Methods and compositions related to evolving botulinum toxin proteases for targeted substrate specificities

Abstract

This invention provides libraries of Botulinum neurotoxin A (BoNT / A) protease variants for stepwise evolution of the enzyme to generate BoNT / A variants that can specifically cleave a desired cleavage site in a target protein. Related methods for performing stepwise evolution of BoNT / A with the BoNT / A variant libraries and simultaneous stepwise evolution of a desired substrate sequence in a target protein are also provided by the disclosure. Additionally provided in the disclosure are specifically evolved BoNT / A variant enzymes and conjugate proteins that specifically degrade intrinsically disordered proteins (IDPs) that are involved in human deceases, e.g., a-Synuclein. Polynucleotide sequences encoding the engineered BoNT / A proteases, expression vectors and related pharmaceutical compositions are also provided in this disclosure. Further encompassed by the invention are therapeutic methods that utilize the engineered BoNT / A enzymes in the treatment of various synucleinopathies.

Description

PATENT Docket No.: TSRI 2277.1PC Methods and Compositions Related to Evolving Botulinum Toxin Proteases for Targeted Substrate SpecificitiesCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U. S.Provisional Patent Application Number 63 / 760,152 (filed February 19, 2025; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.STATEMENT CONCERNING GOVERNMENT SUPPORT

[0002] This invention was made with government support under GM145323 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] This application includes by incorporation of reference a sequence listing in the XML format, 2277_lPC_Sequence_Listing, which was created on February 16, 2026 and contains 36 KB in content.BACKGROUND OF THE INVENTION

[0004] The ability to evolve proteases that selectively and efficiently cleave a desired protein in vivo would provide novel therapeutic agents to degrade proteins contributing to human disease. Indeed, the protease from botulinum neurotoxin A (BoNT / A), by virtue of its exquisite sequence specificity for its native target SNAP25, is used clinically for local deactivation of peripheral neurons and can undergo repeated administration without loss in efficacy due to anti-drug antibodies (1, 2). Structurally, like many other bacterial toxins (3), BoNT / A consists of a “light chain” catalytic domain tethered to a “heavy chain” that is responsible for endocytosis by acting as a receptor-binding and translocation domain. The heavy chain thus provides efficient means for the protease to cross cellular membranes with selective tropism for a specific cell type and proteolyze an intracellular target. After uptake into lysosomes, the disulfide bond connecting the twosubunits is cleaved and the light chain is released into the cytosol. The target of the BoNT / A light chain, SNAP25, is an intracellular, intrinsically disordered protein (IDP). SNAP25 is an integral part of SNARE assemblies required for neurotransmitter exocytosis (4) and selective proteolysis of SNAP25 by BoNT / A leads to synaptic deactivation. Unfortunately, other such highly selective proteases that can be used to degrade targets of clinical interest are lacking.

[0005] The extended >30 amino acid substrate binding site of the BoNT / A Zn2+metalloprotease and distinct catalytic and substrate binding sites (5) makes it an ideal starting point for evolving proteases to other targets of interest. SNAP25 binds to BoNT / A through numerous polar and nonpolar interactions, both C- and N-terminal to the substrate cleavage site. These interactions are mediated by unstructured loops and P-sheets of the protease, which position the substrate in a favorable conformation for hydrolysis in close proximity to the active site Zn2+ion. Previous work on evolving clinically used BoNT / A (6-8) or other BoNT serotypes (9, 10) has demonstrated the possibility of partially changing substrate selectivity. However, the complex interplay of evolving selective substrate binding with concomitant retention of enzymatic activity and protein stability has made the evolution of altered substrate selectivity in BoNT / A challenging task, particularly for non-homologous targets. Consequently, previous efforts focused on evolving specificity only towards highly homologous target sequences, e.g., SNAP23 (6-8), used BoNT serotypes with less complex surface interactions than BoNT / A and SNAP25 (10), or both (9).

[0006] There is an unmet need in the art for evolving the enzymatic activity of the clinically used BoNT / A protease for the selective degradation of proteins that bear little homology to its native substrate. The present invention is directed to this and other unmet needs.SUMMARY OF THE INVENTION

[0007] In one aspect, the present disclosure provides libraries of Botulinum neurotoxin A (BoNT / A) protease variants that can be employed for stepwise evolution of the enzyme towards a desired substrate specificity, e.g., specifically cleaving a selected substate sequence in a target protein (e.g., an intrinsically disordered protein). For example, the selected substate sequence can be present in an intrinsically disordered region of the target protein. Each of the BoNT / A variant libraries can contain a plurality of BoNT / A variants that,relative to the wildtype BoNT / A sequence or a BoNT / A variant identified during an evolution campaign, contain randomized mutations at each residue selected from (1) R363, L367, N368, F369, D370, and A372, (2) F243, K244, V245, G255, L256, and E257, (3) C165, K166, S167, F168, Q184, and R231, (4) K23, 124, P25, A27, G28, and Q29, (5) G169, H170, E171, V172, N174, and T176, (6) D131, T132, C134, N136, Y144, and S146, or (7) V304, G305, T306, T307, A308, and Y312. For reference of the noted residues, the wildtype BoNT / A sequence is based on UniProt ID Q7B8V4 (SEQ ID NO: 1).

[0008] In a related aspect, the disclosure provides methods for simultaneous stepwise evolution of the BoNT / A enzyme and its natural substrate sequence in its native substrate SNAP25. These methods are intended to engineer and obtain BoNT / A variants that can specifically cleave a target protein of interest at a desired or selected site in the target protein. In general, these methods involve first selecting in the target protein a substrate sequence that is to be cleaved. At the minimum, the selected substrate sequence in the target protein will contain the residues from position -7 to position +6 around the desired cleavage site. Once a desired cleavage sequence in the target protein is selected, 3 rounds of primary screening are performed iteratively without a defined order. These screening rounds employ (i) a first library of BoNT / A variants that, relative to a first reference enzyme, contain randomized mutations at each residue selected from R363, L367, N368, F369, D370, and A372, and a first corresponding substrate sequence that, relative to a first reference substrate sequence, has residues at -1, +1, +2, +3 and +6 positions being respectively identical to the corresponding residues in the selected substrate sequence in the target protein, (ii) a second library of BoNT / A variants that, relative to a second reference enzyme, contain randomized mutations at each residue selected from F243, K244, V245, G255, L256, and E257, and a second corresponding substrate sequence that, relative to a second reference substrate sequence, has residues at +4 and +5 positions being respectively identical to the corresponding residues in the selected substrate sequence in the target protein, and (iii) a third library of BoNT / A variants that, relative to a third reference enzyme, contain randomized mutations at each residue selected from C165, K166, S167, F168, Q184, and R231, and a third corresponding substrate sequence that, relative to a third reference substrate sequence, has residues at -7, -6, -5, -4, -3, and -2 positions being respectively identical to the corresponding residues in the selected substrate sequence in the target protein. In performing these 3 rounds of primary screening, each round employs a separate library of BoNT / Avariants and the corresponding substrate sequence to identify a BoNT / A variant that cleaves the corresponding substrate sequence. It is noted that (i) in the first round of primary screening, the reference enzyme for the employed library of BoNT / A variants is the wildtype BoNT / A, and the reference substrate sequence for the employed corresponding substrate sequence contains the native BoNT / A cleavage site in the SNAP25 protein; (ii) in the second round of primary screening, the reference enzyme for the employed library of BoNT / A variants is the BoNT / A variant identified from the first round of primary screening, and the reference substrate sequence for the employed corresponding substrate sequence is the corresponding substrate sequence used in the first round of primary screening, and (iii) in the third round of screening, the reference enzyme for the employed library of BoNT / A variants is the BoNT / A variant identified from the second round of primary screening, and the reference substrate sequence for the employed corresponding substrate sequence is the corresponding substrate sequence used in the second round of primary screening. The BoNT / A variant identified from the third round of primary screening is a protease that can specifically cleave the target protein at a substrate sequence that contains the residues from position -7 to position +6.

[0009] Some of the simultaneous stepwise-evolution methods for BoNT / A described herein can additionally include secondary screening with additional BoNT / A variant libraries. The secondary screening is intended to fine tune substrate specificity of the evolved BoNT / A variants, i.e., to identify BoNT / A variants that specifically cleaves a desired substrate sequence that contains, in addition to the residues from position -7 to +6 around the selected cleavage site, residues from position -8 to position -11 around the cleavage site. The secondary screening typically entails one or more rounds of screening with one of 4 additional libraries of BoNT / A variants that, relative to a reference enzyme (e.g., a BoNT / A variant identified from the primary screening), contain randomized mutations at each residue selected from (i) K23, 124, P25, A27, G28, and Q29, (ii) D131, T132, C134, N136, Y144, and S146, (iii) G169, H170, E171, V172, N174, and T176, or (iv) V304, G305, T306, T307, A308, and Y312. Each of these rounds of secondary screening uses the same corresponding substrate sequence that, relative to the corresponding substrate sequence used in the last round of the primary screening, has residue at positions -8, -9, -10 and -11 being respectively changed to the corresponding residues in the selected substrate sequence in the target protein. These rounds of secondary screening will allow the identification of a final BoNT / A variantthat cleaves a substrate sequence that additionally includes residues from position -8 to position -11 around the intended cleavage site. In some embodiments, the one or more rounds of the secondary screening are performed sequentially. In these embodiments, the reference enzyme for the employed library of BoNT / A variants in each round is the BoNT / A variant identified from the previous round of the screening. In some embodiments, the secondary screening includes screening of each of the 4 additional libraries of BoNT / A variants. In some other embodiments, the one or more rounds of the secondary screening are performed in parallel. In these embodiments, the reference enzyme for the employed library of BoNT / A variants is the BoNT / A variant identified from last round of the primary screening. Once the in parallel rounds of secondary screening are completed, all mutations present in the BoNT / A variants identified from each round of the secondary screening, relative to the reference enzyme, are combined into the reference enzyme by gene shuffling to generate the final evolved BoNT / A variant.

