Targeted protein degradation using bacterial e3 ligase

Abstract

The present disclosure relates to a novel platform for targeted degradation of target protein. The disclosure relates to degradation of target proteins comprising peptide expression tags of small size, such as 25 amino acids or less, by fusion polypeptides comprising an E3 ubiquitin ligase and a binding polypeptide that recognizes the peptide expression tag of the target protein. In some embodiments, the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase, and in some cases does not comprise the native substrate binding domain of the E3 ubiquitin ligase.

Description

[0001] TARGETED PROTEIN DEGRADATION USING BACTERIAL E3 LIGASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 620,732, filed January 12, 2024, and U.S. Provisional Application No. 63 / 620,729, filed January 12, 2024, the disclosures of which are hereby incorporated by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing in computer readable form entitled “01164- 0031-00PCT”, created January 10, 2025, having a size of 22, 109 bytes, which is incorporated by reference herein.

[0006] FIELD

[0007] The present disclosure relates to a novel platform for targeted degradation of target proteins in cells. The disclosure relates to degradation of target proteins comprising peptide expression tags of small size, such as 25 amino acids or less, by fusion polypeptides comprising an E3 ubiquitin ligase and a binding polypeptide that recognizes the peptide expression tag of the target protein. In some embodiments, the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase, and in some cases does not comprise the native substrate binding domain of the E3 ubiquitin ligase.

[0008] BACKGROUND

[0009] Various platforms have been constructed for the purpose of targeting the degradation of a protein of interest. For example, PROTACs (proteolysis targeting chimeras) are small-molecule constructs intended to target cytosolic proteins to the 26S proteosome for degradation. PROTAC systems may involve a binding domain that binds to a protein intended for degradation (protein of interest, POI, or target protein), fused to a “molecular glue” or recognition domain that binds to an E3 ubiquitin ligase, thus bringing the E3 ubiquitin ligase into a complex with the target protein for degradation. PROTACs, however, can be difficult to develop and use effectively. Another option is to express the target protein with a heterologous domain as a fusion protein and to contact the target protein in a cell with E3 ubiquitin ligase conjugated or bound to a domain that binds to the heterologous domain on the target protein. Such approaches, however, typically involve large binding domains and therefore large protein complexes, making targeting of the protein for degradation inefficient.

[0010] A few different tag-based methods have been developed as a more generalized approach to degrade any POI. For example, the mapping of the IMiD-interacting motif to a 25-amino-acid long degron in IKZF3 makes it possible to direct fusion proteins with this degron to be degraded by the E3 ligase Cereblon (CRBN)21. Alternatively, degradation of a POI fused to a Halotag could be induced with a HaloPROTAC22,23. Similarly, Nabet and colleagues developed a FKBP12F36V-based dTAG system, in which different PROTAC molecules, dTAG-13 and dTAGv-l, could be employed to engage the interaction of FKBP12F36V-POI with E3 ligase CRBN or VHL, respectively24,25. The degradation of POI in all these methods relies on the endogenous E3 ligases, specifically, CRBN and VHL, which may not be highly expressed or functional in all cell types (particular tumor cells) or tissues26. In contrast, the auxin-inducible degron (AID) system utilizes an exogenously expressed plant-derived auxin receptor F-box protein TIR1, which forms a functional chimeric SCF E3 complex with endogenously expressed Skpl, Cull and Rbxl. In this case, the POI needs to be fused to an AID tag derived from IAA17. Auxin and its derivatives function as molecular glues to mediate the binding of SCF- TIR1 E3 ligase complex to the AID tagged POI, leading to the ubiquitination and degradation of the POI27. The original version of the AID system suffers from severe basal degradation of AID tagged POI in the absence of auxin and inefficient degradation upon auxin-induction. However, these issues may have been resolved in the improved versions by employing either an mutant form of the TIR1 and 5-Ph-IAA28or a different plant F-box protein (AtAFB2) and a different AID tag (miniIAA7)29.

[0011] In principle, as many E3 ubiquitin ligases have well defined substrate binding domains, another approach would be to replace the substrate binding domain with a domain targeting the POI or an epitope tag comprised on the POI. To date, however, reported tags for such purpose are relatively large and could interfere with function of the POI. For example, the AID tag is 68 amino acids29, FKBP12F36V dTAG is 108 amino acids25, EGFP is 240 amino acids43, and Halotag is 297 amino acids22. The bulky size of the tag may interfere with the normal function of the fused POI44; second, in order to use all the targeted protein degradation methods to investigate the function of a POI, it is generally mandatory to tag all the copies of the endogenous gene in a cell instead of working with tagged transgenes; the tagging of endogenous genes is often achieved through CRISPR-based homologous recombination-mediated knock-ins, which in most cells is not very efficient and often involves labor-intensive screening of hundreds of clones. This is particularly challenging for tumor cell lines that have more than two copies of the genes encoding POI. In general, the larger the tag, the less efficient the knock-in45; besides, it is often more cumbersome to make donor vectors for large tags with larger homology arms.

[0012] Hence, further approaches are needed for development of protein degradation platforms that can be used in cell lines, for instance, to assist with target discovery and validation in drug development and other applications.

[0013] SUMMARY

[0014] The present disclosure relates, for example, to the use of relatively small peptide expression tags and their recognition by binding polypeptides fused to an E3 ubiquitin ligase. Further, in some cases, the binding polypeptides are themselves relatively small, such as 170 amino acid residues or less, while in some cases the platform may use bacterial E3 ubiquitin ligases, which may also be smaller and less complex than mammalian ligases.

[0015] The present disclosure relates, for example, to a fusion polypeptide comprising an E3 ubiquitin ligase and a binding polypeptide that specifically recognizes a peptide expression tag wherein the peptide expression tag comprises 25 amino acids or less, 20 amino acids or less, or 15 amino acids or less. In some cases, the peptide expression tag comprises 6-25, 6-24, 8-24, 6- 20, 8-20, 10-20, 10-15, 6-15, or 8-15 amino acids. In some cases, the peptide expression tag is an ALFA tag (SEQ ID NO: 1, optionally further comprising an N-terminal and / or C-terminal proline), a HiBiT tag (SEQ ID NO: 2), or a SPOT tag (SEQ ID NO: 3), or BC2 tag (SEQ ID NO: 4).

[0016] In some embodiments, the binding polypeptide is an antibody. In other embodiments, it is not an antibody. In some cases, the antibody does not comprise a full length constant region. In some cases, the antibody is a single chain antibody, monobody, camelid antibody, or an antigen binding fragment such as an Fv, Fab, F(ab)2, or ScFv. In some cases, the antibody is a camelid antibody (also called a nanobody or VHH) or an antigen binding fragment derived from a camelid antibody. In some cases, the binding polypeptide comprises 170 amino acid residues or less, such as 100-170 amino acid residues.

[0017] In some cases, the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase. In some cases, the E3 ubiquitin ligase does not comprise a native substrate binding domain. For example, lack of a native substrate binding domain may avoid ubiquitination of native cellular targets, so that the fusion polypeptide can be used to target the peptide epitope binding tag and its associated target protein. In some cases, the E3 ubiquitin ligase is monomeric, while in other cases, it comprises multiple subunits. In some cases, the catalytic domain comprises a NEL, RING / Ubox, Fbox, or HECT catalytic domain. In some cases, the E3 ubiquitin ligase comprises a NEL domain of a bacterial E3 ubiquitin ligase. In some cases, the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase derived from a Shigella, Pseudomonas, Legionella, E. coli or Salmonella species. In some cases, the E3 ubiquitin ligase comprises the NEL domain of Shigella flexneri IpaH9.8. In some cases, the E3 ubiquitin ligase comprises the catalytic domain of AvrPtoB, IpaH0722, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, LegAU13, LegUl, LubX, NleG2-3, HleG5-l, NleL, SidC, SirP, SopA, SspHl, SspH2, or XopL. In some cases, there is no linker between the binding polypeptide and the E3 ubiquitin ligase. In some cases, the fusion polypeptide comprises a linker between the binding polypeptide and the E3 ubiquitin ligase.

[0018] In some cases, the binding polypeptide specifically binds to a peptide expression tag on an extracellular membrane protein, endoplasmic reticulum membrane protein, Golgi membrane protein, mitochondrial outer membrane protein, nuclear protein, or cytosolic protein.

[0019] The disclosure also relates to a complex comprising the fusion polypeptide as described above and elsewhere herein and a protein comprising a peptide expression tag that is specifically recognized by the binding polypeptide. In some such complexes, the protein comprising the peptide expression tag is an extracellular membrane protein, endoplasmic reticulum membrane protein, Golgi membrane protein, mitochondrial outer membrane protein, nuclear protein, or cytosolic protein.

[0020] The present disclosure also relates to a polynucleotide comprising a coding sequence for the fusion polypeptide described above and elsewhere herein. In some cases, the coding sequence for the fusion polypeptide is under the control of an inducible promoter. In some such cases, the inducible promoter comprises a doxycycline (Dox) inducible promoter or a tetracycline (Tet) inducible promoter. In some cases, the polynucleotide further encodes a reporter polypeptide, wherein the reporter polypeptide acts as a marker for cells transfected with the polynucleotide. In some cases, the polynucleotide is RNA, optionally wherein the RNA further comprises a poly-adenosine segment at the 3’ terminus. The present disclosure also relates to a composition comprising the polynucleotide and at least one excipient or carrier, such as lipids, surfactants, or proteins such as albumin or poly A binding protein. The disclosure further encompasses a cell comprising the polynucleotide, wherein the cell is an eukaryotic cell, such as a mammalian cell.

[0021] The disclosure also encompasses a composition comprising the fusion polypeptide and at least one excipient or carrier, such as lipids, surfactants, or proteins. In some cases, the polypeptide is covalently or noncovalently attached to another molecule such as a lipid, surfactant, or another protein.

[0022] The disclosure also relates to a method of degrading a target protein in a cell, wherein the target protein comprises a peptide expression tag, wherein the peptide expression tag comprises 25 amino acids or less, 20 amino acids or less, or 15 amino acids or less, the method comprising introducing to the cell a fusion polypeptide or a polynucleotide encoding the fusion polypeptide or the composition as described herein, wherein the fusion polypeptide comprises a binding polypeptide that specifically recognizes the peptide expression tag. The disclosure further relates to a method of degrading a target protein in a cell, wherein the target protein comprises a peptide expression tag, wherein the peptide expression tag comprises 25 amino acids or less, 20 amino acids or less, or 15 amino acids or less, the method comprising introducing to the cell a polynucleotide encoding the fusion polypeptide as described herein, wherein the fusion polypeptide comprises a binding polypeptide that specifically recognizes the peptide expression tag, and inducing expression of the fusion polypeptide in the cell. In some methods herein, the peptide expression tag comprises 6-25, 6-24, 8-24, 6-20, 8-20, 10-20, 10-15, 6-15, or 8-15 amino acids. In some cases, the peptide expression tag is an ALFA tag (SEQ ID NO: 1, optionally further comprising an N-terminal and / or C-terminal proline), a HiBiT tag (SEQ ID NO: 2), or a SPOT tag (SEQ ID NO: 3), or BC2 tag (SEQ ID NO: 4). Methods herein may further comprise detecting the expression level of the target protein in the cell before and / or after introduction of the fusion polypeptide, polynucleotide, or composition to the cell. In some cases, the method comprises introducing a polynucleotide in which expression of the fusion polypeptide encoded by the polynucleotide is under the control of an inducible promoter. In some cases, the inducible promoter comprises a doxycycline (Dox) inducible promoter or a tetracycline (Tet) inducible promoter. In some cases, the level of the target protein in the cell is reduced upon activation of the inducible promoter. In some cases, the cell is an eukaryotic cell, such as a mammalian cell.

[0023] The disclosure herein also encompasses a kit comprising the fusion polypeptide or a polynucleotide encoding the fusion polypeptide or the composition or the cell as described herein.

[0024] All references cited herein are incorporated by reference herein. Furthter description of certain embodiments is provided in the figures and in the subsequent sections and claims.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Figures 1A-1C show that the small tag ALFA mediates efficient degradation of overexpressed ALFA tag-containing fusion proteins with a fusion polypeptide comprising an anti-ALFA camelid antibody (NbALFA) and the catalytic domain (NEL) of bacterial E3 ligase IpaH9.8. Fig. 1 A shows the principal of operation of the fusion polypeptide (called Nb ALFAbased artificial bacterial E3 ligase (ABEL)). POI, protein of interest; TIP, tag-interacting partner; AEL, artificial E3 ligase; Ub, ubiquitin; E2 and E3, E2 and E3 ligase. Fig. IB shows that Nb ALFA-based ABEL efficiently degrades an mCherry-ALFAtag-ha-synuclein fusion protein. Such activity is abolished when mutations are introduced to NbALFA (Y42R E58G R59G) to destroy its binding to the ALFA tag (NbALFA- AB EL*) or to the NEL domain of bacterial E3 ligase IpaH9.8 (C337A) to kill the E3 ligase activity (NbALFA-dABEL). Fig. 1C shows statistical data from three independent experiments showing the significant reduction of the relative median fluorescent intensity (MFI) of mCherry in tagBFP+ cells, which are normalized to tagBFP+ cells that are transfected with tagBFP control plasmid. 6XHis corresponds to SEQ ID NO: 7.

