Targeted Degradation of Alpha-Synuclein

Abstract

The present invention relates to a proteasome degradative protein complex comprising an E3 ubiquitin ligase component tethered to an alpha-synuclein specific polypeptide binding agent for the degradation of alpha-synuclein. The present invention also includes mechanisms of action, compositions, and related methods for the treatment of neurological disorders, including synucleinopathies.

Description

[Technical field]

[0001] The present invention relates to a proteasome degradative protein complex comprising an E3 ligase substrate receptor and linked to a specific binding agent for a target protein. In particular, the present invention relates to a proteasome degradative protein complex comprising an E3 ligase and linked to a specific polypeptide binding agent for degrading endogenous alpha-synuclein, and related methods and uses in the treatment of neurodegenerative disorders, etc. [Background technology]

[0002] The following discussion is presented to aid the reader in understanding the present disclosure and does not constitute any admissions as to the content or pertinence of prior art.

[0003] In the UK, Parkinson's disease is the second most common neurodegenerative disorder, affecting 1 in every 500 people. Intracellular proteinaceous inclusions known as Lewy bodies, found primarily within dopaminergic neurons, are a hallmark feature of Parkinson's disease. The main component of Lewy bodies is a small 140 kDa protein called α-synuclein, encoded by the SNCA gene.

[0004] The presence of α-synuclein in intracellular inclusions has also been found in various other neurodegenerative disorders, such as dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies. Synucleinopathies are associated with a variety of SNCA mutations, including variants of the SNCA gene, A53T, A30P, H50Q, E46K, G51D, and A53E, or double and triple duplications. Furthermore, aggregation of α-synuclein in dopaminergic neurons is associated with the pathogenesis and progression of synucleinopathies, and thus represents a potential target for therapeutic intervention. However, α-synuclein is considered to be a "undruggable" target by conventional small molecule approaches. Thus, there is a need for the development of new methods and therapeutic agents that target α-synuclein in synucleinopathies.

[0005] The technology of the AdPROM (affinity-directed protein missile) system was developed to target endogenous proteins for proteolysis (Fulcher LJ et al., 2017, "Targeting endogenous proteins for degradation through the affinity-directed protein missile system", Open Biol. 7: 170066). The AdPROM system exploits the ubiquitin-proteasome system by linking an E3 ligase component, such as the CUL2-CRL substrate receptor, the von Hippel-Lindau (VHL) tumor suppressor gene, to a target-specific polypeptide binder, such as an antibody or fragment thereof. By tethering the E3 ligase component to the target-specific peptide binder, the system can be used to selectively recruit target proteins to the CUL2-CRL machinery, which then facilitates ubiquitination and subsequent degradation of the protein via the proteasome with a high degree of selectivity and specificity. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2014 / 144229 [Patent Document 2] WO2019 / 028306 [Patent Document 3] WO2000 / 28004 [Patent Document 4] WO2001 / 23001 [Patent Document 5] WO2004 / 112727 [Patent Document 6] WO2005 / 005610 [Patent Document 7] WO2005 / 072364 [Patent Document 8] US Patent No. US6204059 [Patent Document 9] US Patent No. US5756283 [Patent Document 10] US Patent No. US6258595 [Patent Document 11] US Patent No. US6261551 [Patent Document 12] US Patent No. US6270996 [Patent Document 13] US Patent No. US6281010 [Patent Document 14] US Patent No. US6365394 [Patent Document 15] US Patent No. US6475769 [Patent Document 16] US Patent No. US6482634 [Patent Document 17] US Patent No. US6485966 [Patent Document 18] US Patent No. US6943019 [Patent Document 19] US Patent No. US6953690 [Patent Document 20] US Patent No. US7022519 [Patent Document 21] US Patent No. US7238526 [Patent Document 22] US Patent No. US7291498 [Patent Document 23] US Patent No. US7491508 [Patent Document 24] US Patent No. US5064764 [Patent Document 25] US Patent No. US6194191 [Patent Document 26] US Patent No. US6566118 [Patent Document 27] US Patent No. US8137948 [Patent Document 28] International Publication No. WO1996039530 [Patent Document 29] International Publication No. WO1998010088 [Patent Document 30] International Publication No. WO1999014354 [Patent Document 31] International Publication No. WO1999 / 015685 [Patent Document 32] International Publication No. WO1999 / 047691 [Patent Document 33] International Publication No. WO2000 / 055342 [Patent Document 34] International Publication No. WO2000 / 075353 [Patent Document 35] International Publication No. WO2001 / 023597 [Patent Document 36] U.S. Patent No. 4,683,195 [Non-patent literature]

[0007] [Non-Patent Document 1] Fulcher LJ et al., 2017, “Targeting endogenous proteins for degradation through the affinity-directed protein missile system”, Open Biol. 7: 170066 [Non-Patent Document 2] Guilliams et al., Journal of Molecular Biology, Volume 425, Issue 14, July 24, 2013, Pages 2397-2411 [Non-Patent Document 3] Sambrook et al., "Molecular Cloning, A laboratory manual", Cold Spring Harbor Laboratory Press, volumes 1-3, 2001 (ISBN-0879695773) [Non-Patent Document 4] Ausubel et al., "Short Protocols in Molecular Biology", John Wiley and Sons, 4th Edition, 1999 (ISBN-0471250929) [Non-Patent Document 5] "Methods In Molecular Biology", edited by Richard, Humana Press, NJ (1995) [Non-Patent Document 6] O'Reilly et al., "Baculovirus Expression Vectors, A Laboratory Manual", Oxford Univ. Press (1994) [Non-Patent Document 7] Samulski et al., J Fir., 63:3822 - 8 (1989) [Non-Patent Document 8] Kajigaya et al., Proc. Nat'l. Acad. Sci. USA, 88: 4646 - 50 (1991) [Non-Patent Document 9] Ruffing et al., J. Vir., 66:6922 - 30 (1992) [Non-Patent Document 10] Kimbauer et al., Vir., 219:37 - 44 (1996) [Non-Patent Document 11] Zhao et al., Vir., 272: 382 - 93 (2000) [Non-Patent Document 12] Selene Ingusci et al. ("Gene Therapy Tools for Brain Diseases", Front. Pharmacol., 10:724. doi: 10.3389) [Non-Patent Document 13] Keiser et al., Curr Protoc Mouse Biol., December 2018, 8(4):e57 [Non-Patent Document 14] "Current Protocols in Molecular Biology" (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA) [Non-Patent Document 15] "Molecular Cloning: A Laboratory Manual", 3rd edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press) [Non-Patent Document 16] "Oligonucleotide Synthesis" (edited by MJ Gait, 1984) [Non-Patent Document 17] "Nucleic Acid Hybridization" (Harries and Higgins, eds., 1984) [Non-Patent Document 18] "Transcription and Translation" (Hames and Higgins, eds., 1984) [Non-Patent Document 19] "Culture of Animal Cells" (Freshney, Alan R. Liss, Inc., 1987) [Non-Patent Document 20] "Immobilized Cells and Enzymes" (IRL Press, 1986) [Non-Patent Document 21] Perbal, “A Practical Guide to Molecular Cloning” (1984) [Non-Patent Document 22] "Methods in Enzymology" (edited by Abelson and Simon, Academic Press, Inc., New York), in particular volumes 154 and 155 (edited by Wu et al.) and volume 185, "Gene Expression Technology" (edited by Goeddel) [Non-Patent Document 23] "Gene Transfer Vectors For Mammalian Cells" (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory) [Non-Patent Document 24] "Immunochemical Methods in Cell and Molecular Biology" (eds. Mayer and Walker, Academic Press, London, 1987) [Non-Patent Document 25] "Handbook of Experimental Immunology", volumes I-IV (Weir and Blackwell, eds., 1986) [Non-Patent Document 26] "Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986) [Non-Patent Document 27] Tatusova and Madden, 1999 (FEMS Microbiol Lett, 174:247~250) [Non-Patent Document 28] Smith and Waterman (1981), Adv. Appl. Math., 2:482 [Non-Patent Document 29] Needleman and Wunsch (1970), J. Mol. Biol., 48:443 [Non-Patent Document 30] Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA, 85:2444 [Non-Patent Document 31] Higgins and Sharp (1988), Gene, 73:237-44 [Non-Patent Document 32] Higgins and Sharp (1989), CABIOS, 5:151-3 [Non-Patent Document 33] Corpet et al. (1988), Nucleic Acids Res., 16:10881~90 [Non-Patent Document 34] Huang et al. (1992), Comp. Appl. Biosci., 8:155~65 [Non-Patent Document 35] Pearson et al. (1994) Methods Mol.Biol., 24:307~31 [Non-Patent Document 36] Tatiana et al. (1999), FEMS Microbiol.Lett., 174:247~50 [Non-Patent Document 37] Altschul et al. (1990), J.Mol.Biol., 215:403~10 [Non-Patent Document 38] Bekes, Langley and Crews, 2022 Summary of the Invention [Problem to be solved by the invention]

[0008] Currently, there are no effective treatments for synucleinopathies, and no known therapeutic agents that effectively target α-synuclein. Thus, there is a need to provide new methods for degrading α-synuclein and provide a therapeutic system for treating synucleinopathies.

[0009] Thus, in at least some aspects, the present disclosure describes proteasomal degradative protein complexes that aim to target endogenous alpha-synuclein for proteasomal degradation, providing a highly selective and specific target for therapeutic treatment for synucleinopathies. [Means for solving the problem]

[0010] In a first aspect of the invention, there is provided a proteasomal degradation protein complex comprising an E3 ubiquitin ligase component tethered to an alpha-synuclein-specific polypeptide binding agent, capable of targeting alpha-synuclein for proteasomal degradation.

[0011] Degradation of alpha-synuclein is achieved by targeting alpha-synuclein for degradation by the proteasome system within the cell. Preferably, in some embodiments, alpha-synuclein is specifically targeted for degradation by the ubiquitin-mediated proteasome degradation system.

[0012] α-Synuclein specific polypeptide binding agents: Suitably, in some embodiments, the alpha-synuclein specific polypeptide binding agent is an antibody, an antibody fragment, a monobody, and / or a nanobody. However, it will be apparent to the skilled artisan that other types of alpha-synuclein specific polypeptide binding agents may be used, for example alpha-synuclein specific polypeptide binding agents based on various scaffold proteins. For use in the present invention, an alpha-synuclein specific polypeptide binding agent shall be capable of binding to alpha-synuclein in a cellular context, i.e., within a cell, thereby presenting alpha-synuclein for ubiquitination by the E3 ubiquitin ligase component of the complex.

[0013] The amino acid sequence of α-synuclein (SEQ ID NO: 11) can be divided into three regions: the N-terminal domain (residues 1-60), the NAC domain (residues 61-95), and the C-terminal domain (residues 96-140) (Farzadfard, A et al., 2022).

[0014] In some embodiments, the alpha-synuclein-specific polypeptide binding agent is an antibody specific for a target epitope within the C-terminal region of the alpha-synuclein protein (residues 96-140 of SEQ ID NO: 11). Suitably, in some embodiments, the alpha-synuclein-specific polypeptide binding agent is a nanobody specific for a target epitope within the C-terminal region of the alpha-synuclein protein.

[0015] In some embodiments, the alpha-synuclein-specific polypeptide binding agent is an antibody specific for a target epitope within the N-terminal region of the alpha-synuclein protein (residues 1-60 of SEQ ID NO: 11). Suitably, in some embodiments, the alpha-synuclein-specific polypeptide binding agent is a nanobody specific for a target epitope within the N-terminal region of the alpha-synuclein protein.

[0016] In some embodiments, the alpha-synuclein-specific polypeptide binding agent is an antibody specific for a target epitope within the NAC domain of the alpha-synuclein protein (residues 61-95 of SEQ ID NO: 11). Suitably, in some embodiments, the alpha-synuclein-specific polypeptide binding agent is a nanobody specific for a target epitope within the NAC domain of the alpha-synuclein protein.

[0017] In some embodiments, the alpha-synuclein specific polypeptide binding agent is the nanobody NbSYN87, or a functional variant thereof. In some embodiments, the alpha-synuclein specific polypeptide binding agent is the nanobody NbSYN87 according to SEQ ID NO: 12, or a functional variant thereof. NbSYN87 has been shown to be a particularly effective alpha-synuclein specific polypeptide binding agent for use in the present invention. A functional variant of NbSYN87 suitably comprises a sequence that is at least 60% identical to wild-type NbSYN87 (SEQ ID NO: 12), more preferably at least 70%, 80%, 90%, 95%, or 99% identical to wild-type NbSYN87. A functional variant of NbSYN87 suitably comprises a sequence that is at least 60% identical to SEQ ID NO: 12, more preferably at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 12. In some embodiments, a functional variant of NbSYN87 may be encoded by a DNA sequence that is at least 60% identical to SEQ ID NO: 1, and more preferably at least 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 1. Preferably, a functional variant of NbSYN87 retains equivalent or higher affinity for alpha-synuclein compared to wild-type NbSYN87, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% affinity for alpha-synuclein compared to wild-type NbSYN87.

[0018] In the present invention, NbSYN87 has been shown to work particularly well, although one of skill in the art will appreciate that other alpha-synuclein-specific polypeptide binding agents may be used. In some embodiments, the alpha-synuclein-specific polypeptide binding agent binds to alpha-synuclein with the same or a higher affinity than NbSYN87 (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the affinity for alpha-synuclein compared to NbSYN87).

[0019] The ability of any putative α-synuclein-specific polypeptide binding agent to function in the context of the present invention can be evaluated using the methods described herein. For example, as an initial screen to examine candidate α-synuclein-specific polypeptide binding agents, the interaction of any α-synuclein-specific polypeptide binding agent with α-synuclein can be performed by immunoprecipitation (IP) (e.g., as described in Example 2 below). Then, assuming that the α-synuclein-specific polypeptide binding agent performs the desired function in the immunoprecipitation, its ability to specifically bind α-synuclein in a cellular context can be evaluated (e.g., using immunofluorescence as described in Example 2 below). The activity of an α-synuclein-specific polypeptide binding agent in the context of the proteasome-degrading protein complex of the present invention can be easily evaluated by replacing NbSYN87 in various embodiments discussed below with this binding agent.

[0020] Without wishing to be bound by theory, it is believed that alpha-synuclein-specific polypeptide binding agents that target the same epitope on alpha-synuclein as NbSYN87 may be particularly beneficial in effectively targeting alpha-synuclein for degradation. Thus, in some embodiments, the specific polypeptide binding agent binds with a similar or higher affinity to the same target epitope as NbSYN87 (e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the affinity of NbSYN87 to the same target epitope as NbSYN87). It is believed that the high selectivity of the polypeptide binding agent for alpha-synuclein is beneficial to the function of the proteasome degradative protein complex. In some embodiments, the alpha-synuclein specific polypeptide competes with NbSYN87 for binding to the same target epitope (e.g., in a competitive binding assay), e.g., has the same or a higher affinity for the target epitope as NbSYN87.

[0021] The putative epitope for NbSYN87 is derived from alpha-synuclein and has the following partial (C-terminal) sequence:

[0022] [ka]

[0023] Namely, amino acids 118-129 of α-synuclein are underlined. Further details of NbSYN87 can be found in Guilliams et al., Journal of Molecular Biology, Vol. 425, No. 14, July 24, 2013, pp. 2397-2411.

[0024] Nanobodies as used herein may refer to single domain antibodies derived from heavy chain only (VHH) antibodies. In some embodiments, nanobodies include any of monomeric, dimeric, bispecific, or multivalent nanobodies that are specific for alpha-synuclein and capable of inducing degradation of alpha-synuclein when part of a proteasomal degradative protein complex of the invention.

[0025] The small size of nanobodies compared to conventional antibodies offers a number of advantages. Small polypeptide binders are ideal for intracellular expression since they do not require complex folding or the formation of disulfide bridges. The small size of nanobodies also allows access to non-exposed and / or groove epitopes. Nanobodies are particularly useful in CNS applications due to their ability to cross the blood-brain barrier.

