Therapeutic fusion proteins that target pathogenic protein aggregates for degradation

Abstract

Novel materials and methods are provided for treating neurodegenerative diseases associated with protein aggregates by selectively targeting the aggregates. The present invention relates to methods and materials for use in treating neurodegenerative diseases associated with pathological protein aggregates, and provides fusion proteins comprising (i) a first portion comprising the sequence of a protein that pathologically aggregates in neurodegenerative diseases, such as tau protein, and (ii) a second portion comprising a RING-type E3 ubiquitin ligase, a component of a RING-type E3 ubiquitin ligase complex, or a domain of either. These fusion proteins are useful for selectively or preferentially targeting pathogenic protein aggregates for degradation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority from GB2217084.9, filed on November 16, 2022, which is incorporated herein in its entirety.

[0002] Technical Field The present invention relates generally to methods and materials for use in treating neurodegenerative diseases associated with pathological protein aggregates through the use of therapeutic polypeptides that inhibit aggregate formation or increase aggregate degradation or clearance. [Background technology]

[0003] Background of the Invention Neurodegenerative diseases (NDs) include highly debilitating illnesses such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, frontotemporal dementia (FTD), corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy, multiple system atrophy, dementia with Lewy bodies, and prion diseases (PrD).

[0004] NDs vary widely in clinical manifestations and prevalence, but share many common features, including their chronic and progressive nature, increasing prevalence with age, destruction of neurons in specific brain regions, damage to networks of synaptic connections, and selective brain volume loss.

[0005] The common etiology in these diseases is the progressive accumulation of misfolded protein aggregates in ordered structures, commonly referred to as "amyloids." Despite the fact that the protein aggregates associated with different NDs are different, the process of protein misfolding, its intermediates, end products, and key features are remarkably similar (Soto, Claudio, and Sandra Pritzkow. "Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases." Nature neuroscience 21.10(2018):1332-1340).

[0006] Targeted inhibition or degradation of these aggregates has considerable therapeutic applications. The use of targeted proteolysis as a therapeutic strategy minimizes off-target drug effects and avoids or reduces systemic drug exposure (Wu, T, et al. (2020) Nature Structural & Molecular Biology, 27:605-614).

[0007] Cells have two main options for disposing of misfolded or dysfunctional proteins: the autophagy-lysosomal network and the ubiquitin-proteasome system (UPS).

[0008] Autolysosomes use acid hydrolases to degrade their contents, releasing the resulting cellular units back into the cytosol for recycling.

[0009] The proteasome targets polyubiquitinated proteins and degrades them into short peptide fragments via proteolysis.

[0010] Several approaches have been explored to cellularly target protein species for degradation. These include intracellular expression of antibody fragments, proteolysis-directing chimeric molecules (PROTACs), lysosomal targeting chimeric molecules (LYTACs), and hydrophobic tagging recognized by the UPS. 6 .

[0011] TRIM21 is a cytoplasmic antibody receptor and E3 ubiquitin ligase. Clustering of TRIM21 after binding to antibody-bound substrates activates the N-terminal RING domain of TRIM21, promoting ubiquitin chain formation. 7 Ubiquitin chain catalysis depends on the E2 enzymes Ube2w and Ube2n, which promote the monoubiquitination of TRIM21 and K63-linked ubiquitin chain elongation, respectively, leading to degradation of antibody-bound targets in the proteasome.

[0012] It has been reported that tau aggregates can internalize anti-tau antibodies into the cytosol, allowing TRIM21 to target tau aggregates for proteasomal degradation. 8 .

[0013] WO 2012 / 010855 relates to compounds comprising: (a) a ligand that specifically binds, directly or indirectly, to an antigen of a pathogen, provided that said ligand is not the PRYSPRY domain of TRIM21; and (b) a RING domain and / or an inducer of TRIM21 expression, which are reported to be useful in treating pathogenic infections. Notwithstanding these prior disclosures, it is recognized that it would be a useful contribution to the art to provide novel materials and methods for treating neurodegenerative diseases associated with protein aggregates by selectively targeting the aggregates. Summary of the Invention [Means for solving the problem]

[0014] Disclosure of the Invention The inventors have devised novel agents, referred to herein (purely for brevity) as "E3 ligase-baits," for selectively or preferentially targeting pathogenic protein aggregates for degradation.

[0015] RING-type E3 ligases are characterized by the presence of a RING domain, which is the minimal element required to recruit E2 ubiquitin and stimulate ubiquitin transfer.

[0016] As explained by Balaji, Vishnu, and Thorsten Hoppe (F1000Research 9 (2020)), regulation of these ligases is provided, in part, by homotypic and heterotypic combinations of E3 ligases into oligomeric (i.e., higher-order aggregates) ubiquitination complexes. As explained by Errington, Wesley J., et al. "Adaptor protein self-assembly drives the control of a cullin-RING ubiquitin ligase." Structure 20.7 (2012):1141-1153, the RING superfamily consists of hundreds of E3s that vary widely in size, structure, subunit stoichiometry, and mode of regulation. However, the RING superfamily generally exhibits a striking tendency to self-assemble into oligomers, enhancing activity.

[0017] Some RING E3s are large multi-subunit complexes. These RING-type E3 ligase complexes typically contain several components or subunits that can self-assemble in vivo, typically including a catalytic E3 ligase component as well as a scaffolding component, an adaptor component, and a substrate recognition component.

[0018] Once assembled, these RING-type E3 ubiquitin ligase complexes can facilitate the direct transfer of ubiquitin from an E2 ubiquitin intermediate to a target protein.

[0019] We reasoned that aggregating proteins should promote E3 ligase assembly and therefore enhance degradation activity against the aggregated proteins.

[0020] In this system, a so-called "bait" protein capable of coaggregating with a target protein in vivo is fused to an E3 ubiquitin ligase, or a component of an E3 ubiquitin ligase complex, or a domain of either. Thus, aggregate formation incorporates both the cellular pool of the target protein and the E3 ligase-bait construct.

[0021] As the fusion protein is incorporated into the growing aggregate, the second moiety comes into proximity where it can oligomerize and / or recruit other parts or cofactors of the complete E3 ubiquitin ligase complex if required, leading to ubiquitination and removal of the assembled target into which the E3 ligase-bait has been incorporated.

[0022] Solely for illustrative purposes, we fused full-length tau with aggregation-promoting mutations to the TRIM21 RING domain, which was chosen because it is activated via clustering. 7 .

[0023] Expression of tau-RING prevented tau seed-induced aggregation and cleared pre-existing tau aggregates via the ubiquitin-proteasome system.

[0024] In contrast, monomeric tau was unaffected by tau-RING expression, consistent with tau-RING specifically binding to and activating in the context of aggregated forms of tau. Intravenous and stereotactic injection of AAV encoding tau-RING into a mouse model of tau pathology substantially reduced the number of cell bodies bearing tau aggregates and the total burden of insoluble tau aggregates in the brain. Furthermore, delivery of a tau-RING construct resulted in behavioral improvements in the same mouse model, demonstrating the therapeutic benefit of clearing aggregates.

[0025] Consistent results were obtained using a RING-bait based on the Fused in sarcoma (FUS) protein carrying the P525L mutation.

[0026] The inventors further demonstrated that components of the E3 ligase complex that may not themselves contain a RING domain within the fusion protein can still be effective in E3 ligase-bait, and aggregation-induced subunit recruitment and clustering leads to disassembly of bait-fusion-containing aggregates. For illustrative purposes only, the inventors fused a tau bait to VHL, the substrate recognition component of cullin ligase, and observed selective removal of tau aggregates from cells.

[0027] The findings described herein demonstrate that localization by using the aggregating protein itself as "bait" represents a novel strategy for localization and aggregation-induced assembly of E3 ubiquitin ligase activity that can be utilized for the removal of aggregated or assembled proteins.

[0028] Importantly, this strategy may be effective against both seed-induced and cell-autonomous protein aggregation. 5 9 Furthermore, the present invention can be applied to intraneuronal populations of aggregates, which may be particularly beneficial. 4 .

[0029] In a first aspect: (i) a first portion comprising the sequence of a protein that pathologically aggregates in a neurodegenerative disease; (ii) a second portion comprising an E3 ubiquitin ligase or a component of an E3 ubiquitin ligase complex, or a domain of either; A fusion protein comprising:

[0030] A "fusion protein" or "fusion polypeptide" (the terms are used interchangeably) refers to a polypeptide having two or more covalently linked moieties, where each of the moieties is a polypeptide having a certain biological property.

[0031] In the present invention, the first portion is derived from a protein that pathologically aggregates in neurodegenerative diseases, and for simplicity may be referred to herein as the "protein aggregating" portion or domain.

[0032] As explained above, the second part comprises an E3 ubiquitin ligase, or a component of an E3 ubiquitin ligase complex, or a domain of either. Preferably, the E3 ubiquitin ligase or complex is of the RING type. The second part may or may not itself have E3 ligase catalytic activity.

[0033] Both parts of the fusion protein are described in detail below.

[0034] The terms "E3 ubiquitin ligase" and "E3 ligase" are used interchangeably herein.

[0035] Some E3 ligases become functional once they form part of a larger complex. These can contain several components or subunits that can self-assemble in vivo: this can include the catalytic E3 ligase component, which is a RING protein (i.e., a RING or RING finger domain-containing protein), such as a TRIM or Rbx protein, as well as a substrate receptor (which in turn can include an adaptor complex and a substrate recognition module, e.g., VHL), a scaffold (e.g., a Cullin 2 polypeptide), and so on. The activated E3 ligase can covalently transfer ubiquitin to itself or another protein.

[0036] Those skilled in the art are well aware of examples of scaffold components, adapter components, substrate recognition components, and catalytic E3 ligase components (see, e.g., Buetow, Lori, and Danny T. Huang. "Structural insights into the catalysis and regulation of E3 ubiquitin ligases." Nature reviews Molecular cell biology 17.10(2016):626-642).

[0037] By way of non-limiting example, scaffold components include: CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7, CUL9; adaptor components include: SKP1, elongin B, elongin C, DDB1, BTB; substrate recognition components include FBXW1, FBXW11, FBXL1, FBXO1, VHL, LRR1, FEM1, KEAP1, SPOP, KLHL40, BTB6A, DCAF1, DCAF14, DDB2, CRBN; and catalytic E3 ligase components include RBX1, RBX2.

[0038] A "domain" is a folded protein structure that retains its tertiary structure independently of the rest of the protein. Generally, domains are responsible for distinct functional properties of a protein and can often be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain. The RING domain of a TRIM polypeptide is one example.

[0039] Such fusion proteins can comprise, consist of, or consist essentially of the moieties described herein.

[0040] Such fusion proteins (polypeptides) can be provided in isolated, purified, or semi-purified form.

[0041] Fusion proteins are non-naturally occurring because they contain heterologous sequences that are not found together in nature.

[0042] The two portions of the fusion protein can be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other.

[0043] Typically, the fusion protein will be prepared by recombinant DNA techniques standard in the art and may be referred to herein as a recombinant fusion protein.

[0044] The term "recombinant" refers to genetic material (i.e., nucleic acids, encoding polypeptides, and vectors and cells containing such polynucleotides) that has been modified to change its sequence or expression characteristics, for example, by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence with that of another gene, placing the gene under the control of a different promoter, expressing the gene in a heterologous organism, expressing the gene at reduced or elevated levels, expressing the gene constitutively or constitutively in a manner that differs from its native expression profile, etc. Generally, recombinant nucleic acids, polypeptides, and cells based thereon have been manipulated by man so that they are not identical to the related nucleic acid, polypeptide, and cell found in nature.

[0045] In one embodiment, the E3 ligase complex is of the multisubunit Cullin-RING E3 ubiquitin ligase type.

[0046] In one embodiment, the component or domain does in fact have E3 ligase catalytic activity.

[0047] In another embodiment, the component or domain does not have E3 ligase catalytic activity (e.g., it is a scaffold component, an adapter component, a substrate recognition component), but has E3 ligase recruitment activity.

[0048] A further aspect of the present invention relates to a nucleic acid molecule encoding the fusion protein as described above. The nucleic acid molecule can be operably linked to an expression control sequence, for example, an expression control sequence that allows the nucleic acid molecule to be expressed in a desired host cell, or an expression control sequence that allows inducible expression. The nucleic acid molecule can be located on a vector, for example, a plasmid, a bacteriophage, a viral vector, a chromosomal integration vector, a lipid nanoparticle, etc., or can be a "naked" nucleic acid, such as mRNA, that can be delivered via lipid nanoparticles.

[0049] A further aspect of the present invention relates to a pharmaceutical composition comprising as an active agent at least one fusion protein as described above.

[0050] Also described are the use of the fusions of the invention in therapeutic methods or methods of using the fusions, for example, to treat diseases of protein aggregation.

[0051] Some of these aspects and embodiments of the invention will now be described in more detail:

[0052] In one embodiment, the entity or domain comprises or is a RING domain.

[0053] Tri-element motif (TRIM) proteins constitute a family of proteins based on a conserved domain structure (known as RBCC) characterized by a RING (Really Interesting New Gene) finger domain, one or two B-box domains, a coiled-coil domain, and a variable C-terminus.