[0010] In another aspect, the present invention provides engineered Botulinum neurotoxin serotype A (BoNT / A) proteases that specifically degrade a-Synuclein. The engineered proteases contain the light chain sequence of a wildtype BoNT / A except for the following amino acid substitutions, K23I, I24L, A27N, G28M, Q29D, Q67K, T109M, D131S, N136V, Y144Q, S146M, K166R, F168R, G169E, H170F, E171S, V172M, N174P, Q184C, Fl 961, F213I, T220L, R231F, V242A, F243I, K244W, V245F, N248D, S254R, G255W, L256M, E257K, N280D, F282L, R363R, N368L, F369L, D370P, and A372L. The amino acid numbering of the noted mutations is based on the sequence of the wildtype BoNT / A protein under UniProt ID Q7B8V4 (SEQ ID NO: 1). In some embodiments, the engineered proteases are derived from a subtype Al BoNT / A protein. In some embodiments, the wildtype BoNT / A protein into which the noted mutations are introduced has an amino acid sequence as set forth in any one of SEQ ID NOs: 1-13. In some embodiments, the engineered BoNT / A protease contains the noted mutations that are introduced into a background sequence that is at least 95% or 99% identical to the wildtype BoNT / A light chain sequence set forth in SEQ ID NO: 14. In some embodiments, the engineered BoNT / A protease contains an amino acid sequence set forth in SEQ ID NO: 15 or a conservatively modified variant thereof.

[0011] In some embodiments, the engineered BoNT / A protease further contains a conjugated targeting moiety. In some of these embodiments, the conjugated targeting moietyis a bacterial toxin heavy chain, an antibody or fragment thereof, or monobody with binding specificity for a desired cell type or tissue type. In some of these embodiments, the conjugated targeting moiety is an engineered BoNT / A heavy chain with a receptor binding specificity for a specific cell type or tissue type (e.g., neuron). In some of these embodiments, the engineered BoNT / A light chain protease is conjugated to the engineered BoNT / A heavy chain via a disulfide bond.

[0012] In a related aspect, the invention provides methods for treating or preventing a synucleinopathy in a subject. The methods entail administering to the subject a pharmaceutical composition that contains a therapeutically effective amount of an engineered Botulinum neurotoxin A (BoNT / A) protease that specifically degrades a-Synuclein. This allows treatment or prevention of the synucleinopathy in the subject. In various embodiments, the subject to be treated with the method is a human patient who is afflicted with or at risk of developing Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or pure autonomic failure (PAF). In some methods, the administered engineered BoNT / A protease contains an amino acid sequence set forth in SEQ ID NO: 15 or a conservatively modified variant thereof. In some of these methods, the administered engineered BoNT / A protease further contains a conjugated BoNT / A heavy chain that targets a specific cell type or tissue type (e.g., neuron or glia in the brain).

[0013] In another related aspect, the invention provides polynucleotides that encodes the engineered Botulinum neurotoxin A (BoNT / A) proteases described herein. These include polynucleotides (DNA and RNA) that encode the engineered Botulinum neurotoxin A (BoNT / A) protease having an amino acid sequence set forth in SEQ ID NO: 15 or a conservatively modified variant thereof. In some related embodiments, expression vectors that harbor the polynucleotides and express the engineered Botulinum neurotoxin A (BoNT / A) proteases of the invention are provided. Some of these expression vectors are viral vectors, e.g., AAV vectors or retroviral vectors.

[0014] In another aspect, the invention provides engineered proteases that specifically degrade a target protein in a sequence specific manner, which are generated by stepwise mutagenesis of a wildtype Botulinum neurotoxin A (BoNT / A) protease and simultaneous stepwise evolution of the BoNT / A protease’s natural substrate sequence towards the substrate sequence of the target protein. In some embodiments, the target protein that is to be degraded by the engineered proteases is an intrinsically disordered protein (IDP)involved in a human decease. Examples of such IDPs include a-Synuclein, c-myc, p53, EWS-FLI1, P-amyloid, TDP43, and TMEM106B. In some embodiments, the engineered protease is generated from a subtype Al wildtype BoNT / A protein, e.g., the wildtype BoNT / A protease having the amino acid sequence set forth in SEQ ID NO: 14. In other embodiments the target protein contains loops, N- and C-terminal disordered regions and other unstructured regions that are cleaved by the protease. Examples include the C-terminus of K-Ras.

[0015] In another aspect, the invention provides methods for targeted degradation of a non- SNARE protein in a subject. The methods involve (1) generating an engineered Botulinum neurotoxin A (BoNT / A) protease with substrate specificity for the non-SNARE protein by stepwise mutagenesis of a wildtype BoNT / A protease and simultaneous stepwise evolution of the BoNT / A protease’s natural substrate sequence towards substrate sequence of the non-SNARE protein, and (2) administering the engineered BoNT / A protease in a pharmaceutical composition to the subject. This enables targeted degradation of the non-SNARE protein in the subject. In some of these methods, the non-SNARE protein to be degraded is an intrinsically disordered protein (IDP) that plays a role in the development of a human disease. Examples of IDPs that are to be degraded with the methods include a-Synuclein, c-myc, p53, EWS-FLI1, P-amyloid, TDP43 and TMEM106B. In some methods, the employed wildtype BoNT / A protease for generating the engineered protease is of subtype Al. In some of these embodiments, the employed wildtype BoNT / A protease contains the amino acid sequence set forth in SEQ ID NO: 14.

[0016] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows screening and mutagenesis strategy. A) Alignment of the C-terminus of SNAP25 (SEQ ID NO: 17) and part of a-Synuclein’s NAC region (SEQ ID NO: 18) highlighting homologous sites with teal boxes and the cleavage sites of WT-BoNT / A on SNAP25 by a triangle. B) In the presence of WT-BoNT / A the SNAP25-based linker connecting T7LZ to T7RNAP is proteolytically cleaved, producing active T7 RNA polymerase. C) Proteolytic activity leading to sfGFP production is demonstratedexperimentally when protease expression is induced with arabinose, resulting in strongly increased production of sfGFP. When activity of the protease pBAD promoter is inhibited with glucose, only low levels of sfGFP production are observed. In the absence of a competent protease the tethered T7 lysozyme induces abortive transcription by T7 RNAP, leading to only basal sfGFP production by E. coli, independent of whether glucose or arabinose is added. D) Partial amino acid sequences (SEQ ID NOs: 19-23, respectively) of different linkers tethering T7 lysozyme to T7 RNA polymerase that were employed in this study.

[0018] Figure 2 shows subsite mutation and screening. A) Co-Crystal structure of deactivated double mutant BoNT / A-LC (E224Q / Y366F) and SNAP25 fragment (PDB: 1XTG) with regions highlighted that mediate substrate binding and cleavage. B) Diversification and screening scheme for evolving substrate selectivity of botulinum protease with the substrates used for each respective step. Clones chosen for subsequent rounds are highlighted in black. C) Mutations observed in selected clones from site- saturation mutagenesis. An asterisk marks sequences that were carried on for further mutagenesis. D) Mutations observed in selected BoNT / A clones isolated over the course of this study. Grey boxes highlight amino acids mutated from the WT sequence. Highlight letters indicate residues mutated a second time during the evolution campaign.apreceded by a 32 amino acid insert between BoNT / A amino acids S254 and T255 with the sequence DSEVSFEELRTFVAINPNRVLQSNTNAYYEMS (SEQ ID NO:25).

[0019] Figure 3 shows aSyn-degrading activities of BoNT / A mutants after several rounds of evolution via fluorescence assay and Western blot analysis. A) Fluorescence assay in E. coli as a measure for proteolytic activity of respective BoNT / A mutant against indicated screening system. Fluorescence data from cells without protease is shown for comparison. B) Western blot showing activity of protease mutants on Flag-tagged screening system substrates with induced (top) or uninduced (bottom) protease expression. Proteases selected from site-saturation mutagenesis libraries are active on only 1-2 homologous substrates without prior negative selection.