[0026] In some embodiments of the invention, Nanobodies may be of camelid origin (e.g. camel, alpaca, and llama) and / or shark origin. Nanobodies are non-endogenous proteins, but are considered to be non-immunogenic or of low immunogenicity due to their high similarity to human variable heavy (VH) sequences. Suitably, therefore, the use of Nanobodies as polypeptide binding agents of the invention may have the additional advantage of reducing the immunogenicity of the complex.

[0027] E3 ubiquitin ligase components: In some embodiments described herein, the E3 ubiquitin ligase component of the proteasome degradative protein complex can be any E3 ubiquitin ligase component that is capable of recruiting and placing a target protein in proximity to the E3 ubiquitin ligase and its cognate E2-Ub conjugate. This facilitates the ubiquitination of the target protein and subsequent degradation of the target protein via the proteasome. Suitably, the proteasome degradative protein complex targets α-synuclein protein for degradation by ubiquitin-mediated proteasome degradation.

[0028] In some embodiments, the E3 ubiquitin ligase component is a substrate receptor of the CRL (cullin ring E3 ligase) complex. CRL (cullin ring ubiquitin ligase) is a multi-subunit E3 ubiquitin ligase that uses a specific CUL (Cullin) as a central scaffold to crosslink E2 enzymes to substrates. In some embodiments, the E3 ubiquitin ligase component is a substrate receptor that recruits alpha-synuclein to CUL2-CRL, e.g., the von Hippel-Lindau (VHL) tumor suppressor gene or a functional variant thereof. Suitably, the E3 ubiquitin ligase component is a wild-type VHL protein (SEQ ID NO: 13) or a functional variant thereof. Suitably, a functional variant comprises a sequence that is at least 60% identical to wild-type VHL (SEQ ID NO: 13), preferably a sequence that is at least 70%, 80%, 90%, 95% or 99% identical to wild-type VHL (SEQ ID NO: 13). Functional variants also include any variant of VHL that retains the ability to recruit the CUL2-CRL machinery to a protein of interest.

[0029] In another embodiment, the E3 ubiquitin ligase component is wild-type KLHL6 (Kelch-like protein 6) (SEQ ID NO: 15), or a functional variant thereof. Suitably, the functional variant comprises a sequence that is at least 60% identical to wild-type KLHL6 (SEQ ID NO: 15), preferably at least 70%, 80%, 90%, 95%, or 99% identical to wild-type KLHL6 (SEQ ID NO: 15). Functional variants also include any variant of KLHL6 that retains the ability to recruit CUL3 to a protein of interest.

[0030] In another embodiment, the E3 ubiquitin ligase component is wild-type KEAP1 (Kelch-like ECH-associated protein 1) (SEQ ID NO: 16), or a functional variant thereof. Suitably, the functional variant comprises a sequence that is at least 60% identical to wild-type KEAP1 (SEQ ID NO: 16), preferably at least 70%, 80%, 90%, 95%, or 99% identical to wild-type KEAP1. Functional variants also include any variant of KEAP1 that retains the ability to recruit CUL3 to a protein of interest.

[0031] In another embodiment, the E3 ubiquitin ligase component is wild-type CRBN (Cereblon) (SEQ ID NO: 17), or a functional variant thereof. Suitably, the functional variant comprises a sequence that is at least 60% identical to wild-type CRBN (SEQ ID NO: 17), preferably at least 70%, 80%, 90%, 95%, or 99% identical to wild-type CRBN. Functional variants also include any variant of KEAP1 that retains the ability to recruit CUL4 to a protein of interest.

[0032] In another embodiment, the E3 ubiquitin ligase component is wild-type KLHDC2 (Kelch Domain Containing 2) (SEQ ID NO: 18), or a functional variant thereof. Suitably, the functional variant comprises a sequence that is at least 60% identical to wild-type KLHDC2 (SEQ ID NO: 18), preferably at least 70%, 80%, 90%, 95%, or 99% identical to wild-type KLHDC2. Functional variants also include any variant of KEAP1 that retains the ability to recruit CUL2 to a protein of interest.

[0033] In another embodiment, the E3 ubiquitin ligase component is wild type TRAF3d56 (SEQ ID NO: 19), or a functional variant thereof. Suitably, the functional variant comprises a sequence that is at least 60% identical to wild type TRAF3d56 (SEQ ID NO: 19), preferably at least 70%, 80%, 90%, 95%, or 99% identical to wild type TRAF3d56. Without wishing to be bound by theory, it is believed that TRAF3d56 may or may not require a CULLIN protein, which is a RING E3 ligase.

[0034] The present invention is supported in the specific examples below showing the VHL (von Hippel-Lindau) tumor suppressor gene as the most potent (i.e., VHL results in the greatest reduction in total intracellular α-synuclein protein content) E3 ubiquitin ligase component, however, it should be noted that it will be clear to one skilled in the art that other E3 ubiquitin ligase components may also be used. This is supported in the examples below, where KLHL6, KEAP1, CRBN, KLHDC2, and TRAF3d56 are suitable E3 ligases for use in the present invention. The suitability of any putative E3 ligase for use in the present invention can be easily assessed by replacing VHL in the examples described below with a candidate E3 ubiquitin ligase. Those skilled in the art will understand that in some embodiments, the total amount of alpha-synuclein degraded by the proteasome degradative protein complex of the present invention in a cell may vary depending on the alpha-synuclein-specific polypeptide binding agent selected for use in the complex, the E3 ligase selected for use in the complex, and / or the orientation of the component parts. If it is desired to degrade all or a significant proportion of the total intracellular alpha-synuclein protein, it may be desirable to select VHL, KEAP1, or KLHL6 as the E3 ligase component. In alternative embodiments, it may be desirable to degrade a low proportion of the total intracellular alpha-synuclein protein. In such embodiments, those skilled in the art may use any one of CRBN, KLHDC2, or TRAF3d56 as the E3 ligase component.

[0035] Humans have an estimated 500-1000 E3 ubiquitin ligases, classified into four families: HECT, RING finger, U-box, and PHD finger. Alternative E3 ubiquitin ligase components suitable for use in the present invention include the following (or functional variants thereof): APPBP2, KLHDC10, KLHDC3, KLHDC2, LRR1, LRRC58, LRRC28, LRRC14, PRAME, VHL, LRRC42, MED8, RACK1, ARID1A, ARID1B, FEM1A, FEM1C, FEM1B, ZER1, ZYG11A, ZYG11B, ZSWIM5, ZSWIM8, ANKRD9, NEURL2, ASB1, ASB12, ASB11, ASB5, ASB9, ASB13, ASB7, ASB10, ASB18, ASB16, ASB4, ASB8, ASB14, ASB15, ASB2, ASB 3、SPSB1、SPSB4、SPSB2、SPSB3、ASB17、ASB6、PCMTD1、PCMTD2、CISH、SOCS2、SOCS3、SOCS1、SOCS6、SOCS7、SOCS4、SOCS5、RAB40A、RAB40AL、RAB40B、RAB40C、WSB1、WSB2、ELOA、ELOA2、ELOA3、LRRC41、TULP4、BTRC、FBXW11、FBXW7、FBXW4、FBXW2、FBXW12、FBXW9、FBXW5、FBXW8、CDRT1、FBXW10、CCNF、ECT2L、FBX O16、FBXO36、FBXO43、FBXO5、FBXO17、FBXO27、FBXO2、FBXO44、FBXO6、NCCRP1、FBXO28、FBXO45、FBXO22、FBXO4、FBXO25、FBXO32、FBXO39、FBXO33、FBXO8 、FBXO48、FBXO7、FBXO9、FBXO47、FBXO3、TSPAN17、FBXO15、FBXO31、FBXO24、FBXO21、FBXO34、FBXO46、FBXO30、FBXO40、FBXO42、FBXO41、FBXO10、FBXO11 、FBXO18、FBXO38、LMO7、FBXL12、FBXL7、FBXL14、FBXL2、FBXL20、LRRC29、FBXL15、FBXL16、FBXL6、SKP2、FBXL13、FBXL17、FBXL4、FBXL21、FBXL3、FBXL8、 FBXL22、FBXL19、KDM2A、KDM2B、FBXL5、FBXL18、DCAF8L2、DCAF8L1、DCAF8、WDTC1、DCAF6、DCAF5、CRBN、ERCC8、DDB2、RBBP7、RBBP4、GRWD1、NUP43、DCAF7、DCAF4L1, DCAF4, DCAF4L2, TLE3, TLE1, TLE2, TRPC4AP, TOR1AIP2, DCAF17, DCAF12L2, DCAF12L1, DCAF12, AHR, DCAF10, RBBP5, DCAF13, WDR53, WDR26, WDR61, SMU1, PAFAH1B1, NLE1, GNB2, WDR82, ATG16L1, SNRNP40, DCAF11, WDR5B, WDR5, POC1B, EED, WDR12, PWP1, DTL, KATNB1, CIAO1, DCAF1, DCAF16, DCAF15, WDR76, DET1, AMBRA1, WDR59, PHIP, BRWD1, KLHL4, KLHL1, KLHL5, KLHL8, KLHL20, KLHL3, KLHL2, KLHL17, KLHL18, KLHL12, KLHL7, IVNS1ABP, KLHL28, KLHL10, KEAP1, IPP, KLHL23, KLHL34, BCL6B, KLHL32, KLHL15, KLHL36, KLHL22, KLHL9, KLHL13, KLHL26, KLHL14, KLHL31, KLHL42, KBTBD11, KBTBD13, KLHL33, GAN, KLHL35, KLHL24, KLHL6, KLHL29, KLHL38, KLHL25, ENC1, CCIN, KLHL11, KBTBD4, KBTBD3, KLHL30, KLHL21, KBTBD7, KBTBD6, KBTBD8, KBTBD2, KBTBD12, KLHL41, KLHL40, BTBD6, BTBD3, BTBD2, BTBD1, TNFAIP1, KCTD13, KCTD10, KCTD7, KCTD14, KCTD8, KCTD12, KCTD16, KCTD18, KCTD15, KCTD1, KCTD6, KCTD4, KCTD21, SHKBP1, KCTD3, KCTD5, KCTD17, KCTD2, KCTD9, KCNRG, KCTD19, KCTD11, ARMC5, KCTD20, BTBD10, SLX4, RCBTB2, RCBTB1, IBTK, SPOPL, SPOP, LZTR1, RHOBTB2, RHOBTB1, RHOBTB3, ABTB2, ABTB1, CDC20, FZR1, Anaphase-Promoting Complex (APC), BC-box, eloBC, CUL5, RING, LNXp80, CBX4, PIAS1, PIAS2, PIAS3, PIAS4,or RANBP2, but are not limited to these.

[0036] Alternative E3 ubiquitin ligase components may include single polypeptide E3 ligases. Single polypeptide E3 ligases suitable for use in the present invention include the following (or functional variants thereof): UBR1、UBR2、UBR7、UBR3、UBR4、NOSIP、PPIL2、STUB1、PRPF19、WDSUB1、UBE4A、UBE4B、UBOX5、MEFV、TRIM10、TRIM15、TRIM26、TRIM31、TRIM11、TRIM58、TRIM21、TRIM68、TRIM38、TRIML1、TRIM17、TRIM27、TRIM7、TRIM39、TRIM60、TRIM61、TRIM4、TRIM62、TRIM69、TRIM22、TRIM34、TRIM6、TRIM5、TRIM43、TRIM48、TRIM49D1、TRIM49、TRIM49B、TRIM51、TRIM64、TRIM77、TRIM35、TRIM50、TRIM73、TRIM74、TRIM72、TRIM41、TRIM52、TRIM40、TRIM13、TRIM59、TRIM65、TRIM14、TRIM25、TRIM8、TRIM16、TRIM47、TRIM29、MID1、MID2、TRIM44、TRIM54、TRIM55、TRIM63、TRIM67、TRIM9、TRIM36、TRIM46、TRIM2、TRIM3、TRIM71、TRIM45、TRIM56、TRIM32、TRIM24、TRIM33、TRIM28、TRIM23、TRIM42、PML、TRIM37、TRAF2、TRAF3、TRAF5、TRAF4、TRAF6、TRAF7、TRAF3d56、ANKIB1、ARIH1、ARIH2、RNF144A、RNF144B、RNF217、RNF14、PRKN、RBCK1、RNF19A、RNF19B、RNF216、RNF31、NEURL3、NEURL1B、NEURL1、RNF34、RFFL、MUL1、CGRRF1、UNKL、UNK、RNF123、VPS8、VPS18、MIB1、MYLIP、DCST1、RNF220、LTN1、RNF214、RAPSN、RNF32、RNF213、RNF26、BRAP、NSMCE1、VPS11、CBLB、CBL、CBLC、RNF113B、RNF113A、RNF8、RNF208、PHF7、LRSAM1、FANCL、TMEM129、LONRF2、LONRF1、PEX10、RNF139、RNFT1、PJA2、PJA1、RNF4、LNX2、LNX1、TTC3、RNF157、MGRN1、PELI2、PELI1、PELI3、RNF180、RNF181、RNF175、RNF121、RNF24、RNF122、DZIP3、PEX2、RNF150、RNF130、RNF149、RNF148、RNF133、RNF128、RNF167、RNF13、ZNRF4 RNF44、RNF38、RNF6、RLIM、ZNRF2、ZNRF1、RNF11、RNF103、RNF145、RNF126、RNF115、RNF141、SHPRH、RNF186、LONRF3、ZNRF3、RNF43、RNF215、RNF165、RNF1 11、RFWD3、PEX12、ANAPC11、RNF112、RNF187、RNF169、RNF168、RNF40、RNF20、RNF219、CCNB1IP1、RFPL3、RFPL1、RFPL4A、RFPL4B、RNF39、PDZRN4、PDZRN3 RNF41、BRCA1、BARD1、RC3H2、RC3H1、SYVN1、AMFR、UHRF2、UHRF1、RNF17、RSPRY1、RNF135、NHLRC1、TOPORS、RNF207、RNF183、SIAH2、SIAH1、IRF2BPL、MAP3 K1、CNOT4、RNF166、ZNF598、GID4、SH3RF2、RNF146、RNF151、SH3RF1、RNF182、RNF152、TRAIP、RNF10、RAG1、BFAR、MIB2、RNF25、RNF138、RNF114、RNF125、H LTF、RNF170、RNF5、RNF185、MNAT1、COP1、PCGF5、PCGF3、PCGF6、PCGF2、BMI1、PCGF1、RNF2、RING1、PHRF1、CHFR、ZSWIM2、MSL2、RNF212、RBBP6、RAD18、ZFP L1、MYCBP2、VPS41、CBLL2、CBLL1、RCHY1、RNF7、RBX1、MEX3A、MEX3B、MEX3C、MEX3D、MDM2、MDM4、MARCH1、MARCH8、MARCH2、MARCH3、MARCH11、MARCH4、MARCH1 H9、MARCH5、MARCH10、MARCH7、MARCH6、MKRN1、MKRN3、MKRN2、AREL1、HECTD2、UBE3A、HERC3、HERC4、HERC5、HERC6、HACE1、HECW1、HECW2、ITCH、WWP1、WWP2、These include, but are not limited to, SMURF1, SMURF2, NEDD4, NEDD4L, HUWE1, UBE3D, G2E3, HECTD3, UBE3B, UBE3C, TRIP12, HECTD1, UBR5, HECTD4, HERC1, HERC2, DTX1, DTX4, DTX2, DTX3, DTX3L, BIRC2, BIRC3, BIRC8, XIAP, or BIRC7.