[0054] In one embodiment, the component or domain is derived from a TRIM polypeptide.

[0055] In one embodiment, the component or domain comprises or is a RING-B-box (see SEQ ID NO: 6).

[0056] The optional incorporation of a Box domain can provide an additional layer of control over monomer / aggregate selectivity by virtue of its ability to self-associate or bind to a RING.

[0057] Replacement of RING domains with heterologous TRIM domains, exchanging them between IM proteins, is known in the art. See Li et al., J. Virol. (2006) 6198-6206. The RING domain was described by Freemont et al., Cell. 1991 Feb 8;64(3):483-4. This domain is thought to function as an E3 ligase; see Meroni & Roux, BioEssays 27(11):1147-1157(2005).

[0058] The RING domain has a "cross-brace" motif: Cys-X2-Cys-X (9-39) -(Cys / His)-X (1-4) -(His / Asn / Cys)-X (2-4) -(Cys / His)-X2-Cys-X (4-48) -Cys-X2-(Cys / Asp) They contain a special type of zinc finger of 40 to 60 residues that binds to two atoms of zinc, as defined by (Deshaies RJ et al and Joazeiro C et al, RING Domain E3 Ubiquitin Ligases, Annu. Rev. Biochem(2009) 78:399-434).

[0059] Within this family, there are two variants with different cysteine / histidine patterns: C3HC4 and C3H2C3 (RING-H2 finger). C3HC4 has the motif: C-X2-CX(9-39)-CX(1-3)-HX(2-3)-C-X2-CX(4-48)-C-X2-C.

[0060] Examples of RING class E3 ubiquitin ligases are provided in Balaji, Vishnu, and Thorsten Hoppe, cited above, and are individually incorporated herein by reference.

[0061] Preferred RING domains are derived from TRIM proteins, hi one embodiment the RING domain is derived from the group consisting of TRIM5α, TRIM7, TRIM19, TRIM21 and TRIM28.

[0062] The RING domain of TRIM21 is responsible for the degradation of bound antibody / antigen complexes, due to the E3 ubiquitin ligase activity of the RING domain.

[0063] The sequence of TRIM21 is shown in SEQ ID NO:1.

[0064] RING E3 ligase domains are found in a variety of proteins. Other RING domains include those from mammalian protein X-linked inhibitor of apoptosis (XIAP) and the DER3 / Hrd1 RING domain. Thus, the use of RING domains from other protein families in fusion proteins is also encompassed.

[0065] Examples of RING domains that can be utilized directly or modified for use in the present invention are shown below in Table 1. Fusion proteins can include proteins listed in Table 1, or the RING-containing sequences shown, or portions thereof, or active variants thereof. The RING domain portion will typically be at least 25, 30, or 35 amino acids in length or longer.

[0066] As explained above, RING domains for use in the present invention will have E3 ubiquitin ligase activity, i.e., the ability to catalyze the covalent transfer of ubiquitin to the RING-containing protein itself or to another protein, which activity is induced or enhanced for aggregated protein targets by clustering, e.g., when at least two RING domains are brought into close proximity.

[0067] In one embodiment of the present invention, the RING domain is derived from amino acids 1 to 85 of SEQ ID NO: 1 (=SEQ ID NO: 2).

[0068] In one embodiment, the domain comprises amino acids 3 to 81 of SEQ ID NO: 2, preferably amino acid residues 1 to 81 of SEQ ID NO: 2, more preferably the sequence of SEQ ID NO: 2 or a variant thereof.

[0069] As explained above, in one embodiment, the components or domains do not actually have E3 ligase catalytic activity per se, but may serve another purpose within the complex, such as recognizing or recruiting natural substrates or forming a structural scaffold.

[0070] As demonstrated in the examples below, the fusion protein will remain effective provided that E3 ligase activity can be recruited to the vicinity of the aggregate.

[0071] Thus, in one embodiment, the component is a VHL ("Von Hippel-Lindau") domain or protein. VHL is an E3 ubiquitin ligase component that recruits substrates for ubiquitination and subsequent proteasomal degradation.

[0072] In one embodiment, the VHL polypeptide comprises SEQ ID NO: 5, or a variant having at least 70% identity thereto.

[0073] Aggregation of naturally occurring proteins, such as tau, α-synuclein, and TDP43, drives neurodegeneration in several common proteopathic diseases. Diseases caused by aggregates of the protein tau are the most common neurodegenerative disorders and are characterized by the progressive appearance of tau aggregates over time throughout the brain.

[0074] Disease-associated aggregated states can expand by monomer addition into larger fibrils and macromolecular inclusions such as neurofibrillary tangles (tau) and Lewy bodies (α-synuclein).

[0075] The molecular structures of some of these assemblies, for tau, α-synuclein, and TDP43, have now been resolved by cryoEM, revealing characteristically folded but highly ordered fibrillar structures (e.g., Shi, Y., Zhang, W., Yang, Y. et al. Structure-based classification of tauopathies. Nature598, 359-363(2021). https: / / doi.org / 10.1038 / s41586-021-03911-7; Yang, Y., Shi, Y., Schweighauser, M. et al. Structures of α-synuclein filaments from human brains with Lewy pathology. Nature(2022). https: / / doi.org / 10.1038 / s41586-022-05319-3; Arseni, D., Hasegawa, M., Murzin, AG et al. al. Structure of pathological TDP-43 filaments from ALS with FTLD. Nature 601, 139-143(2022). https: / / doi.org / 10.1038 / s41586-021-04199-3)

[0076] As explained above, the present invention utilizes fusion proteins having a first portion comprising the sequence of a protein that pathologically aggregates in a neurodegenerative disease, which targets pathological protein aggregates, inhibiting their formation and / or increasing their degradation or clearance.

[0077] Those skilled in the art recognize that the common etiology of these diseases is the progressive accumulation of misfolded, insoluble, and non-functional protein aggregates (Fink, Anthony L. "Protein aggregation: folding aggregates, inclusion bodies, and amyloid." Folding and design 3.1 (1998): R9-R23). This aggregation can occur through homotypic interactions of corresponding segments of proteins. These aggregates can be ordered structures, such as fibers, which can be referred to as "amyloids" (see, for example, Chiti, Fabrizio, and Christopher M. Dobson. "Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade." Annual review of biochemistry 86 (2017): 27-68 - Table 1). In some cases, these insoluble protein precipitates may be more irregular and are sometimes referred to as "non-amyloid" (see, eg, Chiti, Fabrizio, and Christopher M. Dobson, supra, Table 2).

[0078] The fibrillar nature of tau inclusions and those seen in other neurodegenerative diseases 23 provides consistent properties for therapeutic use.

[0079] Non-limiting examples of proteins that can be used in the E3 ligase-bait fusions of the present invention, and examples of diseases in which proteins pathologically aggregate, include those shown in Table 2:

[0080] [Table 1]

[0081] Example sequences are provided in Table 3 below.

[0082] In one embodiment, the protein that pathologically aggregates in a neurodegenerative disease is selected from or derived from all or part of one of the proteins listed in Table 2 or Table 3, or a variant thereof that has the same biological activity (here, the ability to incorporate into aggregates of native proteins to form co-aggregates).

[0083] In particular, in one embodiment, the protein-aggregating portion of the fusion protein comprises all or part of a sequence shown in Table 3, or a variant having at least 70% identity thereto.

[0084] Typically, the protein-aggregating portion of the fusion protein comprises at least 20, 25, 30, 40, or 50 amino acids. However, this portion can be longer, e.g., at least 100, 200, 300, 350, or 400 amino acids. In some embodiments, it is less than 400, 450, or 500 amino acids.

[0085] As illustrated in the Examples below, the E3 ligase-bait system of the present invention can be used to target aggregates for clearance without significantly affecting the native (soluble), non-pathogenic activity of these proteins.

[0086] The utility of these exemplary proteins in the present invention is discussed in more detail below:

[0087] Tau protein 4R tauopathies are characterized by fibrillar deposition with enrichment of 4R tau isoforms. Familial FTLD-tau can be caused by rare mutations in MAPT, indicating the important involvement of tau (Bonner 2005 PMID 16014652). In progressive supranuclear palsy (PSP), tau plays a causative role, exemplified by the example of a familial variant with a MAPT mutation (Morris et al. 2002 PMID: 11861703). The MAPT H1 / H2 haplotype is associated with disease risk in corticobasal degeneration (CBD) and argyrophilic grain dementia (AGD), strongly supporting the causative role of tau in these conditions (Houlden et al. 2001 PMID: 11425937; Conrad et al. 2004 PMID: 15030402).

[0088] Rare mutations in MAPT, such as G389R, cause Pick's disease and FTLD-tau with 3R fibrils, strongly supporting a causative role for the 3R tau isoform in disease pathogenesis (Murrell et al 1999 PMID:10604746).

[0089] Some tau pathologies contain both 3R and 4R tau isoforms, which closely correlates with disease progression. In AD, pathological tau contains both 3R and 4R isoforms and is closely associated with cognitive decline (Nelson 2013 PMID:22487856). In chronic traumatic encephalopathy (CTE), similar but not identical tau folds containing all six tau isoforms are observed (Falcon et al. 2019 PMID:30894745). Rare familial mutations, such as V337M and R406W, can cause FTLD-tau with both 3R and 4R fibrils (Goedert et al. 2017 PMID:28772101).

[0090] TDP-43 TAR DNA-binding protein 43 (TDP-43) is a 414-amino acid protein found in ALS and FTD-TDP43 inclusions (Arai 2006 PMID:17084815). Its role as a cause of neurodegeneration is exemplified by mutations in TARDBP, which are causative of ALS and FTLD-U (Sreedharan 2008 PMID:18309045, Neumann et al. 2006 PMID:17023659). A recent structure of the TDP43 fibrillar core from a patient with type B pathology reveals a core extending from amino acids 282–360 (Arseni et al. 2022, https: / / doi.org / 10.1038 / s41586-021-04199-3).

[0091] Antibody therapy against TDP43 in vivo appears to act via TRIM21 (PMID: 33021970), supporting the beneficial effects of the E3 ligase-bait strategy.

[0092] In some embodiments of the present invention, the TDP43 sequence in the E3 ligase bait may lack a nuclear localization signal to target cytoplasmic aggregates. In one embodiment, a RING domain is present at the C-terminus of the fusion with the TDP43 sequence.

[0093] α-synuclein Alpha-synuclein is the core component of Lewy bodies (found in PD and DLB) and inclusions found in MSA (Spillantini et al., 1997, PMID:9278044; Spillantini et al., 1998, PMID:9726379). Its causative role in pathogenesis is exemplified by the fact that SNCA copy number variants and point mutations cause inherited forms of PD (Ibanez, 2004, PMID:15451225; Singleton et al., 2003, PMID:14593171). The fibrillar structures from multiple system atrophy (MSA) have been characterized and shown to be distinct from DLB by Schweighauser et al., 2020 (https: / / doi.org / 10.1038 / s41586-020-2317-6).

[0094] In one embodiment, the RING domain is present at the C-terminus of the fusion with the synuclein sequence.

[0095] Huntingtin (HTT) Expanded CAG repeats encoding poly-glutamine have been reported to cause Huntington's disease (Lee et al. 2019 PMID:31398342). In one embodiment, the RING domain is fused directly or indirectly to exon 1 or a portion of HTT containing a polyglutamine repeat.

[0096] Rhodopsin Mutations in rhodopsin (RHO) lead to retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably leads to vision loss. P23H is the most common variant and is inherited in an autosomal dominant manner.

[0097] In one embodiment, the RING domain is present at the C-terminus of rhodopsin carrying a specific causative mutation (e.g., P23H). It has been reported that the P23H RHO mutant can be specifically inactivated in vitro while maintaining the WT allele using a CRISPR / Cas9 strategy (Giannelli et al., 2018 PMID 29281027).

[0098] superoxide dismutase Mutations in SOD1 can cause amyotrophic lateral sclerosis (Deng et al., 1993, 10.1126 / science.8351519) (Saccon et al., 2013, doi:10.1093 / brain / awt097). Mutations have been found throughout the entire length of the protein, and all reduce native SOD1 activity. SOD1 has been reported to form ThT-positive aggregates (Chattopadhyay et al., 2008 https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC2585484 / ), which can be propagated in mice (Ayers et al., 2014 https: / / link.springer.com / article / 10.1007 / s00401-014-1342-7).

[0099] Antibody therapy against SOD1 improves lifespan in mice (DOI: 10.1126 / scitranslmed.aah3924) and reduces motor neuron toxicity in other models (Benkler et al. 2018 PMID: 30401824), supporting the fact that E3 ligase-bait strategies can be effective against these aggregates.

[0100] FUS FUS mutations cause ALS (Kwiatkowski Jr et al., 2009, doi:10.1126 / science.1166066) and FTD (Langenhove et al., 2010, DOI:https: / / doi.org / 10.1212 / WNL.0b013e3181ccc732).

[0101] ASO-mediated knockdown of FUS levels in mutant mice (and human subjects) has been reported to reduce aggregates and improve lifespan, supporting the use of an E3 ligase-bait approach to achieve this (https: / / doi.org / 10.1038 / s41591-021-01615-z).

