as described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, e.g. Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, a polypeptide as described herein can comprise alternative amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine, parabenzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid, 7-hydroxy- tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, aminoisobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-834926-6123-2472 2Attorney Docket No: 701586-000144WGPT amino-1 -cyclopentanecarboxylic acid, 1-amino-l -cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl -alanine, t-butylglycine, trifluoro valine; hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0275] In some embodiments, a polypeptide can be modified, e.g. by addition of a moiety to one or more of the amino acids that together comprise the peptide. In some embodiments, a polypeptide as described herein can comprise one or more moiety molecules, e.g. 1 or more moiety molecules per polypeptide, 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per polypeptide. In some embodiments, a polypeptide as described herein can comprise one more types of modifications and / or moieties, e.g. 1 type of modification, 2 types of modifications, 3 types of modifications or more types of modifications. Non-limiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-capping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N- terminal acylation, and N-terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

[0276] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0277] It is contemplated that the polypeptides, proteins, targeting moieties, and effector domains described herein can contain so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, and which has little or essentially no influence on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions are substitutions in which one amino acid within the following groups (a)-(e) is substituted by another amino acid within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Other conservative substitutions include: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu844926-6123-2472 2Attorney Docket No: 701586-000144WGPT into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into He or into Leu.

[0278] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established. Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Khudyakov et al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0279] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, z.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope -binding portion thereof, and / or bifiinctional hybrid antibodies. Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain. The heavy chain constant region consists of three domains CHI, CH2 and CH3. Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said854926-6123-2472 2Attorney Docket No: 701586-000144WGPT light chain. The light chain constant region consists of a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0280] As used herein, the term “antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments as well as complete antibodies.

[0281] Antibodies and / or antibody reagents can include an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope -binding portion thereof.

[0282] As used herein, the term “nanobody” or single domain antibody (sdAb) refers to an antibody comprising the small single variable domain (VHH) of antibodies obtained from camelids and dromedaries. Antibody proteins obtained from members of the camel and dromedary (Camelus baclricinus and Calelus dromade rius) family including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT / EP93 / 02214 (WO 94 / 04678 published 3 Mar. 1994; which is incorporated by reference herein in its entirety).

[0283] A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a “camelid nanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003864926-6123-2472 2Attorney Docket No: 701586-000144WGPTBioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J. 17: 3512-3520; each of which is incorporated by reference herein in its entirety. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be “humanized”. Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

[0284] The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody. The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. See U.S. patent application 20040161738 published Aug. 19, 2004; which is incorporated by reference herein in its entirety. These features combined with the low antigenicity to humans indicate great therapeutic potential.

[0285] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0286] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized i.e., not874926-6123-2472 2Attorney Docket No: 701586-000144WGPT worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0287] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to, e.g., metastasis of a cancer. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0288] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0289] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0290] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one substrate or cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.884926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0291] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0292] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” In some embodiments of any of the aspects, the term “about” when used in connection with percentages can mean ±1%.

[0293] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0294] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0295] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0296] As used herein, the term “specific binding” refers to an interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second entity with greater specificity and affinity than it binds to a third entity. A first entity specifically bound to a second entity is not displaced by a non-similar competitor. In certain embodiments, a first entity is said to specifically bind a second entity when it preferentially recognizes the second entity in a complex mixture of proteins and / or macromolecules . In some embodiments, specific binding can refer to an affinity of the first entity for the second entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third entity. In some embodiments, specific binding refers to the ability of a first entity to bind to a second entity with a KDICT5M (10000 nM) or less, e.g., 106M. 107M. 107M, 10 ' M. 10|UM. 101 1M. 1012M. or less. The person of ordinary skill in the art can determine appropriate conditions under which a first entity (e.g., an targeting domain described herein) selectively binds a second entity (e.g., a substrate) using any suitable methods, such as titration of an entity in a suitable binding assay. Specific binding can comprise ionic bonding, hydrogen bonds, ionic bonds, van der Waals interactions, and / or London dispersion forces. In some embodiments, specific binding does not refer to covalent bonding. In some embodiments, specific binding does not refer to a peptide bond. In some embodiments, specific binding does not refer to a phosphodiester bond.