[0020] Figure 4 shows aSyn-degrading activities of a final evolved BoNT / A mutant (Protease 5) via various assays. A) Fluorescence assay using E. coli in the absence and presence of Protease 5 as an indicator for its activity and selectivity against substrate screening systems used in this study. Substrate cleavage was also evaluated by Western Bloton Flag-tagged substrates with F-actin serving as a loading control. Only the SS-aSyn substrate was significantly degraded by the final Protease 5. B) Heatmap visualizing the selectivity trajectory of our evolution campaign by plotting the fluorescence increase in the presence of respective substrate screening systems and proteases in E. coli. White boxes highlight the substrate screening system the respective protease was selected against. C) Western blot showing substrate degradation by WT-BoNT (Flag-SNAP substrate) or evolved Protease 5 (Flag-SS-aSyn substrate). Cells were grown for 3h total and arabinose was added at the indicated time prior to harvest. The high contrast view shows traces of SS-aSyn substrate when no arabinose was added (0 minute timepoint). D) Western blot of HEK293T cells transfected with the indicated ratios of CMV plasmids encoding either full length human a-Syn or Protease 5. E) Viability of HEK239T measured 24h after being transfected with CMV plasmids encoding the indicated proteins. Viability of cells transfected with SNAP25 plasmid was normalized to 100% viability.

[0021] Figure 5 shows deconvoluted MALDI-TOF data of a reaction between evolved Protease 5 and a substrate. The substrate is a fusion protein consisting of “YFP-a-Synuclein(amino acids l-140)-CFP”. Both YFP and CFP lose a molecule of water upon chromophore maturation. Calculated neutral average masses assuming a substrate cleavage site between Synuclein residues T92 and G93 are: 38076.09 amu (N-terminal fragment without starting methionine and after chromophore maturation). 32259.13 (C-terminal fragment after chromophore maturation). 56975.00 (GBl-Protease 5-“LPETGG” (SEQ ID NO:24), evolved Protease 5 with N-terminal GB1 tag and C-terminal sortase tag without starting methionine).DETAILED DESCRIPTIONI. Overview

[0022] The present disclosure is predicated in part on studies undertaken by the inventors to develop novel strategies for targeted degradation of proteins implicated in human diseases. As detailed herein, the inventors developed a structure-guided, stepwise strategy to evolve the enzymatic activity of the clinically used BoNT / A protease for the selective degradation of proteins that bear little homology to its native substrate. The current use of sequence-specific proteases for this purpose is severely limited by the difficulty in engineering the numerous enzyme-substrate interactions required to yield highly selectiveproteases while maintaining catalytic activity. As exemplifications, the inventors evolved a protease for the programmed degradation of a-Synuclein, a presynaptic protein closely linked to Parkinson’s disease. As detailed herein, the structure-guided evolution campaign uses the protease from botulinum neurotoxin and showcases the stepwise change of specificity from its native substrate SNAP25 to the selective degradation of a-Synuclein. The protease’s selectivity is further demonstrated in human cells where near complete degradation of overexpressed human a-Synuclein is observed with no significant effects on cell proliferation. This stepwise strategy can serve as a general approach to evolve highly selective proteases targeting dysregulated proteins.

[0023] The ability to evolve proteases that selectively cleave a desired protein in vivo provides access to useful new therapeutic agents. This would be especially powerful when targeting intrinsically disordered proteins, a hard-to-drug class of proteins involved in many human diseases including cancer and neurodegenerative diseases. As exemplification, the inventors demonstrated the stepwise evolution of clinically used botulinum protease to proteolyze the intrinsically disordered protein a-Synuclein which forms plaques in the brains of patients suffering from Parkinson’s disease.

[0024] In accordance with these studies, the invention provides a generalizable method for evolving BoNT / A proteolytic cleavage against any target protein of interest. The invention also provides specific libraries of BoNT / A variant enzymes that can be used in the stepwise evolution of the wildtype enzyme. These libraries contain BoNT / A mutants that contain randomized residues in several specific regions of the BoNT / A light chain that are important for the protease-substrate interaction. The invention further provides evolved a-Synuclein proteases with specific sequences and related sequences of the a-Synuclein specific protease. Also provided are methods to evolve proteases for degradation of sequence-specific substrates, such as intrinsically disordered proteins that are implicated in human diseases or proteins containing unstructured loops or other regions. This is exemplified with the stepwise evolvement of the natural substrate sequence of a BoNT / A protease to a new target sequence (a-Synuclein) with simultaneous stepwise mutagenesis of the protease. The evolved a-Synuclein proteases inventions described herein (e.g., the BoNT / A proteases) have specificity to hydrolyze the new target protein sequences in a sequence specific manner.

[0025] There are many advantages associated with the inventions described herein. For example, the described methods allow changing BoNT / A specificity stepwise to target substrates that bear no sequence homology to its natural substrate SNAP25. For example, the methods can be used to generate enzymes that can degrade other intrinsically disordered proteins and proteins that contain unstructured regions that are involved in human diseases. BoNT / A has an extended substrate binging site affording the protease very high specificity relative to other proteases. BoNT / A has a mechanism for cell entry allowing the protease to efficiently target intracellular proteins. Also, proteases can be targeted in a cell-specific manner by changing the heavy chain specificity, through conjugation to another toxin heavy chain, as an antibody conjugate, or by delivery with a viral vector or lipid nanoparticle.BoNT / A protease is catalytic, allowing much lower dosing than for example using stoichiometric reagents such as antibodies.II. Definitions

[0026] Unless otherwise indicated, the present invention can be practiced in accordance with the techniques exemplified herein and other standard procedures well known and routinely practiced in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (eds.), Oxford University Press (revised ed., 2000); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3PrdP ed., 2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4PthP ed., 2000). Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information. In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0027] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to compound, comprising "an extracellular domain" includes compounds with one or a pluralityof extracellular domains. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0028] As used herein, the term "amino acid" of a peptide refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxy glutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The engineered BoNT proteases of the invention encompass derivative or analogs which have been modified with non-naturally coding amino acids.

[0029] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0030] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0031] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0032] The term "conservatively modified variant" or “conservatively substituted variant” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0033] For polypeptide or protein sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0034] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0035] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c, 1970; by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI); or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

[0036] Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0037] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence for an engineered BoNT / A protease in the expression vectors of the invention. Due to the operable linking, the promoter or enhancer sequence can stimulate or modulate the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0038] The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-humanprimates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, subjects as used in the present invention refer to human.

[0039] The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” are used interchangeably herein.

[0040] Administration "in conjunction with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0041] As used herein, "treating," “treatment” or "ameliorating" refers to (i) preventing a pathologic condition (e.g., a disorder such as a synucleinopathy) from occurring (e.g., prophylaxis); (ii) inhibiting the pathologic condition or arresting its development; and (iii) relieving symptoms associated with the pathologic condition. Thus, "treatment" includes the administration of the therapeutic compounds or compositions described herein to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease described herein, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. " Treatment" further refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder described herein, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. Detailed procedures for the treatment or amelioration of a disorder or symptoms thereof can be based on objective or subjective parameters, including the results of an examination by a physician.

[0042] As used herein, the term "variant" refers to a molecule (e.g., a polypeptide or polynucleotide) that contains a sequence that is substantially identical to the sequence of a reference molecule. For example, the reference molecule can be a wildtype BoNT / A protein or a polynucleotide encoding the protein. In some embodiments, the variant can share at least 50%, at least 70%, at least 80%, at least 90, at least 95% or more sequence identity with the reference molecule. In some other embodiments, the variant differs from the referencemolecule by having one or more conservative amino acid substitutions. In some other embodiments, a variant of a reference molecule has altered amino acid sequences (e.g., with one or more conservative amino acid substitutions) but substantially retains the biological activity of the reference molecule. Conservative amino acid substitutions are well known to one skilled in the art.

[0043] The term “vector” is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).

[0044] Phage-assisted continuous evolution (PACE) is a phage-based technique for the automated directed evolution of proteins. It relies on relating the desired activity of a target protein with the fitness of an infectious bacteriophage which carries the protein's corresponding gene. Proteins with greater desired activity hence confer greater infectivity to their carrier phage. More infectious phage propagates more effectively, selecting for advantageous mutations. Genetic variation is generated using error-prone polymerases on the phage vectors, and over time the protein accumulates beneficial mutations. This technique is notable for performing hundreds of rounds of selection with minimal human intervention. PACE has been used to evolve proteases to cut different peptides. In these systems, the desired protease cut site is used to link a T7 RNA polymerase and a T7 lysozyme. The T7 lysozyme prevents the T7 polymerase from transcribing gill. When the peptide linker is cleaved, the T7 polymerase is activated, allowing for the transcription of the pill gene.

[0045] As used herein, a library of protease variants or a combinatorial library of protease variants refers to a collection of protease mutants or variants having distinct and diverse amino acid mutations in its sequence with respect to the sequence of a startingtemplate or wild type protease. The mutations represented in the collection can be across the sequence of the starting protease or can be in a specified region or regions of the starting protease. The mutations can be made randomly or can be targeted mutations designed empirically or rationally based on structural or functional information.

[0046] As used herein, a "substrate" is a molecule that binds to the active site of a protease and is cleaved by the protease. After cleavage, part of the substrate may remain bound to the protease. As used herein, desired specificity with reference to a substrate protein or target protein refers to cleavage specificity for a predetermined or preselected substrate sequence in the target protein, e.g., a-Synuclein.