[0037] In some embodiments, the E3 ubiquitin ligase component of the proteasome degradative protein complex is an E3 ubiquitin ligase component that is functional in the nervous system, suitably the peripheral nervous system or the central nervous system. In a preferred embodiment, the E3 ubiquitin ligase component of the proteasome degradative protein complex is an E3 ubiquitin ligase component that is functional in the central nervous system. Suitably, in some embodiments, the E3 ubiquitin ligase is a HECT-type ligase, including Nedd4 family ligases, HERC family ligases, and other HECT-type ligases. In some embodiments, the E3 ubiquitin ligase is RNF183.

[0038] Without wishing to be bound by theory, any alpha-synuclein-specific polypeptide binding agent and any E3 ubiquitin ligase component that selectively binds alpha-synuclein and properly positions it for ubiquitination is suitable for use in the present invention. Suitable combinations of alpha-synuclein-specific polypeptide binding agents and E3 ubiquitin ligase components can be readily identified using the methods described herein.

[0039] One skilled in the art will appreciate that in some embodiments, the orientation of the alpha-synuclein specific polypeptide binding agent and any E3 ubiquitin ligase component of the proteasome degradation protein complex may affect the activity or efficacy of the complex. Suitably, in some embodiments, the alpha-synuclein specific polypeptide binding agent is positioned at the N-terminus of the protein complex. Alternatively, in some embodiments, the alpha-synuclein specific polypeptide binding agent is positioned at the C-terminus of the protein complex. In some embodiments, the E3 ubiquitin ligase component is positioned at the N-terminus of the protein complex. In alternative embodiments, the E3 ubiquitin ligase component is positioned at the C-terminus of the protein complex. The optimal orientation may be readily identified using the methods described herein.

[0040] It will be appreciated that the component parts of the proteasome degrading protein complex of the present invention may be tethered or interconnected via a linker protein. Suitable linkers are known in the art and are described in the examples below. In one embodiment, the linker protein comprises the amino acid sequence 5'-GGGGGG-3' (SEQ ID NO: 28). In alternative embodiments, the E3 ligase component and the alpha-synuclein specific polypeptide binding agent described herein may be provided as separate components that conjugate, for example when expressed in a cell. Suitably, in such embodiments, the E3 ligase component and the alpha-synuclein specific polypeptide binding agent are modified or tagged with a suitable protein conjugation system known in the art. Suitably, the proteasome degrading protein complex may be provided as a protein complex through the interaction of a domain with its binding partner. For example, in one embodiment, the E3 ligase component may be biotinylated and the alpha-synuclein specific polypeptide binding agent may have a streptavidin tag, or vice versa. Alternatively, the E3 ligase component may be streptavidin tagged and the alpha-synuclein specific polypeptide binding agent may be biotinylated. Other protein binding pairs are known in the art, such as SH3 domains, PDZ domains, GK domains, GB domains, etc., with their binding partners. In yet another embodiment, an intein may be provided on a component of the complex to fuse the E3 ligase component to the alpha-synuclein specific polypeptide binding agent to form a complex.

[0041] Nucleic acids and vectors: In a second aspect of the invention, there is provided one or more nucleic acid constructs encoding the proteasome degrading protein complex of the first aspect.

[0042] In some embodiments, the nucleic acid construct comprises a nucleic acid encoding an E3 ubiquitin ligase component linked to a nucleic acid encoding an alpha-synuclein-specific polypeptide binding agent.

[0043] It will be clear to the skilled artisan that in some embodiments, for example when the complex is not a fusion protein, the individual components of the complex are provided on separate nucleic acid constructs and references herein to nucleic acid constructs shall be read in this manner (i.e., without excluding the possibility that there may be more than one nucleic acid construct, where appropriate). Thus, in some embodiments, one or more expression constructs may be provided that comprise nucleic acid sequences encoding the individual components of the complex. In some embodiments, a first nucleic acid construct comprises a first nucleic acid that encodes an E3 ubiquitin ligase component and a second nucleic acid construct comprises a second nucleic acid that encodes an alpha-synuclein-specific polypeptide binding agent. Suitably, in such embodiments, the first and second nucleic acids encode an E3 ligase component and an alpha-synuclein-specific polypeptide binding agent component linked to suitable elements that allow the components to conjugate and form an active complex.

[0044] In some embodiments, the nucleic acid construct comprises a nucleic acid encoding VHL or a functional variant thereof linked to a nucleic acid encoding an alpha-synuclein specific polypeptide binding agent. Suitably, in some embodiments, the alpha-synuclein specific polypeptide binding agent is a nanobody, and in some embodiments, the alpha-synuclein specific polypeptide binding agent may be NbSYN87, or a functional variant or biological equivalent thereof.

[0045] In one embodiment, the nucleic acid construct comprises a nucleic acid encoding a VHL according to SEQ ID NO:2, or a functional variant thereof, and a nucleic acid encoding a nanobody, NbSYN87, according to SEQ ID NO:1, or a functional variant thereof.

[0046] In some embodiments, the nucleic acid construct comprises a nucleic acid sequence according to SEQ ID NO:3, or a functional variant thereof. Suitably, in some embodiments, the nucleic acid construct comprises a nucleic acid sequence that is at least 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO:3.

[0047] In one embodiment, the nucleic acid construct comprises a nucleic acid encoding KLHL6 according to SEQ ID NO: 6, or a functional variant thereof, and a nucleic acid encoding the nanobody NbSYN87 according to SEQ ID NO: 1, or a functional variant thereof.

[0048] In one embodiment, the nucleic acid construct comprises a nucleic acid encoding KEAP1 according to SEQ ID NO: 7, or a functional variant thereof, and a nucleic acid encoding NbSYN87, a nanobody according to SEQ ID NO: 1, or a functional variant thereof.

[0049] In one embodiment, the nucleic acid construct comprises a nucleic acid encoding CRBN according to SEQ ID NO:8, or a functional variant thereof, and a nucleic acid encoding the nanobody NbSYN87 according to SEQ ID NO:1, or a functional variant thereof.

[0050] In one embodiment, the nucleic acid construct comprises a nucleic acid encoding KLHDC2 according to SEQ ID NO: 9, or a functional variant thereof, and a nucleic acid encoding the nanobody NbSYN87 according to SEQ ID NO: 1, or a functional variant thereof.

[0051] In one embodiment, the nucleic acid construct comprises a nucleic acid encoding TRAF3d56 according to SEQ ID NO: 10, or a functional variant thereof, and a nucleic acid encoding the nanobody NbSYN87 according to SEQ ID NO: 1, or a functional variant thereof.

[0052] Nucleic acids encoding the proteasome degrading protein complexes of the invention may be fully synthetic or partially synthetic and may include, but are not limited to, DNA, cDNA, and RNA. Nucleic acid sequences encoding the proteasome degrading protein complexes of the invention may be readily prepared by those of skill in the art using techniques well known to those of skill in the art, such as those described in Sambrook et al., "Molecular Cloning, A laboratory manual," Cold Spring Harbor Laboratory Press, Vols. 1-3, 2001 (ISBN-0879695773); and Ausubel et al., "Short Protocols in Molecular Biology," John Wiley and Sons, 4th Edition, 1999 (ISBN-0471250929). Such techniques include (i) the use of polymerase chain reaction (PCR) to amplify a sample of nucleic acid, (ii) chemical synthesis, or (iii) the preparation of cDNA sequences. The DNA encoding the proteasome degrading protein complex of the present invention can be made and used in any suitable manner known to those skilled in the art, including the steps of taking the encoding DNA, identifying suitable restriction enzyme recognition sites on both sides of the portion to be expressed, and excising the portion from the DNA. The excised portion can then be operably linked to a suitable promoter and expressed in a suitable expression system, such as a commercially available expression system. Alternatively, the desired portion of the DNA can be amplified by using suitable PCR primers. Modifications to the DNA sequence can be made by using site-directed mutagenesis.

[0053] The nucleic acid sequence encoding the proteasome degrading protein complex of the present invention may be provided as an expression construct in the form of a plasmid, vector, transcription cassette or expression cassette, comprising at least one nucleic acid as described above, operably linked to one or more expression control sequences, such as promoters, enhancers, polyA sequences, introns, etc. Suitably, the expression control sequence is sufficient to effect expression of the proteasome degrading protein complex in the target cell. Expression may be constitutive or regulatable.

[0054] Thus, in a third aspect of the present invention there is provided an expression construct comprising a nucleic acid construct as defined above. Suitably the expression construct is a vector, such as an expression vector, adapted for expression in a eukaryotic or prokaryotic cell.

[0055] Suitably, in some embodiments of the invention, the vector is a viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. In some preferred embodiments, the vector is an AAV vector.

[0056] In some embodiments, the vector is a gene therapy vector, suitably an AAV vector, an adenovirus vector, a retrovirus vector, a herpes simplex virus vector, or a lentivirus vector.Lentivirus vectors have been widely used as gene transfer tools in the CNS, and are known to enable successful transduction of neurons, astrocytes, and oligodendrocytes.Lentivirus vectors are advantageous because they have a relatively large cloning capacity and do not express viral genes.In particular, a preferred lentivirus vector system is based on HIV-1.Herpes simplex virus vectors and adenovirus vectors also show potential for use as gene transfer tools in the CNS, as they have shown successful transduction of CNS cells, but are less preferred due to their toxicity.

[0057] AAV vectors have been extensively discussed in the art. AAV vectors are particularly interesting vectors because they typically do not integrate into the genome and do not induce immune responses. AAV serotypes 1, 2, 4, 5, 8, 9, and 2g9 (AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, and AAV2g9) have been noted to achieve effective transduction in the CNS. Thus, AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, and derivatives thereof are particularly preferred AAV serotypes. In some embodiments, AAV9 is a particularly preferred AAV vector. In other embodiments, AAV2g9 is a particularly preferred AAV vector (WO2014 / 144229). In yet other embodiments, a particularly preferred AAV vector is AAVDJ8 (Hammond et al., 2017). Suitably, the AAV vector comprises a viral genome comprising the nucleic acid sequence of the invention arranged between two ITRs (inverted terminal repeats). For example, WO2019 / 028306 discloses a variety of wild-type and modified AAV vectors that can be used in the CNS. In one embodiment, the AAV vector is capable of penetrating the blood-brain barrier after delivery of the AAV vector. In one embodiment, the AAV vector of the invention is a recombinant AAV viral vector that is replication-deficient and lacks sequences encoding functional Rep and Cap proteins in their viral genome. These defective AAV vectors may lack most or all parental coding sequences, and essentially carry only one or two AAV ITR sequences and a nucleic acid of interest for delivery to a cell, tissue, organ, or organism. Suitably, the AAV vector for use herein comprises a virus that has been reduced to the minimum components necessary for transduction of a nucleic acid payload or cargo of interest. In this way, AAV vectors are engineered as vehicles for specific delivery while lacking deleterious replication and / or integration characteristics found in wild-type viruses. In one embodiment, the AAV particles of the present invention are scAAV. In another embodiment, the AAV particles of the present invention are ssAAV.Methods for making and / or modifying AAV particles are widely disclosed in the art (see, e.g., WO2000 / 28004; WO2001 / 23001; WO2004 / 112727; WO2005 / 005610, and WO2005 / 072364, which are incorporated herein by reference). In one embodiment, the AAV vector comprises a capsid that enables penetration of the blood-brain barrier upon intravascular (e.g., intravenous or intra-arterial) administration (e.g., see WO2014 / 144229, which discusses capsids or peptide inserts engineered for effective crossing of the blood-brain barrier, including, e.g., VOY101, VOY201, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, and variants thereof).

[0058] Methods for producing AAV vectors are well known in the art and are described in, for example, U.S. Pat. Nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US Nos. 7238526, 7291498, and 7491508, 5064764, 6194191, 6566118, and 8137948; or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999 / 015685, WO1999 / 047691, WO2000 / 055342, WO2000 / 075353, and WO2001 / 023597; In Molecular Biology, edited by Richard, Humana Press, NJ (1995); O'Reilly et al., "Baculovirus Expression Vectors, A Laboratory Manual", Oxford Univ. Press (1994); Samulski et al., J Fir., 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA, 88: 4646-50 (1991); Ruffing et al., J. Vir., 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir., 272: 382-93 (2000). Viral replicating cells commonly used for the production of recombinant AAV viral particles include, but are not limited to, HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines.

[0059] In some embodiments, the vector is a non-viral vector, e.g., using cationic polymers or cationic lipids, as known in the art. Various non-viral vectors are discussed in Selene Ingusci et al. ("Gene Therapy Tools for Brain Diseases", Front. Pharmacol., 10:724. doi: 10.3389).

[0060] In some embodiments, there is provided a virion (viral particle) comprising a vector, suitably a viral vector, according to the invention. In some embodiments, the virion is an AAV virion.

[0061] Thus, the present invention further provides a recombinant virion (virus particle) comprising the above-described vector.

[0062] Pharmaceutical Compositions: In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a proteasome degrading protein complex as defined above. Such a composition typically comprises at least one pharma- ceutically acceptable diluent or carrier.

[0063] As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like, and all of the above. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not cause allergic or similar adverse reactions when administered to a host. A suitable carrier may be readily selected by one of skill in the art, bearing in mind the application for which the pharmaceutical composition is intended. For example, one suitable carrier includes saline (e.g., phosphate buffered saline), which may be formulated with various buffers. Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not limited by the present disclosure. The pharmaceutical composition may also provide various other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0064] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., can be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the transgene delivered by the vector can be formulated for delivery encapsulated in a lipid particle, liposome, vesicle, nanosphere, nanoparticle, etc.

[0065] Suitably, in one aspect of the invention, there is provided a pharmaceutical composition comprising the proteasome degrading protein complex, nucleic acid, expression construct, vector, or virion discussed above, and a pharma- ceutically acceptable carrier or diluent. Suitably, in one embodiment, the composition is suitable for acting as an inhibitor of alpha-synuclein in a target cell. In a further embodiment, the composition is suitable for modulating the level of alpha-synuclein in a target cell. Modulating protein levels may refer to reducing or increasing the level of alpha-synuclein. Suitably, the pharmaceutical composition results in the degradation of alpha-synuclein.

[0066] Suitably, in one embodiment, the proteasome degradation protein complex or pharmaceutical composition according to any of the aspects and embodiments presented herein, α-synuclein is targeted for proteasome degradation in target cells. Optionally, the α-synuclein has an SNCA double duplication, an SNCA triple duplication, and / or a point mutation selected from the group including A53T, A30P, H50Q, E46K, G51D, and / or A53E. Suitably, in some embodiments, the α-synuclein is a monomer, oligomer, protofibril, mature fiber, or aggregate of α-synuclein.

[0067] Suitable target cells include any eukaryotic cell. Preferably, the target cell is a mammalian cell, more preferably a human cell. In some embodiments, the target cell is a neuronal cell, preferably a cell derived from the central nervous system. The neuronal cell can be a primary neuronal cell, or a cell of a neuronal-derived cell line, such as an immortalized cell line. In some embodiments, the target cell is a neuron, an astrocyte, an oligodendrocyte, a microglia, and / or an ependymal cell.

[0068] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is generally equivalent to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0069] Treatment and Related Methods: In a further aspect, the present invention provides a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a proteasome degrading protein complex, expression construct, vector, virion, or pharmaceutical composition discussed above.

[0070] Suitably, the method comprises the step of introducing into a cell of the subject a proteasome degrading protein complex, an expression construct, a vector, a virion, or a pharmaceutical composition as discussed above. Suitable target cells are discussed above.

[0071] In some embodiments, the method comprises administering to a subject a vector or virion according to the invention. Suitably, the vector is a gene therapy viral vector, such as an AAV vector.

[0072] The invention also provides a proteasome degrading protein complex, expression construct, vector, virion, or pharmaceutical composition as described herein for use in a method of treating or preventing a disease in a subject, suitably comprising administering to said subject a therapeutically effective amount of a proteasome degrading protein complex or pharmaceutical composition of the invention.