[0297] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."894926-6123-2472 2Attorney Docket No: 701586-000144WGPT

[0298] All reference to NCBI database information such as Ref Seq’s and Gene ID’s herein, refer to the sequence information present in the database as of August 20, 2025.

[0299] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0300] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in biological sciences can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978- 0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al. (eds.), The Dictionary of Cell and Molecular Biology, 5thEdition, published by Academic Press, 2013 (ISBN 0123849314, 978-0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell and Molecular Biology, 3rdEdition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898); Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8thEdition, published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C. King et al. (eds.), A Dictionary of Genetics, 8thEdition, published by Oxford University Press, 2013 (ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2ndEdition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8thEdition, published by W.H. Freeman, 2016 (ISBN 1464183392, 978-1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10thEdition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10thEdition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and904926-6123-2472 2Attorney Docket No: 701586-000144WGPTJoseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0301] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0302] In all embodiments where a sample is obtained or has been obtained or provided, the sample can be sample taken, obtained, or provided via minimally invasive methods and / or involves only a minor intervention. In some embodiments of any of the aspects, a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and / or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc), fecal sampling, semen / seminal fluid sampling, or clippings (e.g., of hair or nails). In some embodiments of any of the aspects, the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and / or hair or nail clippings.

[0303] Other terms are defined herein within the description of the various aspects of the invention.