[0047] As used herein, a target protein or substrate protein refers to a protein that is specifically recognized and cleaved at a substrate sequence or substrate recognition site by a protease. Cleavage, e.g., amide bond hydrolysis, typically occurs at a cut site between residues -1 and +1 (aka Pi and Pi'). See, e.g., Schechter and Berger (1968) Biochem.Biophys, Res. Commun. 27: 157-62). For wildtype BoNT / A, the substrate protein is SNAP25, which is cleaved by BoNT / A at residues Q197 / R198. Minimally, a target substrate includes the amino acids that make up the substrate sequence or cleavage sequence, which encompasses the two cut site residues (residues -1 and +1) and several residues flanking the cut site. For example, the substrate sequence can include the two cut site residues, one or more of the +2, +3, +4, +5, and +6 residues located immediately C-terminal to the cut site, and / or one or more of the -2, -3, -4, -5, -6, and -7 residues located immediately N-terminal to the cut site. In some embodiments, the substrate sequence can further include one or more of the -8, -9, -10, and -11 residues located N-terminal to the cut site as exemplified herein.Optionally, a target substrate or substrate sequence can include a peptide containing the cleavage sequence and any other amino acids. A full-length protein, allelic variant, isoform, or any portion thereof, containing a cleavage sequence recognized by a protease, is a target substrate for that protease. Additionally, a target substrate includes a peptide or protein containing an additional moiety that does not affect cleavage of the substrate by a protease.

[0048] The term "therapeutically effective amount" or "dosage" as used herein refers to an amount of an engineered BoNT / A protease that is capable of alleviating a symptom of a given synucleinopathy disease (or pathology) and, preferably, is capable of partially or completely normalizing the physiological response, such as astrocytic network coupling, of a subject suffering from the given synucleinopathy disease (or pathology).Optionally, the therapeutically effective amount is determined based on various factors including, but not limited to, the potency of the engineered enzyme, the age and constitution of the subject, the body weight of the subject, pharmacokinetic characteristics of the compound, and the route of administration, by a person skilled in the art.III. Evolving BoNT / A for targeted substrate specificities

[0049] The invention provides libraries of BoNT / A variant enzymes that can be used to evolve its specific proteolytic activity in a stepwise manner towards a selected cleavage site in a target protein of interest. Related methods for carrying out the stepwise evolution are also provided in the invention. The methods for evolving BoNT / A described herein are intended to generate BoNT / A variants that are capable of specifically cleaving a selected substrate sequence located in a target protein that bears little or no sequence homology to the wildtype substrate sequence in SNAP25 that is recognized and digested by the wildtype BoNT / A enzyme. Typically, the substrate sequence (or cleavage sequence) in the target protein or substrate protein recognized by an evolved BoNT / A enzyme contains a cleavage site (also referred to as “cut site” herein) and several residues flanking the cut site. In some embodiments, the selected substrate sequence in a target protein can include the two cleavage site residues (-1 and +1 residues), one or more of the +2, +3, +4, +5, and +6 residues located immediately C-terminal to the cut site, and / or one or more of the -2, -3, -4, -5, -6, and -7 residues located immediately N-terminal to the cut site. In some embodiments, the substrate sequence to be recognized and cleaved by the evolved BoNT / A variant includes all residues from the -7 position to the +6 position flanking the cleavage site in the target protein. In some methods, the selected substrate sequence can further include one or more of the -8, -9, -10, and -11 residues located N-terminal to the cut site as exemplified herein.

[0050] The full-length sequence of human SNAP25 (SEQ ID NO: 16) is based on UniPro ID and Accession No. P60880. BoNT / A cleavage site residues (- 1 / +1) in the protein sequence are Q197 / R198. C-terminal sequence of SNAP25 encompassing residues 177-206 of the protein, i.e., from residue -21 to residue +9, is shown in SEQ ID NO: 17. Screening BoNT / A variants with evolved substrate specificity can employ a substrate (a peptide or polypeptide) that contains this SNAP25 fragment sequence at the beginning point.

[0051] Full length sequence of human SNAP25 (SEQ ID NO: 16):MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGE QLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQD GVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALD MGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG

[0052] SNAP25 C-terminal sequence flanking BoNT / A cut site (SEQ ID NO: 17): QIDRIMEKADSNKTRIDEANQ-RATKMLGSG

[0053] As described herein, the enzyme libraries contain BoNT / A variants that contain randomized mutations in specific regions that are involved in BoNT / A interaction with its native substrate, SNAP25. Utilizing an iterative evolution strategy, BoNT / A variants with randomized key residues in those regions are screened against substrate sequences that have been changed, also in a stepwise manner, from the wild-type target (SNAP25) to the target sequence at defined positions around the intended cleavage site. Specifically, 7 libraries site saturation libraries of BoNT / A have been designed, here referred to as SSM1 (containing randomized residues R363X, L367X, N368X, F369X, D370X, and A372X), SSM2 (containing randomized F243X, K244X, V245X, G255X, L256X, and E257X), SSM3 (containing randomized C165X, K166X, S167X, F168X, Q184X, and R231X), and SSM4a (containing randomized K23X, I24X, P25X, A27X, G28X, and Q29X), SSM4b (containing randomized G169X, H170X, E171X, V172X, N174X, and T176X), SSM4c (containing randomized D13 IX, T132X, C134X, N136X, Y144X, and S146X), and SSM4d (containing randomized V304X, G305X, T306X, T307X, A308X, and Y312X). “X” in these randomized positions refers to any amino acid residue.

[0054] Each of these libraries is screened to affect changes in BoNT / A cleavage specificity for defined residues in the wild-type SNAP25 target sequence to the desired new recognition sequence in a target protein. The primary screening involves screening of libraries SSM1, SSM2 and SSM3. Specifically, the SSM1 library is screened for the identification of BoNT / A mutants for which the sequence specificity of residues at -1, +1, +2, +3, and +6, relative to the original cut site in SNAP25 has been changed. The substrate sequence to be used in the screening of the SSM1 library will contain the original SNAP25 substrate sequence except for residues -1, +1, +2, +3, and +6, which have been changes to the corresponding residues in the selected cleavage site in the target protein. For example, the substrate can be the peptide shown in SEQ ID NO: 17, except that residues Q197, R198, Al 99, T200, and L203 are respectively changed to the corresponding residues at the chosencleavage site in the target protein. The SSM2 library is screened for the identification of BoNT / A mutants for which the sequence specificity of residues at +4 and +5 relative to the original cut site in SNAP25 has been changed. The SSM3 library is screened for the identification of BoNT / A mutants for which the sequence specificity of residues at -7, -6, -5, -4, -3, and -2 relative to the original cut site in SNAP25 has been changed. Stepwise screening of these 3 primary libraries of BoNT / A variants against the corresponding substrate sequences with the noted residues changed around the cleavage site can lead to identification of evolved BoNT / A variants that specifically cleave a new substrate sequence containing the changed residues from positions -7 to +6.

[0055] It is important to note that the process of changing the cleavage site from the original SNAP25 cleavage site to the desired target sequence using these 3 primary BoNT / A variant libraries (i.e., SSM1, SSM2, and SSM3) is carried out in an iterative manner in no defined order. Both BoNT / A mutations identified in any step of this process, as well as changes to the recognition sequence that have been carried out are carried over to screening of consecutive libraries. For stepwise evolution of the BoNT / A protease, the 3 rounds of primary screening of the stepwise evolution of BoNT / A variants can start with either library SSM1, library SSM2 or library SSM3 in the first round, followed by any of the other two libraries in the next round, and the remaining library in the third round. The actual mutations present in a BoNT / A variant identified from one round of screening will be carried over to the library of BoNT / A variants to be used in the subsequence round of screening. In other words, in addition to the specific residues noted above for randomization, each of the BoNT / A variant libraries of the invention can also contain changes of residues that are present in the BoNT / A variants identified from the previous rounds of screening. The same rationale also applies to sequential screening involving secondary libraries SSM4a-SSM4d described below. For stepwise evolution of the substrate sequence, amino acid changes made to the employed substrate sequence for one round of screening, relative to that of the wildtype SNAP25 cleavage site (or that of the substrate sequence employed in the previous round of screening) are also present in the substrate sequence to be used for the next round of screening.