[0073] Suitably, the proteasome degrading protein complex, expression construct, vector, virion, or pharmaceutical composition discussed above is used for treating, preventing, alleviating, or improving neurological disease and / or neurological disorder.In one embodiment, the proteasome degrading protein complex, expression construct, vector, virion, or pharmaceutical composition is a proteasome degrading protein complex, expression construct, vector, virion, or pharmaceutical composition for use in treating a subject with a neurodegenerative disorder.In some embodiments, the neurodegenerative disorder is synucleinopathy.In some embodiments, the neurodegenerative disorder is any of PD, dementia, and / or multiple system atrophy.

[0074] In some embodiments, the method includes systemically administering the proteasome degrading protein complex, expression construct, vector, virion, or pharmaceutical composition. Systemic administration can be enteral (e.g., oral, sublingual, and rectal) or parenteral (e.g., injection). Suitable administration methods can be enteral (e.g., oral, sublingual, and rectal) or parenteral (e.g., injection), including intravenous, intraarterial, intracranial, intramuscular, subcutaneous, intraarticular, intrathecal, and intradermal injections. Preferred administration methods are intravenous, intraarterial, intracranial, and intrathecal injections.

[0075] In some embodiments, the method comprises introducing a pharmaceutical composition described herein into the CNS of a subject. A particular difficulty with the introduction of vectors, virions, or pharmaceutical compositions into the CNS is the blood-brain barrier. The blood-brain barrier is a semi-permeable boundary of endothelial cells that prevents certain chemicals and molecules in the bloodstream from crossing into the extracellular fluid of the central nervous system. In animal studies, this obstacle has been overcome by direct injection into the animal's brain, such as intracranial injection, suitably intracerebroventricular (ICV) injection (see, for example, Keiser et al., Curr Protoc Mouse Biol., December 2018, 8(4):e57). This method of administration can be disadvantageous for gene therapy in humans, as it is difficult to perform and can be dangerous to the subject.

[0076] Instead, in a human gene therapy context, the expression cassettes described herein are preferably introduced into the CNS by intravenous or intra-arterial (e.g., intracarotid) administration of a viral vector containing the expression cassette. Suitably, the viral vector is an AAV vector. Intravenous or intra-arterial administration of some serotypes of AAV allows penetration of the AAV vector into the brain. Intravenous or intra-arterial administration is safer and less invasive than intracranial administration, while still allowing penetration through the blood-brain barrier.

[0077] In some embodiments, the gene therapy viral vector is administered contemporaneously or sequentially with one or more additional therapeutic agents or one or more saturating agents designed to prevent clearance of the vector by the reticuloendothelial system.

[0078] When the vector is an AAV vector, the dosage of the vector is 1 × 10 per kg. 10 Genome copies: 1 x 10 per kg 15 Doses of genome copies or more, suitably 1 x 10 per kg 12 Genome copies: 1 x 10 per kg 14 Genome copies, appropriately, 5 x 10 per kg 12 Genome copies: 5 x 10 per kg 13 It may be a genome copy.

[0079] Generally, the subject in need of treatment will be a mammal, preferably a primate, more preferably a human. Typically, the subject in need thereof will exhibit symptoms characteristic of a disease, such as those discussed above, most preferably a synucleinopathy. The method typically includes the step of ameliorating the symptoms exhibited by the subject in need thereof by expressing a therapeutic amount of a therapeutic product of the invention.

[0080] Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo are well known in the art and will not be discussed in detail here. Briefly, gene therapy protocols include intramuscular injection, intrainterstitial injection, intratracheal instillation, endothelial application, intrahepatic parenchymal administration, and intravenous or intraarterial administration (e.g., intrahepatic artery, intrahepatic vein) of plasmid DNA vectors (naked or in liposomes) or viral vectors. A variety of devices have been developed to enhance the availability of DNA to target cells. A simple approach is to physically contact the target cells with a catheter or implantable material containing the desired vector, while more complex approaches may use jet injection devices and the like. Gene transfer into mammalian cells has been performed using both ex vivo and in vivo procedures. Ex vivo methods typically require cell harvesting, transduction in vitro with the appropriate expression vector, followed by reintroduction of the transduced hepatocytes into the liver. In vivo gene transfer is achieved by injection of DNA or viral vectors.

[0081] In a further aspect of the invention there is provided a proteasome degrading protein complex, expression construct, vector, virion or pharmaceutical composition according to any aspect of the invention for use as a medicament, e.g. for the treatment of a patient. Suitably the patient is suffering from a synucleinopathy.

[0082] Alpha-synuclein: α-synuclein is a member of the intrinsically disordered protein (IDP) family, and α-synuclein itself does not have a tertiary structure, and its conformation is influenced by many different factors, such as the presence of interactors or lipid membranes (15). Its role in PD is further complicated by the fact that six different mutations of the protein found in patients with familial PD have been shown to favor differential oligomeric and fibrillar morphologies during the aggregation process (16). Interestingly, α-synuclein in these Lewy bodies is almost exclusively phosphorylated at serine 129 (17), which can be used as a marker to confirm the presence of these aggregated structures, since there are only trace levels of phosphorylated protein in the brains of healthy individuals compared to patients with PD. It is unclear whether this phosphorylation promotes protein aggregation or occurs subsequent to aggregate formation. It also remains to be determined whether the toxicity induced on cells is due to the aggregates themselves, or through a toxic gain-of-function, or due to recruitment of α-synuclein from its endogenous site of action, inducing a loss-of-function.

[0083] Suitably, in one embodiment, the proteasomal degradation protein complex according to any aspect presented herein targets alpha-synuclein for proteasomal degradation. Suitably, the alpha-synuclein has an SNCA double duplication, an SNCA triple duplication, and / or a point mutation selected from the group comprising A53T, A30P, H50Q, E46K, G51D, and / or A53E.

[0084] According to any embodiment of the invention, the alpha-synuclein targeted for degradation is alpha-synuclein monomers, oligomers, protofibrils, mature fibers, or aggregates.

[0085] In a further aspect, the invention provides methods for targeting alpha-synuclein for degradation using the proteasome degradative protein complex of any of the aspects described herein.

[0086] In one embodiment, the method comprises: Administering, either in vitro or in vivo, a proteasome degrading protein complex, an expression construct, a vector, a virion, or a pharmaceutical composition of the invention to a cell. Includes; an alpha-synuclein-specific polypeptide binder component of the proteasome degradation protein complex binds to alpha-synuclein; An E3 ligase component tethered to an α-synuclein-specific polypeptide binder recruits α-synuclein protein to the E3 ligase system in the cell; An E3 ligase system within the cell ubiquitinates α-synuclein so that it is degraded.

[0087] Those skilled in the art will appreciate that the E3 ubiquitin ligase component of the proteasome degradation protein complex dictates which Cullin protein is recruited to the complex. Each E3 ubiquitin ligase uses a specific Cullin as a central scaffold when recruiting an E2 ligase. Suitably, in some embodiments, the E3 ligase component of the present invention will recruit its cognate Cullin, e.g., any one of CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, or CUL7. Suitably, in some embodiments, the E3 ligase system comprises CUL2-CRL. In another embodiment, the E3 ligase system comprises CUL3. In yet another embodiment, the E3 ligase system comprises CUL4A. In another embodiment, the E3 ligase system comprises CUL4B. In some embodiments, the E3 ligase system comprises CUL5. In another embodiment, the E3 ligase system comprises CUL7.6. Suitably, in one embodiment of the proteasome degradation protein complex of the invention, the E3 ubiquitin ligase component is capable of recruiting alpha-synuclein to either CUL2-CRL, CUL3, or CUL4. Suitably, in an embodiment where the E3 ligase component is TRAF3d56, a variant of TRAF3, the Cullin protein may not be recruited to the complex.

[0088] In one embodiment, the proteasome degrading protein complex, expression construct, vector, virion, or pharmaceutical composition is administered to a cell. Suitably, the cell is any of the target cells described above.

[0089] In preferred embodiments, α-synuclein is targeted for proteasomal degradation and degraded via ubiquitin-mediated proteasomal degradation. In some embodiments, the α-synuclein targeted for degradation described herein comprises an SNCA double duplication, an SNCA triple duplication, and / or any point mutation selected from the group comprising A53T, A30P, H50Q, E46K, G51D, and / or A53E. Suitably, in some embodiments, the α-synuclein is a monomer, oligomer, protofibril, mature fiber, or aggregate of α-synuclein.

[0090] Research tools: In a further aspect of the present invention, the proteasome degrading protein complex of the present invention is used as a tool for exploring a pathway of interest in a cell. Suitably, the proteasome degrading protein complex is applicable as a research tool. In some embodiments, the proteasome degrading protein complex comprises an E3 ligase tethered to a polypeptide binding agent specific for a protein of interest (POI). Suitably, the proteasome degrading protein complex degrades the POI. In some embodiments, the proteasome degrading protein complex controls the expression of the POI. Suitably, the proteasome degrading protein complex is delivered to a cell, preferably a mammalian cell. In some embodiments, the cell is a central nervous system (CNS) cell. In some embodiments, the POI is alpha-synuclein.

[0091] In some embodiments, when the proteasome degrading protein complexes of the present invention are used as research tools, the proteasome degrading protein complexes can be delivered to cells in vitro, ex vivo, or in vivo.

[0092] kit: In one aspect, there is provided a kit for use in any of the aspects and embodiments of the invention, comprising a proteasome degrading protein complex, an expression construct, a vector, a virion, or a pharmaceutical composition as discussed above, and instructions for use.

[0093] In any aspect and / or embodiment described herein, the proteasome degradative protein complex is suitably an affinity-directed protein missile (AdPROM). [Brief description of the drawings]

[0094] [Figure 1] FIG. 1 is a schematic depicting the AdPROM system that uses NbSYN87, a nanobody tethered to VHL to induce polyubiquitination of α-synuclein. [Figure 2A] VHL tethered to a GFP-specific nanobody degrades α-synuclein, and no α-synuclein degradation was observed using the E3 ligases RIM32, RNF126, SIAH3, RNF144A, and RNF125. [Figure 2B] FIG. 13 shows that U2OS Flp-In-T-Rex GFP-alpha-synuclein cells were retrovirally transduced to express constructs for the E3 ligases Ubch5a, WDR5, KLHDC2, MDM2, KLHL6, KLHL7, TRAF3d52, TRAF3d56, TRAF4, LONRF2(P430-N500), LONRF2(339-754), TRAF3, WWP1, WWP2, VHL, TRIM24, KEAP1, PHIP, and CRBN, as well as relevant negative controls. [Figure 2C]U2OS Flp-In-T-Rex GFP-alpha-synuclein cells were treated with the indicated concentrations of NEDDylation inhibitor MLN4924, proteasome inhibitor MG-132 or bortezomib, lysosomal inhibitor bafilomycin A1, or DMSO as a negative control for 14 hours prior to cell lysis. U2OS Flp-In-T-Rex GFP-alpha-synuclein cells expressing FLAG-KLHL6-anti-GFP16 or FLAG-anti-GFP16-KLHL6. U2OS Flp-In-T-Rex GFP-alpha-synuclein cells expressing FLAG-VHL-anti-GFP16 or FLAG-KEAP1-anti-GFP16. U2OS Flp-In T-Rex GFP-alpha-synuclein cell lines expressing FLAG-CRBN-anti-GFP16 or FLAG-TRAF3d56-anti-GFP16. [Figure 3A] Figure 1. Targeted degradation of GFP-α-synuclein by the AdPROM system through the use of both anti-GFP and anti-α-synuclein nanobodies. A) U2OS Flp-In T-REX cells expressing wild-type or A53T mutant α-synuclein tagged with GFP under the control of the Tet On promoter were treated with 20ng / ml doxycycline for the indicated time points. Cells were lysed and 20μg of protein was resolved by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with the indicated antibodies. U2OS Flp-In T-REX cells expressing α-synuclein tagged with GFP were retrovirally infected to express the AdPROM constructs VHL-anti-GFP, or VHL and anti-GFP single controls. Cells were lysed and 20 μg of lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with the indicated antibodies. [Figure 3B](B) Targeted degradation of GFP-α-synuclein by the AdPROM system through the use of both anti-GFP and anti-α-synuclein nanobodies. B) U2OS Flp-In T-REX cells expressing wild-type α-synuclein tagged with GFP were retrovirally infected to express the AdPROM constructs VHL-anti-GFP or VHL conjugated to one of two α-synuclein-specific nanobodies (NbSYN87 and NbSYN2). Cells were lysed and 20 μg of lysate was resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. [Figure 3C] (C) As in B, but with cells expressing the A53T mutant of GFP-synuclein. [Figure 4A] 1 shows targeted degradation of untagged α-synuclein via the AdPROM system. A) Schematic depicting the use of NbSYN87 tethered to VHL to induce polyubiquitination of untagged α-synuclein via the AdPROM system. [Figure 4B] Figure 1. Targeted degradation of untagged α-synuclein via the AdPROM system. B) U2OS Flp-In T-REX cells expressing wild type or the A53T mutant of α-synuclein were retrovirally infected to express VHL-NbSYN87 or VHL-anti-GFP along with appropriate controls. Cells were lysed and 20 μg of lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with the indicated antibodies. [Figure 4C]Figure 1 shows targeted degradation of untagged α-synuclein via the AdPROM system. C) HeLa Flp-In T-Rex cells expressing wild-type α-synuclein or A53T α-synuclein were retrovirally infected to express VHL tethered to NbSYN2 or NbSYN87, along with appropriate controls. Cells were lysed and 20 μg of lysate was resolved by SDS-PAGE. [Figure 4D] (D) Schematic depicting the three major regions of α-synuclein that display five familial variants within the N-terminal amphipathic region. The binding site of the nanobody NbSYN87 within the C-terminal domain is depicted in red. [Figure 4E] Figure 1 shows targeted degradation of untagged α-synuclein via the AdPROM system. E) HeLa Flp-In T-Rex cells expressing the A30P or E46K mutants of α-synuclein were retrovirally infected to express VHL-NbSYN87 along with VHL and NbSYN87 controls. Cells were lysed and 20 μg of lysate was resolved by SDS-PAGE. [Figure 4F] Figure 2 shows targeted degradation of untagged alpha-synuclein via the AdPROM system.F) As in E, but with the G51D or H50Q mutants of alpha-synuclein. [Figure 5A] Figure 1. Interaction of NbSyn87 with endogenous alpha-synuclein. A) SKMEL13 WT (infected with Flag-VHL or 3xFlag-NbSYN87 (as indicated)) or KO cell extracts (input), endogenous alpha-synuclein immunoprecipitates (IP) from these extracts, or post-immunoprecipitation extracts (flow-through) were subjected to Western blot analysis with the indicated antibodies. [Figure 5B]Figure 1. Interaction of NbSyn87 with endogenous alpha-synuclein. B) SK-MEL13 cells transiently transfected with a control vector (empty) or a vector encoding GFP-NBSYN87 were processed for immunofluorescence and analyzed for colocalization of GFP-NbSyn87 with endogenous alpha-synuclein by fluorescence microscopy. Representative images are attached. DAPI staining was performed for nuclear DNA staining. [Figure 6A] Figure 1. Targeted degradation of endogenous α-synuclein in melanoma cells. A) A panel of five different melanoma cells was lysed and 20 μg of lysates were resolved by SDS-PAGE and immunoblotted for α-synuclein. [Figure 6B] (B) Targeted degradation of endogenous α-synuclein in melanoma cells. B) SK-MEL13 and G-361 cells were retrovirally infected to express VHL-NbSYN87 or VHL-anti-GFP with appropriate controls. Cells were lysed and 20 μg of lysates were resolved by SDS-PAGE. [Figure 6C] Figure 1. Targeted degradation of endogenous α-synuclein in melanoma cells. C) G-361 cells expressing VHL-NbSYN87 or NbSYN87 alone were treated with 1 uM of the NEDDylation inhibitor MLN4924 for 24 hours and then lysed. 20 μg of lysates were resolved by SDS-PAGE. [Figure 7A] SK-MEL-13 cells were retrovirally transduced to express FLAG-tagged NbSYN87-KEAP1, KEAP1-NbSYN87, KLHDC2-NbSYN87, or KLHL6-NbSYN87. Cells were lysed and 20 μg of lysate protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane for immunoblotting with the indicated antibodies. Quantification of alpha-synuclein normalized to GAPDH control ± SD for N=2 independent replicates. [Figure 7B]SK-MEL-13 cells were retrovirally transduced to express FLAG-tagged NbSYN87-KEAP1, KEAP1-NbSYN87, KLHDC2-NbSYN87, or KLHL6-NbSYN87. Cells were lysed and 20 μg of lysate protein was resolved by SDS-PAGE and transferred to nitrocellulose membrane for immunoblotting with the indicated antibodies. Quantification of alpha-synuclein normalized to GAPDH control ± SD for N=2 independent replicates. [Figure 8A] Figure 1 shows that α-synuclein degradation is comparable to CRISPR KO levels and reproducible in neuroblastoma cell lines. Using the CRISPR / CAS9 strategy, SNCA knockout SK-MEL13 cells were generated. Wild-type cells were infected with retrovirus to express AdPROM constructs. Cells were lysed together with KO cells, and 20 μg of lysate was resolved by SDS-PAGE. [Figure 8B] (A) Quantification of alpha-synuclein levels normalized to loading control ± SD from n=3 independent experiments. [Figure 8C] Figure 1 shows that α-synuclein degradation is comparable to CRISPR KO levels and reproducible in neuroblastoma cell lines. SK-MEL13 empty, VHL-NbSYN87, and two SNCA KO clones (clone 14 and clone 22) were subjected to anti-α-synuclein immunofluorescence (red) and DNA was stained with DAPI (blue). [Figure 8D]Figure 1 shows that α-synuclein degradation is comparable to CRISPR KO levels and reproducible in neuroblastoma cell lines. SH-SY5Y cells were retrovirally infected to express VHL-NbSYN87, VHL, NbSYN87, or with empty vector. Cells were lysed and 20 μg of lysate was resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with the indicated antibodies. [Figure 8E] (D) Quantification of α-synuclein levels compared to loading control ± SD from n=3 independent experiments. [Figure 9] Immunoblot analysis of samples subjected to total proteomics.SK-MEL13 cells transduced with empty vector or VHL-NbSYN87 were treated with DMSO or 40 uM of the proteasome inhibitor MG132 for 24 hours and then lysed. [Figure 10] Figure 1 shows that VHL-NbSYN87-mediated targeted degradation of alpha-synuclein is highly specific. Volcano plots of VHL-NbSYN87 and control proteomes. Fold change in protein abundance (log2) is plotted against t-test p-value (-log10). Cut-off curve indicating significant proteins. Alpha-synuclein is the only protein whose abundance is significantly lower in cells transduced with VHL-NbSYN87 AdPROM compared to controls. [Figure 11] FIG. 1 shows that VHL-NbSYN87-mediated targeted degradation of alpha-synuclein is specific for the human form of the protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] Although the making and using of various embodiments of the invention are discussed in detail below, it should be understood that the invention presents many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention, and do not limit the scope of the invention.