[0304] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0305] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based914926-6123-2472 2Attorney Docket No: 701586-000144WOPT on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0306] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0307] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0308] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A chimeric molecule comprising: a degradation domain; and a targeting domain capable of specifically directing said degradation domain to a substrate, wherein said targeting domain is heterologous to said degradation domain.2. The chimeric molecule of paragraph 1, wherein the degradation domain is coupled to the targeting domain via a linker.3. The chimeric molecule of paragraph 1, wherein the chimera achieves targeted protein catabolism which depletes target protein(s) without requiring covalent conjugation of ubiquitin(s) to the target protein.4. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Midnolin (Gene ID: MIDN, SEQ ID NO: 1) including the ubiquitin-like domain (“Ubl”, residues 31 to 105), Catchl domain (residues 112 to 156), Catch2 domain (residues 266 to 332), nuclear localization sequence domain (“NLS”, residues 402 to 413), or C-terminal a helix domain (“aHelix-C”, residues 377 to 413).924926-6123-2472 2Attorney Docket No: 701586-000144WOPT5. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 4A (Gene ID: UBL4A, SEQ ID NO: 2).6. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 4B (Gene ID: UBE4B, SEQ ID NO: 3).7. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 5 (Gene ID: UBL5, SEQ ID NO: 4).8. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 7 (Gene ID: UBL7, SEQ ID NO: 5).9. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog A (Gene ID: RAD23A, SEQ ID NO: 6).10. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog B (Gene ID: RAD23B, SEQ ID NO: 7).11. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-associated domain-containing protein 1 (Gene ID: UBAC1, SEQ ID NO: 8).12. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ISG15 (Gene ID: ISG15, SEQ ID NO: 9).13. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 1 (Gene ID: DDI1, SEQ ID NO: 10).14. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 2 (Gene ID: DDI2. SEQ ID NO: 11).15. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-related modifier 1 (Gene ID: URM1, SEQ ID NO: 12).16. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ATG12 (Gene ID: ATG12, SEQ ID NO: 13).17. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin D (Gene ID: UBD, SEQ ID NO: 14).18. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Polyubiquitin-C (Gene ID: UBC, SEQ ID NO: 15).19. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-1 (Gene ID: UBQLN1, SEQ ID NO: 16).20. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-2 (Gene ID: UBQLN2, SEQ ID NO: 17).21. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-3 (Gene ID: UBQLN3, SEQ ID NO: 18).934926-6123-2472 2Attorney Docket No: 701586-000144WGPT22. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-4 (Gene ID: UBQLN4, SEQ ID NO: 19).23. The chimeric molecule of paragraph 3, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-like protein (Gene ID: UBQLNL, SEQ ID NO: 20).24. The chimeric molecule of paragraph 3, wherein the target-binding moiety comprises at least one monobody, fibronectin type III domain (FN3), antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab')2, Fv, scFv, tascFvs, bis-scFvs, sdAb, VH, VL, Vnar, scFvDIO, scFvl3R4, scFvDIO, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibody, minibody, non-antibody protein scaffold, Adnectin, Affibody and their two-helix variants, Anticalin, camelid antibody, VHH, knottin, DARPin, or Sso7d.25. The chimeric molecule of paragraph 24, wherein the target-binding moiety is a specific binder for an antigen listed in Table 1.26. A method of paragraph 3, wherein the produced protein degrader is expressed and localized to the cytoplasm and the target protein is localized in the cytoplasm or is associated with the cell membrane.27. A method of paragraph 3, wherein the produced protein degrader is expressed and localized to the nucleus and the target protein is localized in the nucleus or is associated with the nuclear membrane.28. A method of paragraph 3, wherein the produced protein degrader is expressed and secreted extracellularly and the target protein is localized extracellularly or is associated with the cell membrane.29. An expression vector comprising one or more polynucleotide(s) encoding the chimeric molecule or a portion of the chimeric molecule of any previous paragraphs.30. A cell comprising the chimeric molecule of any one of paragraphs 1 to 28 or the expression vector of paragraph 29.31. A method of paragraph 3, wherein the production of one or more synthetic chimera(s) from exogenously delivered polynucleotides results in the degradation of a specific protein target in a ubiquitin-independent fashion with a therapeutic effect.32. A composition comprising: the chimeric molecule(s) of paragraph 3; or the expression vector(s) of paragraph 29; and a pharmaceutically-acceptable carrier.33. The composition of paragraph 32 further comprising: a second agent selected from the group consisting of an anti-inflammatory agent, an antidiabetic agent, a hypolipidemic agent, a chemotherapeutic agent, an antiviral agent, an antibiotic, a metabolic agent, a small molecule inhibitor, a protein kinase inhibitor, adjuvants, apoptotic agents, a proliferative agent, and organotropic targeting agents, and any combination thereof.944926-6123-2472 2Attorney Docket No: 701586-000144WGPT34. A method of treating a disease comprising: selecting a subject having a disease and administering the composition of paragraph 32 to the subject to deplete the expression of said target relative to a subject not afflicted with said disease.35. The method of paragraph 34, wherein said disease is selected from the group consisting of cancer, metastatic cancer, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson’s disease, Alzheimer’s disease, cystic fibrosis diabetes, ALS, pathogenic diseases, idiopathic diseases, viral diseases, bacterial, diseases, prionic diseases, fungal diseases, parasitic diseases, arthritis, wound healing, immunodeficiency, inflammatory disease, aplastic anemia, anemia, genetic disorders, congenital disorders, type 1 diabetes, type 2 diabetes, gestational diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, and obesity.36. A method of any of the preceding paragraphs, wherein the nucleotides are delivered intracellularly by a carrier; wherein that carrier is a lipid nanoparticle; wherein the carrier is a peptide conjugated to the nucleotides; wherein the carrier is a cationic polymer; wherein the carrier is of bacterial origin; wherein the carrier is of viral origin; wherein the carrier is of phage origin.37. A method of any of the preceding paragraphs, wherein the therapeutic effect is antifibrotic; wherein the therapeutic effect is vasodilatory; wherein the therapeutic effect is hemodynamic; wherein the therapeutic effect is angiogenic; wherein the therapeutic effect is apoptotic; wherein the therapeutic effect is antiviral; wherein the therapeutic effect increases cell proliferation; wherein the therapeutic effect is antifibrogenic; wherein the therapeutic effect is cytotoxic; wherein the therapeutic effect is proliferative; wherein the therapeutic effect is regenerative; wherein the therapeutic effect regulates cell cycle; wherein the therapeutic results in epigenetic alterations;38. The chimeric molecule of paragraph 3, wherein said targeting domain binds to a non-native substrate.39. The chimeric molecule of paragraph 38, wherein the substrate is a fluorescent protein selected from the group consisting of green fluorescent protein, emerald fluorescent protein, venus fluorescent protein, cerulean fluorescent protein, red fluorescent protein, mCherry, and enhanced cyan fluorescent protein.40. Substrate is a Cas9 / gene editor, or modified variant which has a degron targetable by the degrader