[0027] Figures 2A-2C show that bacterial E3 ligase consistently shows better degradation efficiency when compared to a list of selected mammalian ones, including those that are commonly used. HEK293T cell lines expressing a range of fusion proteins tagged with ALFA are transfected with various Nb ALFA-based artificial E3 ligases and the degradation of the fusion protein is assessed by quantifying the decrease of the fluorescent signal in tagBFP+ (transfected) cells. While all artificial mammalian E3 ligases show dramatic variation of degradation capacity with different fusion protein substrates, the bacterial one shows consistent better degradation efficiency with all tested fusion protein substrates. 6XHis corresponds to SEQ ID NO: 7.

[0028] Figures 3A-3F show reversible temporal control of the degradation of target protein with Dox induced expression of ABEL. Fig. 3 A shows the dox inducible ASTABEL-TDP system. HEK293T cells expressing mCherry- ALFAtag- ha-synuclein fusion protein are engineered to harbor a Dox-inducible cassette that drives the expression of the Nb ALFA-based ABEL upon the administration of Dox. Fig. 3B shows that the percentage of cells that lose the mCherry - ALFAtag- ha-synuclein fusion protein is proportional to the concentration of Dox. Statistical data shown in Fig. 3C. However, at single cell level, the loss of the fusion protein is binary. Figs. 3D, 3E, and 3F show that the degradation of the target protein is reversible. 6XHis corresponds to SEQ ID NO: 7.

[0029] Figures 4A-4E show tagging of both copies of PD-L1 in MC38 cells and its degradation with ASTABEL. Fig. 4A shows CRISPR-Cas9-based tagging of both copies of the endogenous PD-L1 loci in MC38 cells. A mixture of Cas9 / sgRNA RNP complex (SEQ ID NO: 21, sgRNA) and oligo donor (SEQ ID NO: 22), which contains the Hibit tag and 50bp homology arm on either side, was nuclefected into MC38 cells. To prevent the recombined alleles from being recut by the Cas9 / sgRNA complex, a single nucleotide mutation (G to A, red) was introduced into the oligo donor to destroy the PAM sequence (AGG, underlined). Single clones with correctly knocked-in ALFA right before the stop codon (TAA, red) in both PD-L1 alleles were screened with PCR and verified with Sanger sequencing. The cutting site by the RNP complex is indicated by a red arrow. Fig. 4B shows that to inducibly degrade ALFA-tagged PD-L1, a Dox-inducible ABEL cassette was further introduced into the homozygote clones and wild type cells, which is used as control (parental cells). Fig. 4C shows flow cytometry analysis of ASTABEL-mediated degradation of PD-L1 in parental cells, four homozygote clones and PD-L1 knock-out cell lines with DMSO or Dox treatment. To increase the sensitivity of the flow cytometry, all cells were stimulated with interferon-y to stimulate the expression of PD-L1. Fig. 4D and 4E show Western blot confirmation of the flow cytometry data with anti-PD-Ll antibody. 6XHis corresponds to SEQ ID NO: 7.

[0030] Figures 5A-5C show efficient Degradation of OMM (outer membrane of mitochondria) and ER Proteins with ASTABEL. ALFA-tagged mCherry protein were targeted to either OMM or ER by fusing with the C-terminal domain of Fisl or ER retention signal KDEL in HEK293T cells. The organelle-specific localization of the fusion protein was confirmed with immunostaining using OMM and ER-specific markers (ATP0 and calnextin). Their degradation with Nb ALFA-based ABELs were quantified with flow cytometry.

[0031] Figure 6A-6D show that the HiBiT tag could be employed to not only mediate protein degradation, but also quantify the residual protein. Fig. 6A shows that various LgBiT-based ABELs were tested for their ability to degrade a mCherry- ALFAtag-Hibit fusion protein. Similarly, the degradation efficiency was quantified by measuring the relative median fluorescent intensity of mCherry in TagBFP+ cells with flow cytometry. The tag of the gene of interest with Hibit not only makes it degradable with LgBiT-based ABELs, but also facilitates its quantification with Hibit blot. Fig. 6B shows that HEK293T cells stably expressing a mCherry- ALFAtag-Hibit fusion protein were transiently transfected with NbALFA, LgBiT-based ABELs and control plasmids, lysed 24 hours later and quantified with with regular Western blot or Hibit blot. Fig. 6C shows that HeLa cells with one copy of the SMARCA2 tagged with Hibit were stably transfected with a Dox-inducible LgBiT-ABEL expression cassette and treated with DMSO or Dox for 24 hours. The level of Hibit-tagged SMARCA2 was detected with Hibit blot after the cell lysate is resolved on SDS-PAGE and transferred to the membrane. Fig. 6D shows that HEK293 cells with Hibit-tagged GSPT1 were transiently transfected with various ABEL plasmids and total cell lysates (without enrichment of transfected cells) were separated on SDS- PAGE, transferred to the membrane and blotted with LgBiT and luciferase substrate. Note that the expression of LgBiT itself in this cell line does not seem to have dramatic effect on the degradation mediated by LgBiT-based ABELs. 6XHis corresponds to SEQ ID NO: 7.

[0032] Figure 7A-7B show general schemes of experiment and flow cytometry data analysis to test the degradation of a tagged protein of interest with ABELs. Fig. 7A shows the experiment workflow. A piggyBAC plasmid encoding a protein of interest consisting of a fluorescent protein and a tag was transfected into HEK293T cells together with a plasmid encoding the piggyBAC transposase; cells with stably integrated plasmids were sorted after 10 days of culture based on the fluorescent fusion protein expression. To test the degradation activity of ABELs, these cells were transiently transfected with plasmids encoding desired ABELs and the degradation of the target fusion protein was analyzed 24 or 48 hour later with flow cytometry. TIP, tag-interacting partner; HiBiT, HiBiT tag, see Fig. 6. Figure 7B shows a typical flow cytometry data analysis workflow. 6XHis corresponds to SEQ ID NO: 7.

[0033] Figure 8A-8B: Fig. 8A shows confirmation of the degradation of mCherry- ALFAtag- ha-Synuclein with Western blot. Fig. 8B shows that the ALFA tag works efficiently when it is placed at the N-terminus of the fusion protein.

[0034] Figure 9A-9C: Fig. 9A shows assessment of the degradation of mCherry with various LgBiT-based ABELs when its N-terminus was tagged with Hibit tag. Fig. 9B and 9C show that both ALFA and HiBiT tags worked efficiently when placed in the middle of fusion proteins. 6XHis corresponds to SEQ ID NO: 7.

[0035] Figures 10A-10G show degradation of human a-synuclein in cultured cells with fusion polypeptides comprising camelid antibodies (nanobodies) against a-synuclein and a bacterial E3 ligase catalytic domain (NEL). Fig. 10A shows the principle of nanobody guided targeted degradation of human a-synuclein with the fusion polypeptide (also termed an “artificial E3 ligase” or AEL). Ub, ubiquitin; E2 and E3, E2 and E3 ligase. Fig. 10B shows a diagram of an artificial bacterial E3 ligase (ABEL) and the protein target used to test the idea in cultured cells. Fig. 10C shows the amino acid sequence of human a-synuclein and the epitopes that are believed to be bound by two synuclein nanobodies (NbSyn2 and NbSyn87) and a scFv (VH14) tested in the working examples herein. Fig. 10D shows that HEK293T cells stably expressing a mCherry- ALFAtag-human a-synuclein fusion protein were transiently transfected with various ABELs and their degradation capability assessed 24 hours later by examination of the median fluorescent intensity (MFI) of mCherry in the transfected cells (tagBFP+). Cells transfected with a control plasmid that only encodes the tagBFP were used to normalize the MFI. Typical results are shown here (Fig. 10D) and statistical data are shown in Fig. 10E. Fig. 10F shows Western blot confirmation of the degradation of the fusion protein with NbSyn87-based ABELs. Fig. 10G shows degradation of human a-synuclein in cultured primary neurons derived from BAC transgenic mice by NbSyn87-based ABEL when delivered with AAV. mCherry, instead of tagBFP, was used in these viruses after the 2A peptide. 6XHis corresponds to SEQ ID NO: 7.

[0036] Figures 11A-11B show degradation of overexpressed or endogenous human a-synuclein in cultured cells. Fig. 11 A shows that overexpressed human a-synuclein was efficiently degraded. HEK293 cells stably expressing human a-synuclein were nucleofected with control plasmid encoding only TagBFP or plasmid encoding various ABELs, lysed 24 hours later, separated on SDS-PAGE, and blotted with anti-synuclein antibody. Note the addition of proteasome inhibitor MG132 blocks the degradation of human a-synuclein. Fig. 1 IB shows that endogenous expressed human a-synuclein was efficiently degraded. Two human malignant melanoma-derived cell lines that express high level of endogenous a-synuclein, WeMo and SK- Mel-28, were transiently transfected with TagBFP, NbSyn87-ABEL or NbSyn87-deadABEL with Lipofectamine LTX. TagBFP+ cells (transfected cells) were enriched with FACS 24 hours later for Western blot analysis.

[0037] Figures 12A-12C show degradation of human a-synuclein in transgenic mice. Fig. 12A shows diagrams of AAV-PHP.eB viruses. Fig. 12B shows the work-flow of the experiments. Fig. 12C shows results of ELISA measurement of human a-synuclein level in the brain of all mice injected with AAV-PHP.eB viruses. 6XHis corresponds to SEQ ID NO: 7.

[0038] Figures 13A-13B show Western blot confirmation of the degradation of human a- synuclein in the brain of mice injected with NbSyn87-based ABEL. Fig. 13A shows a Western blot result from the experiments performed. Fig. 13B shows quantification of the Western blot data.

[0039] DETAILED DESCRIPTION

[0040] I. DEFINITIONS

[0041] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0042] An “E3 ubiquitin ligase” or “E3 ligase” refers to a class of protein or multi-protein complex that has E3 ubiquitin ligase activity. E3 ubiquitin ligases catalyze transfer of ubiquitin from a ubiquitin-conjugating enzyme (E2) to a target protein that is intended for degradation. In fusion polypeptides herein, an “E3 ubiquitin ligase” comprises or consists of at least the “catalytic domain” of such a ligase protein. Accordingly, an E3 ubiquitin ligase may not be a complete or full length ligase, but may comprise a portion of a native ligase protein comprising the catalytic domain. The “catalytic domain” refers to the portion of the ligase that is sufficient for catalyzing the transfer of ubiquitin to a target protein. For example, in some embodiments, the E3 ubiquitin ligase may lack native domains that bind to native target proteins, so that the fusion polypeptide does not act on such native targets of the ligase but instead is engineered to facilitate degradation of other target proteins. As noted herein, E3 ubiquitin ligases may have different catalytic domain structures and mechanisms. E3 ubiquitin ligases herein may also come from a variety of sources such as vertebrates, mammals, and bacteria, such as pathogenic bacteria. For example, certain pathogenic bacteria include E3 ubiquitin ligases that mimic host E3 ubiquitin ligases and thus hijack the ubiquitination pathway. Vertebrate sources include mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), or domestic mammals (e.g., dogs, cats, horses, livestock such as cattle, pigs, sheep, goats, etc.), avians (e.g., foul, chickens, turkey), or fish. An E3 ubiquitin ligase also includes variants of native ligases, for example, which may include truncations of portions of the protein not necessary for E3 ubiquitin ligase activity, as well as substitutions or insertions or deletions of amino acid residues, or other modifications, for example in order to affect E3 ubiquitin ligase activity.

[0043] A “target protein” or “protein of interest” or “POI” or a “protein targeted for degradation” or a “degradation target protein” or the like refer to a protein that is intended to be degraded.

[0044] In some cases herein the target protein is a “membrane protein.” A “membrane protein” includes proteins that span a membrane (i.e., transmembrane proteins) as well as proteins that are associated with a membrane, such as through binding to transmembrane proteins. In some cases, a target protein is accessible to the cytosol, meaning that it contains domains or portions that are located in the cytosol. In some embodiments herein, a target protein is an “extracellular membrane protein.” This term broadly refers to a protein that is located at the cell surface, either as a transmembrane protein possessing an extracellular domain, or as a protein that is otherwise localized to the cell surface, such as a protein that is bound to a transmembrane protein. In other cases, the target protein is a “cytosolic protein,” which refers to a protein that is found in the cytosol of a cell. In other cases, the protein is an “endoplasmic reticulum (ER) membrane protein,” a “Golgi membrane protein,” or a “mitochondrial outer membrane protein,” which terms refer to proteins that either span such a membrane and include segments accessible to the cytosol, or proteins that are localized to the cytosolic face of such a membrane. In some cases, a target protein is a “nuclear protein,” which includes proteins found in the cell nucleus, and nuclear membrane proteins, which may span the nuclear membrane or be localized to the cytosolic face of the nuclear membrane. In some embodiments, a target protein is engineered to include a “peptide expression tag.” A “peptide expression tag” or “peptide epitope tag” or “expression tag” or “epitope tag” refers to a peptide sequence that is found on a target protein herein. In some embodiments, the tag is 25 amino acid residues in length or shorter. In some embodiments, the target protein is engineered to include the tag, such as at the C-terminal or N-terminal of the target protein.

[0045] A “fusion polypeptide” as used herein refers to a protein molecule that includes amino acid sequences from two different proteins, such as an E3 ubiquitin ligase and another protein such as a binding polypeptide, and is thus a chimeric polypeptide.

[0046] A “binding polypeptide” as used herein refers to a polypeptide molecule or polypeptide domain that is intended to bind to a peptide expression tag. In some cases, the binding polypeptide is an antibody. In other cases, the binding polypeptide is not an antibody.