[0096] The practice of the present invention employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art and are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, 3rd ed. (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins, eds., 1984); Transcription and Translation (Hames and Higgins, eds., 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Methods in Enzymology (Abelson and Simon, eds., Academic Press, 1988). Press, Inc., New York), in particular volumes 154 and 155 (Wu et al., eds.) and volume 185, "Gene Expression Technology" (Goeddel, ed.); "Gene Transfer Vectors For Mammalian Cells" (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); "Immunochemical Methods in Cell and Molecular Biology" (Mayer and Walker, eds., Academic Press, London, 1987); "Handbook of Experimental Immunology", volumes I-IV (Weir and Blackwell, eds., 1986); and "Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0097] In order to facilitate understanding of the present invention, a number of terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art in the areas pertaining to the present invention. Terms such as "a," "an," and "the" are not intended to refer to a singular entity only, but are intended to include the general class of which their specific examples may be used for illustration. Although the terminology used herein is used to describe specific embodiments of the present invention, their usage does not limit the present invention, except as generally outlined in the claims.

[0098] The discussion of the background to the invention herein is incorporated to explain the context of the invention and is not to be understood as an admission that any of the material mentioned was published, publicly known, or part of the general common knowledge in any country as of the priority date of any of the claims.

[0099] Throughout this disclosure, various publications, patents, and patent specification disclosures are referenced by an identifying citation. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such documents that are specifically mentioned herein are incorporated by reference.

[0100] Disclosed herein is a proteasome degradative protein complex that includes an E3 ubiquitin ligase component and is tethered to a target-specific polypeptide binder, such as the von Hippel-Lindau (VHL) tumor suppressor gene, which is a CUL2-CRL substrate receptor. This complex may be referred to as an affinity-directed protein missile or "AdPROM." As used herein, the term "affinity-directed protein missile" or "AdPROM system" refers to a proteasome degradative system that includes an E3 ubiquitin ligase component and is "tethered" or "interconnected" to a polypeptide binder for recognizing a target protein.

[0101] A target-specific polypeptide binding agent refers to any polypeptide that recognizes and binds to a target epitope. Suitably, a target-specific polypeptide binding agent can bind to its target when it is expressed in a cell or introduced into a cell. Target-specific polypeptide binding agents can include antibodies, antibody fragments, monobodies, nanobodies, and / or other types of binding agents, for example, based on scaffold proteins. Any suitable target-specific polypeptide binding agent can be used in the present invention, and the suitability of any given target-specific polypeptide binding agent can be evaluated using the methods described herein, for example by replacing the nanobody used in the methods specified in the examples with another target-specific polypeptide binding agent.

[0102] As used herein, the term "antibody" is a broad term and is given its ordinary and accustomed meaning to those of skill in the art (without being limited to any special or unique meaning) and refers, without limitation, to an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact polyclonal or intact monoclonal antibodies, but also, unless otherwise specified, any antigen-binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen-binding portion, and any other modified configuration of an immunoglobulin molecule that comprises an antigen recognition site. Antigen-binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark antibodies and camelid antibodies), fragments containing the complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, as well as polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen-binding properties to the polypeptide. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0103] As used herein, the term "nanobody" may refer to a single domain antibody derived from a heavy chain only (VHH) antibody. VHH antibodies may be derived from camelids, including camels, alpacas, and llamas. VHH antibodies may be derived from sharks. As used herein, "nanobody" includes any of monomeric, dimeric, bispecific, or multivalent nanobodies that are specific for a target protein.

[0104] As used herein, the term "monobody" may refer to a synthetic binding protein constructed using the fibronectin type III domain (FN3) as a molecular scaffold. This class of binding protein is constructed based on a diversification library for the tenth FN3 domain of human fibronectin. A variety of other synthetic binding proteins based on scaffold proteins are known in the art and may be used in the present invention.

[0105] As used herein, the term "protein complex" refers to the association of two or more polypeptide chains. Thus, two or more proteins of the invention are "tethered" or "interconnected". Thus, these terms refer to the connection of an E3 ligase component to a target-specific polypeptide binding agent. Those skilled in the art will understand that these terms refer to a direct conjugate or a connection via a linker protein.

[0106] E3 ubiquitin ligase components (also called E3 ubiquitin ligases, E3 ligases, or ubiquitin ligases) are proteins that recruit ubiquitin-loaded E2 ubiquitin-conjugating enzymes and assist or directly catalyze the transfer of ubiquitin from the E2 to protein substrates. Ubiquitin is conjugated to lysines on target proteins by isopeptide bonds. E3 ligases typically confer substrate specificity to the E2 because they interact with both the target protein and the E2 enzyme. However, in the context of the present invention, substrate specificity is conferred by a target-specific polypeptide binder. In general, E3 ligases polyubiquitinate their substrates by the Lys-linked chains of ubiquitin and target the substrates for destruction by the proteasome. Any suitable E3 ligase may be used in the present invention, and the suitability of any given E3 ligase may be assessed using the methods described herein, for example by substituting another E3 ligase for VHL in the methods set out in the specific examples.

[0107] The term "ubiquitin-mediated proteasomal degradation" or similar terms refers to a key cellular process that controls protein turnover in cells to maintain protein homeostasis. The evolutionarily conserved cullin ring E3 ubiquitin ligase (CRL) family, which consists of seven members (CUL1 / 2 / 3 / 4A / 4B / 5 / 7), plays a central role in the ubiquitination and degradation of many intracellular proteins (20, 21). Each CRL machinery consists of a substrate receptor (e.g., von Hippel-Lindau (VHL)), a unique adaptor (e.g., Elongin A / B), and a RING E3 ligase (Rbx1 / 2) (21). The substrate receptor subunit recruits substrate proteins and places them in close proximity to E3 ligases and their cognate E2-Ub conjugates, which facilitates their ubiquitination and subsequent degradation via the proteasome. For example, under normoxic conditions, VHL protein recruits proline-hydroxylated HIF1α transcription factor to CUL2-CRL for its ubiquitination and degradation. In the present invention, the substrate receptor of the CUL2-CRL machinery can be utilized to recruit, ubiquitinate, and degrade a protein of interest. Indeed, when VHL is tethered to a nanobody or monobody and introduced into different cells, selective recruitment of target proteins to CUL2-CRL for efficient and rapid destruction (18, 19). Those skilled in the art will understand that different E3 ligases, whether more or less than the reference E3 ligase, can result in the degradation of all target proteins in the cell. Suitably, as described herein, VHL, when complexed with NbSYN87, is a potent E3 ligase and may therefore be a reference E3 ligase.

[0108] As used herein, the term "affinity" refers to the binding strength of a single antigen-binding site with an antigenic determinant. Affinity depends on the closeness of the stereochemical fit between the binding site of an antibody or antigen-binding protein and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups, etc. Affinity may be measured by equilibrium analysis or by surface plasmon resonance ("SP") methods, such as BIACORE™. SPR methods rely on the phenomenon of surface plasmon resonance (SPR), which occurs when a surface plasmon wave is excited at a metal / liquid interface. Light is directed to and reflected from the surface that is not in contact with the sample, and SPR causes a reduction in the reflected light intensity at a specific combination of reflection angle and wavelength. A binding event between two molecules causes a change in the refractive index at the surface layer, which is detected as a change in the SPR signal. In the context of the present invention, a high degree of affinity may refer to the strong binding of a polypeptide binding agent to a target antigen when compared to a competitive binding agent. A low affinity may refer to weak binding of a polypeptide binding agent to a target antigen as compared to a competing binding agent.

[0109] As used herein, "selectivity" refers to the binding preference of a polypeptide binding agent to a target epitope. A high degree of selectivity may refer to a polypeptide binding agent that binds exclusively or preferentially to a target epitope. In some embodiments, the more selective the polypeptide binding agent, the less cross-reactive the polypeptide binding agent is with any proteins present. A low degree of selectivity may refer to a polypeptide binding agent that binds to an epitope shared with other proteins or that is not unique to the target protein.

[0110] A "functional variant" of a nucleic acid construct or amino acid sequence, in the context of the present invention, is a variant of the reference sequence that retains the ability to function in the same manner as the reference sequence. Alternative terms for such functional variants include "biological equivalents" or "equivalents."

[0111] Terms such as "identity" and "identical" refer to sequence similarity between two nucleic acid molecules, such as between two polymeric molecules, e.g., between two DNA molecules. Sequence alignment and sequence identity determination can be performed using BLAST (Basic Local Alignment Search Tool), originally described by Altschul et al., 1990 (J Mol Biol, 215:403-10), such as the "Blast 2 sequences" algorithm described, for example, by Tatusova and Madden, 1999 (FEMS Microbiol Lett, 174:247-250).

[0112] Methods for aligning sequences for comparison are well known in the art. For various programs and alignment algorithms, see, e.g., Smith and Waterman (1981), Adv. Appl. Math., 2:482; Needleman and Wunsch (1970), J. Mol. Biol., 48:443; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA, 85:2444; Higgins and Sharp (1988), Gene, 73:237-44; Higgins and Sharp (1989), CABIOS, 5:151-3; Corpet et al. (1988), Nucleic Acids Res., 16:10881-90; Huang et al. (1992), Comp. Appl. Biosci., 8:155-65; Pearson et al. (1994) Methods Mol. Biol., 24:307-31; Tatiana et al. (1999), FEMS Microbiol. Lett., 174:247-50. A detailed discussion of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990), J. Mol. Biol., 215:403-10.

[0113] BLAST™ (Basic Local Alignment Search Tool; Altschul et al. (1990)) by the National Center for Biotechnology Information (NCBI) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and is also available on the Internet for use in conjunction with several sequence analysis programs. A description of how to use this program to determine sequence identity is available on the Internet under the "Help" section for BLAST™. For comparison of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program using default parameters may be utilized. Nucleic acid sequences with even greater similarity to the reference sequence will show increasing percentage identity when assessed by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.

[0114] For example, a global optimal alignment is suitably found by the Needleman-Wunsch algorithm, with the following scoring parameters: match score: +2, mismatch score: -3; gap penalties: gap start: 5, gap extension: 2. The percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, including both matches and mismatches, multiplied by 100.

[0115] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids connected to each other by peptide bonds. As used herein, the terms "peptide", "polypeptide" and "protein" also refer to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, e.g., biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0116] The term "vector" is well known in the art and is used herein to refer to a nucleic acid molecule, e.g., double-stranded DNA, into which a nucleic acid sequence according to the invention may be inserted. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all the necessary elements that allow the transcription of the inserted nucleic acid molecule and, preferably, the translation of the transcript into a polypeptide. A vector typically, once inside a host cell, is capable of replicating independently of or simultaneously with the host chromosomal DNA; it contains all the necessary elements so that several copies of the vector and its inserted nucleic acid molecule can be made. A vector of the invention may be an episomal vector (i.e., a vector that does not integrate into the genome of the host cell) or may be integrated into the genome of the host cell. This definition includes both non-viral and viral vectors. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, Bluescript vectors (pBS), and pBR322, or derivatives thereof lacking bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors, or Sleeping Beauty (SB) vectors), and the like. Large vectors such as artificial chromosomes (bacterial (BAC), yeast (YAC), or human (HAC)) can be used to accommodate large inserts. Viral vectors are derived from viruses and include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, hepatitis virus vectors, and the like. Viral vectors are typically, but not necessarily, replication-deficient because viral genes essential for replication have been deleted from the viral vector, rendering it unable to propagate in a given cell. However, some viral vectors may also be adapted to replicate specifically in given cells, e.g. cancer cells, and are typically used to induce (cancer) cell-specific (onco)lysis.Virosomes are a non-limiting example of a vector that contains both viral and non-viral elements, in particular, virosomes combine liposomes with inactivated HIV or inactivated influenza virus (Yamada et al., 2003). Another example includes viral vectors mixed with cationic lipids.

[0117] As used herein, the term "CNS cells" or "CNS cells" includes neurons, astrocytes, oligodendrocytes, microglia, and / or ependymal cells.

[0118] As used herein, the term "pharmaceutically acceptable" refers, without limitation, to an entity or component that is included in the compositions presented herein and that does not cause significant adverse toxic effects in patients at the levels specified, or, if no levels are specified, at levels known to be acceptable by those skilled in the art. All components in the compositions described herein are provided at pharmaceutically acceptable levels. For clarity, active ingredients may cause one or more side effects, and the inclusion of components whose side effect profile is acceptable from a regulatory perspective for such components shall be considered to be at "pharmaceutically acceptable" levels for those components.

[0119] As used herein, the term "treatment" or "treating" refers to reducing, ameliorating, or eliminating one or more signs, symptoms, or effects of a disease or condition. As used herein, "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who is predisposed to or at risk for the disease, but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., halting its development; (c) relieving the disease, i.e., causing regression of the disease; and (d) relieving or alleviating any symptoms of the disease.