[0309] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A chimeric molecule comprising: a degradation domain; and a targeting domain capable of specifically directing said degradation domain to a substrate, wherein said targeting domain is heterologous to said degradation domain.2. The chimeric molecule of paragraph 1, wherein the molecule is a polypeptide.954926-6123-2472 2Attorney Docket No: 701586-000144WOPT3. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain is coupled to the targeting domain via a linker.4. The chimeric molecule of any one of the preceding paragraphs, wherein the targeting domain is N- terminal to the degradation domain.5. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises a ubiquitin domain or ubiquitin-like domain.6. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain contains the full-length, or a portion of the protein Midnolin (Gene ID: MIDN, SEQ ID NO: 1) including the ubiquitin-like domain (“Ubl”, residues 31 to 105), Catchl domain (residues 112 to 156), Catch2 domain (residues 266 to 332), nuclear localization sequence domain (“NLS”, residues 402 to 413), or C-terminal a helix domain (“aHelix-C”, residues 377 to 413).7. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises a ubiquitin-like domain and a C-terminal a helix domain.8. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises a midnolin ubiquitin-like domain and a midnolin C-terminal a helix domain.9. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise a nuclear localization signal.10. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise a midnolin nuclear localization signal.11. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise a nuclear localization signal, a Catchl domain, or a Catch2 domain.12. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise a midnolin nuclear localization signal, a midnolin Catchl domain, or a midnolin Catch2 domain.13. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 1.14. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises a sequence with at least 80% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1.15. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises one or more sequences with a) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.16. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises one or more sequences with a) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100, 103, and 106 or b) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100, 104, and 106.964926-6123-2472 2Attorney Docket No: 701586-000144WGPT17. The chimeric molecule of any one of the preceding paragraphs, wherein the sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 106 is the most C-terminal portion of the chimeric molecule.18. The chimeric molecule of any one of the preceding paragraphs, wherein the sequence with the sequence of SEQ ID NO: 106 is the most C-terminal portion of the chimeric molecule.19. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise a sequence with at least 80% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1.20. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises SEQ ID NO: 1.21. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises residues 31-105 and 377-413 of SEQ ID NO: 1.22. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) each of SEQ ID NO: 100 and SEQ ID NO: 104.23. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain comprises a) each of SEQ ID NO: 100, 103, and 106 or b) each of SEQ ID NO: 100, 104, and 106.24. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1.25. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105.26. The chimeric molecule of any one of the preceding paragraphs, wherein the degradation domain does not comprise the sequences of each of SEQ ID NO: 101, 102, and 105.27. The chimeric molecule of any one of the preceding paragrap...

Claims

Attorney Docket No: 701586-000144WOPTWhat is claimed herein is:

1. A chimeric molecule comprising: a degradation domain; and a targeting domain capable of specifically directing said degradation domain to a substrate, wherein said targeting domain is heterologous to said degradation domain.

2. The chimeric molecule of claim 1, wherein the molecule is a polypeptide.

3. The chimeric molecule of any one of the preceding claims, wherein the degradation domain is coupled to the targeting domain via a linker.

4. The chimeric molecule of any one of the preceding claims, wherein the targeting domain is N- terminal to the degradation domain.

5. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises a ubiquitin domain or ubiquitin-like domain.

6. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Midnolin (Gene ID: MIDN, SEQ ID NO: 1) including the ubiquitin-like domain (“Ubl”, residues 31 to 105), Catchl domain (residues 112 to 156), Catch2 domain (residues 266 to 332), nuclear localization sequence domain (“NLS”, residues 402 to 413), or C-terminal a helix domain (“aHelix-C”, residues 377 to 413).

7. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises a ubiquitin-like domain and a C-terminal a helix domain.

8. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises a midnolin ubiquitin-like domain and a midnolin C-terminal a helix domain.

9. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise a nuclear localization signal.

10. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise a midnolin nuclear localization signal.

11. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise a nuclear localization signal, a Catchl domain, or a Catch2 domain.

12. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise a midnolin nuclear localization signal, a midnolin Catchl domain, or a midnolin Catch2 domain.

13. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises a sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 1.

14. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises a sequence with at least 80% sequence identity to residues 31-105 and 377-413 of SEQ ID NO: 1.1674926-6123-2472 2Attorney Docket No: 701586-000144WOPT15. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises one or more sequences with a) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100 and SEQ ID NO: 104.

16. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises one or more sequences with a) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100, 103, and 106 or b) at least 80% sequence identity to the entirety of each of SEQ ID NO: 100, 104, and 106.

17. The chimeric molecule of any one of the preceding claims, wherein the sequence with at least 80% sequence identity to the entirety of SEQ ID NO: 106 is the most C-terminal portion of the chimeric molecule.

18. The chimeric molecule of any one of the preceding claims, wherein the sequence with the sequence of SEQ ID NO: 106 is the most C-terminal portion of the chimeric molecule.

19. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise a sequence with at least 80% sequence identity to residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1.

20. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises SEQ ID NO: 1.

21. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises residues 31-105 and 377-413 of SEQ ID NO: 1.

22. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises each of SEQ ID NO: 100 and SEQ ID NO: 103, or b) each of SEQ ID NO: 100 and SEQ ID NO: 104.

23. The chimeric molecule of any one of the preceding claims, wherein the degradation domain comprises a) each of SEQ ID NO: 100, 103, and 106 or b) each of SEQ ID NO: 100, 104, and 106.

24. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise residues 112-156, 266-332, and 402-413 of SEQ ID NO: 1.

25. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise: a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 101, a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 102, and / or a sequence with more than 80% sequence identity to the entirety of SEQ ID NO: 105.

26. The chimeric molecule of any one of the preceding claims, wherein the degradation domain does not comprise the sequences of each of SEQ ID NO: 101, 102, and 105.

27. The chimeric molecule of any one of the preceding claims, wherein the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of:Midnolin; Ubiquitin-like protein 4A; Ubiquitin-like protein 4B; Ubiquitin-like protein 5;1684926-6123-2472 2Attorney Docket No: 701586-000144WOPTUbiquitin-like protein 7; RAD23A; RAD23B; UBAC1; ISG15; DDI1; DDE; Ubiquitin-related modifier 1; ATG12; Ubiquitin D; Polyubiquitin-C; Ubiquilin-1; Ubiquilin-2; Ubiquilin-3; Ubiquilin-4; Ubiquilin-like protein; USP14, USP7; PARK2; BAG6; UBUCP1; and TMUB1.

28. The chimeric molecule of any one of the preceding claims, wherein the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin-like domain of a protein selected from the group consisting of:Midnolin; Ubiquitin-like protein 4A; Ubiquitin-like protein 4B; Ubiquitin-like protein 5;Ubiquitin-like protein 7; RAD23A; RAD23B; UBAC1; ISG15; DDI1; DDE; Ubiquitin-related modifier 1; ATG12; Ubiquitin D; Polyubiquitin-C; Ubiquilin-1; Ubiquilin-2; Ubiquilin-3; Ubiquilin-4; Ubiquilin-like protein; USP14, USP7; PARK2; BAG6; UBUCP1; and TMUB1.

29. The chimeric molecule of any one of the preceding claims, wherein the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20 and 200-205.

30. The chimeric molecule of any one of the preceding claims, wherein the ubiquitin domain or ubiquitin-like domain comprises a sequence with at least 80% sequence identity to the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1-20.