[0102] As explained above, the two parts of the fusion protein can be linked directly by a single peptide bond or via a peptide linker containing one or more amino acid residues, such as a flexible linker.

[0103] Furthermore, the first and second portions can be of either polarity, for example, the protein aggregation domain can be C-terminal and N-terminal to the RING domain.

[0104] Fusion proteins according to the invention may have additional N- and / or C-terminal amino acids or sequences, and / or additional domains (e.g., fluorescent protein domains) located at the termini or between the RING domain and the protein aggregation domain.

[0105] In other embodiments, standard linker sequences known in the art can also be used, for example, polyglycine or polyserine amino acid sequences can be used, or mixed glycine / serine linkers can be used. Preferred linkers are shown in SEQ ID NOs: 3 or 4, such as GSGGGSG, or GSSS-based linkers, such as (GSSS)3.

[0106] The linker length can vary in size. In one embodiment, the linker sequence between the protein aggregation domain and the RING domain is 1 to 200, 1 to 100, or 1 to 50 amino acids in length, preferably 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, or 1 to 10 amino acids in length. More preferably, the linker is 1 to 6 amino acids in length, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.

[0107] Non-limiting examples of E3 ligase baits based on tau protein domains are shown in SEQ ID NOs: 3 and 4. In one embodiment, the E3 ligase bait is one of these or a variant thereof, e.g., substantially identical to one of these. In other embodiments, the protein aggregation domain is selected according to the target protein or relevant disease (non-limiting examples are given in Tables 3 and 4) and is arranged in a suitable polarity with the RING domain, optionally via one or more linkers.

[0108] As explained above, the fusion proteins of the present invention are typically prepared by recombinant DNA techniques standard in the art.

[0109] For example, a polynucleotide encoding a fusion protein can be provided by a process comprising: (a) providing a first nucleic acid encoding a first portion; (b) providing a second nucleic acid encoding a second portion; (c) combining said first and second nucleic acids, for example in a single vector, to provide a single ORF.

[0110] Generally speaking, those skilled in the art are fully capable of constructing vectors and designing protocols for recombinant gene expression. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, marker genes, and other sequences, as appropriate, in addition to the elements of the invention described above. For further details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press, or Current Protocols in Molecular Biology, Second Edition, Ausubel et al., eds., John Wiley & Sons, (1995, and periodic supplements).

[0111] The term "expression vector" refers to a DNA construct containing a DNA sequence encoding a designated polypeptide and operably linked to suitable control sequences capable of effecting the expression of the polypeptide in a suitable host. Such control sequences include a promoter to initiate transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. A vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, a vector can replicate and function independently of the host genome, or in some cases, may be integrated into the genome itself.

[0112] Expression vectors encoding the fusion proteins of the present invention can be for the in vitro production of these fusion proteins or for in vivo gene therapy.

[0113] The present invention also provides host cells comprising such nucleic acid constructs. The present invention also provides host cells transgenic transformed with such nucleic acid constructs.

[0114] The terms "transfect," "transfection," "transgenic," and similar terms are used herein to mean Refers to the introduction of genes into eukaryotic cells, such as neurons or keratinocytes, and includes "transduction," which is viral-mediated gene transfer, for example, by use of recombinant AAV, adenovirus (Ad), retrovirus (e.g., lentivirus), or any other applicable viral-mediated gene transfer platform.

[0115] "Transformation" refers to a transient or permanent genetic change that occurs in a cell following the incorporation of new DNA (i.e., DNA exogenous to the cell). When the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.

[0116] The present invention also provides a method of preparing a fusion protein of the present invention, comprising culturing or maintaining a host cell containing (or transfected or transformed with) a nucleic acid construct or vector as described above under conditions such that said host cell produces the fusion protein, and optionally further isolating the fusion protein.

[0117] In one embodiment, the E3 ligase-bait polypeptides (or other agents) of the present invention are directly introduced into cells or subjects to provide their effect or medical benefit, which can be achieved by inhibiting the formation of pathological aggregates of aggregating proteins within the cells or by increasing the degradation or clearance of pathological aggregates.

[0118] As explained above, the fusion protein is incorporated into coaggregates with the aggregating protein, and clustering of domains (e.g., RING domains) within the coaggregates and / or assembly of the complete E3 ligase complex leads to ubiquitination of the coaggregates, thereby resulting in their clearance from the cell. The proximity and potentiation achieved by clustering means that normal cytosolic proteins (not present in the aggregates) are less likely to be degraded by the E3 ligase-bait system.

[0119] In one aspect, the invention provides a method of therapeutic treatment of an individual (e.g., for a disease or disorder (these terms are used interchangeably)), comprising administering (directly or indirectly) to the individual a therapeutically effective amount of a fusion protein of the invention. Relevant diseases are discussed in Table 2 above, along with proteins of interest from which aggregation protein domains can be obtained.

[0120] Also provided is the use of a fusion protein of the invention for therapeutic treatment of the human or animal body, or the use of a fusion protein of the invention or other agents described herein for the preparation of a medicament for this purpose.

[0121] Thus, the present specification defines methods of treating diseases as discussed herein (e.g., proteopathic diseases) using the fusion proteins of the present invention or other agents described herein.

[0122] Further disclosed herein are the fusion proteins of the invention or other agents described herein for use in treating these diseases.

[0123] Also disclosed herein is the use of the corresponding agents, or combinations of agents, in the preparation of medicaments for use in treating these diseases.

[0124] It will be understood that discussion of methods of treatment herein applies mutatis mutandis to these uses, and vice versa. Similarly, it will be understood that methods of treatment herein apply mutatis mutandis to methods used in non-human test animals.

[0125] While it is possible for the fusion protein to be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation, e.g., together with a pharmaceutically acceptable carrier or diluent, which will contain an amount or dosage effective to achieve the intended purpose when appropriately administered to an individual.

[0126] The precise nature of the carrier or other material with which the fusion protein of the invention is combined for pharmaceutical use will depend on the route of administration, which may be by bolus, infusion, injection, or any other suitable route as discussed below.

[0127] Determining an effective dose is well within the capabilities of a skilled person. For any compound, the therapeutically effective dose can be initially estimated in cell culture assays, for example, of cell lines, or in animal models, usually, but not exclusively, mice. Animal models can also be used to determine appropriate concentration ranges and administration routes. Based on these pilot experiments, useful doses and routes for administration in humans can be determined. A therapeutically effective dose refers to the amount of the active ingredient, e.g., fusion protein, of the present invention that is sufficient to treat a specific condition (therapeutic efficacy), offsetting any potential toxicity.

[0128] Long-acting pharmaceutical compositions can be administered every 3 to 4 days, or weekly, or once every two weeks, depending on the half-life and clearance rate of the particular formulation. Typical dosages can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending on the route of administration. Guidance regarding specific dosages and delivery methods is provided in the literature and is generally available to those skilled in the art.

[0129] Generally, a daily dose of between 0.01 μg / kg and 100 mg / kg of body weight of the agent according to the present invention can be used to treat, ameliorate, or prevent a disease. More preferably, the daily dose of the agent is between 1 mg / kg and 100 mg / kg of body weight, more preferably between 10 mg / kg and 10 mg / kg of body weight, and most preferably between about 100 mg / kg and 10 mg / kg of body weight.

[0130] The duration of treatment is: 1 to 14 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 1 to 4 weeks, e.g., 1, 2, 3, or 4 weeks, or It can be 1 to 12 months, for example 1, 2, 3, 4, 5, 6, 7, 9, 9, 10, 11, or 12 months.

[0131] For prophylaxis, treatment may be continued.

[0132] In some embodiments, one or more agents can be administered to a subject or individual during late stage disease.

[0133] In all cases, the duration of treatment will generally be subject to medical advice and review.

[0134] In one embodiment, the dosage of the fusion protein can be 20-500 mg twice a week, once a week, every 10 days, every two weeks, every three weeks, or every four weeks.

[0135] Most preferably, a given dose will be between 50 mg and 200 mg twice a week, once a week, or once every two weeks.

[0136] The pharmaceutical compositions detailed in this invention can be administered by any number of routes, including but not limited to intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, intraocular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0137] The preferred mode of administration will be parenteral, eg, subcutaneous or intravenous.

[0138] For parenteral administration, e.g., by injection, pharmaceutical compositions containing the fusion proteins of the present invention may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic excipients such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be used as needed, including buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins such as serum albumin, gelatin, etc. hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0139] In one embodiment, the E3 ligase bait polypeptide of the present invention is expressed in vivo to provide its medical benefit. This is achieved by the use of a nucleic acid (polynucleotide) encoding the E3 ligase bait polypeptide. Typically, the polynucleotide is in the form of or contained within a genetic construct comprising an open reading frame encoding the E3 ligase bait polypeptide under the transcriptional control of a transcriptional control element.

[0140] In one aspect, the invention provides a method for therapeutic treatment of an individual (e.g., for a disease or disorder (these terms are used interchangeably)), comprising administering to the individual a therapeutically effective amount of a nucleic acid encoding a fusion protein of the invention. Relevant diseases are discussed above.

[0141] Also provided is the use of a nucleic acid encoding a fusion protein of the invention for the treatment of the human or animal body by therapy, or for the preparation of a medicament for this purpose.

[0142] It will be understood that discussions herein of methods of treatment apply mutatis mutandis to these uses, and vice versa.

[0143] Preferably, a nucleic acid construct for use in gene therapy contains a promoter to drive expression of the DNA encoding the E3 ligase-bait polypeptide in target cells.

[0144] "Promoter" means a minimal DNA sequence sufficient to direct transcription of a DNA sequence operably linked to it. "Promoter" is also meant to encompass promoter elements sufficient for regulatable, promoter-dependent gene expression that is cell-type specific, tissue-specific, or inducible by external signals or agents; such elements can be located in the 5' or 3' regions of the native gene.

[0145] The promoter can be any known in the art that is suitable for gene therapy—see, for example, Papadakis, ED, et al. “Promoters and control elements: designing expression cassettes for gene therapy.” Current gene therapy 4.1(2004):89-113; and Joshi CR, Labhasetwar V, Ghorpade A. “Destination Brain: the Past, Present, and Future of Therapeutic Gene Delivery.” J Neuroimmune Pharmacol. 2017;12(1):51-83. The promoter can be a naturally occurring nucleotide sequence or a synthetic combination of a minimal promoter sequence and other regulatory elements, such as enhancers. Examples of commonly used promoters include hSyn, mdl, CBA, Ef1a, TH, CMV, mDlx5 / 6, DRD2, Drd1a, SSFV, and TRE3GS.

[0146] Specificity of expression can be achieved, for example, by region- and cell-type-specific expression of the fusion protein using only tissue- or region-specific promoters.

[0147] Alternatively, the promoter can be an inducible promoter that can be activated, for example, by an exogenous agent administered to the subject. For example, the promoter can direct cell-specific expression in neurons, eg, spinal cord cells, or CNS neurons such as brain or nerve cells, or can direct expression in glial cells.

[0148] A promoter is "specific" for a designated cell if, in the context of use, e.g., therapeutic use, it causes gene expression in that cell to a degree sufficient to produce a useful or therapeutically effective amount of the described E3 ligase-bait polypeptide in the designated cell, and relatively low (and non-detrimental) expression elsewhere.

[0149] One example is the Camk2a (alpha CaM kinase II gene) promoter, which drives expression relatively specifically in pre-cancer cells - see, e.g., Sakurada et al. (2005) "Neuronal cell type-specific promoter of the alpha CaM kinase II gene is activated by Zic2, a Zic family zinc finger protein." Neurosci Res. 2005 Nov;53(3):323-30. Epub 2005 Sep 12.

[0150] Other neuronal cell type-specific promoters include the NSE promoter (Liu H. et al., Journal of Neuroscience. 23(18):7143-54, 2003); the tyrosine hydroxylase promoter (Kessler MA. et al., Brain Research. Molecular Brain Research. 112(l-2):8-23, 2003); the myelin basic protein promoter (Kessler MA. et al., Biochemical & Biophysical Research Communications. 288(4):809-18, 2001); the glial fibrillary acidic protein promoter (Nolte C. et al., GLIA. 33(l):72-86, 2001); and the neurofilament gene (heavy, medium, and light chain) promoter (Yaworsky PJ. et al., Journal of Biological Chemistry. 272(40):25112-20, 2003). 1997) (all of which are incorporated herein by reference, at least for the promoter sequence and related sequences). The NSE promoter is disclosed in Peel AL. et al., Gene Therapy. 4(1):16-24, 1997) (pTR-NT3myc; Powell Gene Therapy Center, University of Florida, Gainesville FL). Another suitable promoter is the synapsin 1 promoter (see Kuegler et al. "Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area." Gene Ther. 2003 Feb; 10(4):337-47). Another suitable promoter is the cd68 promoter, which is expressed in microglia.Suitable promoters for general expression include the EF1a or CAG promoter.

[0151] In one embodiment, the vector encoding the E3 ligase-bait polypeptide can include any of these promoters.

[0152] Vectors can be used to effect permanent transformation or can be expressed only transiently in the brain.