[0120] As used herein, the terms "inhibit," "reduce," and similar terms refer to a decrease of at least about 5%, 10%, 15%; 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, or more.

[0121] As used herein, unless otherwise stated, the term "subject" is used interchangeably with "individual" or "patient" and refers to any individual subject suffering from a disease or condition requiring prevention or treatment. For purposes of this disclosure, the subject may be a mammal, preferably a human.

[0122] advantage: As will be discussed in detail in the following examples, the present inventors have confirmed that the proteasome degradation protein complex of the present invention is effective and exquisitely selective. By using unbiased quantitative proteomic methods, it was revealed that in the targeted degradation of α-synuclein protein by AdPROM, α-synuclein is the only target to be degraded among more than 10,000 proteins identified (Figure 9). This selectivity is more advantageous than protein silencing methods via genome modification or transcript inhibition, which may result in truncated forms of proteins that are still translated, or in other cases, off-target silencing. The highly selective proteasome degradation protein complex provides additional advantages for use as a research tool. The proteasome degradation protein complex can be used to study the effect of removing target proteins without relying on genome or transcription modulation.

[0123] This system has the added advantage over other targeted protein degradation methods, such as auxin-inducible degrons, which require the insertion of an IAA degron sequence into the protein of interest (POI) locus: the present invention does not require insertion into the POI locus, thereby reducing off-target effects.

[0124] Other E3 ligase systems combined with nanobodies have also been proposed, such as the ZIF1 proteasomal degradation system, but because ZIF1 itself is regulated at different developmental stages, POI degradation competes with endogenous substrates and is therefore less effective.

[0125] The proteasome degradation protein complexes presented herein can be applied in any cell to rapidly target POI degradation, opening up new therapeutic options not previously available.

[0126] By way of non-limiting example, embodiments of the present invention will now be described with reference to the accompanying drawings, in which: EXAMPLES

[0127] Introduction: In the UK, Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting approximately 1 in 500 people (1). PD is characterized by the presence of intracellular proteinaceous inclusions, known as Lewy bodies, found primarily within dopaminergic neurons of the substantia nigra pars compacta (2, 3). PD is a movement-related disorder, and the main symptoms include bradykinesia, resting tremor, and postural instability, as well as reported cognitive symptoms such as depression and dementia (4). Although the underlying cause of disease development remains unknown, these Lewy bodies are a hallmark feature of the disease and contain potential therapeutic targets. These Lewy bodies have been reported to be composed of a number of different proteins, but their major component was found to be a small 140 kDa protein called α-synuclein, encoded by the SNCA gene (5). Its involvement in the pathogenesis of PD has been further confirmed by the discovery of many different mutations of the protein in familial PD: most commonly the variant A53T (6), as well as the mutations A30P, H50Q, E46K, G51D, and A53E (7-10). Gene duplications and triplications have also been shown in patients with familial PD (11, 12), strengthening the evidence for the involvement of α-synuclein in PD, as well as its potential as a therapeutic target. Intracellular inclusions of α-synuclein are also found in many different neurodegenerative disorders, such as dementia with Lewy bodies and multiple system atrophy (MSA), collectively referred to as synucleinopathies. Effective treatments for synucleinopathies remain elusive, but with increasing life expectancy worldwide, the need for effective treatments is becoming urgent.

[0128] Despite intensive research, the function of α-synuclein remains unknown, although it has been found to be enriched in presynaptic terminals (13), and research suggests that α-synuclein plays a role in the formation of the SNARE complex that allows the release of synaptic vesicles (14). As a member of the intrinsically disordered protein (IDP) family, α-synuclein itself does not have a tertiary structure, and its conformation can be influenced by a number of different factors, such as the presence of interactors or lipid membranes (15). Its role in PD is also further complicated by the fact that six different mutations of the protein found in patients with familial PD have been shown to favor differential oligomeric and fibrillar forms during the aggregation process (16). Interestingly, α-synuclein in these Lewy bodies is almost exclusively phosphorylated at serine 129 (17), which can be used as a marker to confirm the presence of these aggregated structures, since there are only trace levels of phosphorylated protein in the brains of healthy individuals compared to patients with PD. It is unclear whether this phosphorylation promotes protein aggregation or occurs subsequent to aggregate formation. It also remains to be determined whether the toxicity induced on cells is due to the aggregates themselves, or through a toxic gain-of-function, or due to recruitment of α-synuclein from its endogenous site of action inducing a loss-of-function. However, evidence suggests the involvement of aggregated α-synuclein in the pathogenesis and progression of PD, characterizing aggregated α-synuclein as a potential target for therapeutic intervention. α-synuclein is considered to be an “undruggable” target by conventional small molecule drug discovery. Thus, this study aimed to explore whether a targeted protein degradation approach through the use of the AdPROM (affinity-directed protein missile) system, developed in the Sapkota lab, could effectively degrade α-synuclein and open up new avenues for therapeutic strategies (18, 19).

[0129] The affinity-directed protein missile system, or AdPROM system, is a novel targeted protein degradation strategy developed to target ubiquitination and subsequent proteasomal degradation of both tagged and endogenous proteins. Ubiquitin-mediated proteasomal degradation is a key cellular process that controls protein turnover in cells to maintain protein homeostasis. The evolutionarily conserved cullin ring E3 ubiquitin ligase (CRL) family, which consists of seven members (CUL1 / 2 / 3 / 4A / 4B / 5 / 7), plays a central role in the ubiquitination and degradation of many intracellular proteins (20, 21). Each CRL machinery consists of a substrate receptor (e.g., von Hippel-Lindau (VHL)), a unique adaptor (e.g., Elongin A / B), and a RING E3 ligase (Rbx1 / 2) (21). Substrate receptor subunits recruit substrate proteins and place them in close proximity to E3 ligases and their cognate E2-Ub conjugates, facilitating their ubiquitination and subsequent degradation via the proteasome. For example, under normoxic conditions, VHL protein recruits proline-hydroxylated HIF1α transcription factor to CUL2-CRL for its ubiquitination and degradation. We can utilize the substrate receptor of the CUL2-CRL machinery together with our AdPROM system to recruit, ubiquitinate, and degrade POIs. Indeed, by tethering VHL to nanobodies or monobodies, which are small, high-affinity polypeptide binders for specific proteins, and introducing them into different cells, we were able to selectively recruit target proteins to CUL2-CRL for effective and rapid destruction (18, 19). Thus far, we have demonstrated the efficacy of AdPROM in disrupting different endogenous intracellular target proteins in different human cells (18, 19).

[0130] AdPROMs consist of two components: an "affinity" probe for a target protein (e.g., a nanobody against α-synuclein) and an interconnected E3 ligase component, such as VHL, the CUL2-CRL substrate receptor. For the affinity probe, we used a nanobody against GFP (which degrades GFP-tagged α-synuclein overexpressed in cells as a proof of concept) and a nanobody made using wild-type α-synuclein protein antigen (a single-chain VHH antibody made from alpaca) (22, 23). One of these nanobodies, NbSYN87, showed particular promise in degrading alpha-synuclein when conjugated to the PEST proteasome targeting sequence (24). We show that the use of both anti-GFP nanobodies to degrade GFP-tagged alpha-synuclein, and NbSYN87 to degrade untagged, endogenous alpha-synuclein, is achieved via the AdPROM system.

[0131] Materials and Methods: Plasmids: All DNA constructs were generated by MRC PPU Reagents and Services and sequenced by MRC PPU DNA Sequencing and Services. All plasmids used in this study are available on request from https: / / mrcppureagents.dundee.ac.uk. For SK-MEL13 SNCA KO cells, the following guide RNAs (gRNAs) were generated: sense gRNA (DU64505) and antisense gRNA (DU64516).

[0132] Retroviral generation of stable cell lines: Retrovirus was generated using pBABED puro vector. HEK 293FT cells in a 10 cm dish at approximately 70% confluency were transiently transfected with 6 μg of vector, along with 2.2 μg of pCMV5-VSV-G and 3.8 μg of pCMV5-GAG / POL. Briefly, 6 μg of vector, 2.2 μg of VSV-G, and 3.8 μg of GAG / POL were added to 300 ul of Opti-MEM in one tube. 24 ul of 1 mg / ml PEI was added to 300 ul of Opti-MEM in a second tube. Both tubes were left to incubate for 5 minutes, mixed, and left for an additional 20 minutes. The contents of the tube were then added to 9 ml of DMEM medium and poured onto the HEK 293FT cells. Fresh medium was added to the cells 16 hours after transfection. After 24 hours, the viral medium was collected and passed through a 0.45 μm sterile syringe filter. The retroviral medium with optimized titer, diluted in fresh medium containing 8 μg / ml polybrene (Sigma-Aldrich), was transduced into the target cells at a confluency of about 60% for 24 hours. The retroviral medium was then replaced with a selection medium containing 2 μg / ml puromycin to select the cells that had integrated the construct. After complete death of the non-transduced cells that were placed under selection in parallel, the pool of transduced cells was utilized for subsequent experiments.

[0133] Generation of Flp-In T-Rex cell line: Flp-In T-Rex U2OS and Flp-In T-Rex HeLa cells were maintained in complete medium supplemented with 15 μg / ml blasticidin and 100 μg / ml zeocin to maintain expression of the Tet repressor and integrity of the Flp-recombination sites, respectively. Cells at 60-70% confluency were transfected with 1 μg of pcDNA5-FRT / TO vector encoding the POI and 9 μg of pOG44 Flp recombinase plasmid in 1 ml of Opti-MEM with 20 μl of 1 mg / ml PEI. The transfection mixture was incubated at room temperature for 20 min and then added dropwise to the cells. After 24 h, the medium was replaced with fresh selection medium containing 15 μg / ml blasticidin and 50 μg / ml hygromycin B. Selection medium was replaced every 2-3 days for approximately 2-3 weeks until positive clones were selected. Clones were expanded and verified by immunoblotting. Cells were incubated with 20 ng / ml doxycycline for 24 hours prior to use to induce protein expression, unless otherwise stated in the figure captions. These were used to reduce any artifacts potentially caused by overexpression of the POI in cases where leakage of expression levels was observed.

[0134] Cell lysis: First, cells were washed twice in ice-cold PBS and scraped on ice in lysis buffer (50 mM Tris-HCl pH 7.5, 0.27 M sucrose, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium β-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, and 1% NP-40) supplemented with 1× cOmplete™ protease inhibitor cocktail (Roche). Lysates were transferred to Eppendorf tubes and incubated by rotation at 4° C. for 30 min to 1 h. Lysates were clarified by centrifugation at 17,000 rpm for 20 min at 4° C. The supernatant was transferred to a fresh Eppendorf tube and the pellet was discarded. Samples were either processed for immediate use or else flash frozen in liquid nitrogen and stored at -80°C.

[0135] SDS-PAGE and Western Blotting: Cell lysates containing equal amounts of protein (10-20 μg) were resolved by SDS-PAGE using Bis-Tris gels. Gels were transferred to nitrocellulose membranes and blocked with 5% (w / v) nonfat dry milk (Marvel) / TBS-T for 1 h at room temperature. Membranes were incubated overnight at 4°C in 5% (w / v) milk / TBS-T containing the appropriate primary antibody dilutions. The primary antibodies and their dilutions used were: anti-α-synuclein (Ab6162, Abcam, 1:500), anti-FLAG HRP (A8592, Sigma, 1:1000), anti-HIF1α (610959, BD Biosciences, 1:1000), anti-GAPDH (2118S, CST, 1:5000), anti-GFP (11814460001, Sigma, 1:1000), anti-Cullin2 (51-1800, Invitrogen, 1:1000), anti-ubiquitin (Z0458, DAKO, 1:1000). The membrane was then washed with TBS-T and incubated with HRP-conjugated or fluorescent secondary antibodies for 1 hour at room temperature. The secondary antibodies used and their dilutions were rabbit anti-sheep IgG (31480, Thermo Fisher Scientific, 1:2500), goat anti-rabbit IgG (7074, CST, 1:5000), goat anti-mouse IgG (31430, Thermo Fisher Scientific, 1:5000), StarBright Blue 700 goat anti-rabbit IgG (12004161, Bio-Rad, 1:5000). The membrane was washed again with TBS-T and signal detection was performed using ECL (Merck) and ChemiDoc MP System (Bio-Rad). Protein bands were analyzed by densitometry using Image Lab (Bio-Rad).

[0136] Immunofluorescence microscopy: For immunofluorescence imaging, cells were first seeded on 16 mm diameter round sterile coverslips in 12-well culture plates and left to adhere overnight. Before use, all coverslips were sterilized with 100% (v / v) ethanol and dried. Cells were washed twice in PBS and then fixed in 4% (w / v) paraformaldehyde (diluted in PBS) at room temperature for 10 min. PFA was removed and coverslips were washed twice in PBS. Coverslips were then permeabilized with 0.2% (v / v) NP40 in PBS for 3 min. Cells were then blocked by washing twice and incubation in 1% (w / v) BSA / PBS for 15 min. The coverslips were then incubated with anti-α-synuclein antibody (610786, BD Biosciences) at a dilution of 1:100 for 1.5 hours at room temperature in a humidified chamber. The coverslips were then washed three times (10 minutes each) in 0.2% (w / v) BSA / PBS, and then incubated with goat anti-mouse IgG alexa-fluor 594-conjugated secondary antibody (A-11005, Thermo Fisher Scientific) at a dilution of 1:500 for 1 hour at 37° C., protected from light. The coverslips were then washed three times, 10 minutes each, in 0.2% (w / v) BSA / PBS, with DAPI at a dilution of 1:15,000 for the first wash. The coverslips were then briefly dipped into deionized water using tweezers and placed on a paper towel to air dry. Once dry, approximately 5 μl of Vectashield was dropped onto a glass slide and a coverslip was gently added (cell side down) to the solution and then sealed with clear nail polish. Cells were imaged on a Deltavision system (Applied Precision) with a 60× or 40× oil immersion objective and processed with SoftWoRx (Applied Precision). Where applicable, Z-series images were acquired and deconvolved using SoftWoRx.The software Adobe Photoshop or OMERO was used to process the images and create the drawings.

[0137] Proteomic analysis: Cell lysis, in-solution digestion, TMT (tandem mass tag) labeling, and fractionation Cells were lysed in lysis buffer (8M urea, 20mM HEPES pH 8.0, 1mM sodium orthovanadate, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate), sonicated, and centrifuged at 16,000×g for 20 min. Protein concentrations were determined using a BCA assay (Pierce, Waltham, MA). 100 μg of protein from each sample was reduced with 5mM DTT at 60° C. for 20 min and alkylated with 10mM iodoacetamide at room temperature for 10 min. For trypsin digestion, samples were diluted with 20 mM HEPES, pH 8.0 to reduce the urea concentration to <2M and subjected to digestion with TPCK (tosyl phenylalanyl chloromethyl ketone)-treated trypsin in 1:20 enzyme-to-substrate (Worthington Biochemical Corp, Lakewood, NJ) for 12-16 h at room temperature. Digested peptides were acidified with 1% trifluoroacetic acid (TFA), desalted using a C18 Sep-Pak cartridge (Waters, model number: WAT051910), and dried in a vacuum concentrator. These peptides from the corresponding samples were labeled with 16-plex TMT reagent according to the manufacturer's instructions. The labeled peptides were pooled together and subjected to fractionation at basic pH. Briefly, peptides were dissolved in Buffer A (10 mM ammonium formate, pH 10) and resolved into a total of 96 fractions on an XBridge BEH RPLC column (Waters XBridge BEH C18 Column, 130, 5 μm, 4.6 mm×250 mm; model no. 186003010) using a flow rate of 0.3 ml / min and a 7-40% gradient (90% acetonitrile by volume in solvent B, 10 mM ammonium formate, pH 10) over 80 min. Adjacent fractions were pooled together to generate 48 fractions for mass spectrometry analysis.