[0047] A “linker” as used herein, when referring to a segment between two domains of a fusion polypeptide, for example, refers to an amino acid sequence that differs from that of the two domains being joined together. In some cases, a linker is flexible, such as comprised of glycineserine repeats or a similar sequence, thus allowing the joined domains of the fusion polypeptide to have flexibility to perform their different functions.

[0048] The term “antibody” herein refers to a molecule comprising at least complementaritydetermining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain or alternatively comprising at least CDR1, CDR2, and CDR3 of a camelid antibody heavy chain, wherein the molecule is capable of binding to antigen. The term “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to full length antibodies, single-chain antibodies, camelid antibodies (e.g. VHH domains or nanobodies), antibody conjugates, antibody fusion polypeptides, monobodies, and a varieity of antibody fragments, so long as they exhibit the desired binding activity.

[0049] An “isolated” antibody has been separated from at least one component of its natural environment. In some aspects, an antibody is also “purified,” such as to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0050] An “antigen” refers to the target of an antibody, i.e., the molecule to which the antibody specifically binds. The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes on a protein can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e. by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents.

[0051] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule and its binding partner. Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0052] In this disclosure, “binds” or “binding” or “recognizes” or “specific binding” or “specific recognition” and similar terms, when referring to a binding polypeptide and the molecule to which it binds, or an antibody and its antigen target for example, means that the binding affinity is sufficiently strong that the interaction between the members of the binding pair cannot be due to random molecular associations (i.e. “nonspecific binding”). Such binding typically requires a dissociation constant (KD) of IpM or less, and may often involve a KD of 100 nM or less.

[0053] The term “heavy chain” refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0054] The term “light chain” refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0055] The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable region which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”). Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 or heavy chain CDR1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0056] (a) “Chothia CDRs”: hypervariable loops occurring at amino acid residues 26-32 (LI), SO- 52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) “Kabat CDRs”: CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 3 l-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0057] (c) “McCallum CDRs”: antigen contacts occurring at amino acid residues 27c-36 (LI), 46- 55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

[0058] Unless specifically indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to McCallum, or any other scientifically accepted nomenclature system.

[0059] “Framework” or “FR” refers to the residues of the variable region residues that are not part of the complementary determining regions (CDRs). The FR of a variable region generally consists of four FRs: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR- H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4. An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0060] The term “variable region” or “variable domain” interchangeably refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). See, e.g., Kindt et al. Kuby Immunology, 6thed., W.H. Freeman and Co., page 91 (2007). A variable domain may comprise heavy chain (HC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and / or FR4; and light chain (LC) CDR1-FR2-CDR2-FR3-CDR3 with or without all or a portion of FR1 and / or FR4. That is, a variable domain may lack a portion of FR1 and / or FR4 so long as it retains antigen-binding activity. A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol.

[0061] 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0062] The term “VHH” or “VHH domain” or “VHH region” refers to the heavy chain variable region of a camelid antibody, which may comprise the associated heavy chain (HC) CDR1-FR2- CDR2-FR3-CDR3 with or without all or a portion of FR1 and / or FR.

[0063] The light chain and heavy chain “constant regions” of an antibody refer to additional sequence portions outside of the FRs and CDRs and variable regions. Certain antibody fragments may lack all or some of the constant regions. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, which in most cases is followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

[0064] The term “Fc region” or “Fc domain” herein is used to define a C-terminal region of an immunoglobulin G (IgG) heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0065] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgGi, IgG4, IgAi, and IgA2. In certain aspects, the antibody is of the human IgGi IgG?, IgGi, or IgG4isotype. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, 8, y, and p, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0066] An “antibody fragment” or “antigen binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (sdAbs, VHH, nanobodies); and multispecific antibodies formed from antibody fragments. Antibody fragments in some cases may also be fusion polypeptides with polypeptide segments from other proteins fused to all or part of a variable region antibody segment, e.g., monobodies. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23 : 1126-1136 (2005). The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure, for example, in the case of an IgG antibody, having heavy chains that contain an Fc region as defined herein.

[0067] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the disclosure in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017, published online 12 June 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 Bl).

[0068] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from at least one component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0069] “Isolated nucleic acid encoding a polypeptide” and the like refers to one or more nucleic acid molecules encoding polypeptides herein (or fragments thereof), which may be included in a single nucleic acid molecule in a single vector or in separate nucleic acid molecules in separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0070] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In some embodiments, the vector is a viral vector, such as an adenoassociated virus vector (AAV) or lentivirus vector.

[0071] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0072] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU5 10087 and is described in WO 2001 / 007611.

[0073] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www. ebi.ac.uk / Tools / sss / fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare the sequences, using the ggsearch (global protein: protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

[0074] An “inducible promoter” herein refers to a promoter for expression of a polypeptide that may be upregulated or activated by a change in the surrounding medium, such as addition or an increase in concentration of a particular chemical substance, such as an antibiotic, which may result in activation of the promoter through associated cell signaling events. An inducible promoter, therefore, may be controlled by a researcher by changing the concentration or amount of one or more substances in the environment, such as in the extracellular medium.

[0075] A “reporter polypeptide” as used herein refers to a polypeptide for which changes in concentration can be detected, thus allowing it to ack as a marker. An example is a fluorescent protein, for which fluorescence levels may be detected. Another example is an enzyme, which may be detected through its enzymatic activity. Another is an antibody or a polypeptide that binds to a particular ligand, through which changes in its level may be detected by changes in the degree of ligand binding.

[0076] An “excipient or carrier” herein refers to one or more substances that facilitate the formulation of a polypeptide or polynucleotide. Examples include surfactants, lipids, proteins, amino acids, sugars, sugar alcohols, polymers, and the like.

[0077] In some embodiments, a target protein is human “alpha-synuclein,” also referred to as “a- synuclein.” As used herein, the terms “alpha-synuclein” and “a-synuclein” and “ha-synuclein” refer to the human protein unless expressy stated otherwise. An exemplary amino acid sequence of the protein is also provided in Fig. IOC and comprises the amino acid sequence of SEQ ID NO: 17.

[0078] As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., + / -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as “at least” and “about” precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

[0079] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0080] In this application, the use of “or” means “and / or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0081] Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are described, e.g., in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among other places.

[0082] Additional definitions may be found in the sections that follow.

[0083] II. EXEMPLARY FUSION POLYPEPTIDES AND METHODS OF USE

[0084] The disclosure herein relates, inter alia, to fusion polypeptides that comprise an E3 ubiquitin ligase and a binding polypeptide, wherein the binding polypeptide specifically recognizes a peptide expression tag that comprises about 25 amino acids or less. The disclosure herein also relates to methods of using such fusion polypeptides, for example, to degrade a target protein in a cell, wherein the target protein comprises the peptide expression tag that is recognized by the binding polypeptide portion of the fusion polypeptide. Such methods may be conducted, for example, in vitro, such as in cell culture, in some embodiments. In some embodiments, the cells may be eukaryotic cells that are capable of degrading proteins via the ubiquitination pathway, such as mammalian cells. Alternatively, in some embodiments, the methods herein may be conducted in vitro, such as where the target protein comprising the peptide expression tag is expressed in vivo.

[0085] To degrade a target protein in a cell, for example, one may introduce a fusion polypeptide as described herein to a cell, wherein the cell expresses a target protein for degradation comprising a peptide expression tag. In some embodiments, one may introduce a polynucleotide expressing the fusion polypeptide to the cell. In some cases, the polynucleotide comprises an inducible promoter for expression of the fusion polypeptide, such as a doxycycline (Dox) or tetracycline (Tet) inducible promoter. Thus, in some such cases, one may induce expression of the fusion polypeptide in the cell.

[0086] In some embodiments, the fusion polypeptide reduces the level of a target protein in a cell in vitro compared to the level observed in the absence of the fusion polypeptide. In some cases, the change in the level of the target protein is observed by flow cytometry, compared to the level observed in the absence of the fusion polypeptide or the level observed with a mutant fusion polypeptide that lacks E3 ubiquitin ligase activity or that lacks the ability to bind to the peptide expression tag. In some cases, the change in the level of the target protein is observed by attaching the target protein to a reporter molecule such as a reporter polypeptide, and detecting the level or activity of the reporter. In other cases, the change in the level of the target protein is observed by detecting the level or activity of the target protein directly, such as by a binding or activity assay.

[0087] In another aspect, the disclosure herein also relates to fusion polypeptides comprising an E3 ubiquitin ligase and a binding polypeptide that specifically recognizes a target protein, e.g., alpha-synuclein (a-synuclein) such as human a-synuclein, and to methods of using such fusion polypeptides, for example, to degrade a target protein such as a-synuclein in a cell, as well as methods of treating neurodegenerative diseases using such fusion polypeptides or polynucleotides, compositions, or cells encoding them.

[0088] A. Exemplary Peptide Expression Tags and Binding Polypeptides

[0089] In some embodiments, a peptide expression tag that is recognized by the binding polypeptide of a fusion polypeptide herein comprises 25 amino acids or less. In some cases, the peptide expression tag comprises 6-25, 6-24, 8-24, 6-20, 8-20, 10-20, 10-15, 6-15, or 8-15 amino acids. In some cases, the peptide expression tag comprises 20 amino acids or less. In some cases, the peptide expression tag comprises 15 amino acids or less. In some cases, the peptide expression tag comprises 6-15 amino acids or 10-15 amino acids. Examples include, for instance, an ALFA tag (SEQ ID NO: 1), a HiBiT tag (SEQ ID NO: 2), or a SPOT tag (SEQ ID NO: 3), or BC2 tag (SEQ ID NO: 4), as well as a FLAG, myc, His, ID4 tag, or HA tag (SEQ ID NOs: 5-9, respectively), each of which comprise about 6-15 amino acids in length. In some embodiments, a peptide expression tag comprises a molecular weight of 3 KDa or less, such as 2 KDa or less. Exemplary peptide expression tags are also disclosed, for example, in Dixon et al., ACS Chemical Biology, 11 : 400-408 (2016), Gotzke et al., Nature Communications, doi.org / 10.1038 / s41467-019-12301-7 (2019), or Burgstaller et al., iScience, 25, 104907 (2022), doi.org / 10.1016 / j.isci.2022.104907, all of which are incorporated by reference herein. In cases where an ALFA tag is used, a proline amino acid may in some embodiments be added either at the N-terminal end or at the C-terminal end, or at both the N-terminal end and the C-terminal end of the ALFA tag. For example, if the tag is placed at the N-terminal end of the target protein, it may be preceded by a Met-Pro stretch, and then followed by a Pro residue. If it is placed internally in the target protein, it may be preceded and followed by a Pro residue. And if it is placed at the C-terminal of the target protein, it may be preceded by a Pro residue. For example, it is possible that such linking proline residues may help to separate the ALFA tag structurally from the domains of the target protein for easier recognition.

[0090] Peptide expression tags may be specifically recognized by binding polypeptides. In some cases, the binding polypeptide is an antibody. In some cases, the antibody does not comprise a full length constant region. In some cases, the antibody is an antigen binding fragment, such as an Fv, Fab, F(ab)2, or ScFv fragment. In some cases, the antibody is a single chain antibody, monobody, or camelid antibody (i.e., nanobody or VHH), or an antigen binding fragment derived from a camelid antibody. In some embodiments, the binding polypeptide is not an antibody. In some embodiments, the binding polypeptide has an amino acid sequence of 170 amino acid residues or less, such as 50-170 amino acid residues, such as 100-170 amino acid residues or 100-160 amino acid residues, or 120-160 amino acid residues. In some cases, the binding polypeptide specifically recognizes a particular peptide epitope tag, e.g., an anti-ALFA tag binding polypeptide or an HiBiT binding polypeptide (also called LgBiT), or an anti-SPOT binding polypeptide, or the like. Exemplary binding polypeptides that recognize peptide epitope tags such as ALFA, HiBiT, SPOT, BC2, FLAG, myc, His, ID4, or HA, among others, are commercially available or have been described, for example, in Dixon et al., ACS Chemical Biology, 11 : 400-408 (2016), Gotzke et al., Nature Communications, doi.org / 10.1038 / s41467- 019-12301-7 (2019), or Burgstaller et al., iScience, 25, 104907 (2022), doi.org / 10.1016 / j.isci.2022.104907. A HiBiT / LgBiT system is also available from Promega Corp. (Madison, WI, USA). For example, HiBiT and LgBiT are interacting fragments of a nucleotransferase protein, which form a tag (HiBiT) and a binding polypeptide for that tag (LgBiT). In some cases, binding polypeptides that recognize peptide epitope tags such as ALFA, HiBiT, SPOT, BC2, FLAG, myc, His, ID4, or HA, for example, are camelid antibodies, monobodies, single chain antibodies, or antigen binding fragments, thus allowing the complex between the tag and the binding polypeptide to be small in size, in comparison to targeted protein degradation systems that utilize larger components such as full length antibodies and / or larger target antigens. In some cases, the peptide expression tag is located at the N-terminal of the target protein to be degraded. In other cases, the peptide expression tag is located at the C-terminal of the target protein. In yet other cases, the peptide expression tag is located in an internal segment of the target protein. In some cases, the target protein is a protein that is heterologous to the cell; i.e., the cell has been engineered to express the target protein comprising the peptide epitope tag. In other cases, the target protein is a native cellular protein. For example, in such cases, a cell can be engineered such that the native gene encoding the protein is silenced or disrupted, and replaced with a gene encoding the protein comprising the peptide epitope tag.