[0153] Any of a variety of vectors can be used in accordance with the present invention to produce cells expressing E3 ligase-bait polypeptides. Vectors for use in the therapeutic methods of the present invention will be suitable for in vivo gene therapy protocols. The vectors can be stable integrating vectors or stable non-integrating vectors. Preferred vectors are viral vectors, such as lentivirus or AAV (adeno-associated virus) vectors.

[0154] The use of both of these types of viral vectors is well known in the art for gene therapy. By way of example only, WO2008011381 describes the use of these and other vectors to express polypeptides in a subject. The contents of that application, regarding the description of the preparation and properties of AAV and lentiviral vectors, are specifically incorporated herein by reference.

[0155] Briefly, AAV is a preferred vector because it is a defective parvovirus, capable of infecting many cell types, and nonpathogenic to humans, as described in WO 2008011381. AAV-type vectors can transfer approximately 4 to 5 kb, and wild-type AAV is known to stably integrate into chromosome 19.

[0156] In another type of AAV vector, AAV contains a pair of inverted terminal repeats (ITR) adjacent to at least one cassette that contains a promoter that induces cell-specific expression, and is operably linked to heterologous gene (herein, E3 ligase-bait polypeptide).More information can be found in US Patent No. 6,261,834.AAV vectors are discussed in International Publication No. 2018 / 175443.

[0157] Viral vectors are commercially available, for example, from Viralgen, Parque Cientifico y Tecnologico de Gipuzkoa, Paseo Mikeletegi 83, 20009 San Sebastian, Spain.

[0158] Lentiviral vectors are a special type of retroviral vector that are typically characterized by a long incubation period for infection. Furthermore, lentiviral vectors can infect non-dividing cells. Lentiviral vectors are based on the viral nucleic acid backbone from the lentivirus family of viruses. Typically, lentiviral vectors contain the 5' and 3' LTR regions of lentiviruses such as SIV and HIV. Lentiviral vectors also typically contain the Rev response element (RRE) of lentiviruses such as SIV and HIV. Examples of lentiviral vectors include those described in Dull, T. et al., "A Third-generation lentivirus vector with a conditional packaging system," J. Virol 72(11):8463-71 (1998).

[0159] Thus, in one embodiment, there is provided an expression vector comprising the polynucleotide of the invention described above. The vector may be a viral vector, such as an adenoviral vector and / or an adeno-associated vector (AAV), which is optionally selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof.

[0160] One preferred AAV is AAV-9P31, a derivative of AAV9 (see WO2020072683A1 and PMID: 33553485).

[0161] AAV vectors capable of crossing the blood-brain barrier are known in the art (see, for example, Nonnenmacher, Mathieu, et al. "Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning." Molecular Therapy-Methods & Clinical Development 20(2021):366-378), and may be preferred. Examples include AAV-F.

[0162] Alternatively, the vector may be a herpes virus vector, a retroviral vector, or a lentiviral vector.

[0163] In methods of delivering a vector or nucleic acid encoding an E3 ligase-bait polypeptide to a cell or patient according to any of the embodiments described herein, the vector or nucleic acid can be delivered in any useful form and by any useful method, as recognized by those skilled in the art of gene therapy.

[0164] For example, liposomes or nanoparticles containing nucleic acids can be injected at a desired site, such as within or adjacent to a specific neural tissue. In other embodiments, recombinant viral particles (transduction particles) are delivered, e.g., injected, at a desired site, such as within or adjacent to a specific neural tissue, or other targeted specific neural tissue. The nucleic acid can be injected once or multiple times to establish sufficient expression of the E3 ligase-bait polypeptide in the target neuron.

[0165] Delivery can be via direct injection into the brain, using known methodologies such as direct interstitial injection, burr-hole craniotomy, and stereotactic injection, among others (see, e.g., "Stereotactic and Functional Neurosurgery" Editors: Nikkhah & Pinsker; Acta Neurochirurgica Supplement Volume 117, 2013).

[0166] AAV can be advantageously used for intraocular gene administration (see, for example, Ail, Divya, et al. "Systemic and local immune responses to intraocular AAV vector administration in non-human primates." Molecular Therapy-Methods & Clinical Development 24(2022):306-316).

[0167] In gene therapy embodiments using viral delivery of E3 ligase-bait polypeptides, as described in WO2008096268, a unit dose can be calculated in terms of the dose of viral particles administered. A viral dose includes a specific number of viral particles or plaque-forming units (pfu). In embodiments involving AAV, a specific unit dose may include 10 3 , 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 pfu or vector genome. The particle dose may be somewhat higher (10 to 100 times) due to the presence of non-infectious particles.

[0168] In one embodiment, the vector is 5×10 11 vg / ml (=2.5×10 11 The virus genome) is injected as a 500 μL suspension.

[0169] Agents utilized in the present invention can be provided as compositions, e.g., "pharmaceutical compositions" (e.g., formulations, preparations, medicaments), comprising at least one agent of the invention (i.e., a fusion protein, or a nucleic acid encoding the fusion protein, or a corresponding vector or capsid or particle) described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0170] The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, and the like, which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0171] In some embodiments, the composition is a pharmaceutical composition comprising at least one agent of the present invention together with one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweetening agents.

[0172] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks, see, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th Edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994.

[0173] In some embodiments, a pharmaceutical composition comprises, consists essentially of, or consists of an agent of the invention and a pharmaceutically acceptable carrier, diluent, or excipient, hi some embodiments, the composition further comprises another active agent, e.g., another therapeutic or prophylactic agent.

[0174] In some embodiments, the methods or treatments of the present invention can be combined with other therapies, whether symptomatic or disease-modifying, for example, a second therapeutic agent believed to exhibit therapeutic benefit in the cancer in question.

[0175] The term "treatment" includes combination therapies and therapies, in which two or more treatments or therapies are combined, for example sequentially or simultaneously.

[0176] For example, it may be beneficial to combine treatment using a fusion protein of the invention as described herein with one or more other (eg, 1, 2, 3, 4) drugs or therapies.

[0177] Suitable examples of co-therapeutics will be known to those skilled in the art based on the disclosure herein. Typically, the co-therapeutic can be any known in the art that is believed to be capable of providing a therapeutic benefit in treating the disease in question.

[0178] It will be understood by those skilled in the art that whenever amino acid and nucleic acid sequences are discussed herein (e.g., with respect to encoding fusion proteins or portions thereof), functional variants of the amino acids disclosed herein, and therefore the encoding nucleic acid sequences, are also contemplated. Typically, such variants will be "substantially identical" to the reference sequences disclosed herein. Typically, they will have the biological activity of the reference sequences.

[0179] For example, the biological activity of a RING domain is its ability to catalyze the covalent transfer of ubiquitin to the RING-containing protein itself or to another protein, a property that can be enhanced when at least two RING domains are brought into close proximity after assembly into a multimeric complex. The biological activity of a protein-aggregating moiety is its ability to be incorporated into aggregates of natural proteins to form multimeric coaggregates.

[0180] In the context of at least two nucleic acids or polypeptides, "substantially identical" means that the polynucleotides or polypeptides include sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a parent or reference sequence, or any other sequence that contains amino acid substitutions, insertions, deletions, or modifications that are made without adding functionality and that merely circumvent the description of the invention.

[0181] The percentage identity between different amino acid / polypeptide / nucleic acid sequences can be calculated as follows: A multiple alignment is first generated by the ClustalX program (pairwise parameters: gap initiation 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiplex parameters: gap initiation 10.0, gap extension 0.2, delay divergent sequence 30%, DNA transition weight 0.5, negative matrix off, protein matrix Gonnet series, DNA weight IUB; protein gap parameters: residue-specific penalty on, hydrophilic penalty on, hydrophilic residue GPSNDQERK, gap separation distance 4, terminal gap separation off). The percentage identity is then calculated from the multiplex alignment as (N / T) * 100, where N is the number of positions where the two sequences have identical residues, and T is the total number of positions compared. Alternatively, the percentage identity can be calculated as (N / S)*100, where S is the length of the shorter sequence being compared. The amino acid / polypeptide / nucleic acid sequence can be synthesized de novo or can be a naturally occurring amino acid / polypeptide / nucleic acid sequence or a derivative thereof.

[0182] Alternatively, a substantially similar nucleotide sequence will be encoded by a sequence that hybridizes under stringent conditions to any of the nucleic acid sequences referred to herein or their complements. Stringent conditions refer to hybridization of the nucleotides to filter-bound DNA or RNA in 6x sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 5-65°C. Alternatively, a substantially similar polypeptide may differ from a peptide sequence according to the invention by at least one, but fewer than 5, 10, 20, 50, or 100, amino acids.

[0183] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence can be altered or changed to provide functional variants thereof without substantially affecting the sequence of the protein encoded thereby. Of course, degenerately equivalent (e.g., codon-optimized) nucleotide sequences to any of those described herein can be used instead.

[0184] Therefore, suitable nucleotide variants include those having sequences modified by substitution of different codons that encode the same amino acid within the sequence, thus resulting in a silent change. Other suitable variants include those having a homologous nucleotide sequence but having all or part of the sequence modified to produce a conservative change by substitution of different codons that encode amino acids with side chains with similar biophysical properties to the amino acid they replace. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0185] It will be understood that the fusion proteins used or provided in accordance with the present invention may be derivatives of the native or original sequence, and thus include derivatives that increase the efficacy or half-life of the drug in vivo. Examples of derivatives that can increase the half-life of polypeptides according to the present invention include peptoid derivatives, D-amino acid derivatives, and peptide-peptoid hybrids.

[0186] Fusion proteins according to the present invention may be subject to degradation by many means, including protease activity at the target site. Such degradation may limit their bioavailability and therefore their therapeutic utility. There are many well-established techniques by which peptide derivatives can be designed and produced with enhanced stability in biological contexts. Such peptide derivatives may have improved bioavailability as a result of increased resistance to protease-mediated degradation. Preferably, derivatives suitable for use according to the present invention are more protease-resistant than the protein or peptide from which they are derived. The protease resistance of peptide derivatives and the protein or peptide from which they are derived can be assessed using well-known proteolysis assays. The relative values ​​of protease resistance for the peptide derivative and the peptide can then be compared.

[0187] Peptoid derivatives of the fusion proteins of the present invention can be readily designed from knowledge of the structure of the active moiety. Commercially available software can be used to develop peptoid derivatives according to well-established protocols.

[0188] Retropeptoids, in which all amino acids are replaced by peptoid residues in reverse order, can also mimic proteins or peptides according to the invention. Retropeptoids are predicted to bind in the opposite direction in the ligand-binding groove compared to peptides or peptoid-peptide hybrids containing a peptoid residue. As a result, the side chains of the peptoid residues can point in the same direction as the side chains in the original peptide.

[0189] Further embodiments of modified forms of peptides or proteins according to the present invention include D-amino acid forms, in which the order of amino acid residues is reversed. The preparation of peptides using D-amino acids rather than L-amino acids greatly reduces any unwanted degradation of such derivatives by normal metabolic processes and reduces the amount of derivative that needs to be administered, as well as the frequency of its administration.

[0190] Other modifications in the protein sequence are also contemplated and within the scope of the claimed invention, i.e., those that occur during or after translation, for example, by acetylation, amidation, carboxylation, phosphorylation, proteolytic cleavage, or binding to a ligand.

[0191] The term "derived from" encompasses the terms "originating from," "obtained from," "obtainable from," "isolated from," and "made from," and generally indicates that a specified material has characteristics that allow its origin to be found in, or described with respect to, another specified material (which may be referred to as a "reference" or "parent"). The E3 ligase-bait polypeptides herein may be derived from reference or parent sequences, such as wild-type aggregation protein sequences and TRIM proteins.

[0192] The term "polynucleotide" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single- or double-stranded and can contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0193] As used herein, "polypeptide" refers to a molecule comprising multiple amino acids linked via peptide bonds. The terms "polypeptide," "peptide," and "protein" are used interchangeably. Proteins can optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, and sulfonated) to add functionality. Conventional one-letter or three-letter codes for amino acid residues are used, and amino acid sequences are presented in the standard amino to carboxy terminal direction (i.e., N→C).

[0194] The terms "subject," "individual," or "patient" to be treated (these terms are used interchangeably unless the context requires otherwise) typically refer to a mammal, e.g., a human or a non-human mammal. Thus, in one embodiment, the individual is a human subject, e.g., a patient. Alternatively, the mammal can be a non-human mammal, e.g., a test animal, such as a rodent (e.g., mouse, rat) or a primate. Non-human subjects include rabbits, pigs, monkeys, chimpanzees, cats, dogs, horses, goats, and guinea pigs. The non-human mammal can be a transgenic mammal. Alternatively, the subject or organism can be a bird, fish, reptile, or amphibian.

[0195] The term "therapeutically effective amount," as used herein, relates to an amount of a compound of the invention, or a material, composition, or dosage form comprising said compound, that, when administered in accordance with a desired treatment regimen, is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0196] "Therapeutic efficacy" and "toxicity" can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as LD50 / ED50. Pharmaceutical compositions exhibiting large therapeutic indices are preferred. Dosages preferably lie within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages will vary within this range depending on the dosage used, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the physician, taking into account factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors that can be taken into account include the severity of the condition, the subject's general health, the subject's age, weight, and sex, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to treatment.