31. The chimeric molecule of any one of the preceding claims, wherein the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1- 20 and 200-205.

32. The chimeric molecule of any one of the preceding claims, wherein the ubiquitin domain or ubiquitin-like domain is the ubiquitin domain or ubiquitin-like domain of one of SEQ ID NOs: 1- 20.

33. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 4A (Gene ID: UBL4A, SEQ ID NOE).

34. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 4B (Gene ID: UBL4B, SEQ ID NO: 3).

35. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 5 (Gene ID: UBL5, SEQ ID NO: 4).

36. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein 7 (Gene ID: UBL7, SEQ ID NO: 5).

37. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog A (Gene ID: RAD23A, SEQ ID NO: 6).1694926-6123-2472 2Attorney Docket No: 701586-000144WOPT38. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein UV excision repair protein RAD23 homolog B (Gene ID: RAD23B, SEQ ID NO: 7).

39. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-associated domain-containing protein 1 (Gene ID: UBAC1, SEQ ID NO: 8).

40. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ISG15 (Gene ID: ISG15, SEQ ID NO: 9).

41. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 1 (Gene ID: DDI1, SEQ ID NO:10).

42. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein DDI1 homolog 2 (Gene ID: DDI2. SEQ ID NO:11).

43. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-related modifier 1 (Gene ID: URM1, SEQ ID NO: 12).

44. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin-like protein ATG12 (Gene ID: ATG12, SEQ ID NO: 13).

45. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquitin D (Gene ID: UBD, SEQ ID NO: 14).

46. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Polyubiquitin-C (Gene ID: UBC, SEQ ID NO:15).

47. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-1 (Gene ID: UBQLN1, SEQ ID NO:16).

48. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-2 (Gene ID: UBQLN2, SEQ ID NO:17).

49. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-3 (Gene ID: UBQLN3, SEQ ID NO:18).1704926-6123-2472 2Attorney Docket No: 701586-000144WGPT50. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-4 (Gene ID: UBQLN4, SEQ ID NO: 19).

51. The chimeric molecule of any one of the preceding claims, wherein the degradation domain contains the full-length, or a portion of the protein Ubiquilin-like protein (Gene ID: UBQLNL, SEQ ID NO: 20).

52. The chimeric molecule of any one of the preceding claims, wherein the targeting domain binds specifically to the substrate.

53. The chimeric molecule of any one of the preceding claims, wherein the targeting domain comprises an antibody reagent.

54. The chimeric molecule of any one of the preceding claims, wherein the targeting domain or targetbinding moiety comprises at least one monobody, fibronectin type III domain (FN3), antibody, polyclonal antibody, monoclonal antibody, recombinant antibody, antibody fragment, Fab', F(ab')2, Fv, scFv, tascFvs, bis-scFvs, sdAb, VH, VL, Vnar, scFvDIO, scFvl3R4, scFvDIO, humanized antibody, chimeric antibody, complementary determining region (CDR), IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgM antibody, nanobody, intrabody, unibody, minibody, non-antibody protein scaffold, Adnectin, Affibody and their two-helix variants, Anticalin, camelid antibody, VHH, knottin, DARPin, or Sso7d.

55. The chimeric molecule of any one of the preceding claims, wherein the targeting domain comprises a single -domain antibody.

56. The chimeric molecule of any one of the preceding claims, wherein the targeting domain comprises a fibronectin type III monobody.