[0056] To further illustrate, if the SSM1 library is screened with a corresponding substrate sequence in the first round, the mutations in the identified BoNT / A variant are also introduced into a second enzyme library (e.g., SSM2) that will be screened in the next round.In this case, the library of BoNT / A variants to be used in the next round will contain randomized mutations at the above-noted residues for the SSM2 library, plus the actual mutations in the identified BoNT / A variant from the first round of screening at one or more of the above-noted randomization positions for the SSM1 library. For the substrate sequence in the same illustrated example, the employed substrate sequence for screening with library SSM1 in the first round has residues at positions -1, +1, +2, +3, and +6 changed to the corresponding residues at the selected cleavage site in the target protein. Therefore, in the next round of screening with the SSM2 library, the employed substrate sequence should contain the same changed residues at positions -1, +1, +2, +3, and +6, plus residues at positions +4 and +5 changed to corresponding residues at the selected cleavage site in the target protein. By using this iterative and simultaneous stepwise evolution of both the enzyme and the substrate, BoNT / A variants can be identified that specifically cleave a selected substrate sequence in the target protein that bears little or no resemblance to the original cleavage site in SNAP25 substrate. For screening with each of the secondary libraries described below, the employed substrate sequence will additionally contain residues at the -8, -9, -10, and -11 positions respectively changed to corresponding residues around the selected cleavage site in the target protein

[0057] For fine tuning of substrate specificity, evolved BoNT / A variants that specifically cleave a new substrate sequence containing the chosen residues from positions -7 to +6 from the target protein can be further screened in secondary screening to identify BoNT / A mutants with specificity for a substrate sequence that contains additional residues around the cleavage site. As exemplified herein, these additional residues can include residues at the -8, -9, -10, and -11 positions. BoNT / A variants recognizing a substrate sequence that additionally contains changed residues at these more remote sites of secondary importance can be screened with one or more BoNT / A variant libraries selected from the SSM4a, SSM4b, SSM4c, and SSM4d libraries. If more than one of these 4 libraries are employed in the secondary screening, the different libraries can be screened sequentially, similarly to the screening of the SSM1, SSM2 and SSM3 libraries described above.Alternatively, these secondary libraries can be screened in parallel using the evolved BoNT / A variant identified from the last round of the primary screening of the SSM1-SSM3 libraries. Once the in parallel secondary screenings are completed, additional mutations in theidentified BoNT / variants from the different secondary libraries are combined via "gene shuffling" into one final BoNT / A variant.

[0058] The selected cleavage site for evolving the BoNT / A enzyme may be at any location of the target protein. In some preferred embodiments, the selected cleavage site in located in an intrinsically disordered region (IDR) of the protein. As described in more detail below, IDRs are regions in proteins that lack a stable secondary or tertiary structure. They are highly abundant in nature and their functional repertoire complements the functions of ordered proteins. Proteins that contain IDRs, called intrinsically disordered proteins (IDPs), are involved in regulation, signaling, and control, where binding to multiple partners and high-specificity / low-affinity interactions play a crucial role.

[0059] Stepwise evolution of the BoNT / A enzyme as described herein can be performed with any suitable protocols known in the art for screening proteases. To perform each round of screening of the stepwise evolution method, the BoNT / A variant library can be co-expressed in a cell with a selection system. As exemplified herein, the selection system expresses a sfGFP reporter under the control of a T7 promoter, as well as a fusion protein that links T7 RNA polymerase to an inhibitor (T7 lysozyme) via the substrate sequence. Cleavage at the substrate sequence by a BoNT / A variant will relieve T7 RNA polymerase from the inhibition, which can then activate expression of the reporter molecule. The detectable signal from the reporter molecule (e.g., green fluorescence) in a cell can then be correlated with the proteolytic activity of the BoNT / A variant expressed in the same cell.IV. Intrinsically disordered proteins for targeting with engineered proteases

[0060] The invention provides novel methods and agents for specifically degrading non-SNARE intrinsically disordered proteins of clinical relevance. Intrinsically disordered proteins (IDPs) contain regions that lack a stable secondary or tertiary structure, called 'Intrinsically Disordered Regions' (IDRs). They are highly abundant in nature and their functional repertoire complements the functions of ordered proteins. IDPs are involved in regulation, signaling, and control, where binding to multiple partners and high-specificity / low-affinity interactions play a crucial role. Functions of IDPs are tuned via alternative splicing and posttranslational modifications. Intrinsic disorder is a unique structural feature that enables IDPs to participate in both one-to-many and many-to-one signaling. Numerous IDPs are associated with human diseases, including cancer,1cardiovascular disease, amyloidoses, neurodegenerative diseases, and diabetes, including Alzheimer’s and Parkinson’s diseases. Overall, intriguing interconnections among intrinsic disorder, cell signaling, and human diseases suggest that protein conformational diseases may result not only from protein misfolding, but also from mis-identification, miss-signaling, and unnatural or nonnative folding. IDPs, such as a-Synuclein, tau protein, p53, and BRCA1, are attractive targets for drugs modulating protein-protein interactions.

[0061] As demonstrated herein, the methods of the invention allow changing BoNT / A specificity stepwise to degrade other none-SNARE IDPs that are involved in human diseases. A notable example of such none-SNARE IDPs is alpha-synuclein (a-Synuclein or aSyn). a-Synuclein is a protein that, in humans, is encoded by the SNCA gene. It is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release. It is abundant in the brain, while smaller amounts are found in the heart, muscle and other tissues. In the brain, a-Synuclein is found mainly in the axon terminals of presynaptic neurons. Within these terminals, a-Synuclein interacts with phospholipids andproteins. Presynaptic terminals release chemical messengers, called neurotransmitters, from compartments known as synaptic vesicles. The release of neurotransmitters relays signals between neurons and is critical for normal brain function.

[0062] a-Synuclein is a presynaptic neuronal protein that is linked genetically and neuropathologically to the development of synucleinopathies (e.g., Parkinson's disease). Using Parkinson's disease (PD) as an example, a-Synuclein may contribute to the pathogenesis of the disorder in a number of ways, but it is generally thought that its aberrant soluble oligomeric conformations, termed protofibrils, are the toxic species that mediate disruption of cellular homeostasis and neuronal death, through effects on various intracellular targets, including synaptic function. Furthermore, secreted a-Synuclein may exert deleterious effects on neighboring cells, including seeding of aggregation, thus possibly contributing to disease propagation. Although the extent to which a-Synuclein is involved in all cases of PD is not clear, targeting the toxic functions conferred by this protein when it is dysregulated may lead to novel therapeutic strategies not only in PD, but also in other neurodegenerative conditions, termed synucleinopathies.

[0063] The human a-Synuclein protein is made of 140 amino acids. An a- Synuclein fragment, known as the non-amyloid beta (non-Abeta) component (NAC) of Alzheimer's disease amyloid, originally found in an amyloid-enriched fraction, was shownto be a fragment of its precursor protein, NACP. It was later determined that NACP is the human homologue of synuclein in electric rays, genus Torpedo. Therefore, NACP is now referred to as human a-Synuclein.

[0064] In addition to a-Syn, other IDPs involved in the development of human diseases are also suitable for targeted proteolysis with engineered proteases that can be generated with the methods described herein. These include, e.g., c-myc (18, 19), p53 (20), and EWS-FLI1 (21) in oncology and P-amyloid, TDP43(22), and TMEM106B(23) in neurodegenerative diseases.V. Evolved botulinum toxin proteases with substrate specificities for IDPs

[0065] For targeted degradation of IDPs, the invention provides novel engineered Botulinum neurotoxin (BoNT) proteases with catalytic specificities for these non-natural substrates. This is achieved by stepwise evolution of a BoNT’s substrate specificity from its natural protein substrate towards an IDP. BoNTs are neurotoxic proteins produced by the bacterium Clostridium botulinum and related species. They prevent the release of the neurotransmitter acetylcholine from axon endings at the neuromuscular junction, thus causing flaccid paralysis. Botulinum toxins are acetylcholine release inhibitors and neuromuscular blocking agents. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was reported to produce BoNT serotype B (BoNT / B) and a novel BoNT, designated as serotype BoNT / H. Types A and B BoNTs are capable of causing disease in humans, and are also used commercially and medically. Types C-G are less common; types E and F can cause disease in humans, while the other types cause disease in other animals. Botulinum neurotoxin serotype A (BoNT / A) comprises eight subtypes, A1-A8. Among the BoNT / A subtypes, BoNT / Al possesses high potency and a long duration of action, BoNT / A4 shows low potency, and BoNT / A3 has a short duration of action.

[0066] BoNTs are produced as 150-kDa single-chain proteins that are post-translationally cleaved into disulfide-linked di-chains by a host or endogenous proteases that are classic AB-type bacterial toxins. The BoNT chains consist of an N-terminal ~50 kDa catalytic A subunit or light chain (LC), and a C-terminal -100 kDa host-receptor binding / translocation B subunit or heavy chain (HC). BoNT LCs are zinc-metalloproteases containing an (H-E-X-X-H) motif for zinc coordination and cleave neuronal Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors (SNARE) at unique sites. BoNT HC possesses two domains, the translocation domain (HN) and the receptor binding domain (Hc). HNis responsible for binding specifically to presynaptic nerve terminals, and Hcis responsible for mediating translocation of the light chain into the cell cytoplasm as the vacuole acidifies.

[0067] Botulinum toxins (e.g., BoNT / A) exert their effect by cleaving key proteins required for nerve activation. First, the toxin binds specifically to presynaptic surface of neurons that use the neurotransmitter acetylcholine. Once bound to the nerve terminal, the neuron takes up the toxin into a vesicle by receptor-mediated endocytosis. As the vesicle moves farther into the cell, it acidifies, activating a portion of the toxin that triggers it to push across the vesicle membrane and into the cell cytoplasm. Botulinum neurotoxins recognize distinct classes of receptors simultaneously (gangliosides, synaptotagmin and SV2). Using BoNT / A as an example, once inside the cytoplasm the light chain (BoNT / A LC) cleaves the substrate SyNaptosomal Associated Protein of 25 kDa (SNAP-25), a protein which mediates the fusion of vesicles with the target membrane. As a result, the acetylcholine vesicles cannot bind to the intracellular cell membrane, preventing the cell from releasing vesicles of neurotransmitter. This stops nerve signaling, leading to flaccid paralysis.