[0197] The term "treatment," as used herein with respect to treating a condition, generally relates to human or animal (e.g., veterinary) care and therapy to achieve some desired therapeutic effect, such as inhibition of the progression of the condition, including slowing the rate of progression (prolonged survival), halting the rate of progression, regression of the condition, amelioration of the condition, and curing the pathology. The present invention also encompasses treatment as an included preventative measure, and "treating" will be understood accordingly. "Prevention" can utilize a "prophylactically effective amount," which, as used herein, relates to the amount of agent that, when administered in accordance with a desired treatment regimen, is effective to produce some desired preventative effect commensurate with a reasonable benefit / risk ratio. "Prevention" in this context should not be understood to encompass complete success, i.e., complete protection or complete prevention. Rather, prevention in this context refers to a measure taken prior to the detection of a symptomatic condition, with the goal of maintaining health by helping to delay, alleviate, or avoid a particular condition.

[0198] As used herein, the terms "wild-type," "native," or "reference" refer to a polypeptide or polynucleotide found in nature. With respect to polypeptides, the term refers to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. With respect to polynucleotides, the term refers to a naturally occurring polynucleotide that does not contain an artificial substitution, insertion, or deletion at one or more nucleosides. However, it should be noted that a polynucleotide encoding a wild-type, or native, or reference polypeptide is not limited to naturally occurring polynucleotides, but encompasses any polynucleotide that encodes that polypeptide.

[0199] In order to more fully describe and disclose the present invention and the prior art to which it pertains, several patents and publications are cited herein. Each of these references is incorporated by reference in its entirety into the present disclosure herein to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0200] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0201] Wherever present in this specification the phrase "selected from the group comprising" may be replaced with the phrase "selected from the group consisting of," and vice versa.

[0202] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0203] Ranges are often expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Any subheadings herein are included for convenience only and should not be construed as limiting the disclosure in any way.

[0204] The invention will now be further described with reference to the following non-limiting figures and examples, in light of which other embodiments of the invention will occur to those skilled in the art. [Brief explanation of the drawings]

[0205] drawing [Figure 1a] Figure 1: Tau-RING prevents seed-induced aggregation and removes existing aggregates. (a) Schematic and representative images of a HEK293 reporter cell line expressing tau-venus (TV cells) + / - tau-RING seeded with + / - 10 nM tau aggregates in LF2000. White arrows point to examples of aggregates. [Figure 1b] (b) Quantification of cells treated as in (a). N=3. [Figure 1c] (c) Western blot of cells expressing tau-venus + / - tau-RING + / - 10 nM tau aggregates (delivered with LF2000). The blot was probed for tau and the loading control CypB. [Figure 1d](d) Quantification of Western blot tau-venus in (c) normalized to CypB. N=3. [Figure 1e] (e) Quantification of Western blot tau-RING in (c) normalized to CypB. N=3. [Figure 1f] (f) Western blot of cells expressing tau-venus + / - tau-RING + / - 10 nM tau aggregates (delivered with LF2000). Blots were probed with AT8 and loading control CypB. [Figure 1g] (g) Quantification of AT8-positive tau-venus in (f) Western blot normalized to CypB. N=3. [Figure 1h] (h) TEM of P301S tau or P301S tau-RING aggregates. [Figure 1i] (i) Tau aggregates (TVA). A HEK293 reporter cell line constitutively expressing tau-venus was infected with lentivirus containing tau-RING and assessed over a 3-day period. [Figure 1j] (j) Time course of tau aggregation in TVA reporter cells with and without tau-RING. N=3. [Figure 1k] (k) Quantification of tau aggregates in TVA cells 72 hours after infection with tau-RING lentivirus. N=3. Statistical significance for (b) was determined by two-way ANOVA and Sidak's multiple comparison test. Statistical significance for (d), (e), and (g) was determined by one-way ANOVA and Tukey's multiple comparison test. Statistical significance for (j) and (k) was determined by unpaired t-test with Welch's correction. ***, p<0.001. ****, p<0.0001. ns, not significant. [Figure 2a]Figure 2: Tau-RING colocalizes with aggregates, initiating their complete disassembly and eliminating seed-competent species. (a) BHK cells expressing tau-venus were infected with doxycycline-inducible tau-mCherry-RING. Upon seeding with preformed tau aggregates, tau-venus and tau-mCherry-RING were observed to colocalize. [Figure 2b] (b) Quantification of colocalization of tau-venus and tau-mCherry-RING. [Figure 2c] (c) Time course of tau-mCherry-RING causing disassembly of tau-venus-positive aggregates in BHK cells. [Figure 2d] (d) Schematic of the secondary seeding assay. TVA cells were infected with lentivirus carrying tau-mCherry-RING. The cells were then sorted for infected cells, and a pure population expressing tau-venus and tau-mCherry-RING was cultured for 2 weeks. One million cells were harvested, lysed, and applied to a HEK293 reporter cell line expressing tau-venus to assess the amount of seed-competent species present in the lysate. Lysates were also collected from uninfected TVA and TV cells. [Figure 2e] (e) Representative images of secondary seeding from TVA, TVA + tau-mCherry-RING, and TV cell lysates. [Figure 2f] (f) Quantification of secondary seeding in reporter TV cells from TVA, TVA + Tau-mCherry-RING, and TV cells. Statistical significance for (f) was determined by one-way ANOVA and Tukey's multiple comparison test. ****, p<0.0001. [Figure 3a] Figure 3: RING dimerization and VCP recruitment are essential for tau-RING function. (a) Schematic of RING dimerization between two TRIM21 dimers. The M72 residue is shown in orange. The M72E mutation inhibits RING dimerization. [Figure 3b](b) Western blots of cells expressing tau-venus+tau-RING or tau-RING-M72E run on the same gel. [Figure 3c] (c) TV cells + / - tau-RING or tau-RING-M72E, + / - 10 nM tau aggregates (delivered with lipofectamine) N=3, n=9. [Figure 3d] (d) TVA cells + / - lentivirus (expressing tau-RING or tau-RING-M72E). Aggregates were analyzed 72 hours post-infection. [Figure 3e] (e) Schematic of the neuronal tau entry assay. P301S 0N4R tau, tau-RING, or tau-RING-M72E proteins were generated and aggregated using the HiBiT tag. Primary neurons were infected with AAV to express LgBiT, and the aggregated tau species were then incubated with the neurons. The amount of tau in the cytosol was quantified by luminescence signal. Once in contact with the cytosol, tau-RING becomes available for proteasomal degradation, allowing real-time visualization of degradation. [Figure 3f] (f) Aggregation of Tau-HiBiT, Tau-RING-HiBiT, and Tau-RING-M72E-HiBiT proteins in the presence of heparin, visualized with thioflavin T (ThT). [Figure 3g] (g) Neuronal entry assay of tau-HiBiT, tau-RING-HiBiT, and tau-RING-M72E-HiBiT aggregates into the cytosol using a HiBiT-LgBiT split luciferase assay. N=3. [Figure 3h] (h) Representative Western blot of TV cells + / - tau-RING, after 48 hours of addition of NTC or VCP siRNA, probed for VCP and loading control GAPDH. [Figure 3i](i) Quantification of Tau-RING in TV cells seeded with 30 nM tau aggregates using lipofectame and assessed at 24 h. Cells were either untreated or treated with NTC or VCP siRNA for 48 h prior to seeding. N=3, n=9. [Figure 3j] (j) TV+Tau-RING data from graph (i) plotted on a linear scale to visualize the increase in seeding mediated by VCP siRNA knockdown. N=3, n=9. Statistical significance for (c), (i), and (j) was determined by two-way ANOVA and Sidak's multiple comparison test. Statistical significance for (d) and (g) was determined by one-way ANOVA and Tukey's multiple comparison test. **, p<0.01; ***, p<0.001; ****, p<0.0001. ns, not significant. [Figure 4a] Figure 4: Tau-RING is effective with linkers of different sizes. (a) HEK293 cells expressing tau-venus and tau-CFP-RING are positive for both venus and CFP by live cell imaging. [Figure 4b] (b) Western blot of cells expressing tau-venus+ / -tau-RING or tau-CFP-RING probed for tau and the loading control CypB. [Figure 4c] (c) Quantification of tau-RING and tau-CFP-RING levels in the Western blot in (b). Statistical significance was determined by unpaired t-test. [Figure 4d] (d) Quantification of tau-venu levels in the Western blot in (b). Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test. [Figure 4e] (e) Quantification of HEK293 cells expressing tau-venus+ / -tau-CFP-RING seeded with 10 nM tau aggregates and analyzed at 72 hours. N=3, n=9. Statistical significance was determined by two-way ANOVA and Tukey's multiple comparison test. [Figure 4f] (f) Quantification of HEK293 cells expressing tau-venus+ / -tau-RING seeded with 10 nM tau aggregates and analyzed at 72 hours. N=3, n=9. Statistical significance was determined by two-way ANOVA and Tukey's multiple comparison test. ****, p<0.0001. ns, not significant. [Figure 5a] Figure 5: Tau-RING reduces seed-induced aggregation in primary neurons. (a) Schematic of the Venus-P2A-Tau-RING construct packaged into an AAV vector. Primary neurons were infected on day 2, and full expression of the construct was observed by day 6. [Figure 5b] (b) Western blot of primary neurons from P301S mice infected with venus AAV, venus-P2A-tau-RING AAV, or untreated. Blots were probed for tau, venus, and the loading control CypB. [Figure 5c] (c) Quantification of endogenous tau levels in P301S primary neurons infected with + / -venus or venus-P2A-tau-RING AAV. [Figure 5d] (d) Primary neurons infected with venus or venus-P2A-tau-RING AAV seeded with 100 nM P301S tau aggregates on day 7. Cells were fixed on day 14. [Figure 5e] (e) AT8-positive puncta at day 14 in neurons treated as in (d). [Figure 5f] (f) AT8-positive neuronal cell bodies at day 14 in neurons treated as in (d). [Figure 5g](g) Neuronal viability at the time of tau aggregate seeding, as determined by NeuN counts on day 14, in neurons treated as in (d). Statistical significance for (e), (f) was determined by two-way ANOVA and Sidak's multiple comparison test. Statistical significance for (c), (g) was determined by one-way ANOVA and Tukey's multiple comparison test. ****, p<0.0001. ns, not significant. [Figure 6a] Figure 6: Tau-RING clears tau aggregates in vivo. (a) Four-month-old P301S mice were injected with 1 x 10 vg of venus or venus-P2A-tau-RING AAV 9P31. No adverse side effects were observed during treatment (exemplified by stable body weight over a 2-month period). [Figure 6b] (b) Sarkosyl Insoluble (SI) extracts from one hemisphere of each mouse. Total tau and SI tau were probed with pan-tau antibody A0024 (Dako). [Figure 6c] (c) Quantification of SI tau normalized to total tau present in N = 6 mice per group. [Figure 6d] (d) Representative images from the frontal cortex showing colocalization of AAV and tau aggregates visualized by the venus fluorophore and stained with AT8, respectively. [Figure 6e] (e) Quantification of venus+AT8-positive neurons in the frontal cortex. N = 6 mice per group. [Figure 6f] (f) Quantification of neuronal nuclei in the frontal cortex as a measure of toxicity. N=6 mice per group. [Figure 6g](g) Five-month-old P301S mice were stereotaxically injected into the frontal cortex with 5 × 10 vg of venus-P2A-tau-RING or venus-only AAV PHP.eB and sacrificed one month later. Coronal sections were sliced ​​and assessed for AT8 positivity. White arrows point to the injected hemisphere. Quantification of AT8- and venus-positive areas. AT8 positivity was normalized to the uninjected hemisphere of each mouse. Three sections were analyzed per mouse. N = 5 mice. Statistical significance for Figures 6(c), 6(e), and 6(f) was determined by an unpaired t-test with Welch's correction. Statistical significance for Figure 6(h) was determined by a nested t-test. **, p < 0.01; ****, p < 0.0001. ns, not significant. [Figure 7a] Figure 7: Tau fusions with other RING constructs or VHL E3 ligase remove pre-existing intracellular tau aggregates. (a) Quantification of the number of tau aggregates in TVA cells 72 h after infection with lentiviruses encoding the indicated genes. [Figure 7b] (b) Quantification of the number of tau aggregates in a HEK293 reporter cell line expressing Tau-venus (TV cells) + / - Tau-VHL, seeded with + / - 30 nM tau aggregates with LF2000. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test. ***, p<0.001. [Figure 7c] (c) Western blot of cells expressing tau-venus + / - tau-VHL. The blot was probed for GFP to detect tau-venus and the loading control CoxIV. [Figure 7d] Quantification of Western blots shown in (c). [Figure 8a] a, Schematic of RING domain activation via dimerization. The I18R and M72E mutations are highlighted in green and purple, respectively. [Figure 8b] b Time course of tau-RING+ / -I18R, M72E, and I18R / M72E RING mutations in TVA cells. [Figure 8c]c, Quantification of the number of aggregates in TVA cells 72 hours after infection with Tau-RING lentivirus. N=3. [Figure 8d] d. Schematic representation of inhibitors of degradation pathway components utilized by TRIM21. TAK-243 is an E1 inhibitor, NMS-873 is a VCP inhibitor, and MG-132 is a proteasome inhibitor. [Figure 8e] e. Time course of TVA cells treated with Tau-RING+ / -TAK-243 and quantification at the endpoint 48 hours post-infection. N=3. [Figure 8f] f. Time course of TVA cells treated with Tau-RING+ / -NMS-873 and quantification at 48 hours post-infection. N=3. [Figure 8g] g. Time course of TVA cells treated with Tau-RING+ / -MG-132 and quantification at 48 hours. N=3. Statistical significance for c, e, f, and g was determined by one-way ANOVA and Tukey's multiple comparison test. **, p<0.01; ***, p<0.001; ****, p<0.0001. ns, not significant. [Figure 9a] a, Schematic of the TVA cell assay, with lentiviruses used to express different isoforms of tau-RING as examples of different "baits." Incorporation of the "bait" into aggregates leads to proteasomal degradation and a reduction in the number of puncta observed by high-content microscopy. [Figure 9b] b. Time course of lentivirus carrying P301S 0N4R tau-RING, WT 0N3R tau-RING, or WT 0N4R tau-RING applied to TVA cells. [Figure 9c] c, Quantification of the number of aggregates in TVA cells treated as in b 72 h after infection. N=3. [Figure 9d] d. Quantification of the number of aggregates in HEK293T cells expressing venus-3R tau seeded with AD-derived tau aggregates + / - 0N3R tau-RING. N=3. [Figure 9e]Representative images of cells treated as in. White arrows point to tau aggregates. [Figure 9f] f. Quantification of the number of aggregates in HEK293T cells expressing venus-4R tau seeded with PSP-derived tau aggregates + / - 0N4R tau-RING. N=3. [Figure 9g] Representative images of cells treated as in gf. White arrows point to tau aggregates. Statistical significance for c was determined by one-way ANOVA and Tukey's multiple comparison test. Statistical significance for d,f was determined by unpaired t-test. ****, p<0.0001. ns, not significant. [Figure 10a] a. Schematic of intravenous injection of AAV 9P31 into 4-month-old P301S mice. At 6 months, one half of the brain was homogenized to extract sarkosyl-insoluble (SI) tau aggregates, and the other half was fixed and analyzed for AT8-positive tau aggregates by immunofluorescence staining. [Figure 10b] b. Representative immunofluorescence images of mice infected with AAV 9P31 hSyn. Mice were injected with VPTR or hSyn:VPTR I18R / M72E (at 4 months), or PBS and assessed for tau aggregates via AT8 staining at 6 months. Venus fluorescence was detected from virally expressed proteins. Neuronal nuclei were probed using an antibody against NeuN. Magnified cortical regions are shown illustrating tau aggregate levels. [Figure 10c] c Quantification of AT8-positive tau aggregates in the frontal cortex as shown in b. [Figure 10d] d, Western blot of sarkosyl-insoluble (SI) fractions of mouse brains treated as in b, stained for total human tau (HT7) and hyperphosphorylated tau at serine 422 (pS422). Mouse brain homogenates were also stained with HT7 and probed for Venus protein in addition to GAPDH as a loading control. [Figure 10e] e. Quantification of SI HT7. [Figure 10f] f. Quantification of SI pS422. [Figure 10g] g. Quantification of homogenate Venus protein levels from viral infection. [Figure 10h] h. Median mouse footprints from each condition (VPTR, VPTR I18R / M72E, PBS) on the MouseWalker apparatus. [Figure 10i] i. Quantification of the time to cross the aisle for mice from each condition (VPTR, VPTR I18R / M72E, PBS) from 4 to 6 months of age. N = 6–8 mice per group. Statistical significance for c, e, f, g, and i was determined by unpaired t-test. *, p<0.05. **, p<0.01. ****, p<0.0001. ns, not significant. [Figure 11a] a. Quantification of the higher HT7 band in the homogenate Western blot of Figure 10c. [Figure 11b] b. Quantification of the major HT7 band in the homogenate Western blot of Figure 10c. [Figure 11c] c. Western blot of mice treated as in Figure 10 probed for neuron-specific β-III-tubulin and loading control GAPDH. [Figure 11d] d Quantification of β-III-tubulin levels in c. [Figure 11e] e. Mice infected with hSyn:Venus AAV 9P31. [Figure 12a] a. Analysis of frames per video in which the foot was in contact with the platform 4 months before treatment and 6 months after treatment. [Figure 12b] b. Time taken for mice to cross the MouseWalker platform at 4 and 6 months. [Figure 12c] c. Number of front paw steps across the MouseWalker platform at 4 and 6 months. [Figure 12d]d. Number of hind paw steps across the MouseWalker platform at 4 and 6 months. Statistical significance for a, b, c, d was determined by two-way ANOVA and Sidak's multiple comparison test. [Figure 13a] a) Representative image of HEK293 cells co-transfected with mGreenLantern-tagged P525L FUS along with RING-525L FUS-T2A-mCherry. Doxycycline was used to drive expression of the RING-bait. The P525L variant of FUS forms cytosolic aggregates that are degraded upon transfection of the RING-FUS P525L construct. [Figure 13b] b) Quantification of cytosolic aggregates 24 hours after addition of dox to drive expression of RING-bait or RING-only control. N=3. Statistical significance was determined by two-way ANOVA. DETAILED DESCRIPTION OF THE INVENTION