57. The chimeric molecule of any one of the preceding claims, wherein the targeting domain or targetbinding moiety is a specific binder for an antigen selected from the group consisting of:RAS family (KRAS / HRAS / NRAS); RAF family (A / B / c-RAF); Tau (MAPT); MEK family (MEK1 / 2); ERK family (ERK1 / 2); PI3K complex (PIK3C3); AKT; (AKT1 / 2 / 3); RSK (RSK1 / 2 / 3 / 4); PIM1; PKA; PKCI; PKCE; PRKD1; PKC; p38; BIM; NOXA; c-myc; BAD; BAK; BOK; CDK5; AMPK; GSK3P; CK1; MARKs; Dyrk-IA; FYN; ABL; SYK; Insulin Receptor (IR); IRS1; mTOR; FOXOs; JNK; c-JUN; IKKP; Nf-Kp (NFKB1 / 2); S0S1; Pyruvate Kinase (PKM); a-Synuclein (SNCA); STAT3; YAP; EGFR; PDK1; GYS1 / 2; HER2; Huntingtin (HTT); VHL; ITK; FGFR1 / 2 / 3 / 4; BRD4; MDM2; TBK1; Pyruvate Kinase PKLR; PIP (PIP2 / 3); Bruton’s tyrosine kinase (BTK); SMAD (SMAD1-7); p-catenin (CTNNB1); Programmed death-ligand 1 (PD-L1); Estrogen receptor (ER); Androgen receptor (AR); B-cell lymphoma 6 (BCL6); Hematopoietic progenitor kinase 1 (HPK1); Leucine-rich repeat kinase 2 (LRRK2); IRAK-4; STAT family; STAT6; BCL-XL; IKZF1, IKZF3, and BRPD9.

58. The chimeric molecule of any one of the preceding claims, wherein said targeting domain binds to a non-native substrate.1714926-6123-2472 2Attorney Docket No: 701586-000144WGPT59. The chimeric molecule of any one of the preceding claims, wherein said targeting domain binds to a substrate which is exogenous to a human cell.

60. The chimeric molecule of any one of the preceding claims, wherein the substrate is a fluorescent protein selected from the group consisting of: green fluorescent protein, emerald fluorescent protein, venus fluorescent protein, cerulean fluorescent protein, red fluorescent protein, mCherry, and enhanced cyan fluorescent protein.

61. The chimeric molecule of any one of the preceding claims, wherein the substrate is a Cas9 / gene editor, or modified variant of a Cas9 / gene editor which has a degron targetable by the chimeric molecule.

62. The chimeric molecule of any one of the preceding claims, wherein the chimeric molecule achieves targeted protein catabolism which depletes target protein(s) without requiring covalent conjugation of ubiquitin(s) to the target protein.

63. A mRNA encoding the chimeric molecule of any one of the preceding claims.

64. A nucleic acid molecule encoding the chimeric molecule of any one of the preceding claims.

65. The nucleic acid molecule of claim 64, wherein the nucleic acid molecule is a mRNA or saRNA.

66. An expression vector comprising one or more polynucleotide(s) encoding the chimeric molecule of any one of the preceding claims.

67. A composition comprising the chimeric molecule, mRNA, or expression vector of any one of the preceding claims and at least one of:A) a lipid nanoparticle encapsulating the chimeric molecule, mRNA, or expression vector,B) an AAV encoding the chimeric molecule or mRNA,C) a polymer based delivery vehicle, and / orD) an extracellular contractile injection system.

68. A cell comprising the chimeric molecule or expression vector of any one of the preceding claims.

69. A composition comprising the chimeric molecule or expression vector of any one of the preceding claims; and a pharmaceutically-acceptable carrier.

70. The composition of claim 69, further comprising a second agent selected from the group consisting of: an anti-inflammatory agent, an antidiabetic agent, a hypolipidemic agent, a chemotherapeutic agent, an antiviral agent, an antibiotic, a metabolic agent, a small molecule inhibitor, a protein kinase inhibitor, adjuvants, apoptotic agents, a proliferative agent, organotropic targeting agents, and any combination thereof.

71. A method comprising expressing and localizing the chimeric molecule of any one of the preceding claims to the cytoplasm of a cell, wherein the substrate or the target protein is localized in the cytoplasm or is associated with the cell membrane.1724926-6123-2472 2Attorney Docket No: 701586-000144WGPT72. A method comprising expressing and localizing the chimeric molecule of any one of the preceding claims to the nucleus of a cell, wherein the substrate or the target protein is localized in the nucleus or is associated with the nuclear membrane.

73. A method comprising expressing and secreting extrace llularly the chimeric molecule of any one of the preceding claims, wherein the substrate or the target protein is localized extracellularly or is associated with a cell membrane.