[0068] As detailed in the Examples herein, the engineered BoNT / A proteases of the invention are obtained via stepwise mutagenesis of a wildtype BoNT / A light chain sequence (e.g., SEQ ID NO: 14) and simultaneous stepwise evolution of its natural substrate sequence towards the desired target substrate sequence (e.g., a-Synuclein). Preferably, the starting wildtype BoNT / A sequence is of subtype Al. As specific exemplification, the employed wildtype BoNT / A sequence is a subtype Al sequence under UniProt ID Q7B8V4. The full-length amino acid sequence of this protein is provided under Accession No.WP_011948511.1 (SEQ ID NO: 1). It is noted that the same identical BoNT / A sequence is found in several type Al Clostridium botulinum strains, including Hall A-hyper (Accession AAM75961.1), Allergan-Hall A (Accession AAQ06331.1), Hall A (Accession ABD65472.1 ), ATCC 25763 (Accession ABM73968.1), 32A (Accession ACS52165.1), BS-A (Accession ACS52166.1), Cam2A (Accession ACS52167.1), 69A (Accession AC S52163.1), 73A (Accession ACS52164.1), CDC 21547 (Accession ACS52168.1 ), CDC220726 (Accession ACS35618.1), CDC220727 (Accession ACS35619.1), CDC36923 (Accession ACS35616.1), CDC36924 (Accession ACS35617.1), CDC36955 (Accession ACS35614.1), CDC36956(Accession ACS35615.1), Nan 2006 (Accession AFV92905.1), A Hall (Accession AIR72304.1), and Hall (Accession AIQ77655.1). As described herein, stepwise evolution of the light chain sequence (SEQ ID NO: 14) of this BoNT / A protein, which contains residues 1-424 of the full-length wildtype sequence, resulted in the generation of a reprogrammed BoNT / A protease. This engineered BoNT / A protease, “Proteases 5” as termed herein (SEQ ID NO: 15), is capable of specifically degrading a-Synuclein. Relative to the wildtype light chain sequence (SEQ ID NO: 14), this exemplified engineered BoNT / A protease (SEQ ID NO: 15) contains a number of amino acid substitutions in several sequence motifs throughout of the BoNT / A light chain sequence. Specifically, the substitutions include K23I, I24L, A27N, G28M, Q29D, Q67K, T109M, D131S, N136V, Y144Q, S146M, K166R, F168R, G169E, H170F, E171S, V172M, N174P, Q184C, F196I, F213I, T220L, R231F, V242A, F243I, K244W, V245F, N248D, S254R, G255W, L256M, E257K, N280D, F282L, R363R, N368L, F369L, D370P, and A372L.Full length wildtype BoNT / A sequence under UniProt ID Q7B8V4 (SEQ ID NO: 1):MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKF ATDPAVT LAHELIHAGH RLYGIAINPN RVFKVNTNAY YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLA AN FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSKTK SLDKGYNKAL NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIQ QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG KKYELDKYTM FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS KRNEKWDEVY K YIVTNWLAK VNTQIDLIRK KMKEALENQ A EATKAIINYQ YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP KYFNSISLNN EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK YSQMINISDY INRWIFVTIT NNRLNNSKIY INGRLIDQKP ISNLGNIHAS NNIMFKLDGC RDTHRYIWIK YFNLFDKELN EKEIKDLYDN QSNSGILKDF WGDYLQYDKP Y YM LNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQAGVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPLWildtype light chain sequence of BoNT / A under UniProt ID Q7B8V4 (SEP ID NO: 14): MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTE IYTEDNFVI< FFI< VLNRI< TYLNFDI< AVFI< INIVPI< VNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLKNFTGLFEa-Synuclein-degrading engineered BoNT / A enzyme (“Protease 5”; SEQ ID NO: 15) (residues mutated from wildtype sequence are italicized)MPFVNKQFNY KDPVNGVDIA YI / ZPNA DM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKAVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLA / S IVRGIPFWGG STIDTELKVI A’TNCI EVIQP DGS0RMEELN LVIIGPSADI IQFEC / ? S / ? EF LPLTRNGY GSTCYIRFSP DFTFG7EESL EVDTNPLLGA GK7ATDPAVZ LAHELIHAGH FLYGI AINPN R.1 / I17'N I7)A Y YEMEJWAVSF EELRTFGGHD AKFIDSLQED EZRLYYYNKF KDIASTLNKA KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV LNRKTYLZZP KLVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT GLFE

[0069] Other than the exemplified light chain (SEQ ID NO: 14) of botulinum neurotoxin A (subtype Al; Accession WP 011948511; SEQ IDNO:1), many other botulinum neurotoxin sequences can also be employed to generate an engineered protease that can specifically degrade a-Synuclein. In particular, different BoNT / A subtype Al sequences known in the art can be readily employed to produce the a-Synuclein-degrading proteases of the invention. Examples of such sequences include, Accession Nos. 9ARL A (SEQ ID NO:2), 3BTA A (SEQ ID NO:3), 6XCD A (SEQ ID NO:4), 3QW8 A (SEQ ID NO:5), 6XCC_A (SEQ ID NO:6), 7N18 B (SEQ ID NO:7), 3DS9 A (SEQ ID NO:8), EDT83034.1 (SEQ ID NO:9), EKN40638 (SEQ ID NO: 10), EPS48830 (SEQ ID NO: 11), and UOJ22069.1 (SEQ ID NO: 12). The light chain portions of these BoNT / A subtype Al sequences are all substantially identical to the wildtype light chain sequence of theexemplified BoNT / A subtype Al enzyme (SEQ ID NO: 14), e.g., at least 95% or even 99% identical. Importantly, the residues mutated in the engineered enzyme exemplified herein (SEQ ID NO: 15) are essentially all conserved in these additional wildtype BoNT / A subtype Al sequences. Thus, by introducing the same mutations into these wildtype subtype Al sequences, engineered BoNT / A proteases that specifically degrade a-Synuclein can be similarly generated. Further, the reference or background BoNT / A sequence into which the mutations are to be introduced also include variant sequences that are conservatively modified variants of the exemplified wildtype BoNT / A sequences (e.g., SEQ ID NOs:l-12), as well as variant sequences that are substantially identical (e.g., at least 95% or at least 99% identical) to one of these exemplified sequences.

[0070] In addition to subtype Al BoNT / A enzymes, various other subtype sequences of BoNT / A known in the art may also be employed in the practice of the invention. Sequence accession numbers of representative strains of these subtype BoNT / A sequences include, e.g., CAL82360 (subtype Al, strain ATCC 3502), CAA51824 (subtype A2, strain Kyoto-F), ACA57525 (subtype A3, strain Loch Maree), ACQ51417 (subtype A4, strain Ba657), ACG50065 (subtype A5, strain H04402065), ACW83608 (subtype A6, strain CDC 41370), AFV13854 (subtype A7, strain 2008-148), and AJA05787 (subtype A8, strain Chemnitz). It is known that amino acid differences between BoNT / A subtype Al and the other BoNT / A subtypes are about 10.1% (Subtype A2), 15.4% (Subtype A3), 10.6% (Subtype A4), 2.9% (Subtype A5), 4.3% (Subtype A6), 6.2% (Subtype A7), and 6.7% (Subtype A8), respectively. See, e.g., Peck et al., Toxins 2017, 9(1), 38. With such high degrees of sequence homology, residues corresponding to the residues mutated in the engineered enzyme exemplified herein (SEQ ID NO: 15) are also mostly conserved in the other BoNT / A subtype sequences. Such residues in the other BoNT / A subtype sequences can be readily identified via sequence alignment. This can be illustrated with ACA57525 (SEQ ID NO: 13) as an example, which is the representative BoNT / A subtype A3 sequence. Among the other BoNT / A subtype sequences, subtype A3 has the lowest sequence homology with subtype Al. Nevertheless, a sequence alignment of this subtype A3 sequence (SEQ ID NO: 13) with the exemplified subtype Al sequence (SEQ ID NO: 1) indicates that most of the residues mutated in the evolved BoNT / A enzyme (SEQ ID NO: 15) are conserved in this A3 subtype sequence. This suggests that introducing identical or similar amino acid substitutionsat the same positions in the other subtypes of BoNT / A sequences can lead to engineered BoNT proteases that are similarly capable of degrading a-Synuclein.