[0206] Example Methods HEK cell line production Tau-RING was cloned into the lentiviral plasmid smppv2 and transfected with the helper plasmid spmmv2 into HEK293 cells to generate lentivirus. After three days, the virus was harvested, filtered, and titrated against HEK293 cells (TV cells) that already express tau-venus (McEwan et al., 2017). Stably expressing cells were selected with puromycin for more than three passages. Colonies were expanded and pooled for use in tau seeding assays. Aggregate-bearing tau-venus cells (TVA cells) were generated by seeding these cells with preformed tau aggregates and stably expanding the cells that constitutively bear aggregates.

[0207] HEK293 tau seeding assay HEK293 cells expressing tau-venus + / - tau-RING were plated at 20,000 cells per well in a black 96-well plate containing 50 μl of low-serum OptiMEM. Preformed P301S tau aggregates (aggregated with heparin) were diluted with an equal volume of OptiMEM and mixed with the transfection reagent Lipofectamine 2000. After a 20-minute incubation, 50 μl of tau aggregates were added to each well and incubated for 1 hour. After 1 hour, the reaction was quenched with 100 μl of complete DMEM. The plate was then transferred to an Incucyte for live-cell imaging every 2 hours for 72 hours. The number of tau aggregates was analyzed at 72 hours.

[0208] Neuronal tau seeding assay Primary neurons were generated from P1 / 2 P301S mice and plated in black 96-well plates. On day 2, cultures were infected with AAV PHP.eB expressing Venus or Venus-P2A-tau-RING under the CAG promoter. On day 7, preformed P301S tau aggregates were added to the medium to a final concentration of 100 nM. On day 14, cultures were fixed and stained for the neuronal nucleus marker NeuN to obtain neuronal cell counts and for AT8 (phospho-tau) to obtain aggregate counts.

[0209] Removal of endogenous aggregates The TVA cell line, which constitutively harbors tau aggregates, was plated in a 96-well format and challenged with lentivirus expressing P301S tau-RING. The number of aggregates was assessed at 72 hours.

[0210] Secondary seeding assay TVA cells treated with + / - tau-RING lentivirus were lysed, and the whole cell homogenate was used as "seeding" material. 1 μl of the homogenate was applied to fresh TV cells using lipofectamine as described in the "HEK293 tau seeding assay" section. After 72 hours, the number of aggregates in TV cells was quantified.

[0211] WT tau seeding assay 0N3R venus-tau was expressed in HEK293T cells using lentiviral transduction. Tau aggregates derived from Alzheimer's disease (AD) brains were applied to the cells as described in the "HEK293 Tau Seeding Assay" section. Lentivirus encoding 0N3R tau-RING was applied to the cells, and the number of aggregates was quantified 72 hours later. The same assay was performed using the 0N4R system to test RING-bait against aggregates derived from progressive supranuclear palsy (PSP) brains.

[0212] Quantification of aggregates Aggregates from both the HEK293 and neuronal assays were quantified using the Fiji plugin comdet and normalized to cell number.

[0213] In vivo experiments 5 × 10 10 The venus of vg or venus-P2A-tau-RING AAV PHP.eB was stereotactically injected in a volume of 1.5 μl. Mice were sacrificed one month later and perfused with 4% PFA. Brains were dehydrated in 30% sucrose and sliced ​​into 30 μm-thick sections using a vibratome. The venus-positive zone was selected for analysis of tau aggregates by AT8 staining. Alternatively, 1 × 10 Tau-RING AAVs were injected into the tail vein of 4-month-old mice. 11 vg or 4×10 11 Mice were injected with Venus or Venus-P2A-tau-RING AAV 9P31 at 100 mg / kg and sacrificed at 6 months. One hemisphere was fixed in 4% PFA for 48 hours and then prepared for staining as previously described. One hemisphere was used for extraction of sarkosyl-insoluble tau, which was analyzed by Western blot.

[0214] MouseWalker phenotyping test Mice were videotaped biweekly from 4–6 months of age walking along the MouseWalker platform, and the videos were analyzed using a neural network trained using DeepLabCut software. [Example]

[0215] Example Example 1 - E3 ligase-bait efficiently disassembles aggregates while leaving soluble protein To demonstrate that the "E3 ligase-bait" strategy can be successfully applied to degrade aggregated proteins, we selected tau as both a target for degradation and a bait. For demonstration purposes, we used the P301S mutation, which causes early-onset familial dementia and is pro-aggregation, increasing the rate of tau fibril elongation by approximately 50-fold compared to wild-type tau. 10 .

[0216] For the bait, we fused the TRIM21 RING domain to the C-terminus of ON4R P301S tau, termed tau-RING. We co-expressed tau-RING together with the target protein, P301S ON4R tau-venus. This construct has previously been shown to respond to the presence of exogenous tau aggregates by forming aggregates, which appear as bright spots that can be quantified by high-content fluorescence microscopy. 8 HEK293 cells expressing P301S 0N4R tau-venus are hereafter referred to as "TV cells."

[0217] We first tested whether tau-RING can prevent the de novo formation of new tau aggregates induced by the addition of exogenous misfolded tau seeds. Recombinant 0N4R P301S tau was aggregated with heparin and transfected into cells to seed intracellular tau-venus aggregation in TV cells. After 72 hours, the resulting tau-venus aggregates were quantified by high-content microscopy. The tau-RING construct reduced target, P301S tau-venus seed-induced aggregation by <95% compared to cells not expressing tau-RING (Fig. 1a, b). Thus, tau-RING is capable of inhibiting tau seed-induced aggregation.

[0218] To investigate whether Tau-RING specifically acts on nascent tau aggregates or simply reduces the available pool of intracellular tau, we analyzed the total levels of Tau-venus upon coexpression with Tau-RING (Fig. 1c). No reduction in soluble Tau-venus occurred in the presence of Tau-RING, suggesting that the construct was incapable of degrading monomeric forms of tau, consistent with the clustering mechanism of TRIM21 RING domain activation (Fig. 1d). The reduction in Tau-RING and Tau-venus upon addition of aggregates was also insignificant, indicating that Tau-RING, when activated, is highly specific for degrading aggregated species (Fig. 1d, e). In the presence of Tau-RING, addition of tau aggregates resulted in a clear reduction in the level of staining with an antibody detecting AT8, i.e., tau phosphorylated at S202 / T205 (Fig. 1f, g). AT8 phosphorylation of tau-RING was not detectable, consistent with the rapid degradation of the assembled form of this protein before phosphorylation. Tau-RING protein immediately formed aggregated seeds that were visualized by TEM. Thus, tau-RING does not inhibit seed-induced aggregation by preventing the formation of fibrillar aggregates (Fig. 1h). Having demonstrated that tau-RING can disassemble tau aggregates resulting from seed-induced aggregation, we investigated whether tau-RING could deplete preformed cytoplasmic tau aggregates. HEK293 P301S tau-venus cells were seeded with the aggregates and constitutively expressed P301S tau-venus aggregates. This cell line is hereafter referred to as "TVA cells." The cells were infected with a lentivirus encoding tau-RING, and the presence of tau-venus puncta was monitored over time (Fig. 1i). After transduction, the percentage of cells containing tau-venus-positive aggregates decreased over 72 hours until only 10% of cells contained aggregated tau-venus (Fig. 1j, k).We hypothesize that because tau fibril elongation occurs during both seeded and cell-autonomous aggregation, this dual mechanism of tau-RING incorporation is the mechanism behind the successful disassembly of new and pre-existing intracellular fibrils.