[0091] B. Exemplary E3 Ubiquitin Ligases

[0092] In some embodiments, in order to further reduce the size and complexity of the components of the protein degradation system, the E3 ubiquitin ligase comprises the catalytic domain of an E3 ubiquitin ligase, such as the catalytic domain of a bacterial E3 ubiquitin ligase. For example, in some embodiments, the E3 ubiquitin ligase is engineered to lack domains or segments that are not necessary for catalytic activity or that are intended to bind to native proteins. Thus, in some embodiments, the E3 ubiquitin ligase does not comprise a native substrate binding domain. In other embodiments, the E3 ubiquitin ligase comprises the complete, native E3 ubiquitin ligase protein or protein complex. In some embodiments, the native E3 ubiquitin ligase is a monomeric protein; thus, the catalytic domain of the E3 ubiquitin ligase is a portion of the native monomeric protein, for example. In other cases, the native E3 ubiquitin ligase is a multimeric protein, and the catalytic domain may comprise one or more subunits of the protein or domains within such subunits.

[0093] E3 ubiquitin ligases may have a variety of structures and catalytic domains. See, e.g., Buetow and Huang, Nature Rev. Mol. Cell. Biol., 17: 626-642 (2016). E3 ubiquitin ligases can have several catalytic domain structures, such as NEL, RING / Ubox, Fbox, or HECT catalytic domains. See Id. Thus, in some embodiments, the E3 ubiquitin ligase comprises a NEL, RING / Ubox, Fbox, or HECT catalytic domain. For example, an E3 ubiquitin ligase with a HECT or RBR (RING between RING) domain typically is a monomeric protein. E3 ubiquitin ligases of the cullin-RING ligase (CRL) family are typically multi-subunit proteins, comprising a RING subunit that interacts with E2, and other components such as a native substrate binding domain, which are located on different subunits of the protein complex. In a multi-subunit E3 ubiquitin ligase, for example, the catalytic domain may comprise one or more subunits or domains that are sufficient for transfer of ubiquitin from E2 to the target protein. See, e.g., X-M. Li et al., Biomedicine & Pharmacotherapy, 149 (2022) 112882; J. Barankiewicz et al., Cancers 2022, 14, 4492. In some embodiments, the E3 ubiquitin ligase is a monomeric protein (i.e., comprising a single subunit). In some embodiments, the E3 ubiquitin ligase is a bacterial E3 ubiquitin ligase, which is a monomeric protein. In some embodiments, the E3 ubquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase. In some embodiments, the E3 ubquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase derived from a Shigella, Pseudomonas, Legionella, E. coli or Salmonella species. In some embodiments, the E3 ubiquitin ligase comprises the NEL domain of Shigella flexneri IpaH9.8. In some embodiments, the E3 ubiquitin ligase comprises the catalytic domain of AvrPtoB, IpaH0722, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, LegAU13, LegUl, LubX, NleG2-3, HleG5-l, NleL, SidC, SirP, SopA, SspHl, SspH2, or XopL. In some embodiments, the E3 ubiquitin ligase comprises, for example, residues 1-436 (U-box domain) of AvrPtoB from Pseudomonas syringae, residues 295- 587 (NEL domain) of IpaH0722 from Shigella flexneri, residues 285-575 (NEL domain) of IpaH1.4 from Shigella flexneri, 292-570 (NEL domain) of IpaH2.5 from Shigella flexneri, 296- 574 (NEL domain) IpaH4.5 from Shigella flexneri, 274-565 (NEL domain) IpaH7.8 from Shigella flexneri, 254-545 (NEL domain) IpaH9.8 from Shigella flexneri, 1-50 (F-box domain) ofLegAU13 of L. pneumophila, 1-56 (F-box domain) of LegUl of L. pneumophila, 1-215 (U- box domain) of LubX of L. pneumophila, 90-191 (U-box) ofNleG2-3 ofEHEC O157:H7, 371- 782 (HECT domain) of HleG5-l ofEHEC O157:H7, NleL (HECT domain) ofEHEC O157:H7, 1-542 of SidC of L. pneumophila, 465-765 (NEL) of SirP ofEHEC O157:H7, 370-782 (HECT) of SopA of Salmonella typhimurium, 404-710 (NEL) of SspHl of S. typhimurium, 492-788 (NEL) of SspH2 of S. typhimurium, or 474-660 of Xop of Xanthomonas campestris. See Ludwicki et al., ACS Cent. Sci. 5: 852-866 (2019), Supplemental Table 1.

[0094] In some embodiments, the E3 ubiquitin ligase comprises a NEL domain. In some embodiments, the E3 ubiquitin ligase is a bacterial E3 ubiquitin ligase comprising a NEL domain. In some embodiments, the E3 ubiquitin ligase comprises residues 254-545 (NEL domain) IpaH9.8 from Shigella flexneri.

[0095] C. Fusion Polypeptides and Polynucleotides Encoding Fusion Polypeptides

[0096] In some embodiments, the fusion polypeptide comprises a linker between the E3 ubiquitin ligase and the binding polypeptide. In some cases, the linker is a glycine-serine linker, for example, to provide flexibility between the two components of the fusion polypeptide. In other embodiments, there is no linker between the E3 ubiquitin ligase and the binding polypeptide. In some embodiments, the E3 ubiquitin ligase is arranged N-terminal to the binding polypeptide. In other embodiments, the E3 ubiquitin ligase is arranged C-terminal to the binding polypeptide. For example, in the native IpaH9.8 E3 ubiquitin ligase, a native substrate binding domain precedes the catalytic NEL domain of the ligase. Thus, a fusion protein herein may be constructed so as to replace the native substrate binding domain with a binding polypeptide that recognizes a peptide expression tag.

[0097] Such a fusion polypeptide may be expressed, for example, from a polynucleotide vector. For example, a plasmid vector may be constructed that includes coding regions for the fusion polypeptide of the E3 ubiquitin ligase and binding polypeptide behind appropriate promoter and / or enhancer elements so that the fusion polypeptide may be expressed in a cell. In some embodiments, an open reading frame for a reporter polypeptide is also included in such a vector in order to identify cells that have been properly transformed with the vector. Accordingly, the disclosure herein also encompasses polynucleotides encoding the fusion polypeptides herein, such as expression vectors, such as plasmid vectors and viral vectors. In some embodiments, a polynucleotide is a DNA vector. In other embodiments, the polynucleotide is an RNA vector, such as a viral RNA vector or mRNA vector. An RNA vector, for example, may further comprise a poly-adenosine segment at the 3’ terminus following the open reading frame(s).

[0098] In some embodiments, a polynucleotide encoding a fusion polypeptide herein expresses the fusion polypeptide under the control of an inducible promoter. For example, in some uses, it may be desirable to control the expression of the degradation-inducing fusion polypeptide by varying the environmental conditions. Examples of inducible promoters that may be used in the disclosure include, for instance, doxycycline (Dox) or tetracycline (Tet) inducible promoters whose activity is dependent on the presence or absence of Dox or Tet in the cellular medium.

[0099] The present disclosure also comprises compositions and kits that comprise a polynucleotide encoding the fusion polypeptide, which may for example be used to transform an appropriate cell in order to control the degradation of a peptide epitope tagged target protein, as well as cells expressing the fusion polypeptides. In some cases, cells expressing the fusion polypeptides are eukaryotic cells, such as mammalian cells or insect cells.

[0100] D. Methods of Use

[0101] In another aspect of the disclosure, fusion polypeptides or polynucleotides expressing fusion polypeptides, or compositions comprising such polypeptides or polynucleotides, may be used to degrade one or more target proteins by way of the ubiquitin pathway, such as in cells. In some cases, the cells are eukaryotic cells, such as mammalian cells. Cells, for example, may comprise substrates for E3 ubiquitin ligases, thus allowing for degradation of target proteins brought into complex with the E3 ubiquitin ligase through the binding of the binding polypeptide to the peptide expression tag on the target protein. In some cases, the polypeptides or polynucleotides may be used in vitro, such as in cell culture, or in a tissue sample or biological fluid sample. In other cases, they may be used in vivo to degrade a target protein. For instance, there are numerous instances in which it may be beneficial to assay the behaviour of a cell or tissue sample in which the level of a particular cell surface signaling molecule is artificially reduced. Employing such a fusion polypeptide herein, or transfecting cells with a polynucleotide expressing the fusion polypeptide herein, may provide a relatively simply way to modulate the level of such a target protein.

[0102] Thus, the present disclosure also encompasses methods of reducing the level of a cell surface protein in a cell or tissue sample in vitro, comprising incubating the sample with the multispecific binding protein, wherein the sample comprises a target protein comprising a peptide expression tag recognized by the binding polypeptide of the fusion polypeptide. The present disclosure also encompasses kits for this purpose. Kits may comprise the fusion polypeptide, optionally also with instructions for use, appropriate buffers, and / or labeling molecules. Kits may also or alternatively comprise vectors or host cells that comprise polynucleotides encoding the fusion polypeptide, so that the fusion polypeptide may be expressed by the vectors or host cells. In some methods or kits herein, a polynucleotide vector expresses the fusion polypeptide under an inducible promoter, such as a Dox or Tet inducible promoter, so that one can control degradation of the target protein.

[0103] In some embodiments, the methods further comprise detecting the expression level, activity, or function, or concentration of the target protein before and / or after introduction of the fusion polypeptide. In some embodiments, the target protein further comprises a detectable moiety, such as a fluorescent protein, such as green fluorescent protein (GFP), tdTomato, mCherry, or EGFP or the like, that may be used to detect its concentration or expression. In other embodiments, an activity assay may be performed to detect degradation of the target protein. In other embodiments, the expression or level of the target protein may be detected by methods such as in situ hybridization, immunohistochemistry, ELISA, Western blotting, flow cytometry, or the like.

[0104] In some embodiments, methods herein may be used, for example, to provide model systems to study the function of a particular protein in a cellular environment in vitro or in vivo. For example, a gene encoding a native, endogenous protein may be knocked-out and replaced with a gene encoding a peptide expression tagged version of the protein to be targeted by fusion polypeptides herein, optionally under an inducible promoter. Such models may be useful, for example, in target validation and a variety of protein function studies and for testing potential drug molecules. In some embodiments, a target protein for degradation is a cytosolic protein. In other embodiments, the target protein is a membrane protein, such as an extracellular membrane protein, endoplasmic reticulum membrane protein, Golgi membrane protein, or mitochondrial outer membrane protein. In other embodiments, the target protein is a nuclear protein such as a nuclear membrane protein. In some embodiments, the membrane protein or the nuclear protein is accessible to the cytosol. In some embodiments, the membrane protein or the nuclear protein is localized to the cytosolic face of the membrane, such that it is accessible to the cytosol. In some embodiments, the peptide expression tag is located at the N-terminus of the target protein. In other embodiments, the peptide expression tag is located at the C-terminus of the target protein. In yet other embodiments, the peptide expression tag is located internally within the target protein, such as between domains of the target protein or in a flexible region of the target protein. In some cases, the target protein comprises more than one peptide expression tag.

[0105] In some embodiments, the methods herein result in at least a 50% reduction in the concentration, activity, or expression level of the target protein. In some embodiments, the methods herein result in at least 60% reduction in the concentration, activity, or expression level of the target protein. In some embodiments, the methods herein result in at least 70% reduction in the concentration, activity, or expression level of the target protein. In some embodiments, the methods herein result in at least 80% reduction in the concentration, activity, or expression level of the target protein. In some cases, the percent reduction is relative to the level in the absence of introduction of the fusion polypeptide. In other cases, the percent reduction is relative to the level with introduction of a control fusion polypeptide that either cannot bind to the peptide epitope tag or that does not have E3 ubiquitin ligase activity. In some embodiments, the reduction in the concentration, activity , or expression level of the target protein is reversible, such as when the method employs an inducible promoter system.

[0106] III. FUSION POLYPEPTIDES RECOGNIZING ALPHA-SYNUCLEIN AND THEIR

[0107] USES

[0108] In another aspect, the present disclosure also relates to fusion polypeptides comprising a binding polypeptide recognizing a-synuclein and an E3 ubiquitin ligase, and their use for example for degrading a-synuclein. In some cases, the binding polypeptide is an antibody that recognizes an epitope on a-synuclein. In some cases, the antibody does not comprise a full length constant region. In some cases, the antibody is an antigen binding fragment, such as an Fv, Fab, F(ab)2, or ScFv fragment. In some cases, the antibody is a single chain antibody, monobody, or camelid antibody (i.e., nanobody or VHH), or an antigen binding fragment derived from a camelid antibody. In some embodiments, the binding polypeptide is not an antibody. In some embodiments, the binding polypeptide has an amino acid sequence of 170 amino acid residues or less, such as 50-170 amino acid residues, such as 100-170 amino acid residues or 100-160 amino acid residues, or 120-160 amino acid residues. In some cases, binding polypeptides, for example, are camelid antibodies, monobodies, single chain antibodies, or antigen binding fragments, thus allowing the complex between the a-synuclein target protein and the binding polypeptide to be small in size, in comparison to targeted protein degradation systems that utilize larger components such as full length antibodies.