[0071] Examples of other suitable BoNT / A sequences: 11 BoNT / A subtype Al and 1 BoNT / A subtype A3 sequences:9ARL A (SEQ ID NO:2)MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKFATDPAVT LAHQLIHAGH RLYGIAINPN RVFKVNTNAY YEMSGLEVSF EELRTF GGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV LNAKTFLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSKTK SLDKGYNKAL NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIQ QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG KKYELDKYTM FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS KRNEKWDEVY K YIVTNWLAK VNTQIDLIRK KMKEALENQ A EATKAIINYQ YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP KYFNSISLNN EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK YSQMINISDY INRWIFVTIT NNRLNNSKIY INGRLIDQKP ISNLGNIHAS NNIMFKLDGC RDTHRYIWIK YFNLFDKELN EKEIKDLYDN QSNSGILKDF WGDYLQYDKP Y YM LNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL3BTA A (SEQ ID NO:3) PFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDL NPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPF WGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTR NGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGI AINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKD IASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY TEDNFVI< FFI< VLNRI< TYLNFDI< AVFI< INIVPI< VNYTIYDGFNLRNTNLAANFNGQNT EINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLF FSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRV YTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPAL NIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEE EKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASL KD ALLKYI YDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNII NTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYN SMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIK QRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIM FKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKP YYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYA SGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVV MKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTL GCSWEFIPVDDGWGERPL3QW8 A (SEQ ID NO:4) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTE IYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLKNFTGLFEHHHHHH6XCC_A (SEQ ID NO: 5) HHHHHHSSGLVPRGSHMQFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHN KIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI YSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDP AVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFI DSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKF SVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYD GFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFE4ZJX A (SEQ ID NO: 6) MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTElYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLKNFTGLFELEHHHHHH3DS9 A (SEQ ID NO:7) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVI< FFI< VLNRI< TYLNFDI< AVFI< INIVPI< VNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLK7N18_B (SEQ IDN0:8) MGSSHHHHHHSSGLVPRGSHMEFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAF KIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTK LFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVII GPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKF ATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHD AKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDT SGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNY TIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEF EDT83034.1 (SEQ ID N0:9) MQFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTE IYTEDNFVI< FFI< VLNRI< TYLNFDI< AVFI< INIVPI< VNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWD LFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSS DIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPS RVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGP ALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYT EEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDA SLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIK NIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIV YNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQ EIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASN NIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYD KPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKK YASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQV VVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSR TLGCSWEFIPVDDGWGERPL EKN40638 (SEQ ID NO: 10) MQFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTE IYTEDNFVI< FFI< VLNRI< TYLNFDI< AVFI< INIVPI< VNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWD LFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSS DIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGP ALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYT EEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDA SLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIK NIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIV YNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQ EIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASN NIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYD KPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKK YASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQV VVM EPS48830 (SEQ ID NO: 11) MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTElYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWD LFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSS DIIGQLELMPNIERFPNGEKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPS RVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGP ALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYT EEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDA SLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIK NIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIV YNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQ EIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASN NIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYD KPYYMLNLYDPNKYIDVNNVGIRGYMYLKGPRGNVMTTNIYLNSSLYMGTKFIIKK YASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQV VVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSR TLGCSWEFIPVDDGWGERPL UOJ22069.1 (SEQ ID NO: 12) MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGI PFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLY GIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKF KDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTElYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQ NTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWD LFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSS DIIGQLELMPNIERFPNGEKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGP ALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYT EEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDA SLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIK NIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIV YNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQ EIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASN NIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYD KPYYMLNLYDPNKYIDVNNVGIRGYMYLKGPRGNVMTTNIYLNSSLYMGTKFIIKK YASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQV VVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKEVASNWYNRQIERSSR TLGCSWEFIPVDDGWGERPL ACA57525 (SUBTYPE A3) (SEQ ID NO: 13):MPFVNKQFNYRDPVNGVDIAYIKIPNAGQMQPVKAFKIHEGVWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVIKLFDRIYSTGLGRMLLSFIVKGI PFWGGSTIDTELKVIDTNCINVIEPGGSYRSEELNLVITGPSADIIQFECKSFGHDVFNL TRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGTFATDPAVTLAHELIHAAHRLY GIAINPNRVLKVKTNAYYEMSGLEVSFEELRTFGGNDTNFIDSLWQKKFSRDAYDNL QNIARILNEAKTIVGTTTPLQYMKNIFIRKYFLSEDASGKISVNKAAFKEFYRVLTRGF TELEFVNPFKVINRKTYLNFDKAVFRINIVPDENYTINEGFNLEGANSNGQNTEINSRN FTRLKNFTGLFEFYKLLCVRGIIPFKTKSLDEGYNKALNDLCIKVNNWDLFFSPSEDN FTNDLDKVEEITADTNIEAAEENIS SDLIQQ YYLTFDFDNEPENISIENLS SDIIGQLEPM PNIERFPNGKKYELDKYTMFHYLRAQEFEHGDSRIILTNSAEEALLKPNVAYTFFSSK YVKKINKAVEAVIFLSWAEELVYDFTDETNEVTTMDKIADITIIVPYIGPALNIGNMVS KGEFVEAILFTGVVALLEFIPEYSLPVFGTFAIVSYIANKVLTVQTINNALSKRNEKWD EVYI< YTVTNWLAI< VNTQIDLIREI< MI< I< ALENQAEATRAIINYQYNQYTEEEI< NNIN FNIDDLSSKLNRSINRAMININKFLDQCSVSYLMNSMIPYAVKRLKDFDASVRDVLLK YIYDNRGTLILQVDRLKDEVNNTLSADIPFQLSKYVNDKKLLSTFTEYIKNIVNTSILSI VYKKDDLIDLSRYGAKINIGDRVYYDSIDKNQIKLINLESSTIEVILKNAIVYNSMYEN FSTSFWIKIPKYFSKINLNNEYTIINCIENNSGWKVSLNYGEIIWTLQDNKQNIQRVVF KYSQMVNISDYINRWMFVTITNNRLTKSKIYINGRLIDQKPISNLGNIHASNKIMFKLD GCRDPRRYIMIKYFNLFDKELNEKEIKDLYDSQSNPGILKDFWGNYLQYDKPYYML NLFDPNKYVDVNNIGIRGYMYLKGPRGSVMTTNIYLNSTLYMGTKFIIKKYASGNED NIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKD DQGIRNKCKMNLQDNNGNDIGFVGFHLYDNIAKLVASNRQVGKASRTFGCSWEFIP VDDGWGESSL

[0072] Beyond engineered proteases targeting a-Synuclein, the strategy described herein, including the described libraries of BoNT / A variants, can also be employed and adapted as necessary to evolve BoNT / A proteases to generate engineered proteases that specifically degrade other non-SNARE proteins. In particular, engineered BoNT / A proteases can be obtained via similar stepwise mutagenesis for degrading other intrinsically disordered proteins (IDPs) that are involved in a human diseases. The level of homology between SNAP25 and a-Synuclein can be found in virtually any IDPs, highlighting the adaptability ofthe method described herein to other IDP targets. In various embodiments, engineered BoNT / A proteases can be generated that specifically degrade other IDPs that include, e.g., c-myc, p53, EWS-FLI1, P-amyloid, TDP43 and TMEM106B.

[0073] To ensure proper targeting and cell entry, the engineered BoNT light chain protease can be conjugated to a targeting moiety or otherwise modified to provide targeted delivery of the evolved proteolytic activities. In some embodiments, the engineered BoNT / A light chain protease (e.g., Protease 5) can be conjugated with (e.g., via a disulfide bond) to a reprogrammed heavy chain which contains a desired receptor-binding and translocation functionalities. By changing the heavy chain’s receptor binding specificity, the modified BoNT / A protein is able to cross cellular membranes with selective tropism for a specific cell type and proteolyze an intracellular target (e.g., an IDP such as a-Synuclein). In some embodiments, the engineered light chain protease can be conjugated to another toxin heavy chain to provide targeted delivery of the evolved BoNT / A protease activity. In some embodiments, the engineered light chain protease can be conjugated to an antibody so that the evolved protease activity can be delivered via the binding specificity of the conjugated antibody. In still some other embodiments, targeted delivery of the engineered BoNT / A protease can be achieved by specific viral expression vectors (e.g., AAV vectors) or by lipid nanoparticles. These modifications to an engineered BoNT light chain protease can all be carried out in accordance with methods that have been known in the art. See, e.g., Miyashita et al., Science Translational Medicine 13, eaaz4197 (2021); McNutt et al., Science Translational Medicine 13, eabd7789 (2021); Tian et al., Cell Reports 38 (2022); Roh et al., J. Am. Chem. Soc. 145, 10220-10226 (2023); Blum et al., Science 371, 803-810 (2021); Naso et al., BioDrugs 31, 317-334 (2017); Wang et al., Nat Rev Drug Discov 18, 358-378 (2019); and Wang et al., Sig Transduct Target Ther 9, 1-33 (2024).