[0219] The above data are consistent with tau-RING directly interacting with tau-venus aggregates, leading to their degradation. To test this, we engineered baby hamster kidney (BHK) cells to express tau-venus and a doxycycline-inducible fluorescent form of tau-RING, tau-mCherry-RING. This cell line was selected for its large cytoplasm, allowing for real-time monitoring of the fate of cytosolic aggregates. We seeded these cells with preformed P301S tau seeds and allowed tau-venus aggregates to establish for 24 h before adding doxycycline. Tau-mCherry-RING was recruited to sites of existing tau-venus aggregates, consistent with the incorporation of both constructs into growing aggregates (Fig. 2a, b). Following incorporation of tau-mCherry-RING, the aggregates subsequently degraded over a period of approximately 5 hours, with tau-venus and tau-mCherry-RING exhibiting similar degradation kinetics (Fig. 2c). These results demonstrate that the E3 ligase-bait construct interacts with its target prior to extensive disruption of the complex.

[0220] To determine whether degradation of microscopically detectable tau-venus aggregates resulted in complete disruption of the aggregates and did not generate smaller, seed-competent species that could continue to grow, a secondary seeding assay was performed. In this assay, lysates from TVA cells were probed for seed-competent species by adding the lysates to TV cells in the presence or absence of tau-RING. Evidence of aggregates was assessed after 72 hours. TVA cells were infected with tau-mCherry-RING lentivirus and then sorted for red and green cells, resulting in pure populations (Fig. 2d). Cells expressing both tau-venus and tau-mCherry-RING were expanded and subsequently lysed. TVA + tau-mCherry-RING lysates were compared with TVA and TV-only lysates. TVA lysates seeded aggregates with abundant seeds in fresh TV cells (Fig. 2e, f). TVA + tau-mCherry-RING lysate seeded aggregates with approximately 90% fewer seeds, demonstrating that the number of visually observable tau-venus aggregates closely correlates with the number of seed-capable species present in the cells (Fig. 2e, f). TVA lysate did not seed aggregates, demonstrating that tau-venus does not aggregate by itself (Fig. 2e, f). This experiment demonstrates that tau-RING induces complete disassembly of tau aggregates in cells and does not induce the formation of small seed-capable species.

[0221] Example 2 - Proposed mechanism of action Clustering of TRIM21 after ligation of multivalent immune complexes stimulates E3 catalytic activity through dimerization of the TRIM21 RING domain 12 To determine whether this clustering activity is required for tau-RING activity, we repeated the experiment using tau-RING with the M72E mutation (tau-RING-M72E), which has been shown to disrupt the RING dimerization interface. 12(Fig. 3a). We stably expressed Tau-RING-M72E or its wild-type equivalent (Tau-RING-WT) in TV reporter cells as described above (Fig. 3b). Unlike Tau-RING-WT, the Tau-RING-M72E variant was unable to effectively prevent seed-induced aggregation or clear pre-existing tau aggregates (Fig. 3c, d). Collectively, the above data support a model in which Tau-RING is incorporated into tau aggregates and stimulates RING multimerization and subsequent ubiquitination and degradation. This model predicts that assemblies composed of Tau-RING will be short-lived, whereas RING-free tau assemblies, i.e., Tau-RING-M72E, will remain intact in the cytosol. Recently, we have shown that entry of exogenously supplied tau assemblies into the cytosol can be quantified by luciferase complementation between cytosolically expressed LgBiT and tau bearing an 11 amino acid, HiBiT tag. 11 Therefore, we treated primary mouse neurons with heparin-assembled HiBiT-tagged forms of tau and tau-RING and measured their persistence in the cytosol. Tau-HiBiT, tau-RING-HiBiT, and tau-RING-M72E-HiBiT proteins were aggregated in the presence of heparin. All three proteins showed comparable aggregation kinetics, as quantified by thioflavin T fluorescence (Fig. 3F). Tau-HiBiT rapidly accumulated in the cytosol of primary neurons, whereas significantly lower levels of tau-RING-HiBiT were observed (Fig. 3G). To verify that this difference was not due to a failure of tau-RING-HiBiT uptake, we also used tau-RING-M72E assemblies. This protein accumulated similarly to tau-HiBiT, demonstrating that an intact dimerization interface is required for efficient disassembly of the aggregates (Fig. 3g).

[0222] TRIM21 can initiate a potent degradation response against virus particles entering the cytosol by antibodies bound to their capsid, termed antibody-dependent intracellular neutralization (ADIN). This pathway relies on the ubiquitin-selective AAA+ ATPase and unfoldase, p97 / valosin-containing protein (VCP), which acts upstream of the proteasome. Inhibition of VCP prevented TRIM21 from efficiently neutralizing adenovirus infection and reduced its neutralization of tau seeding activity in cell-based assays (McEwan, William A et al. "Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation." Proceedings of the National Academy of Sciences of the United States of America vol. 114,3(2017):574-579. doi:10.1073 / pnas.1607215114; Hauler, Felix et al. "AAA ATPase p97 / VCP is essential for TRIM21-mediated virus neutralization." Proceedings of the National Academy of Sciences of the United States of America vol. 109,48(2012):19733-8. doi:10.1073 / pnas.1210659109). We asked whether the same VCP dependence observed for adenovirus neutralization could be observed in the context of tau-RING degradation. TV cells with and without tau-RING were treated with siRNA to deplete VCP or a non-targeting control (NTC) (Fig. 3h). The level of tau-venus seed-induced aggregation was then measured in response to exogenously supplied tau assemblies. We found that VCP depletion significantly reduced tau-RING protection compared to control conditions (Fig. 3i). When VCP was depleted, a threefold increase in seed-induced aggregation was observed (Fig. 3j).These results demonstrate that ADIN and tau-RING share VCP as an essential component for efficient substrate degradation, and the data also suggest that tau-RING participates in the same degradation mechanism as full-length TRIM21.

[0223] Example 3: Tau-RING is effective when spaced with a linker domain To investigate whether linker size is important for the function of tau-RING, we generated a new construct, tau-CFP-RING, for direct comparison with tau-RING. Tau-CFP-RING remained soluble inside cells (Fig. 4a) and was expressed at similar levels to tau-RING (Fig. 4b, c). Cell-autonomous aggregation due to tau overexpression was not observed. Similar to tau-RING, tau-CFP-RING did not reduce endogenous tau-venus levels (Fig. 4d). Tau-CFP-RING was effective, albeit less efficiently, in reducing seed-induced aggregation in TV cells (Fig. 4e, f). Therefore, the domain between RING and the bait can be incorporated as a linker to retain the activity of the construct.

[0224] Example 4 - E3 ligase-bait constructs are effective in neurons To verify the efficacy of the E3 ligase bait in a neural environment, we generated AAV particles encoding tau-RING. Because the efficiency of tau-RING decreased when fused to a fluorescent protein, we decided to add a P2A cleavage site between the fluorophore and tau-RING to maintain tau-RING activity while also allowing for easy visualization of construct delivery (Figure 5a). On day 2, primary P301S neurons were transfected with 1 × 10 12vg. Expression of tau-RING and venus proteins was confirmed by Western blot. Similar to HEK293 cells, expression of tau-RING in neurons did not reduce the levels of native soluble tau (Fig. 5b, c). On day 7, heparin-preassembled P301S tau aggregates were added, and on day 14, aggregation was analyzed by AT8 staining (Fig. 5d). Expression of tau-RING resulted in almost complete prevention of AT8-positive aggregates in cell bodies (Fig. 5e). Substantial reduction of aggregates in neurites was also observed, with approximately 66% reduction achieved (Fig. 5f). Primary cultures were stained for the neuron-specific antigen NeuN to determine whether cell death was caused by delivery of the construct or activation by the addition of tau aggregates. We observed a decrease in cell number in all conditions upon addition of tau aggregates, but there was no independent cell death caused by delivery of the viral construct or by the degradation process initiated by Tau-RING (Fig. 5g). These results demonstrate that Tau-RING can be used to prevent tau seed-induced aggregation in primary neurons.

[0225] Example 5 - E3 ligase-bait is effective in vivo Having determined that E3 ligase baits are effective in reducing seed-induced aggregation in cultured neurons, we delivered E3 ligase baits to animal disease models of tauopathy. P301S mice exhibit aggregation primarily in the spinal cord, brainstem, and frontal cortex. Pathology in these regions appears at 6 months, when mice begin to develop motor symptoms, and this time point was chosen as the endpoint for all experiments. Venus or venus-P2A-tau-RING were packaged into the AAV vector 9P31, which has recently been demonstrated to independently cross the blood-brain barrier. At 4 months, mice were injected with 1 x 10 tau baits into the tail vein. 11Mice were injected with venus-P2A-tau-RING and sacrificed at 6 months to assess tau pathology. No adverse side effects were observed upon delivery of the virus or virus-containing constructs, as evidenced by the stable body weights of all mice over the 2-month experiment (Fig. 6a). Sarkosyl-insoluble (SI) tau was extracted from one hemisphere of each brain and visualized by Western blot (Fig. 6b). A significant reduction in SI tau was observed in mice injected with venus-P2A-tau-RING compared with venus alone (Fig. 6c). Because complete infection of all neurons cannot be achieved, immunofluorescence staining of tau aggregates with AT8 in the brain was performed to understand the relationship between infection and the location of tau aggregates (Fig. 6d). Neurons positive for both AT8 and venus were counted in the frontal cortex. Colocalization was observed in the frontal cortex of all mice infected with venus AAV, but little colocalization occurred between AT8 and venus-P2A-tau-RING (Fig. 6e). This indicates that venus-P2A-tau-RING virus aggregates do not form or are eliminated in infected neurons. Nuclei were counted in the same regions of interest assessed for AT8+venus-positive cells. The same density of neurons was present in both the control venus group and venus-P2A-tau-RING-infected mice, demonstrating that tau-RING-induced degradation does not cause neuronal cell death in vivo (Fig. 6f).

[0226] Natural variation in tau pathology between P301S mice is significant. Therefore, we stereotactically injected tau-RING into one hemisphere of the frontal cortex of 5-month-old P301S mice, allowing us to quantify the number of tau aggregates that were controlled within each mouse. To determine whether tau-RING could clear existing aggregates in vivo, we injected 5 × 10 Tau-RING into the frontal cortex of 5-month-old mice. 10Mice were stereotactically injected with either venus-P2A-tau-RING or venus-only AAV PHP.eB in the vg mice and sacrificed at 6 months (Fig. 6g). The injected hemisphere was compared with the contralateral uninjected hemisphere. Analysis of AT8-positive aggregates showed an approximately 50% reduction in the AT8-positive area upon delivery of Venus-P2A-tau-RING (Fig. 6h). Comparison of individual injected hemispheres with the contralateral hemisphere showed a consistent reduction in the AT8 area in all mice treated with tau-RING compared with mice treated with Venus alone (Fig. 6h). Thus, tau-RING can protect against tau pathology in vivo.

[0227] Example 6: Tau fusions with other RING constructs or E3 recruitment scaffolds allow clearance of pre-existing intracellular tau aggregates We next investigated whether fusing tau with another RING domain could clear pre-existing tau aggregates. To do this, we utilized the RING domain of TRIM5α. TRIM5α can form clustered, cage-like structures that enclose the retroviral core in the cytoplasm, leading to activation of its RING E3 ligase domain and viral particle disassembly. The Box domain of TRIM5α is known to assist in TRIM5α clustering. Therefore, we fused tau to the RING-Box domain of TRIM5α. Lentivirus containing P301S T5-RING-Box-tau was applied to TVA cells. T5-RING-Box-tau reduced pre-existing aggregates by approximately 80% (Figure 7a), further suggesting that other clustered RING domains can be utilized to achieve tau aggregate clearance.

[0228] The data presented so far demonstrate that direct fusion of RING to a bait can promote degradation of an engineered target.

[0229] Next, we tested whether fusing the bait to an E3-recruiting domain was also sufficient. To do so, we fused tau to the von Hippel-Lindau tumor suppressor protein (VHL), which recruits the Cullin-E3 ligase complex. TVA cells infected with lentivirus encoding tau-VHL showed a roughly fivefold reduction in tau aggregates (Fig. 7a). Furthermore, when recombinant 0N4R P301S tau seeds were transfected into TVA cells, expression of tau-VHL (Fig. 7b) inhibited seed-induced tau aggregation without any change in monomeric tau expression (Fig. 7c, d). Thus, both direct fusion to E3 and fusion to an E3-recruiting protein can induce degradation of the engineered target without compromising the stability of its monomeric form.

[0230] Example 7 - Discussion of Examples 1 to 6 The inventors have demonstrated that fusions between E3 catalytic or recruitment proteins or domains and proteins capable of forming pathological aggregates can allow for the selective degradation of the assembled protein. This is exemplified herein with tau, which can adopt a fibrillar conformation during neurodegenerative disease (a property also shared by other proteins involved in neurodegeneration, such as TDP43 and α-synuclein).

[0231] We showed that incorporation of tau-RING into tau fibers accelerates fiber degradation and provides potent protection against seeds. The tau-RING construct was also able to degrade pre-existing aggregates within cells, demonstrating that, provided the correct signals are present, the ubiquitin-proteasome system can process and remove these large aggregates.

[0232] We further showed that this protection was conferred to neurons expressing the tau-RING construct in cell-based assays of seed-induced aggregation and after AAV-mediated expression in mouse brain, suggesting that neurons, like HEK293 and BHK cell lines, possess the necessary machinery to promote clearance of fibrillar tau structures.