[0109] In some cases, a binding polypeptide comprises an antibody recognizing an epitope as shown in Fig. 10C herein, such as comprising residues located within residues 51 and 140 of a- synuclein (see Fig. 10C; SEQ ID NO: 17). In some cases, the binding polypeptide comprises an antibody recognizing an epitope of a-synuclein comprising residues 51-87 of a-synuclein. In some cases, the binding polypeptide comprises an antibody recognizing an epitope of a- synuclein comprising residues 118-136 of a-synuclein (see Fig. 10C; SEQ ID NO: 17). In some cases, the binding polypeptide comprises an antibody recognizing an epitope of a-synuclein comprising residues 130-140 of a-synuclein (see Fig. 10C; SEQ ID NO: 17). In some cases, the binding polypeptide comprises an antibody recognizing an epitope of a-synuclein located within residues 118-140 of a-synuclein (see Fig. 10C; SEQ ID NO: 17). In some cases, the binding polyeptide comprises NbSyn87 (SEQ ID NO: 14).

[0110] In some embodiments, the E3 ubiquitin ligase utilized with these binding polypeptides, for example, in the creation of a fusion polypeptide, comprises at least the catalytic domain of a mammalian or human E3 ubiquitin ligase. For instance, in some embodiments, the E3 ubiquitin ligase comprises the catalytic domain of any one of betaTrCP, CHIP, SPOP, or VHL. See Ludwicki et al., ACS Cent. Sci. 5: 852-866 (2019), Supplemental Table 1. In some cases, such an E3 ubiquitin ligase is a multi-subunit protein, and its catalytic domain may comprise more than one subunit of the protein. For example, the term SPOP refers to the RBXl-Cul3-SPOP E3 ligase complex; VHL refers to the RBXl-Cul2-EloBC-VHL E3 ligase complex; CRBN refers to the RBXl-Cul4A-DDBl-CRBN E3 ligase complex. In such multi-subunit ligases, the catalytic domain comprises the portion of the protein that binds to the scaffold / adaptor protein to recruit the E2 -binding domain. For example, the catalytic domain of SPOP comprises the BTB and BACK domains, which bind to Cul3 and recruit the E2 -binding RBX1 protein.

[0111] A. Methods of Use to Degrade Alpha-Synuclein in Cells

[0112] The fusion polypeptides recognizing a-synuclein may be used, for example, in vitro, such as in cell culture, in some embodiments. In some embodiments, the cells may be eukaryotic cells that are capable of degrading proteins via the ubiquitination pathway, such as mammalian cells. In some cases, the cells are human cells. In some cases, the cells are neural cells, such as cultured neural cells.

[0113] To degrade a target protein in a cell, for example, one may introduce a fusion polypeptide as described herein to a cell, wherein the cell endogenously expresses a-synuclein, or wherein the cell is engineered to express a-synuclein. For example, a non-human animal or mammalian cell, such as a neural cell, may be engineered to express human a-synuclein by introducing heterologous human a-synuclein and, if needed, knocking out the native mammalian a- synuclein. In some cases, a cell may be engineered to express human a-synuclein along with a reporter polypeptide such as a fluorescent protein, so that fluorescence level can be used to track degradation of the human a-synuclein in the cell. In some embodiments, one may introduce a polynucleotide expressing the fusion polypeptide to the cell. In some cases, the polynucleotide comprises an inducible promoter for expression of the fusion polypeptide, such as a doxycycline (Dox) or tetracycline (Tet) inducible promoter. Thus, in some such cases, one may induce expression of the fusion polypeptide in the cell. In some cases, the polynucleotide further encodes a reporter protein in order to confirm transformation of the cells with the polynucleotide.

[0114] In some embodiments, the fusion polypeptide reduces the level of a target protein in a cell in vitro compared to the level observed in the absence of the fusion polypeptide. In some cases, the change in the level of the target protein is observed by flow cytometry, compared to the level observed in the absence of the fusion polypeptide or the level observed with a mutant fusion polypeptide that lacks E3 ubiquitin ligase activity or that lacks the ability to bind to the peptide expression tag. In some cases, the change in the level of the target protein is observed by attaching the target protein to a reporter molecule such as a reporter polypeptide, and detecting the level or activity of the reporter. In other cases, the change in the level of the target protein is observed by detecting the level or activity of the target protein directly, such as by a binding or activity assay.

[0115] In some embodiments, the methods further comprise detecting the expression level, activity, or function, or concentration of the a-synuclein before and / or after introduction of the fusion polypeptide. As noted above, the a-synuclein may be fused to a detectable moiety, such as a fluorescent protein, such as green fluorescent protein (GFP), tdTomato, mCherry, or EGFP or the like, that may be used to detect its concentration or expression in the cell in the presence and absence of the fusion polypeptide. In other embodiments, an activity assay may be performed to detect degradation of the a-synuclein. In other embodiments, the expression or level of the a-synuclein may be detected by methods such as in situ hybridization, immunohistochemistry, ELISA, Western blotting, flow cytometry, or the like. In some embodiments, methods herein may be used, for example, to provide model systems to study the function of a-synuclein in a cellular environment in vitro or in vivo. Such models may be useful, for example, in target validation and a variety of protein function studies and for testing potential drug molecules.

[0116] In some embodiments, the methods herein result in at least a 50% reduction in the concentration, activity, or expression level of the a-synuclein in vitro in the cell. In some embodiments, the methods herein result in at least 60% reduction in the concentration, activity, or expression level of the a-synuclein in vitro in the cell. In some embodiments, the methods herein result in at least 70% reduction in the concentration, activity, or expression level of the a- synuclein in vitro in the cell. In some embodiments, the methods herein result in at least 80% reduction in the concentration, activity, or expression level of the a-synuclein in vitro in the cell. In some cases, the percent reduction is relative to the level in the absence of introduction of the fusion polypeptide. In other cases, the percent reduction is relative to the level with introduction of a control fusion polypeptide that either cannot bind to the a-synuclein or that does not have E3 ubiquitin ligase activity. In some embodiments, the reduction in the concentration, activity, or expression level of the a-synuclein in an in vitro or in vivo model system is reversible, such as when the method employs an inducible promoter system. In some embodiments, the reduction in the level of the a-synuclein in vitro is measured by Western blot.

[0117] B. Methods of Degrading Alpha-Synuclein for Therapeutic Use

[0118] In some embodiments, fusion polypeptides herein, or polynucleotides encoding the fusion polypeptides, or vectors or cells comprising the polynucleotides, may be used for therapeutic treatment of a subject. For example, in some embodiments, a fusion polypeptide or polynucleotide, vector or cell may be used for treating a neurodegenerative disease in a subject.

[0119] A “subject” as used herein refers to a human unless expressly stated otherwise (e.g, a murine subject or the like). In some embodiments, methods of treating other mammals, including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided.

[0120] The term “treating” or “treatment,” as used herein, covers any administration or application of a therapeutic for disease in a human, or other mammal, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting or slowing its development, inhibiting, reducing, or slowing development of at least one symptom of the disease, slowing the time to onset of the disease, preventing onset of at least one disease symptom, slowing the time to onset of at least one disease symptom, partially or fully relieving the disease, or curing the disease, for example, by causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. The terms “inhibition” or “inhibit” refer to a decrease or cessation of any symptom or phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that symptom or characteristic.

[0121] A “neurodegenerative disease” refers to a disease or disorder characterized by loss of structure or function of or damage to neural cells. In some cases, the loss of structure or function of neural cells is progressive over time.

[0122] In some embodiments, the neurodegenerative disease is Alzheimer’s disease, taupathy, synucleinopathy, Huntington’s disease, Parkinson’s disease, frontotemporal dementia, dementia, dementia with Lewy bodies (DLB), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), multiple-system atrophy (MSA), Nasu-Hakola disease, Guillain-Barre Syndrome (GBS), lysosomal storage disease, sphingomyelinlipidosis (Neimann-Pick C), mucopolysaccharidosis II / IIIB, metachromatic leukodystrophy, multifocal motor neuropathy, neuro-Behcet’s disease, neuromyelitis optica (NMO), optic neuritis, polymyositis, dermatomyositis, stroke, transverse myelitis, traumatic brain injury, or spinal cord injury. In some embodiments, the neurodegenerative disease is synucleinopathy, Parkinson’s disease, dementia, dementia with Lewy bodies (DLB), Alzheimer’s disease, amyotrophic lateral sclerosis, multiple-system atrophy, or frontotemporal dementia.

[0123] For example, a-synuclein is a neural protein that may regulate synaptic vesicle trafficking and release of neurotransmitters, and which is found in the brain, such as in presynaptic neurons. In a variety of neurodegenerative diseases such as Parkinson’s disease and dementia, for example, insoluble forms of a-synuclein have been found to accumulate as inclusion bodies and Lewy bodies in affected brain regions. See, e.g., Estaun-Panzano et al., Neurobiol. Dis. 176: 105966 (Jan, 2023); Burre et al., Cold Spring Harbor Persp. Med., 8(3): a024091 (2018). Synucleinopathy, for example, comprises neurodegenerative disorders characterized by inclusion or Lewy bodies comprising such a-synuclein aggregates. See Id. Parkinson’s disease is also associated with mutations in a-synuclein. Accordingly, such diseases may be treated by therapeutic regimens targeting a-synuclein, such as to degrade a-synuclein in the brain.

[0124] In some embodiments, a polypeptide or polynucleotide may be administered to the subject, for instance, in a composition comprising at least one carrier or excipient, such as a surfactant, phospholipid, poly-A binding protein, or albumin. In some cases, a polynucleotide may be comprised within a liposomal nanoparticle (LNP). In such cases, the polynucleotide may be DNA or RNA. In some cases, the polypeptide or polynucleotide is delivered to the subject using a means to transform brain or neural cells, such as a viral vector that is capable of transforming brain or neural cells in vivo. In some embodiments, the viral vector is an adenoassociated virus (AAV) or lentivirus vector. In some embodiments, wherein the viral vector is AAV, the polynucleotide encoding the fusion polypeptide comprises no more than 4500 nucleobases in length, such as no more than 4000 nucleobases in length. In some cases, the polynucleotide may be comprised within a virus-like particle (VLP).

[0125] In some embodiments, administration of the fusion polypeptide herein in vivo in a murine model results in at least a 30% reduction in the level of a-synuclein in the mouse brain, for example, as measured by ELISA. In some embodiments, administration of the fusion polypeptide herein in vivo in a murine model results in at least a 40% reduction in the level of a- synuclein in the mouse brain, for example, as measured by ELISA. In some embodiments, administration of the fusion polypeptide herein in vivo in a murine model results in at least a 45% reduction in the level of a-synuclein in the mouse brain, for example, as measured by ELISA. In some embodiments, administration of the fusion polypeptide herein in vivo in a murine model results in at least a 50% reduction in the level of a-synuclein in the mouse brain, for example, as measured by ELISA. In some cases, the percent reduction is in comparison to control mice treated with a non-functional fusion polypeptide, such as one that does not encode a binding polypeptide for a-synuclein, and / or that does not have a functional E3 ubiquitin ligase catalytic domain.

[0126] C. Additional Exemplary Embodiments

[0127] The disclosure herein also relates to the following exemplary embodiments, related to fusion polypeptides recognizing a-synuclein, such as human a-synuclein, and their uses.

[0128] Embodiment l is a fusion polypeptide comprising an E3 ubiquitin ligase and a binding polypeptide that specifically recognizes human a-synuclein.

[0129] Embodiment 2 is the fusion polypeptide of embodiment 1, wherein the binding polypeptide is an antibody.

[0130] Embodiment 3 is the fusion polypeptide of embodiment 2, wherein the antibody does not comprise a full length constant region.

[0131] Embodiment 4 is the fusion polypeptide of embodiment 2 or 3, wherein the antibody is a single chain antibody, monobody, camelid antibody, or an antigen binding fragment such as an Fv, Fab, F(ab)2, or ScFv.

[0132] Embodiment 5 is the fusion polypeptide of embodiment 2 or 3, wherein the antibody is a camelid antibody or an antigen binding fragment derived from a camelid antibody. Embodiment 6 is the fusion polypeptide of any one of embodiments 1-5, wherein the binding polypeptide comprises 170 amino acid residues or less, such as 100-170 amino acid residues.

[0133] Embodiment 7 is the fusion polypeptide of any one of embodiments 1-6, wherein the E3 ubiquitin ligase comprises the catalytic domain of a mammalian E3 ubiquitin ligase.

[0134] Embodiment 8 is the fusion polypeptide of any one of embodiments 1-6, wherein the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase.

[0135] Embodiment 9 is the fusion polypeptide of embodiment 7 or 8, wherein the E3 ubiquitin ligase does not comprise a native substrate binding domain.

[0136] Embodiment 10 is the fusion polypeptide of embodiment 7, 8, or 9, wherein the catalytic domain comprises a NEL, RING / Ubox, Fbox, or ELECT catalytic domain.

[0137] Embodiment 11 is the fusion polypeptide of embodiment 8, wherein the E3 ubiquitin ligase comprises a NEL catalytic domain.

[0138] Embodiment 12 is the fusion polypeptide of embodiment 8, wherein the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase derived from a Shigella, Pseudomonas, Legionella, E. coli or Salmonella species.

[0139] Embodiment 13 is the fusion polypeptide of embodiment 8, wherein the E3 ubiquitin ligase comprises the NEL domain of Shigella flexneri IpaH9.8.

[0140] Embodiment 14 is the fusion polypeptide of embodiment 8, wherein the E3 ubiquitin ligase comprises the catalytic domain of AvrPtoB, IpaH0722, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, LegAU13, LegUl, LubX, NleG2-3, HleG5-l, NleL, SidC, SirP, SopA, SspHl, SspH2, or XopL.