[0138] mass spectrometry These individual fractions were reconstituted in 0.1% formic acid and analyzed on an Orbitrap Fusion Tribrid Mass spectrometer (Thermo Fisher Scientific, San Jose, USA) interfaced with Dionex 3000 RSLC nano-liquid chromatography. Peptide samples were enriched on a nano viper trap column (C18, 5 μm, 100A, 100 μm × 2 cm; model number: 164562, Thermo Scientific) and separated on a 50 cm analytical column (2 μm, 100A, 75 μm × 50 cm; model number: ES803, Thermo Scientific) with a gradient of solvent B (100% CAN, 0.1% formic acid) over 100 min. The mass spectrometer was operated in data-dependent acquisition mode. Survey full scan MS (m / z: 400-1600) was acquired on the Orbitrap with a resolution of 120,000 at 200 m / z. Maximum scan speed was used, including a cycle time of 3 seconds (MS1 and MS2). Precursor ions were separated with a charge state of ≥2 in the quadrupole, a mass width of 1.6 m / z, fragmented using HCD fragmentation with a normalized collision energy of 32% and detected with a mass resolution of 60,000. AGC target for MS1: 3 × 10 5 and 50 ms; and AGC target for MS2: 5 × 10 4 The following ion acquisition times were used: MS1 ion acquisition time: 50 ms; MS2 ion acquisition time: 250 ms. Dynamic exclusion was set for 30 seconds with a mass range of 10 ppm.

[0139] 4. Data Analysis MS / MS searches were performed using the SEQUEST search algorithm against the UniProt human protein database using Proteome Discoverer 2.4 (Thermo Fisher Scientific, Bremen, Germany). The workflow included the following nodes: spectrum selector, SEQUEST search, peptide validator, reporter ion quantifier, and percolator. Methionine oxidation was set as a variable modification, and carbamidomethylation of cysteine ​​and TMT modifications at the N-terminus and lysine were set as fixed modifications. MS tolerance and MS / MS mass tolerance were set to 10 ppm and 0.02 Da, respectively. Trypsin was designated as the protease, allowing a maximum of one false cleavage. Data were also searched against a decoy database and filtered with a false discovery rate (FDR) of 1%. A “t-test” for two samples was used to identify significantly differential proteins.

[0140] Example 1 Screening for E3 ligases capable of degrading α-synuclein Introduction: The AdPROM system was designed to link E3 ligases with binders of target proteins (see Figure 1). To determine which E3 ligases are most effective in degrading α-synuclein, the E3 ligases TRIM32, RNF126, SIAH3, RNF144A, RNF125, and VHL were tethered to GFP-specific nanobodies and examined for their ability to degrade GFP-α-synuclein.

[0141] Methods: Human osteosarcoma epithelial cells (U2OS cell line) were transfected with 1 μg of pcDNA5-FRT / TO vector encoding GFP-α-synuclein, as well as 9 μg of pOG44 Flp recombinase plasmid along with the appropriate negative control construct. Positive clones were selected for 2-3 weeks. Screens were performed on Flp-IN TRex U2OS cells that had integrated GFP-α-synuclein without induction by tetracycline. Flp-IN TRex U2OS cells that had integrated GFP-α-synuclein expressed low levels of GFP-α-synuclein without induction of expression by tetracycline. Degradation of GFP-α-synuclein protein was measured by SDS-PAGE / Western blot analysis in cells expressing the E3 ligases TRIM32, RNF126, SIAH3, RNF144A, RNF125, and VHL tethered to GFP-specific nanobodies. Cells were lysed with 20 μg of protein resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and immunoblotted with the indicated antibodies against the E3 ligases TRIM32, RNF126, SIAH3, RNF144A, RNF125, and VHL.

[0142] Results: Interestingly, of the E3 ligases tested, only VHL tethered to a GFP-specific nanobody resulted in degradation of α-synuclein. No degradation of α-synuclein was observed using the TE3 ligases TRIM32, RNF126, SIAH3, RNF144A, and RNF125 in the AdPROM system (see Figure 2).

[0143] The methods described herein are suitable tools to investigate functional E3 ligases suitable for use in the present invention.

[0144] Example 2 Further screening for E3 ligases capable of degrading α-synuclein Introduction: To identify additional E3 ligases suitable for use in the degradation of alpha-synuclein when part of an AdPROM conjugate, an extended E3 ligase AdPROM screen was performed to examine the degradation of GFP-alpha-synuclein.

[0145] Methods: The method described in Example 1 was carried out to express one E2 ligase and 18 E3 ligases (Table 1) in both C-terminal and N-terminal orientations with anti-GFP16 by retroviral transduction, followed by lysis and immunoblotting to identify any degrading agents.

[0146] result: The only E2 tested in this screen, Ubch5a, failed to induce degradation of alpha-synuclein when used with the AdPROM system compared to the non-transduced control (Figure 2B). Interestingly, only three of the 18 tested E3 ligase AdPROMs appeared to induce a reduction in alpha-synuclein levels compared to the E3 / anti-GFP16 alone control, as indicated by immunoblotting (Figure 2B). VHL-anti-GFP16, shown in the initial screen (Example 1) to degrade alpha-synuclein, showed a robust reduction in GFP-alpha-synuclein protein levels (Figure 2B). KLHL6 and KEAP1 also showed a robust reduction in alpha-synuclein levels compared to the control (Figure 2B). Both the C-terminal and N-terminal orientations of KLHL6 and KEAP1 relative to anti-GFP16 showed degradation, but the E3-anti-GFP16 orientation seemed to show slightly better degradation for both E3s. CRBN-anti-GFP16 also seemed to show slight degradation of GFP-alpha-synuclein, but the reverse orientation showed no change in protein levels (Figure 2B). This was similar only for the TRAF3d56-anti-GFP16 and anti-GFP16-KLHDC2 orientations, both of which showed slight degradation of alpha-synuclein compared to the control, but the reverse orientation showed no degradation (Figure 2B). Interestingly, MDM2 showed no degradation of alpha-synuclein. This was surprising since MDM2, along with VHL and CRBN, is a commonly used E3 ligase for PROTAC development (Bekes, Langley and Crews, 2022).

[0147] To confirm that the degradation observed by the E3 ligases identified in the screen occurred via the ubiquitin-proteasome pathway, a panel of inhibitors was used: NEDDylation inhibitor MLN4924, proteasome inhibitors MG-132 and bortezomib, and lysosomal inhibitor bafilomycin A1. First, wild-type U2OS Flp-In T-REX cells were treated with these inhibitors for 14 hours to determine whether treatment alone had any effect on GFP-alpha-synuclein levels. Both bortezomib and MG-132 treatment appeared to slightly increase levels, whereas both MLN4924 and bafilomycin A1 appeared to slightly decrease GFP-alpha-synuclein protein levels compared to DMSO-treated controls (Figure 2C). Immunoblots for total ubiquitin in extracts were performed to confirm successful proteasome inhibition, while immunoblots for Cul2 and LC3b were performed to confirm successful inhibition of NEDDylation and lysosomal degradation, respectively (Figure 2C). Since both orientations of KLHL6 against anti-GFP16 showed robust degradation of GFP-alpha-synuclein in the E3 ligase screen, cells expressing either orientation were treated with inhibitors. Both MG-132 and bortezomib resulted in rescue of the degradation of alpha-synuclein caused by KLHL6 AdPROM, confirming that this degradation is proteasome dependent (Figure 2C). Bafilomycin A1 and MLN4924 did not result in rescue of KLHL6-AdPROM-dependent alpha-synuclein degradation, which was unexpected since KLHL6 is a substrate acceptor for Cullin 3 RING E3 ligase and thus MLN4924 treatment has been shown to result in the inactivation of Cul3 and rescue of degradation.However, since the previous 24 hour time point showed rescue of any AdPROM-mediated degradation, it is possible that MLN4924 treatment alone causes a decrease in GFP-alpha-synuclein levels, thus masking the rescue effect and that a longer time point should be used.

[0148] Cells expressing VHL-anti-GFP16 and KEAP1-anti-GFP16 were also treated with these inhibitors, and again, treatment with MG-132 and bortezomib rescued the degradation of GFP-alpha-synuclein caused by these AdPROMs (Figure 2C). Again, no rescue was observed with bafilomycin A1 or MLN4924. This result confirms that the short time point of 14 hours with MLN4924 was insufficient to rescue the degradation of GFP-alpha-synuclein. Two of the E3s that showed little degradation, CRBN-anti-GFP16 and TRAF3d56-anti-GFP16, were also treated with these different inhibitors. The degradation observed with these E3 AdPROMs was not as robust as that seen with VHL, KLHL6 and KEAP1 AdPROMs (Figure 2C), but the degradation was rescued by bortezomib and MG-132 treatment, confirming that this is proteasome-dependent degradation (Figure 2C). No rescue was observed when cells were treated with bafilomycin A1 and MLN4924. While the lack of rescue by MLN4924 is surprising for an AdPROM with a CUL substrate receptor, the rescue observed upon proteasome inhibition confirms that targeted degradation of GFP-alpha-synuclein occurs via the ubiquitin proteasome system (UPS). The screen further establishes KLHL6 and KEAP1 as potent proximity-induced degraders of GFP-POIs, including GFP-alpha-synuclein, when used with the AdPROM system.

[0149] [Table 1]

[0150] Example 3 The AdPROMs VHL-anti-GFP and VHL-NbSYN87 degrade overexpressed, GFP-tagged α-synuclein in U2OS osteosarcoma cells Introduction: To assess whether α-synuclein could be targeted for proteolysis by AdPROM, we generated Flp-IN T-Rex U2OS osteosarcoma cells with single copy N-terminally GFP-tagged α-synuclein integrated into a specific genomic locus containing an upstream Tet-inducible promoter, both wild-type and A53T, a common mutant found in familial PD cases. The ability to degrade alpha-synuclein via the AdPROM system, which uses a nanobody directed against GFP and tethered to VHL, previously utilized to degrade GFP-tagged proteins, was determined.

[0151] Results: Treatment of Flp-IN T-Rex U2OS osteosarcoma cells with doxycycline for 24 hours resulted in a time-dependent increase in the expression of both GFP-α-synuclein and GFP-α-synuclein-A53T (Figure 3A). In both cases, some protein expression was still detected even in the absence of doxycycline, with substantially more GFP-α-synuclein-A53T mutants detected compared to GFP-α-synuclein (Figure 3A). To eliminate any potential degradation masking through massive overexpression of the protein and to test for targeted degradation of the protein, we exploited the leaky basal levels of expression. Retroviral particles encoding the AdPROM constructs were then generated and used to infect U2OS cells stably expressing GFP-α-synuclein and GFP-α-synuclein-A53T. The levels of GFP-α-synuclein in cells infected with VHL-anti-GFP AdPROM were substantially lower than the levels in uninfected cells or in cells infected with VHL or anti-GFP controls (FIG. 3B). Similarly, the levels of GFP-α-synuclein-A53T in cells infected with VHL-anti-GFP AdPROM were much lower than the levels in uninfected cells or in cells infected with VHL or anti-GFP controls (FIG. 3B). These results imply that GFP-tagged α-synuclein or its mutants can be targeted for proteolysis by AdPROM.

[0152] Next, to determine whether the use of the two nanobodies was directed against α-synuclein, NbSYN87 and NbSYN2 could be used instead of the GFP nanobody to degrade GFP-tagged α-synuclein. This would facilitate the degradation of untagged, endogenous α-synuclein, obviating the need to tag the protein. The nanobodies were packaged into a VHL-AdPROM construct (Figure 4B) and infected into U2OS cells stably expressing GFP-α-synuclein or GFP-α-synuclein-A53T. As before, the levels of GFP-α-synuclein in cells infected with VHL-anti-GFP AdPROM were substantially reduced compared to uninfected cells or cells infected with anti-GFP and VHL controls (Figure 4C). Under these conditions, the levels of GFP-α-synuclein were nearly undetectable in cells infected with VHL-NbSYN87 AdPROM compared to uninfected cells or cells infected with VHL and NbSYN87 controls (FIG. 4C). In contrast, infection of cells with VHL-NbSYN2 or NbSYN2 alone did not affect the levels of GFP-α-synuclein compared to uninfected cells (FIG. 4B). Identical results were obtained in cells stably expressing GFP-α-synuclein-A53T, in that the VHL-Nb1 AdPROM resulted in an effective reduction of GFP-α-synuclein-A53T compared to the control (FIG. 4D). These results suggest that VHL-NbSYN87 AdPROM is effective in targeted proteolysis of GFP-α-synuclein and GFP-α-synuclein-A53T in U2OS cells.

[0153] The target-specific polypeptide binding agents of the invention must bind in a cellular context, as exemplified by the ability to degrade α-synuclein by VHL-NbSYN87, but not VHL-NbSYN2 ( FIG. 3C ). Without wishing to be bound by theory, the selectivity of the polypeptide binding agent is important for the function of the proteasome degradative protein complex.

[0154] To examine the interaction of suitable candidate polypeptide binders with the target protein, a first-pass immunoprecipitation (IP) screen was developed to identify candidate binders.

[0155] The interaction of NbSyn87 with alpha-synuclein was determined in extracts and in cells, and alpha-synuclein knockout cells were included as a control for clean immunoprecipitation of alpha-synuclein. Since a single flag tag was undetectable, the insert 3xFlag tag was used to detect the nanobody NbSyn87. NbSYN87 pulled down alpha-synuclein (Figure 5A). The results were confirmed by intracellular immunofluorescence, where GFP-tagged NbSyn87 perfectly colocalized with endogenous alpha-synuclein (Figure 5B).

[0156] This screen can be used to search for suitable selective binders of a target protein of interest. Once a specific binder is identified, it can be tested along with other AdPROM components to determine whether the binder and E3 ligase component are active, e.g., in some cases, the binder may need to interact with the substrate so that the E3 ligase component properly positions the substrate for ubiquitination.

[0157] Example 4 VHL-NbSYN87 AdPROM degrades wild-type and mutant forms of α-synuclein overexpressed in both U2OS osteosarcoma and HeLa cells Introduction: The active degradation of GFP-α-synuclein by VHL-NbSYN87 may be mediated by ubiquitination of lysine residues on the GFP tag, rather than α-synuclein itself. Therefore, to investigate the effectiveness of VHL-Nb1 in degrading untagged α-synuclein or α-synuclein-A53T mutant, we generated Flp-In T-Rex cells stably integrated with untagged α-synuclein or α-synuclein-A53T mutant under the tetracycline promoter in both U2OS and HeLa cell lines to investigate the applicability of this targeted degradation in two different cell lines. Taking advantage of the nanobody NbSYN87 tethered to VHL, we aimed to investigate the ability of degrading untagged α-synuclein via the AdPROM system (Figure 4A).

[0158] Results: When U2OS cells were infected with the VHL-anti-GFP AdPROM, no reduction in the levels of untagged α-synuclein or α-synuclein-A53T was observed compared to uninfected cells or cells infected with the VHL control or anti-GFP control. Cells infected with the VHL-NbSYN87 AdPROM showed a substantial reduction in the levels of both α-synuclein and α-synuclein-A53T compared to uninfected cells or cells infected with the VHL-anti-GFP control, VHL control, anti-GFP control, or Nb1 control (Figure 4B). This degradation of α-synuclein in cells expressing VHL-NbSYN87 was also reproducible in HeLa Flp-In T-Rex cells, which express an untagged form of the protein (Figure 4B). As previously shown for the GFP-tagged α-synuclein cell lines, these cells were also retrovirally infected to express VHL-NbSYN2, and no degradation was observed using this nanobody (Figure 4B). These results suggest that VHL-NbSYN87 is able to degrade untagged α-synuclein and α-synuclein-A53T overexpressed in both the tested cell lines, HeLa and U2OS.