74. A method comprising producing one or more chimeric molecules of any one of the preceding claims from exogenously delivered polynucleotides, whereby a specific protein target is degraded in a ubiquitin-independent fashion with a therapeutic effect.

75. A method of treating a disease in a subject in need thereof, the method comprising administering the chimeric molecule, expression vector, or composition of any one of the preceding claims to the subject.

76. A method of treating a disease comprising: selecting a subject having a disease and administering a chimeric molecule, expression vector, or composition of any one of the preceding claims to the subject to deplete the expression of said target relative to a subject not afflicted with said disease.

77. The method of claim 71, wherein said disease is selected from the group consisting of cancer, metastatic cancer, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, hepatitis, cirrhosis, Parkinson’s disease, Alzheimer’s disease, cystic fibrosis diabetes, ALS, pathogenic diseases, idiopathic diseases, viral diseases, bacterial, diseases, prionic diseases, fungal diseases, parasitic diseases, arthritis, wound healing, immunodeficiency, inflammatory disease, aplastic anemia, anemia, genetic disorders, congenital disorders, type 1 diabetes, type 2 diabetes, gestational diabetes, high blood glucose, metabolic syndrome, lipodystrophy syndrome, dyslipidemia, insulin resistance, leptin resistance, atherosclerosis, vascular disease, hypercholesterolemia, hypertriglyceridemia, non-alcoholic fatty liver disease, overweight, and obesity.

78. A method of any one of the preceding claims, wherein the nucleotides are delivered intracellularly by a carrier; wherein that carrier is a lipid nanoparticle; wherein the carrier is a peptide conjugated to the nucleotides; wherein the carrier is a cationic polymer; wherein the carrier is of bacterial origin; wherein the carrier is of viral origin; or wherein the carrier is of phage origin.

79. A method of any one of the preceding claims, wherein the therapeutic effect is antifibrotic; wherein the therapeutic effect is vasodilatory; wherein the therapeutic effect is hemodynamic; wherein the therapeutic effect is angiogenic; wherein the therapeutic effect is apoptotic; wherein the therapeutic effect is antiviral; wherein the therapeutic effect is antifibrogenic; wherein the therapeutic effect is cytotoxic; wherein the therapeutic effect is proliferative; wherein the therapeutic effect is regenerative; wherein the therapeutic effect regulates cell cycle; or wherein the therapeutic results in epigenetic alterations.

80. A method of degrading a target protein in a cell, comprising: introducing into the cell a chimeric molecule, expression vector, or composition of any one of the preceding claims.1734926-6123-2472 2Attorney Docket No: 701586-000144WGPT81. The method of claim 80, whereby the substrate is selectively degraded in a ubiquitin-independent manner.

82. The method of any one of the preceding claims wherein the chimeric molecule, expression vector, or composition is delivered as mRNA, saRNA, or a DNA transgene.

83. The method of any one of the preceding claims, wherein the chimeric molecule is delivered as a purified protein.

84. The method of any one of the preceding claims, wherein the chimeric molecule is delivered via a lipid nanoparticle or viral vector.

85. The method of any one of the preceding claims, wherein the substrate is a mutant isoform, a post- translationally modified protein, or a protein localized to a specific subcellular compartment.

86. The method of any one of the preceding claims, wherein the cell is a mammalian cell, a plant cell, or a fungal cell.

87. The method of any one of the preceding claims, wherein the chimeric molecule is engineered to avoid the “hook effect” and exhibits a linear dose-response relationship with respect to target protein degradation.

88. The method of any one of the preceding claims, wherein the chimeric molecule is engineered such that modification of the unstructured C-terminal region abrogates degradation activity.

89. The method of any one of the preceding claims, wherein the chimeric molecule is used to treat a disease selected from the group consisting of cancer, genetic disorders, neurodegenerative diseases, and autoimmune diseases.

90. A library of chimeric molecules according to any one of the preceding claims, wherein each chimeric molecule comprises a different binding moiety for targeting a different protein of interest.1744926-6123-2472 2

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