[0141] Embodiment 15 is the fusion polypeptide of embodiment 7, wherein the E3 ubiquitin ligase comprises the catalytic domain of a human E3 ubiquitin ligase.

[0142] Embodiment 16 is the fusion polypeptide of embodiment 15, wherein the E3 ubiquitin ligase comprises the catalytic domain of any one of betaTrCP, CHIP, SPOP, or VHL.

[0143] Embodiment 17 is the fusion polypeptide of any one of embodiments 1-16, wherein there is no linker between the binding polypeptide and the E3 ubiquitin ligase.

[0144] Embodiment 18 is the fusion polypeptide of any one of embodiments 1-16, wherein the fusion polypeptide comprises a linker between the binding polypeptide and the E3 ubiquitin ligase.

[0145] Embodiment 19 is a complex comprising the fusion polypeptide of any one of embodiments 1-18 and a-synuclein. Embodiment 20 is a polynucleotide comprising a coding sequence for the fusion polypeptide of any one of embodiments 1-18.

[0146] Embodiment 21 is the polynucleotide of embodiment 21, wherein the coding sequence for the fusion polypeptide is under the control of an inducible promoter.

[0147] Embodiment 22 is the polynucleotide of embodiment 22, wherein the inducible promoter comprises a doxycycline (Dox) inducible promoter or a tetracycline (Tet) inducible promoter.

[0148] Embodiment 23 is a vector comprising the polynucleotide of any one of embodiments 20- 22.

[0149] Embodiment 24 is the vector of embodiment 23, wherein the vector is a viral vector or a virus-like particle (VLP).

[0150] Embodiment 25 is the vector of embodiment 24, wherein the vector is an AAV vector, or a lentivirus vector.

[0151] Embodiment 26 is the vector of any one of embodiments 23-25, wherein the vector is capable of targeting the polynucleotide to the brain, and / or wherein the vector is capable of transforming brain or neural cells.

[0152] Embodiment 27 is the polynucleotide or vector of any one of embodiments 20-26, wherein the polynucleotide further encodes a reporter polypeptide, wherein the reporter polypeptide acts as a marker for cells transfected with the polynucleotide.

[0153] Embodiment 28 is the polynucleotide or vector of any one of embodiments 20-27, wherein the polynucleotide is RNA, optionally wherein the RNA further comprises a polyadenosine segment at the 3’ terminus.

[0154] Embodiment 29 is a composition comprising the polynucleotide or vector of any one of embodiments 20-28, wherein the composition further comprises at least one excipient or carrier, such as a surfactant, phospholipid, poly-A binding protein, or albumin.

[0155] Embodiment 30 is a composition comprising the polynucleotide or vector of any one of embodiments 20-28, which is a liposomal nanoparticle (LNP) composition.

[0156] Embodiment 31 is a cell comprising the polynucleotide or vector of any one of embodiments 20-28, wherein the cell is a eukaryotic cell, such as a mammalian cell, such as a mammalian neural cell.

[0157] Embodiment 32 is the cell of embodiment 31, wherein the cell is a human cell, such as a human neural cell.

[0158] Embodiment 33 is a method of degrading a-synuclein in a cell, the method comprising introducing to the cell a fusion polypeptide according to any one of embodiments 1-18 or a polynucleotide or vector according to any one of embodiments 20-28 encoding the fusion polypeptide or the composition of embodiment 29 or 30.

[0159] Embodiment 34 is a method of degrading a-synuclein in a cell, the method comprising introducing to the cell a polynucleotide or vector according to any one of embodiments 20-28 encoding a fusion polypeptide or the composition of embodiment 29 or 30 comprising the polynucleotide, and inducing expression of the fusion polypeptide in the cell.

[0160] Embodiment 35 is the method of embodiment 33 or 34, further comprising detecting the expression level of a-synuclein in the cell before and / or after introduction of the fusion polypeptide, polynucleotide, or composition to the cell.

[0161] Embodiment 36 is the method of any one of embodiments 33-35, wherein the method comprises introducing a polynucleotide in which expression of the fusion polypeptide encoded by the polynucleotide is under the control of an inducible promoter.

[0162] Embodiment 37 is the method of embodiment 36, wherein the inducible promoter comprises a doxycycline (Dox) inducible promoter or a tetracycline (Tet) inducible promoter.

[0163] Embodiment 38 is the method of embodiment 36 or 37, wherein the level of a-synuclein in the cell is reduced upon activation of the inducible promoter.

[0164] Embodiment 39 is the method of any one of embodiments 33-38, wherein the cell is an eukaryotic cell, such as a mammalian cell, such as a mammalian neural cell.

[0165] Embodiment 40 is the method of embodiment 39, wherein the cell is a human cell, such as a human neural cell.

[0166] Embodiment 41 is a kit comprising the fusion polypeptide according to any one of embodiments 1-18 or the polynucleotide or vector according to any one of embodiments 20-28 encoding the fusion polypeptide or the composition of embodiment 29 or 30.

[0167] Embodiment 42 is a method of treating a neurodegenerative disease in a subject, comprising administering an effective amount of the fusion polypeptide according to any one of embodiments 1-18 or the polynucleotide or vector according to any one of embodiments 20-28 encoding the fusion polypeptide or the composition of embodiment 29 or 30 to the subject.

[0168] Embodiment 43 is the method of embodiment 42, wherein the subject suffers from synucleinopathy, Parkinson’s disease, dementia, dementia with Lewy bodies (DLB), Alzheimer’s disease, amyotrophic lateral sclerosis, multiple-system atrophy, or frontotemporal dementia.

[0169] Embodiment 44 is the fusion polypeptide according to any one of embodiments 1-18 or the polynucleotide or vector according to any one of embodiments 20-28 encoding the fusion polypeptide or the composition of embodiment 29 or 30 for use in treating a neurodegenerative disease in a subject.

[0170] Embodiment 45 is the fusion polypeptide, polynucleotide, vector, or composition of embodiment 44, wherein the neurodegenerative disease is synucleinopathy, Parkinson’s disease, dementia, dementia with Lewy bodies (DLB), Alzheimer’s disease, amyotrophic lateral sclerosis, multiple-system atrophy, or frontotemporal dementia.

[0171] Embodiment 46 is the use of the fusion polypeptide according to any one of embodiments 1-18 or the polynucleotide or vector according to any one of embodiments 20-28 encoding the fusion polypeptide or the composition of embodiment 29 or 30 in the preparation of a medicament for treating a neurodegenerative disease in a subject.

[0172] Embodiment 47 is the use of embodiment 46, wherein the neurodegenerative disease is synucleinopathy, Parkinson’s disease, dementia, dementia with Lewy bodies (DLB), Alzheimer’s disease, amyotrophic lateral sclerosis, multiple-system atrophy, or frontotemporal dementia.

[0173] Embodiment 48 is a method of treating a neurodegenerative disease in a subject, comprising administering an effective amount of a viral vector or virus-like particle (VLP) encoding the fusion polypeptide of any one of embodiments 1-18 to the subject, wherein the vector or VLP is capable of transforming brain or neural cells.

[0174] Embodiment 49 is the method of embodiment 48, wherein the viral vector is an AAV vector or lentiviral vector.

[0175] Embodiment 50 is the method of embodiment 48 or 49, wherein the neurodegenerative disease is synucleinopathy, Parkinson’s disease, dementia, dementia with Lewy bodies (DLB), Alzheimer’s disease, amyotrophic lateral sclerosis, multiple-system atrophy, or frontotemporal dementia.

[0176] Embodiment 51 is a viral vector or virus-like particle (VLP) encoding the fusion polypeptide of any one of embodiments 1-18 for use in treating a neurodegenerative disease in a subject, wherein the vector or VLP is capable of transforming brain or neural cells.

[0177] Embodiment 52 is the viral vector of embodiment 51, wherein the viral vector is an AAV vector or lentiviral vector.

[0178] Embodiment 53 is the viral vector of embodiment 51 or 52, wherein the neurodegenerative disease is synucleinopathy, Parkinson’s disease, dementia, dementia with Lewy bodies (DLB), Alzheimer’s disease, amyotrophic lateral sclerosis, multiple-system atrophy, or frontotemporal dementia. Embodiment 54 is the use of a viral vector or virus-like particle (VLP) encoding the fusion polypeptide of any one of embodiments 1-18 in the preparation of a medicament for treating a neurodegenerative disease in a subject, wherein the vector or VLP is capable of transforming brain or neural cells.

[0179] Embodiment 55 is the use of embodiment 54, wherein the viral vector is an AAV vector or lentiviral vector.

[0180] Embodiment 56 is the use of embodiment 54 or 55, wherein the neurodegenerative disease is synucleinopathy, Parkinson’s disease, dementia, dementia with Lewy bodies (DLB), Alzheimer’s disease, amyotrophic lateral sclerosis, multiple-system atrophy, or frontotemporal dementia.

[0181] IV. EXAMPLES

[0182] The following are examples of methods and compositions of the disclosure. It is understood that various other embodiments may be practiced, given the general description provided above.

[0183] Example 1: Overexpressed fusion proteins containing the ALFA tag could be efficiently degraded by a fusion polypeptide comprising an anti-ALFA camelid antibody (NbALFA) and the catalytic domain of a bacterial E3 ligase

[0184] To test the idea whether small tags could be used to replace large tags like EGFP to target protein for degradation (Fig. 1A), a newly reported 14-15 amino acid long tag named ALFA (e.g., SEQ ID NO: 1) was used, which has a high affinity nanobody (NbALFA) available46. A bacterial E3 ligase IpaH9.8 was used, which has well defined substrate binding domain at its N-terminus (Leucine-rich repeats; LRR) and catalytic domain (novel E3 ligase; NEL) at C- terminus49. First, HEK293T stable cell pools were derived, expressing a fusion protein comprising mCherry, ALFA tag and human a-synuclein (Fig. 7A); then transiently transfected these cells with a plasmid encoding the artificial bacterial E3 ligase (ABEL), in which the substrate binding domain LRR is replaced with the high affinity nanobody against the ALFA tag (NbALFA). To focus the analysis on transfected cells, a nuclear localized tagBFP fluorescent protein was added to the C-terminus of ABEL via a T2A peptide linker. About 24 hours later, the cells were collected for flow cytometry analysis (Fig. 7B). The median fluorescent intensity (MFI) level of mCherry in tagBFP+ cells was quantified and normalized to cells transfected with a plasmid that only encode tagBFP. As shown in Fig. IB, dramatic reduction of mCherry signal was observed in tagBFP+ cells transfected with Nb ALFA-AB EL, but not in the ones with a control fusion polypeptide called NbALFA*-ABEL, which has point mutations in NbALFA that destroys the binding to the ALFA tag, and the one with a control fusion polypeptide called NbALFA-dABEL, which has a C337A mutation in the catalytic NEL domain that abolishes the catalytic activity. Quantification of the relative MFI showed more than 80% reduction of mCherry signal in Nb ALFA- ABEL transfected cells, but not the mutant controls (NbALFA*- ABEL and NbALFA-dABEL) (Fig. 1C). Western blot with either anti-mCherry or anti-a- synuclein antibodies confirmed the reduction of the mCherry signal is indeed due to protein degradation, but not the conformational change of the mCherry afte the binding of the Nb ALFA- ABEL (Fig. 8A). Similar experiments on fusion proteins with the ALFA tag at the N-terminus or C-terminus showed similar results (Fig. 8B; Fig. 9A). These data suggest that a small tag, like the ALFA tag, could mediate efficient degradation of a protein of interest (POI).

[0185] Example 2: IpaH9.8-based ABEL consistently shows better degradation efficiency when compared to similar mammalian E3-ligase-based artificial E3 ligases

[0186] Artificial mammalian E3 ligases (AMELs) are reported to have substrate selectivity, i.e., while one AMEL may be able to degrade a specific substrate efficiently, it may not be the case for a different substrate50. The degradation efficiency of ABELs against different target proteins was assessed. Six different HEK293T cell pools that express six different fusion proteins were prepared, all with a fluorescent protein (tdTomato, mCherry or EGFP) and the ALFA tag, and transfected with six different plasmids encoding IpaH9.8-based ABEL or SPOP, KLHL6, FBXL15, CRBN, VHL-based AMELs (Fig. 2). The data showed that while SPOP-AMEL, KLHL6-AMEL and VHL-AMEL could degrade the TDP43 Q331K-EGFP-ALFAtag fusion protein efficiently, they were not good at degrading other fusion proteins. In fact, most of the fusion proteins could not be efficiently degraded by most of the AMELs, although these mammalian E3 ligases are either frequently used (SPOP, VHL, CRBN) or found to be very insensitive to substrate change50. In contrast, IpaH9.8-based ABEL consistently showed efficient degradation of all substrates tested, whether they are cytoplasmic, nuclear or membranous (Fig. 2). These data suggest that the IpaH9.8-based ABEL might be better suited as a tool for knocking down most POIs in mammalian cells.