[0159] Alpha-synuclein is a 140 kDa protein that consists of three major domains: an N-terminal amphipathic alpha-helical region, a central hydrophobic core or NAC domain, known as the non-amyloid component, and a C-terminal acidic tail. Six mutations have been found in alpha-synuclein in familial cases of PD, all contained within the N-terminal region of the protein. The epitope of the nanobody NbSYN87 is the C-terminal region of the protein, indicated by the red line (Figure 4D). This implies that this approach can be used to degrade all mutant forms of the protein that have been described so far in clinical cases of familial Parkinson's disease.

[0160] We generated HeLa Flp-In T-Rex cells expressing four other mutants of the protein: A30P, E46K, G51D, and H50Q. When retrovirally infected to express VHL-NbSYN87, VHL alone, or NbSYN87 alone, we observed significant degradation of all four mutants of α-synuclein in cells expressing the VHL-NbSYN87 AdPROM (Figures 4E-4F). This indicates that VHL-NbSYN87 is capable of degrading untagged α-synuclein as well as clinically relevant mutant forms of the protein.

[0161] Example 5 VHL-NbSYN87 AdPROM degrades endogenous α-synuclein To explore whether VHL-NbSYN87 AdPROM targets physiological levels of α-synuclein for degradation, we probed commonly used human melanoma cell lines in the absence of relevant nearby neural cells to find any melanoma cell lines that expressed detectable levels of α-synuclein, since previous studies have reported on endogenous levels of the protein in certain melanoma cell lines (25). Two melanoma cell lines, SK-MEL13 and G-361, indeed expressed levels of α-synuclein protein that were detectable by Western blotting, whereas most other cell lines we examined did not express it (Figure 6A).

[0162] Infection of both SK-MEL13 and G361 cells with VHL-NbSYN87 AdPROM resulted in a near complete loss of endogenous α-synuclein levels compared to uninfected cells or cells infected with VHL-anti-GFP control, VHL control, anti-GFP control, or NbSYN87 control (Figure 6B). These results suggest that VHL-NbSYN87 can degrade physiological α-synuclein protein from cells expressing α-synuclein and has the potential to be involved in therapeutic removal of α-synuclein. To determine whether the loss of endogenous α-synuclein levels by VHL-NbSYN87 is indeed mediated by a CUL2-CRL mechanism, we treated G-361 cells with MLN4924, a pan-Cullin neddylation inhibitor, for 24 hours prior to cell lysis. As expected, treatment of G-361 cells with MLN4924 resulted in robust inhibition of CUL2 neddylation and stabilization of its endogenous target, HIF1α (Figure 6C). Under these conditions, the loss of endogenous α-synuclein levels was partially rescued via treatment of cells with MLN4924 after 24 h (Figure 6C). Importantly, expression of VHL-NbSYN87 did not affect endogenous HIF1α levels compared to controls, suggesting that the AdPROM system does not interfere with the endogenous CUL2 E3 ubiquitin ligase machinery (Figure 6C).

[0163] Example 6 The AdPROMs KLHL6-NbSYN87, KEAP1-NbSYN87, and KLHDC2-NbSYN87 degrade endogenous α-synuclein Introduction: As discussed above, in addition to VHL, KLHL6, KLHDC2, and KEAP1 were also found to be capable of degrading GFP-alpha-synuclein in an E3 ligase screen. The ability of KLHL6, KLHDC2, and KEAP1 to degrade endogenous alpha-synuclein was then determined.

[0164] Methods: KLHL6, KLHDC2, and KEAP1 were each cloned into a vector along with NbSYN87. KEAP1 was examined in both orientations to determine if there were any differences between having NbSYN87 on the N-terminus versus the C-terminus, whereas KLHL6 and KLHDC2 were examined only in the orientation with the nanobody at the C-terminus. SK-MEL-13 cells were retrovirally transduced to express these constructs and, after puromycin selection, lysed and immunoblots were performed to examine any effects on alpha-synuclein levels.

[0165] Results: All AdPROMs, NbSYN87-KEAP1, KEAP1-NbSYN87, KLHL6-NbSYN87, and KLHDC2-NbSYN87, induced degradation of endogenous alpha-synuclein protein in SK-MEL-13 cells compared to empty vector control (Figure 7A). Quantitation of duplicate biological replicates showed that the level of alpha-synuclein degradation was approximately 50% compared to empty vector control (Figure 7B). The E3 screen revealed three additional E3 ligases capable of degrading endogenous alpha-synuclein via proximity induction, which may provide an alternative approach to the complete degradation achieved by VHL-NbSYN87.

[0166] Example 7 Targeted degradation of α-synuclein via VHL-NbSYN87 is comparable to knockout cells and reproducible in neuroblastoma cell lines The extent of reduction in α-synuclein levels in these melanoma cells expressing VHL-NbSYN87 led us to quantify this degradation and compare it to complete KO cells. Previous studies performed in dopaminergic neurons differentiated from iPSCs derived from SNCA triple duplication patients showed that complete KO of α-synuclein reduced disseminated aggregation compared to wild-type cells (26). Other studies using KO cells have shown that abolishment of α-synuclein protein levels results in resistance to certain neurotoxicity models for Parkinson's disease (27-29). Due to the difficulties in using CRISPR / Cas9 strategies for therapeutic development, we wanted to examine the level of α-synuclein degradation compared to complete KO cells to determine whether the AdPROM system could be used as a similar strategy to target α-synuclein.

[0167] Knockout cells were generated in SK-MEL13 cells using a CRISPR / Cas9 strategy. Cells infected with retroviruses to express VHL-NbSYN87, VHL, or NbSYN87 were lysed together with the two resulting clones of SK-MEL13 SNCA KO cells (clones 20 and 22). The reduction in α-synuclein levels obtained by targeted degradation via the AdPROM system was comparable to that of all KO cells (Figure 8A), which was confirmed by three independent experiments and subsequent quantification of relative α-synuclein levels (Figure 8B). Immunostaining of SK-MEL13 cells for α-synuclein shows a marked lack of staining in KO and VHL-NbSYN87 cells compared to wild-type cells (Figure 8C). This highlights the resolving power of the AdPROM system and, with previous studies performed using KO cells, suggests a potential therapeutic value in targeted degradation of this alpha-synuclein by the AdPROM system.

[0168] Due to the unavailability of primary neuronal cells, we next attempted to demonstrate the applicability of this approach in SH-SY5Y cells, a neuroblastoma cell line. When retrovirally infected to express VHL-NbSYN87 or an appropriate control, these cells showed a significant reduction in endogenous α-synuclein levels only in cells expressing the VHL-NbSYN87 construct (Figure 8D). Quantification of relative α-synuclein levels by triplicate independent repetitions of this experiment shows a significant reduction in protein levels only in VHL-NbSYN87 expressing cells (Figure 8E). This confirms the feasibility of this approach in more neuronal-like cell lines, suggesting the potential of this approach in primary neurons.

[0169] Example 8 Targeted degradation of α-synuclein via the VHL-NbSYN87 is highly specific Introduction: To determine the specificity of this targeted degradation of α-synuclein, we investigated global quantitative proteomic changes upon expression of VHL-NbSYN87 AdPROM in SK-MEL13 cells.

[0170] SK-MEL13 cells transduced with pBABED empty vector control or virus encoding the VHL-NbSYN87 AdPROM were compared. In these cells, we confirmed a reduction in alpha-synuclein levels by immunoblotting before processing the samples for proteomic analysis (Figure 9). Samples treated with the proteasome inhibitor MG132 were also prepared for ongoing pan-proteomics to attempt to identify potential ubiquitin sites (Figure 10). Interestingly, alpha-synuclein was the only protein whose levels were significantly reduced in cells expressing the VHL-NbSYN87 construct compared to empty vector control cells (Figure 10). As expected due to transduction of the VHL-NbSYN87 plasmid into the cells, VHL protein levels were significantly elevated. Plasminogen activator inhibitor 2 (PAI2), another protein encoded by the SerpinB2 gene, was also increased in VHL-NbSYN87 cells compared to cells treated with empty vector (Figure 10). Protein levels of the other two members of the synuclein family, β-synuclein and γ-synuclein, did not change across samples, indicating the high specificity of the approach. The nanobody itself has also been shown to be highly specific for human α-synuclein. When U2OS Flp-In T-REX cells expressing mouse alpha-synuclein were retrovirally transduced to express VHL-NbSYN87 or VHL and NbSYN87 alone, no degradation of mouse α-synuclein was observed in cells expressing VHL-NbSYN87, despite the approximately 97% sequence identity between the human and mouse forms of the protein (Figure 11).

[0171] Sequence (5' to 3')

[0172] DNA sequence encoding NbSYN87 (SEQ ID NO:1):

[0173] [ka]

[0174] Amino acid sequence of NbSYN87 (SEQ ID NO:12):

[0175] [ka]

[0176] DNA sequence encoding VHL (SEQ ID NO:2):

[0177] [ka]

[0178] Amino acid sequence of VHL (SEQ ID NO:13):

[0179] [ka]

[0180] DNA sequence encoding VHL-NbSYN87 (SEQ ID NO:3):

[0181] [ka]

[0182] Amino acid sequence of VHL-NbSYN87 (SEQ ID NO: 14)

[0183] [ka]

[0184] DNA sequence encoding KLHL6 (SEQ ID NO:6):

[0185] [ka]

[0186] Amino acid sequence of KLHL6 (SEQ ID NO:15):

[0187] [ka]

[0188] DNA sequence encoding KEAP1 (SEQ ID NO:7):

[0189] [ka]

[0190] Amino acid sequence of KEAP1 (SEQ ID NO:16):

[0191] [ka]

[0192] DNA sequence encoding CRBN (SEQ ID NO:8):

[0193] [ka]

[0194] Amino acid sequence of CRBN (SEQ ID NO:17):

[0195] [ka]

[0196] DNA sequence encoding KLHDC2 (SEQ ID NO:9):

[0197] [ka]

[0198] Amino acid sequence of KLHDC2 (SEQ ID NO:18):

[0199] [ka]

[0200] DNA sequence encoding TRAF3d56 (SEQ ID NO:10):

[0201] [ka]

[0202] Amino acid sequence of TRAF3d56 (SEQ ID NO:19):

[0203] [ka]

[0204] Alpha-synuclein:

[0205] [ka]

[0206] DNA sequence of NbSYN87(5-G linker)KEAP1 (SEQ ID NO:20)

[0207] [ka]

[0208] [ka]

[0209] NbSYN87 (5-G linker) KEAP1 (SEQ ID NO: 21)

[0210] [ka]

[0211] DNA sequence of KEAP1 (5-G linker) NbSYN87 (SEQ ID NO:22)

[0212] [ka]

[0213] Amino acid sequence of KEAP1 (5-G linker) NbSYN87 (SEQ ID NO:23)

[0214] [ka]

[0215] DNA sequence of KLHDC2 (5-G linker) NbSYN87 (SEQ ID NO:24)

[0216] [ka]

[0217] Amino acid sequence of KLHDC2 (5-G linker) NbSYN87 (SEQ ID NO:25)

[0218] [ka]

[0219] DNA sequence of KLHL6 (5-G linker) NbSYN87 (SEQ ID NO:26)

[0220] [ka]

[0221] [ka]

[0222] Amino acid sequence of KLHL6 (5-G linker) NbSYN87 (SEQ ID NO:27)

[0223]

change

[0224] (References) TIFF2025503444000031.tif206159TIFF2025503444000032.tif206159TIFF2025503444000033.tif22715 9TIFF2025503444000034.tif208160TIFF2025503444000035.tif226160TIFF2025503444000036.tif65160

Claims

1. A proteasomal degradation protein complex comprising an E3 ubiquitin ligase component tethered to an alpha-synuclein-specific polypeptide binder, capable of targeting alpha-synuclein for proteasomal degradation.

2. the alpha-synuclein-specific polypeptide binding agent is an antibody, antibody fragment, monobody, and / or nanobody; and / or the alpha-synuclein-specific polypeptide binding agent is capable of binding to a target epitope within the C-terminal region of the alpha-synuclein protein and degrading alpha-synuclein; and / or the alpha-synuclein-specific polypeptide binding agent is NbSYN87 or a functional variant thereof comprising a sequence that is at least 70, 80%, 90%, 95%, or 99% identical to NbSYN87; or whether the α-synuclein-specific polypeptide binding agent targets the same epitope on α-synuclein as NbSYN87; or the alpha-synuclein-specific polypeptide binding agent has an affinity equivalent to or higher than NbSYN87; and / or the E3 ubiquitin ligase component is a substrate acceptor for a cullin ring E3 ligase complex; and / or the E3 ubiquitin ligase component is capable of recruiting alpha-synuclein to either CUL2-CRL, CUL3, or CUL4; and / or the E3 ubiquitin ligase component is the VHL (von Hippel-Lindau) tumor suppressor gene or a functional variant thereof, suitably the functional variant comprises a sequence which is at least 60% identical to wild-type VHL, preferably a sequence which is at least 70%, 80%, 90%, 95% or 99% identical to wild-type VHL; and / or the alpha-synuclein protein is targeted for degradation by the ubiquitin-mediated proteasome degradation system, and / or The proteasome degradation protein complex of claim 1, which is an affinity-directed protein missile (AdPROM).

3. 10. One or more nucleic acid constructs encoding the proteasome degradation protein complex of claim 1.

4. the E3 ligase component is VHL or a functional variant thereof, and the alpha-synuclein-specific polypeptide binding agent is NbSYN87 or a functional variant thereof; and / or 4. The one or more nucleic acid constructs of claim 3, comprising a nucleotide sequence according to SEQ ID NO: 3, or a sequence that is at least 60%, 70%, 80%, 90%, 95%, or 99% identical thereto.

5. 4. An expression construct comprising the nucleic acid construct of claim 3 operably linked to one or more expression control sequences.

6. A vector comprising the expression construct of claim 5.

7. 7. The vector of claim 6, which is a gene therapy vector, suitably a viral vector, suitably an AAV vector, an adenoviral vector, a retroviral vector, or a lentiviral vector.

8. A pharmaceutical composition comprising a proteasome degradation protein complex described in claim 1 or 2, a nucleic acid construct described in claim 3 or 4, an expression construct described in claim 5, or a vector described in claim 6 or 7, and a pharmaceutically acceptable carrier or diluent.

9. 10. The pharmaceutical composition of claim 8 for use in treating a subject with a neurodegenerative disorder.

10. the neurodegenerative disorder is a synucleinopathy, and / or the neurodegenerative disorder is Parkinson's disease, dementia, and / or multiple system atrophy; and / or a proteasomal degradation protein complex targets alpha-synuclein for proteasomal degradation; and / or 9. The pharmaceutical composition of claim 8, wherein the subject has an SNCA double duplication, an SNCA triple duplication, or point mutations A53T, A30P, H50Q, E46K, G51D, and / or A53E.

11. An in vitro or ex vivo method for targeting alpha-synuclein for degradation using the pharmaceutical composition of claim 8.

12. Administering the pharmaceutical composition of claim 8 to cells in vitro. Includes; an α-synuclein-specific polypeptide binder component of the proteasome degradation protein complex binds to α-synuclein; The E3 ligase component tethered to the α-synuclein-specific polypeptide binder recruits the α-synuclein protein to the intracellular E3 ligase system; An intracellular E3 ligase system ubiquitinates α-synuclein so that it can be degraded.

10. An in vitro or ex vivo method for targeting alpha-synuclein for degradation using the pharmaceutical composition of claim 8.

13. 12. The method of claim 11, wherein the alpha-synuclein is a monomer, oligomer, protofibril, mature fiber, or aggregate.

14. 12. The method of claim 11, wherein the alpha-synuclein is an A53T, A30P, H50Q, E46K, G51D, and / or A53E mutant.

15. 12. The method of claim 11, wherein alpha-synuclein is targeted for proteasomal degradation.

16. The method of claim 15, wherein the proteasomal degradation is ubiquitin-mediated proteasomal degradation.

17. 10. The pharmaceutical composition of claim 8 for use as a targeting research tool.

18. A kit comprising the pharmaceutical composition of claim 8 and instructions for use.

Images