[0233] Activation of tau-RING depended on clustering of the RING domain after incorporation of tau-RING into fibrils. Overall, our results demonstrate that effective protection against protein misfolding and aggregation can be achieved using an E3 ligase-bait strategy.

[0234] Example 8 - The mechanism of RING-bait is driven by RING activity Figure 8 shows how the RING-bait technology can drive RING activity through the use of functional mutations and inhibitors. The data demonstrate that this process requires RING to both dimerize (M72E) and bind ubiquitin (I18R) (see Figure 8A-C). Furthermore, the RING-bait mechanism proceeds through ubiquitination (which is blocked by the E1 inhibitor TAK-243), the unfoldase / segregase VCP (blocked by NMS-873), and then the proteasome (blocked by MG132) (Figure 8D-G).

[0235] Example 9 - RING-baits can be utilized with different portions of the bait protein to target different types of aggregates Figure 9 shows how both different isoforms of tau (3R and 4R) can be used as bait fragments—see Figure 9A-C. Furthermore, RING-baits are effective against different types of tau aggregates induced by Alzheimer's disease (AD) or progressive supranuclear palsy (PSP) (Figures 9D-E and 9F-G, respectively).

[0236] Example 10 - RING-baits can be used in vivo to improve clinical phenotypes Figure 10 shows in vivo data demonstrating the effectiveness of RING-bait not only against tau aggregates in the mouse brain but also in achieving positive behavioral effects. Active RING-bait or catalytically inactive RING-bait (I18R / M72E) was delivered by AAV to P301S mice at 4 months, and brains were then analyzed by immunofluorescence (Figures 10B and C) or Western blot (Figures 10D-G; Figure 11). Active RING-bait reduced tau aggregates by both measures. Furthermore, behavioral data show that mice treated with RING-bait had improved motor phenotypes at 6 months compared with untreated or inactive controls (Figures 10H and I; Figure 12).

[0237] Example 11 - Different types of proteins can be used as baits in RING-baits. Figure 13 demonstrates that the aggregating mutant Fused in sarcoma (FUS) protein (P525L) can be degraded using a RING-bait construct (RING-FUS P525L), which was expressed with a self-cleaving T2A peptide and C-terminal mCherry (see SEQ ID NO: 7).

[0238] References 1. Shi, Y. et al. Structure-based classification of tauopathies. Nature 598, 359-363(2021). 2. Arseni, D. et al. Structure of pathological TDP-43 filaments from ALS with FTLD. Nature 1-5(2021) doi:10.1038 / s41586-021-04199-3. 3. Schweighauser, M. et al. Structures of α-synuclein filaments from multiple system atrophy. Nature 585, 464-469(2020). 4. Biogen Shelves Gosuranemab After Negative Alzheimer’s Trial | ALZFORUM. https: / / www.alzforum.org / news / research-news / biogen-shelves-gosuranemab-after-negative-alzheimers-trial. 5. Meisl, G. et al. In vivo rate-determining steps of tau seed accumulation in Alzheimer’s disease. Science Advances(2021) doi:10.1126 / sciadv.abh1448. 6. Hyun, S. & Shin, D. Chemical-Mediated Targeted Protein Degradation in Neurodegenerative Diseases. Life 11, 607(2021). 7. Zeng, J. et al. Target-induced clustering activates Trim-Away of pathogens and proteins. Nat Struct Mol Biol 28, 278-289(2021). 8. McEwan, W. A. et al. Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation. Proc Natl Acad Sci USA 114, 574-579(2017). 9. Croft, C. L. et al. Photodynamic studies reveal rapid formation and appreciable turnover of tau inclusions. Acta Neuropathol 141, 359-381(2021). 10. Kundel, F. et al. Measurement of Tau Filament Fragmentation Provides Insights into Prion-like Spreading. ACS Chem Neurosci 9, 1276-1282(2018). 11. Tuck, B. J. et al. Tau assemblies enter the cytosol in a cholesterol sensitive process essential to seeded aggregation. 2021.06.21.449238 https: / / www.biorxiv.org / content / 10.1101 / 2021.06.21.449238v3(2021) doi:10.1101 / 2021.06.21.449238. 12. Dickson, C. et al. Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21. eLife 7, e32660. 13. Zhang, W. et al. Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases. eLife 8, e43584(2019). 14. Goodwin, M. S. et al. Anti-tau scFvs Targeted to the Cytoplasm or Secretory Pathway Variably Modify Pathology and Neurodegenerative Phenotypes. Molecular Therapy 29, 859-872(2021). 15. Danis, C. et al. Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment. 2021.03.23.436266 https: / / www.biorxiv.org / content / 10.1101 / 2021.03.23.436266v1(2021) doi:10.1101 / 2021.03.23.436266. 16. Gallardo, G. et al. Targeting tauopathy with engineered tau-degrading intrabodies. Molecular Neurodegeneration 14, 38(2019). 17. Lu, M. et al. Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway. Eur J Med Chem 146, 251-259(2018). 18. Chu, T.-T. et al. Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation. Cell Chemical Biology 23, 453-461(2016). 19. Silva, MC et al. Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. eLife 8, e45457(2019).

[0239] [Table 2]

[0240] [Table 3]

[0241] [Table 4]

[0242] [Table 5]

[0243] [Table 6]

[0244] [Table 7]

[0245] Other arrays TRIM21 (SEQ ID NO: 1) [ka] Amino acids 1 to 85 of TRIM21 (SEQ ID NO: 2) MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEARE Tau-RING amino acid sequence: (SEQ ID NO: 3) [ka] [ka] (0N4R P301S) [ka] (EcoR1 site and GSGGGSG flexible region) RING (TRIM21 RING domain) Tau-CFP-RING amino acid sequence (SEQ ID NO: 4) [ka] [ka] [ka] [ka] RING (TRIM21 RING domain) VHL (Gene ID 7428) - (SEQ ID NO: 5) [ka] Trim5-B-box domain - (SEQ ID NO: 6) QKVDHCARHGEKLLLFCQEDGKVICWLCERSQEHRGHHTFLTE RING-FUS construct - (SEQ ID NO: 7) [ka]

Claims

1. (i) a first portion comprising the sequence of a protein that pathologically aggregates in a neurodegenerative disease; (ii) a second portion comprising a RING-type E3 ubiquitin ligase, a component of a RING-type E3 ubiquitin ligase complex, or a domain of either; and A fusion protein comprising:

2. 2. The fusion protein of claim 1, wherein the second portion comprises the RING-type E3 ubiquitin ligase or comprises a component or domain having E3 ligase catalytic activity.

3. 3. The fusion protein of claim 1 or 2, wherein the component or domain comprises or is a RING domain.

4. The fusion protein of claim 3, wherein the component or domain comprises or is a RING-box.

5. 5. The fusion protein of claim 3 or 4, wherein the component or domain comprises or is derived from a polypeptide or RING domain shown in Table 1 and / or is derived from a TRIM polypeptide.

6. The fusion protein of any one of claims 2 to 5, wherein the RING domain comprises at least 30 amino acids.

7. The fusion protein of any one of claims 2 to 6, wherein the RING domain comprises SEQ ID NO: 2 or a variant having at least 70% identity thereto.

8. The fusion protein of claim 1 , wherein the component or domain is selected from a scaffold component, an adapter component, and a substrate recognition component.

9. 9. The fusion protein of claim 8, wherein the component is a VHL polypeptide optionally comprising SEQ ID NO: 5 or a variant having at least 70% identity thereto.

10. The fusion protein of any one of claims 1 to 9, wherein the protein aggregation portion comprises the sequence of a protein selected from the list consisting of: tau; TDP43; alpha-synuclein; huntingtin; rhodopsin; superoxide dismutase; FUS protein.

11. 11. The fusion protein of claim 10, wherein the protein-aggregating portion comprises all or part of a sequence shown or set forth in Table 3, or a variant having at least 70% identity thereto.

12. The fusion protein of any one of claims 1 to 11, wherein the protein-aggregating portion comprises at least 25 amino acids.

13. The fusion protein of any one of claims 1 to 12, wherein the first portion is upstream of the second portion from the N-terminus to the C-terminus.

14. The fusion protein of any one of claims 1 to 12, wherein the first portion is downstream of the second portion from the N-terminus to the C-terminus.

15. 15. The fusion protein of any one of claims 1 to 14, comprising one or more further amino acids or amino acid sequences between the first and second portions or at the N-terminus or C-terminus of the fusion protein.

16. 16. The fusion protein of claim 15, comprising a linker between the first portion and the second portion.

17. 17. The fusion protein of claim 16, wherein the linker comprises or consists of glycine and serine.

18. The fusion protein of any one of claims 1 to 12, comprising SEQ ID NO: 3 or 4, or a variant thereof having at least 70% identity thereto.

19. A polynucleotide encoding the fusion protein of any one of claims 1 to 18.

20. 20. A process for producing the polynucleotide of claim 19, comprising: (a) providing a first nucleic acid encoding the first portion; (b) providing a second nucleic acid encoding the second portion; (c) combining the first nucleic acid and the second nucleic acid; A process involving:

21. 21. A polynucleotide obtained or obtainable by the process of claim 20.

22. 22. An expression vector comprising the polynucleotide of claim 19 or claim 21.

23. 23. The vector of claim 22, wherein the polynucleotide encoding the fusion protein is operably linked to a promoter, which is optionally a tissue- or cell-specific promoter.

24. 24. The vector of claim 23, wherein the promoter is a neural cell type-specific promoter.

25. The vector according to any one of claims 22 to 24, which is a viral vector.

26. 26. The vector of claim 25, which is an adenoviral vector and / or adeno-associated vector (AAV), optionally selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids or derivatives thereof, optionally selected from AAV-F, AAV-9P31, and AAV-9P801.

27. 26. The vector of claim 25, which is a herpesvirus vector, a retrovirus vector, or a lentivirus vector.

28. A host cell comprising, transfected with or transformed with the polynucleotide or vector according to any one of claims 19 or 21, or claims 22 to 27.

29. A method for producing a fusion protein according to any one of claims 1 to 18, comprising the steps of: (a) stably transforming a host cell with an expression vector according to any one of claims 22 to 27; (b) culturing the transformed host cells under conditions suitable for the host cells to produce the fusion protein; (c) optionally recovering the fusion protein; A method comprising:

30. 30. A fusion protein obtained or obtainable by the process of claim 29.

31. A viral particle comprising the vector according to any one of claims 25 to 27.

32. 32. A pharmaceutical composition comprising an agent that is a fusion protein, polynucleotide, vector, or particle according to any one of claims 1 to 19, 21 to 27, or 30 to 31, and optionally a pharmaceutically acceptable carrier, diluent, or excipient.

33. 1. A method for inhibiting the formation or increasing the degradation or clearance of pathological aggregates of an aggregating protein in a cell, comprising: introducing a fusion protein according to any one of claims 1 to 18 or claim 30 into said cell, optionally via expression from a polynucleotide, vector or particle according to any one of claims 19, 21 to 27 or 31; As a result, a plurality of the fusion protein molecules are incorporated into co-aggregates with the aggregation protein, the plurality of fusion proteins clustering into the co-aggregates to cause ubiquitination of the co-aggregates; This results in the inhibition of aggregate formation or the increased degradation or clearance of aggregates in cells. A method comprising:

34. 34. The method of claim 33, wherein the cell is present in an organism, optionally a non-human test animal.

35. 1. A method of treating a neurodegenerative disease in a subject, wherein the neurodegenerative disease is associated with a protein that pathologically aggregates, the method comprising: (1) providing a selected agent that is a fusion protein, polynucleotide, vector, particle, or composition according to any one of claims 1 to 19, 21 to 27, 30 to 31, or 32; (2) administering the agent to the subject; A method comprising:

36. (i) assembling the viral particles of claim 28 in vitro by transducing mammalian cells with the expression vector, expressing in the cells viral packaging and envelope proteins necessary for particle formation, and culturing the transduced cells in a culture medium such that the cells produce viral particles comprising the expression vector that are released into the medium; (ii) administering the viral particles to the subject; 36. The method of claim 35, comprising:

37. 37. The method of claim 35 or claim 36, wherein the subject is a human subject.

38. 38. The method of any one of claims 33 to 37, wherein the protein of the first part of the fusion protein and the respective disease are as defined in Table 2, and optionally, the protein that pathologically aggregates is a tau protein and the disease is Alzheimer's disease or progressive supranuclear palsy, or the protein that pathologically aggregates is a Fused in sarcoma (FUS) protein and the disease is amyotrophic lateral sclerosis or frontotemporal dementia.

39. 38. The method of any one of claims 35 to 37, wherein the subject is a non-human mammal, a bird, a fish, a reptile, or an amphibian.

40. 36. A medicament as defined in claim 35 for use in a method of treatment of the human or animal body.

41. 41. The method of claim 40, wherein the treatment is treatment of a neurodegenerative disease in a subject.

42. 36. Use of the agent of claim 35 in the manufacture of a medicament for the treatment of a neurodegenerative disease in a subject.

43. Use or medicament for use according to any one of claims 40 to 42, wherein the treatment is as defined in any one of claims 35 to 39.

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