[0187] Example 3: Reversible temporal control of the degradation of target protein with Dox induced expression of ABEL

[0188] One of the greatest advantages to modulate gene function at protein level, but not at genomic DNA level, is the opportunity of temporal control and reversibility. To test the temporal control and reversibility of the ASTABEL platform, a Dox-inducible Nb ALFA-based ABEL expression cassette was introduced into HEK293 cells stably expressing the mCherry -ALFAtag- human a-synuclein fusion protein (Fig. 3 A). First, the dosage-response of the degradation of the fusion protein to increasing concentration of Doxycyclin was tested. Indeed, higher concentration of Dox induced the expression of ABEL, reflected by the expression of tagBFP, in more cells and the subsequent loss of the mCherry signal (Fig. 3B, C). However, at single cell level, it seemed that the expression of ABEL and the degradation of the fusion protein was bi- modal. In other words, for a particular cell, it is either induced to express ABEL and degrade the fusion protein or not induced and maintain the fusion protein (Fig. 3B, E). To test the reversibility of the degradation of the fusion protein, cells were treated with lug / ml Doxycyclin (Fig. 3D). Within 12 hours, a significant amount of cells were tagBFP+ and these cells lost mCherry signal; at 24 hours, more cells started to express ABEL and lose mCherry signal (Fig. ID, IE); Doxycyclin was washed away and followed the disappearance of tagBFP signal and the recovery of mCherry signal was observed. Within 48 hours post withdrawal of Doxycyclin, the majority of cells lost tagBFP expression and re-gained mCherry signal. After another 72 hours (120h post Doxycyclin withdrawal), nearly all cells regain mCherry signal. When Dox was readministered into these cells, tagBFP (ABEL) expression was induced again and the fusion protein was degraded again (Fig. 3D-3F). These data suggest that the degradation of the fusion protein is tightly regulated by Doxycyclin and could be temporally controlled in a reversible manner.

[0189] Example 4: The tagging and Dox-controlled degradation of endogenous type I membrane protein PD-L1 in MC38 cells with ASTABEL

[0190] So far, the data showed that ABEL could be employed to efficiently degrade overexpressed fusion proteins containing the ALFA tag. Tagging an endogenous gene with the ALFA tag and then explored to test degradation of such a tagged endogenous protein. First, MC38 cells with the Dox-inducible ABEL-expression cassette were derived and used as parental cells for the tagging of PD-L1 alleles (Fig. 4B). CRISPR / Cas9-meidated HDR-based knock-in strategy was used to tag both alleles of PD-L1 gene at the C-terminus right before the stop codon in mouse MC38 cells. The small size of the ALFA tag (42 bases) made it possible to use short oligo donors as recombination template, which is only 142-base long, including 50-base homology arm on each side (Fig. 4A). Cells nucleofected with a sgRNA / Cas9 RNP complex as well as the oligo donor were sorted into 96-well plate two days post nucleofection, with aims to get one clone per well. Screening of about 50 clones with PCR identified four with both alleles tagged, which were further confirmed with Sanger sequencing (Fig. 4B). Dox-treatment of all four homozygote clones and the parental cells show dramatic reduction of PD-L1 protein, based on flow cytometry analysis post anti-PD-Ll surface staining with fluorescence dye conjugated antibody or Western blot, in three homozygote clones (1F4, 2A6 and 2D1) (Fig. 4. C, D). Clone 2A12 showed dramatic reduction of PD-L1 level in Western blot but much less in flow cytometry and the reason is not further investigated. These data suggest that ASTABEL could be a powerful tool to modulate endogenous protein level.

[0191] Example 5: Efficient Degradation of OMM (outer membrane of mitochondria) and ER Proteins with ASTABEL

[0192] Proteins located in OMM and ER have been shown to be degradable with PROTAC- recruited endogenous E3 ligases such as VHL and CRBN, although the efficiency various dramatically based on the PROTAC and the tag used51. Degradation of OMM and ER proteins with ASTABEL were next explored. HEK293T cell lines that express fusion proteins containing the ALFA tag and mCherry in OMM and ER, respectively, were prepared (Fig. 5). The location of the fusion proteins are directed to the OMM or ER with either the C-terminus domain of Fi852or ER retention signal KDEL53, and their correct location was confirmed by immunostaining with OMM marker ATP0 and ER marker Calnexin (Fig. 5). When these cells were transfected with Nb ALFA-based ABEL, around 90% of mCherry signal was reduced, suggesting extremely efficient degradation of these proteins, comparable or even better than those in the cytosol, nuclear or membrane (Fig. 5).

[0193] Example 6: The HiBiT tag could be used for targeted protein degradation and quantification

[0194] The 11 -amino-acid HiBiT (SEQ ID NO: 2) has been used to tag endogenous protein for quantification purpose since its high affinity to the complementary LgBiT fragment in live cells, in the cell lysate and in the Hibit-blotting buffer reconstitutes the nanoluciferase activity47The strong interactions between HiBiT and LgBiT were used to direct HiBiT-tagged protein for degradation. Stable HEK293T cell pools expressing an mCherry-ALFAtag-HiBiT fusion protein were made and then transfected with various plasmids encoding LgBiT-based ABELs (Fig.6A). All of these LgBiT-based ABELs were able to mediate efficient but different degree of degradation of the fusion protein. Among those, the one with LgBiT at the N-terminus and the NEL domain of IpaH9.8 at the C-terminus had the highest degradation activity. In contrast, a similar plasmid with the dead NEL domain did not show any degradation activity (Fig. 6A). Similar to the ALFA tag, the HiBiT tag could be at the N-terminus or in the middle of the fusion protein (Fig. 9). Next, the use of the same HiBiT tag for protein quantification with LgBiT-based HiBiT blotting post LgBiT-based ABEL-mediated degradation was investigated. 24 hours posttransfection of HEK293T cells stably expressing mCherry-ALFA-HiBiT fusion protein with various ABEL plasmids, cells were lysated, denatured and separated on SDS-PAGE, transferred to nitrocellulose membrane and detected with HiBiT blot or regular Western blot with antibodies against the ALFA tag or 6XHis tag (SEQ ID NO: 7) (which is fused to ABELs) (Fig. 6B). Cells transfected with both NbALFA and LgBiT-based ABELs showed dramatic reduction of the fusion protein with LgBiT-based HiBiT blotting, consistent with the results with anti-ALFA Western blot, suggesting that the same HiBit tag could be used for both mediating the degradation and the detection of the fusion protein. The denaturation and SDS-PAGE steps were sufficient to destroy the strong interactions between HiBiT and LgBiT, as shown in the cells transfected with the LgBiT-based dABEL (note the weak band marked with red star, which is the complex between the fusion protein and LgBiT-dABEL; Fig. 6B). Next, whether the LgBiT- ABEL could be used to degrade HiBiT-tagged endogenous protein was tested. A Dox-inducible LgBiT-ABEL-expressing plasmid was introduced into HeLa cells in which one allele of the SMARCA2 was tagged with the HiBiT tag. Upon Dox treatment, efficient degradation of SMARCA2 was observed when compared to the control cells treated with DMSO, suggesting that LgBiT-ABEL could mediate the efficient degradation of HiBiT-tagged endogenous protein (Fig. 6C). This was further confirmed with HiBiT-tagged GSPT1 protein in HEK293 cells. Note that the presence of the LgBiT protein from the expression of a stably integrated plasmid, which was introduced for easy quantification of HiBiT-tagged GSPT1 in cell lysate, did not prevent the LgBiT-ABEL mediated degradation (Fig. 6D). Similar to what has been observed with the transgenes (Fig. 6A and 9), the HiBiT tag-mediated degradation regardless of the HiBiT tag position (i.e., placed at the N-terminus or C-terminus) (Fig. 6C and 6D).

[0195] Example 7: A fusion polypeptide comprising a nanobody against a-synuclein (NbSyn87) and a bacterial E3 ligase catalytic domain (NEL) mediates degradation of human a- synuclein fusion protein in cultured HEK293 cells

[0196] Fusion polypeptides directed against a-synuclein, which has multiple high affinity nanobodies available in the literature54-57(Fig. 10A), were tested in order to determine if a- synuclein could be degraded by such fusion polypeptides. The fusion polypeptides comprised the bacterial E3 ligase IpaH9.8. Stable HEK293 cell lines expressing a fusion protein between mCherry, a recently reported 13 amino acid long ALFA tag58and the human a-synuclein were prepared. Those cells were then transiently transfected with plasmids encoding various fusion polypeptides comprising the IpaH9.8 E3 ubiquitin ligase and nanobodies or scFv against mCherry (LaM4)59or human a-synuclein (NbSyn87, NbVH14 and NbSyn2) (Fig. 10B, C). To exclude the effect of transfection efficiency, a nuclear localized tagBFP fluorescent protein is added to the C-terminus via a T2A peptide linker so only transfected cells (tagBFP+) would be analyzed. 24 hours post nucleofection, cells were analyzed with flow cytometry; the median fluorescence intensity of mCherry in tagBFP+ cells was quantified and normalized to the cells transfected with a plasmid that only expresses tagBFP to assess the degradation of the mCherry- ALFAtag-ha-synuclein fusion protein. Fusion polypeptides comprising bacterial E3 ligases (ABELs) and the mCherry nanobody LaM4 (LaM4-ABEL) or a-synuclein nanobody NbSyn87 (NbSyn87-ABEL) mediated about 80% reduction of the mCherrry-ALFAtag-a-synuclein fusion protein, compared to fusion polypeptides comprising a-synuclein nanobody VH14 (VH14- ABEL) or NbSyn2 (NbSyn2-ABEL) (Fig. 10D and E). Western blot with either anti-mCherry or synuclein antibody confirmed the loss of the mCherry signal in the flow cytometry indeed reflects the degradation of the fusion protein (Fig. 10F). Furthermore, the degradation relied on the catalytic activity of the E3 ligase domain, as a single amino acid mutation of C337 into alanine in the IpaH9.8 E3 ligase abolished the degradation48. We also noticed that a short 7 amino acid linker (GSGSGSS; SEQ ID NO: 13) abolished the activity of NbSyn87-ABEL, suggesting that the proximity between ABEL and the mCherrry-ALFAtag-a-synuclein fusion protein substrate was critical.

[0197] Example 8: Wild type (non-fusion) a-synuclein could be efficiently degraded by NbSyn87- ABEL

[0198] To examine whether non-fusion a-synuclein could be degraded by NbSyn87-ABEL, stable HEK293T cells overexpressing human a-synuclein were derived and were transfected with various ABELs. Consistent with results of the flow cytometry experiments with the mCherrry-ALFAtag-a-synuclein, significant degradation of the overexpressed a-synuclein was observed with NbSyn87-ABEL, but not NbSyn2-ABEL and VH14-ABEL (Fig. 11 A). Similarly, cells transfected with the dead NbSyn87-ABEL (NbSyn87-dABEL), which possessed the C337A mutation, did not show any reduction of a-synuclein (Fig. 11 A). Consistent with the idea that degradation is mediated by 26S proteasome, treatment of cells with proteasome inhibitor MG132 blocked the reduction of a-synuclein (Fig. 11 A). To test whether the endogenously expressed a- synuclein could be efficiently degraded by NbSyn87-ABEL, two malignant melanoma-derived cell lines that are reported to express high levels of a-synuclein, MeWo and SK-Mel-2860, were transfected with control tagBFP, NbSyn87-ABEL and NbSyn87-dABEL plasmids. Transfected cells were enriched with FACS and the a-synuclein level was assessed with Western blot. Cells transfected with NbSyn87-ABEL, but not NbSyn87-dABEL, showed dramatic reduction of a- synuclein (Fig. 1 IB). Example 9: AAVl-based delivery of NbSyn87-ABEL mediates efficient degradation of human a-synuclein in primary neurons derived from BAC transgenic mice

[0199] To test the degradation of human a-synuclein in neurons, primary neurons were derived and cultured from embryonic brains of BAC transgenic mice expressing human a-synuclein. These neurons were infected with two different dosages of AAV1 viruses (MOI of 10K and 100K) encoding only mCherry, NbSyn87-ABEL or NbSyn87-dABEL. Western blot results showed dramatic reduction of human a-synuclein in primary neurons infected with viruses encoding NbSyn87-ABEL at a MOI of 10K (Fig. 10G). When the MOI was increased to 100K, near complete loss of human a-synuclein was achieved (Fig. 10G). In contrast, no reduction of human a-synuclein was observed in the neurons infected with control viruses (Fig. 10G). These data suggest that AAV-mediated delivery of NbSyn87-ABEL resulted in highly efficient degradation of human a-synuclein in the cultured mouse primary neurons.

[0200] Example 10: PHP.eB-AAV-based delivery of NbSyn87-ABEL into the adult brain of transgenic mouse leads to the reduction of overexpressed human a-synuclein

[0201] To test whether NbSyn87-ABEL could mediate efficient degradation of human a- synuclein in vivo, 1X1012PHP.eB-based AAV viruses encoding only a fluorescent protein mKate2, NbSyn87-ABEL or NbSyn87-dABEL were retro-orbitally injected into adult transgenic mice (Fig. 12A). Four weeks later, these animals were sacrificed and brain tissues were collected for analysis (Fig. 12B). ELISA quantification showed about 43% reduction of average human a- synuclein level in the cohort of 10 animals injected with PHP.eB viruses encoding NbSyn87- ABEL when compared to the ones with mKate2 (1.127ug / ml vs. 0.646ug / ml) (Fig. 12C). In contrast, no obvious reduction of average human a-synuclein level was observed in the 10 animals injected with viruses encoding NbSyn87-dABEL (1.122ug / ml). One way ANOVA analysis showed a p value of 0.0010. These data were further confirmed with Western blot analysis (Fig. 13). These data suggest that AAV-based delivery of NbSyn87-ABEL may be used to degrade human a-synuclein in vivo.

[0202] Example 11: Materials and Methods for Examples 1-4

[0203] Plasmids and AAVs

[0204] All plasmids were synthesized, cloned and purified by Genscript Inc. AAV 1 and PHP.eB -AAV were packaged and prepared by Virovek Inc.

[0205] Cell Culture

[0206] HEK293T, MeWo and SK-MEL-28 cells were obtained from an internal cell line depository. HEK293T and SK-Mel-28 cells were cultured in DMEM supplemented with 10% FBS and 2mM GlutaMAX; MeWo cells were cultured in RPMI-1640 supplemented with 10% FBS and 2mM GlutaMAX. HEK293T cells were either transfected with Lipofectamaine 2000 (Thermo Fisher Scientific Inc) or nucleofected with solution F (Lonza Inc) according to manufacturer’s recommendation; MeWo and SK-Mel-28 cells were transfected with Lipofectamaine 2000 (Thermo Fisher Scientific Inc).

[0207] To make stable HEK293T cell pools expressing mCherry-ALFAtag-ha-synuclein fusion protein, HEK293T cells were co-transfected with a PiggyBac-based plasmid containing the coding cassette for the fusion protein and a plasmid encoding the PiggyBac transposase. The cells were cultured / passaged for 10 days before FACS sorted to enrich for mCherry positive cells.

[0208] Flow cytometry and FACS

[0209] HEK293T cells stably expressing the mCherry-ALFAtag-ha-synuclein fusion protein were nucleofected with plasmids encoding various ABELs and analyzed with flow cytometry 24 hours later. Data were collected on BD FACSCelesta cell analyzer with FACSDiva software (BD Inc) and analyzed with FlowJo. Sorting of transfected MeWo and SK-Mel-28 cells was performed by FACS on a BD FACSAria Fusion cell sorter.

[0210] ELISA

[0211] Left cerebrum of each brain was weighed and lysed in RIPA buffer supplemented with Complete protease inhibitor (Roche Inc) at a ratio of 1ml buffer per lOOmg tissue with Qiagen TissueLyser II (Qiagen Inc). Tissue lysate was centrifuged at 12,500rpm for 15 minutes and supernatant (generally with a total protein concentration around 7ug / ul) was serially diluted by 10 folds to l,000X, which was further diluted by 2 folds to 2,000X, 4, 000X, 8,000Xand 16,000X. The last four dilutions were used for ELISA with Legend Max Human alpha-synuclein (colormetric) ELISA kit (Biolegend Inc.) according to manufacturer’s protocol. Plates were read with SpectralMax i3x (Molecular Devices Inc.) and data analyzed with Graphpad Prism 10.

[0212] Western blot

[0213] Cultured or sorted cells were lysed in IX Laemmli buffer supplemented with 2- mercapitoethanol. Supernatant of brain lysates prepared as above at a total protein concentration around 7ug / ul was mixed with equal volume of water and two times volume of 2XLaemmli buffer supplemented with 2-mercapitoethanol and boiled for 5-10 minutes. Samples were then loaded onto and separated with 4-20% Mini-Protean TGX Precast SDS-PAGE gel (Bio-Rad Inc), transferred to nitrocellulose membrane, blocked and detected with primary antibody followed by HRP-conjugated secondary antibody. To increase the detection sensitivity of human a-synuclein, nitrocellulose membrane was fixed in 0.4% paraformaldehyde for 30 minutes before blocked and detected with primary antibody. The following primary antibody was used: polyclonal Rabbit anti-synuclein antibody (Novus biologicals NBP2-15365; 1 :2, 000 dilution; for Western blot in Fig. 10F); human a-synuclein-specific mouse antibody (in-house developed; 1 : 10, 000 dilution; for the rest of the Western blot); polyclonal Rabbit anti-mCherry antibody (Novus biologicals NBP2-25157; 1 :250 dilution); monoclonal mouse anti-P-actin antibody AC-15 (Sigma- Aldrich A5441; 1 :10, 000); rabbit monoclonal anti-lamin Bl antibody (Invitrogen #702972; 1 : 1, 000 dilution); polyclonal rabbit anti-tubulin antibody (Cell Signaling Technology #2144; 1 : 1,000 dilution). HRP-conjugated goat anti-rabbit (ThermoFisher Scientific #32460; 1 :20,000 dilution) and goat anti-mouse (ThermoFisher Scientific #31430; 1 :20,000 dilution).

[0214] Total Synuclein IHC

[0215] Mice were anesthetized, perfused with cold PBS, followed by isolation and drop fixing of hemi-brains with 4% Paraformaldehyde (PF A). These hemi-brains were then sent to NeuroScience Associates, where they were incubated with 20% glycerol and 2% Dimethyl Sulfoxide overnight to prevent any freeze artifacts. The hemi-brains were embedded and prepared for coronal sectioning within a gelatin matrix utilizing MultiBrain® Technology (NeuroScience Associates, Knoxville, TN). The hemi-brains were sectioned coronally at a thickness of 30 pm via a microtome. Sections comprising the hippocampus and hypothalamus were selected for immunochemistry. After a series of washes with PBS and PBST, sections were blocked for one hour using a room temperature solution of 5% Donkey Serum (DS). Postblocking, the sections were incubated overnight at 4°C with the primary antibody (monoclonal anti human SNCA 31C2 (7.5mg / ml) developed internally at Roche Inc.) at 1 :2000 dilution in 0.5% DS. The following day, sections were washed with TBST and then treated with a donkey anti mouse secondary antibody (1 : 1000, Catalog: A-31571) for two hours at room temperature. Following another pair of PBST washes, sections were mounted with a gelatin solution and airdried. Lastly, a DAPI-containing ProLongTM Gold Antifade mounting medium (Catalog: P36931) was applied, the specimen was overlaid with a cover slip, and left to dry overnight.

[0216] Statistical analysis

[0217] All data were analyzed with Microsoft Excel and Graphpad Prism 10. One way ANOVA analyses were performed with Microsoft Excel.

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[0273] V. SEQUENCES

[0274] The table below provides exemplary sequences referred to in this disclosure.

Claims

WHAT IS CLAIMED IS:

1. A fusion polypeptide comprising an E3 ubiquitin ligase and a binding polypeptide that specifically recognizes a peptide expression tag wherein the peptide expression tag comprises 25 amino acids or less, 20 amino acids or less, or 15 amino acids or less.

2. The fusion polypeptide of claim 1, wherein the peptide expression tag comprises 6-25, 6-24, 8-24, 6-20, 8-20, 10-20, 10-15, 6-15, or 8-15 amino acids.

3. The fusion polypeptide of claim 1 or 2, wherein the peptide expression tag is an ALFA tag (SEQ ID NO: 1, optionally further comprising an N-terminal and / or C-terminal proline), a HiBiT tag (SEQ ID NO: 2), or a SPOT tag (SEQ ID NO: 3), or BC2 tag (SEQ ID NO: 4).

4. The fusion polypeptide of any one of claims 1-3, wherein the binding polypeptide is an antibody.

5. The fusion polypeptide of claim 4, wherein the antibody does not comprise a full length constant region.

6. The fusion polypeptide of claim 4 or 5, wherein the antibody is a single chain antibody, monobody, cam elid antibody, or an antigen binding fragment such as an Fv, Fab, F(ab)2, or ScFv.

7. The fusion polypeptide of claim 4 or 5, wherein the antibody is a camelid antibody or an antigen binding fragment derived from a camelid antibody.

8. The fusion polypeptide of any one of claims 1-7, wherein the binding polypeptide comprises 170 amino acid residues or less, such as 100-170 amino acid residues.

9. The fusion polypeptide of any one of claims 1-8, wherein the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase.

10. The fusion polypeptide of claim 9, wherein the E3 ubiquitin ligase does not comprise a native substrate binding domain.

11. The fusion polypeptide of claim 9 or 10, wherein the catalytic domain comprises a NEL, RING / Ubox, Fbox, or HECT catalytic domain.

12. The fusion polypeptide of claim 9, 10, or 11, wherein the E3 ubiquitin ligase comprises a NEL catalytic domain.

13. The fusion polypeptide of any one of claims 9-12, wherein the E3 ubiquitin ligase comprises the catalytic domain of a bacterial E3 ubiquitin ligase derived from a Shigella, Pseudomonas, Legionella, E. coli or Salmonella species.

14. The fusion polypeptide of claim 13, wherein the E3 ubiquitin ligase comprises the NEL domain of Shigella flexneri IpaH9.8.

15. The fusion polypeptide of any one of claims 9-12, wherein the E3 ubiquitin ligase comprises the catalytic domain of AvrPtoB, IpaH0722, IpaH1.4, IpaH2.5, IpaH4.5, IpaH7.8, IpaH9.8, LegAU13, LegUl, LubX, NleG2-3, HleG5-l, NleL, SidC, SirP, SopA, SspHl, SspH2, or XopL.

16. The fusion polypeptide of any one of claims 1-15, wherein there is no linker between the binding polypeptide and the E3 ubiquitin ligase.

17. The fusion polypeptide of any one of claims 1-15, wherein the fusion polypeptide comprises a linker between the binding polypeptide and the E3 ubiquitin ligase.

18. The fusion polypeptide of any one of claims 1-17, wherein the binding polypeptide specifically binds to a peptide expression tag on an extracellular membrane protein, endoplasmic reticulum membrane protein, Golgi membrane protein, mitochondrial outer membrane protein, nuclear protein, or cytosolic protein.

19. The fusion polypeptide of any one of claims 1-18, wherein the polypeptide is covalently or noncovalently attached to another molecule such as a lipid, surfactant, or another protein.

20. A complex comprising the fusion polypeptide of any one of claims 1-18 and a protein comprising a peptide expression tag that is specifically recognized by the binding polypeptide.

21. The complex of claim 20, wherein the protein comprising the peptide expression tag is an extracellular membrane protein, endoplasmic reticulum membrane protein, Golgi membrane protein, mitochondrial outer membrane protein, nuclear protein, or cytosolic protein.

22. A polynucleotide comprising a coding sequence for the fusion polypeptide of any one of claims 1-18.

23. The polynucleotide of claim 22, wherein the coding sequence for the fusion polypeptide is under the control of an inducible promoter.

24. The polynucleotide of claim 23, wherein the inducible promoter comprises a doxycycline (Dox) inducible promoter or a tetracycline (Tet) inducible promoter.

25. The polynucleotide of any one of claims 22-24, wherein the polynucleotide further encodes a reporter polypeptide, wherein the reporter polypeptide acts as a marker for cells transfected with the polynucleotide.

26. The polynucleotide of any one of claims 22-25, wherein the polynucleotide is RNA, optionally wherein the RNA further comprises a poly-adenosine segment at the 3’ terminus.

27. A composition comprising the polynucleotide of any one of claims 22-26 and at least one excipient or carrier, such as lipids, surfactants, or proteins such as albumin or poly A binding protein.

28. A composition comprising the fusion polypeptide of any one of claims 1-18 and at least one excipient or carrier, such as lipids, surfactants, or proteins.

29. A cell comprising the polynucleotide of any one of claims 22-26, wherein the cell is an eukaryotic cell, such as a mammalian cell.

30. A method of degrading a target protein in a cell, wherein the target protein comprises a peptide expression tag, wherein the peptide expression tag comprises 25 amino acids or less, 20 amino acids or less, or 15 amino acids or less, the method comprising introducing to the cell a fusion polypeptide according to any one of claims 1-19 or a polynucleotide according to any one of claims 22-26 encoding the fusion polypeptide or the composition of any one of claims 27-28, wherein the fusion polypeptide comprises a binding polypeptide that specifically recognizes the peptide expression tag.

31. A method of degrading a target protein in a cell, wherein the target protein comprises a peptide expression tag, wherein the peptide expression tag comprises 25 amino acids or less, 20 amino acids or less, or 15 amino acids or less, the method comprising introducing to the cell a polynucleotide according to any one of claims 22-26 or the composition of any one of claims 27 or 28, encoding the fusion polypeptide of any one of claims 1-18, wherein the fusion polypeptide comprises a binding polypeptide that specifically recognizes the peptide expression tag, and inducing expression of the fusion polypeptide in the cell.

32. The method of claim 30 or 31, further comprising detecting the expression level of the target protein in the cell before and / or after introduction of the fusion polypeptide, polynucleotide, or composition to the cell.

33. The method of any one of claims 30-32, wherein the method comprises introducing a polynucleotide in which expression of the fusion polypeptide encoded by the polynucleotide is under the control of an inducible promoter.

34. The method of claim 33, wherein the inducible promoter comprises a doxycycline (Dox) inducible promoter or a tetracycline (Tet) inducible promoter.

35. The method of claim 33 or 34, wherein the level of the target protein in the cell is reduced upon activation of the inducible promoter.

36. The method of any one of claims 30-35, wherein the cell is an eukaryotic cell, such as a mammalian cell.

37. The method of any one of claims 30-36, wherein the peptide expression tag comprises 6-25, 6-24, 8-24, 6-20, 8-20, 10-20, 10-15, 6-15, or 8-15 amino acids.

38. The method of any one of claims 30-37, wherein the peptide expression tag is an ALFA tag (SEQ ID NO: 1, optionally further comprising an N-terminal and / or C-terminal proline), a HiBiT tag (SEQ ID NO: 2), or a SPOT tag (SEQ ID NO: 3), or BC2 tag (SEQ ID NO: 4).

39. A kit comprising the fusion polypeptide of any one of claims 1-18 or the polynucleotide of any one of claims 21-25 or the composition of claim 26 or 27 or the fusion polypeptide of claim 28 or the cell of claim 29.

Images