[0074] In addition to the engineered BoNT / A light chain proteases and derivative proteins (e.g., conjugates with a bacterial toxin heavy chain), related embodiments of the invention include polynucleotide sequences that encode such engineered proteins, expression constructs for producing the engineered proteins, and host cells that harbor the polynucleotides or expression constructs. The polynucleotide sequences of the invention can be any polynucleotide having a nucleotide sequence that encodes the engineered proteins of the invention, including DNA and RNA (e.g., mRNA). The recombinant constructs or expression vectors of the invention harbor a polynucleotide sequence of the invention that encodes an engineered BoNT / A protease or conjugate protein. The recombinant constructs ofthe invention may be obtained by ligating (inserting) the polynucleotide (DNA) of the invention into a suitable vector (e.g., a viral vector such as AAV vector or retroviral vector). The vector for inserting the polynucleotide sequence is not subject to any particular limitation, provided it is capable of replication in an appropriate host. The expression vectors include viral vectors derived from animal viruses such as CMV as exemplified herein, retroviruses, adeno-associated viruses (AAV), vaccinia viruses and insect viruses (e.g., baculoviruses). The expression vectors also include, e.g., bacteriophages, plasmids, cosmids or phagemids. Examples of recombinant bacteriophage or phagemid vectors include that based on a filamentous phage such as Ml 3. Plasmid vectors include those based on plasmids from, e.g., E. coli (e.g., pET21 as exemplified herein, pBR322, pBR325, pUC118 and pUCl 19), plasmids from Bacillus subtilis (e.g., pUBl 10 and pTP5), and plasmids from yeasts (e.g., YEpl3, YEp24 and YCp50).VI. Therapeutic applications

[0075] The engineered proteases described herein, including their derivative proteins (e.g., conjugates with a heavy chain or an antibody), encoding-polynucleotides, expression vectors and liposomes, can be readily employed in the treatment of synucleinopathies by degrading a-Synuclein aggregates. Synucleinopathies include a group of neurodegenerative disorders characterized by fibrillary aggregates of a-Synuclein protein in the cytoplasm of selective populations of neurons and glia. Clinically, they are characterized by a chronic and progressive decline in motor, cognitive, behavioral, and autonomic functions, depending on the distribution of the lesions. Well known examples of synucleinopathies include Parkinson's disease (PD) (including idiopathic and inherited forms of Parkinson's disease) and Diffuse Lewy Body (DLB) disease (also known as Dementia with Lewy Bodies (DLB), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease (CAPD), pure autonomic failure (PAF) and multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy, Striatonigral Degeneration and Shy -Drager Syndrome)).

[0076] Parkinson's Disease (PD) is a synucleinopathy disorder. There are no well-defined diagnostic criteria for PD. However, there are various symptoms and diagnostic tests that can be used in combination to support a PD diagnosis. Making an accurate diagnosis of Parkinson's, particularly in its early stages, is difficult, but a skilled practitioner can come to a reasoned conclusion that the human subject suffers from PD by using clinical assessmenttools. In some embodiments, the PD diagnosis may be based on the presence of at least two of the four main symptoms of PD: shaking or tremor; slowness of movement (called bradykinesia); stiffness or rigidity of the arms, legs or trunk; and trouble with balance and possible falls, also called postural instability. Imaging techniques such as MRI, ultrasound of the brain, PET scans, and / or specific single-photon emission computerized tomography (SPECT) scan to analyze dopamine transport (DaTscan) may also be useful in supporting the diagnosis of PD.

[0077] Dementia with Lewy bodies (DLB) is a type of synucleinopathy that involves progressive dementia that leads to a decline in thinking, reasoning and independent function. Dementia with Lewy bodies is often hard to diagnose because its early symptoms may resemble those of Alzheimer's disease or a psychiatric illness. There are no definitive diagnostic criteria but some core clinical symptoms of DLB are dementia, movement problems / parkinsonism, cognitive fluctuations, visual hallucinations and REM sleep behavior disorder. Some supportive clinical symptoms are extreme sensitivity to antipsychotic medications, falls or fainting, severe problems with involuntary functions (maintaining blood pressure, incontinence, constipation, loss of smell), changes in personality and mood (depression, apathy, anxiety). A diagnosis of Lewy body dementia requires a progressive decline in ability to think, as well as at least two of the following: fluctuating alertness and thinking function, repeated visual hallucinations, Parkinsonian symptoms, and REM sleep behavior disorder.

[0078] Multiple system atrophy (MSA) is the most rapidly progressive of the synucleinopathies. In the present disclosure, the term MSA is meant to refer to all types of MSA such as the multiple system atrophy parkinsonian type (MSA-P) or multiple system atrophy cerebellar type (MSA-C). MSA is sometimes denoted as Olivopontocerebellar Atrophy, progressive autonomic failure with multiple system atrophy, Striatonigral Degeneration or Shy -Drager Syndrome. The present invention provides dosage regimens for the treatment of MSA in all stages of disease progression including prodromal, early, moderate and advanced stages and all stages between. Diagnosing multiple system atrophy (MSA) can be challenging as certain signs and symptoms of MSA, such as muscle rigidity and unsteady gait, also occur with other disorders, such as Parkinson's disease. A proper clinical examination, with various autonomic tests and imaging studies, may assist in determining whether the diagnosis is probable MSA or possible MSA. Examples of clinicalmethods which can be useful in supporting the MSA diagnosis of patients with suspected MSA is structural and functional brain imaging, cardiac sympathetic imaging, cardiovascular autonomic testing, olfactory testing, sleep study, urological evaluation, and dysphagia, cognitive assessments, skin biopsy, retinal biomarkers, blood and / or cerebrospinal fluid biomarkers, and genetic testing. Diagnosis of possible or probable MSA can for example be facilitated by the so-called Gilman criteria (Gilman et al., Neurology. 2008 Aug. 26; 71(9): 670-676).

[0079] Pure autonomic failure (PAF) is a rare neurodegenerative condition that affects the autonomic nervous system. It is caused by an abnormal buildup of a-Synuclein in autonomic nerves. The most common symptom of PAF is orthostatic hypotension, a significant drop in blood pressure when standing up. But PAF often causes other issues as well. With PAF, one might first notice that a dizzy feeling when getting up from a sitting position. Other symptoms may include a loss of bladder control or blurry vision. PAF tends to develop in adults in their 40s to 60s. PAF is also called idiopathic orthostatic hypotension or Bradbury-Eggleston syndrome. The diagnostic process for PAF mainly involves ruling out other conditions that cause orthostatic hypotension. Beyond physical exams and neurological exams, additional tests that can be performed to achieve this purpose are tilt table test (which shows how blood pressure responds to standing up after lying down), quantitative sudomotor axon reflex test or sweat test (QSART which looks at the nerves that control sweating and help diagnose autonomic nervous system conditions, peripheral neuropathies and some types of pain disorders), imaging tests (e.g., cardiac functional imaging and brain MRI scans to rule out other conditions), laboratory tests (having low levels of norepinephrine in blood and urine while lying down with minimal to no increase on standing typically supports a PAF diagnosis), and thermoregulatory sweat test (which examines how much one sweats in a room that has controlled temperature, humidity and airflow capabilities).

[0080] In various embodiments of the invention, the present invention provides therapeutic methods and suitable dosage regimens for treating any of these synucleinopathies in human subjects. In some embodiments, the synucleinopathy to be treated in the subject is Parkinson's disease (PD), including idiopathic and inherited forms of PD). Some other embodiments of the invention are directed to treating subjects afflicted with dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD), Lewy body variant of Alzheimer's disease (LBV), or combined Alzheimer's and Parkinson's disease. In some otherembodiments, the subject to be treated with an engineered BoNT / A protease of the invention has multiple system atrophy (MSA). In still some other embodiments, the subject to be treated suffers from pure autonomic failure (PAF).

[0081] In some embodiments, the subject to be treated is at the prodromal phase of a synucleinopathy or is otherwise at risk of developing a synucleinopathy. The subject can be identifiable by exhibiting one or more clinical markers of prodromal synucleinopathies. Such clinical markers of prodromal synucleinopathies may be selected from REM sleep behavior disorder (RBD) such as isolated RBD (iRBD), dysfunctional olfaction such as hyposmia, abnormal cognitive performance in neuropsychological testing, subtle motor dysfunction or abnormal motor performance assessed by objective testing, abnormal color vision, autonomic dysfunctions (such as constipation, urinary symptoms, erectile dysfunction, orthostatic hypotension), reduced nigrostriatal dopaminergic binding in the putamen and striatum (abnormal DAT-SPECT), Seborrhoeic dermatitis, and a genotype associated with increasing phenoconversion risk such as mutations in glucocerebrosidase (encoded by the GBA gene).

[0082] In some embodiments, the subject at the prodromal phase of a synucleinopathy can be identified by detecting abnormal accumulation or deposition of a-Synuclein in the central nervous system. In some embodiments, the subject can be identified by in vivo imaging of a-Synuclein (e.g., in the brain). This can be accomplished via, e.g., positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging, magnetic resonance imaging (MRI), dopamine transporter (DAT) imaging, or substantia nigra ultrasonography. In some embodiments, the human subject can be identified by assaying the level of a-Synuclein in a blood, plasma, or cerebrospinal fluid (CSF) sample obtained from the subject and comparing the assayed level of a-Synuclein in the subject to a reference standard. The level of a-Synuclein may be assessed by methods known in the art comprising, e.g., analyzing a-Synuclein by one or more techniques chosen from Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent activated cell sorting (FACS), two-dimensional gel electrophoresis, mass spectroscopy (MS), matrix-assisted laser desorption / ionization-time of flight-MS (MALDI-TOF), surface-enhanced laser desorption ionization-time of flight (SELDI-TOF), high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), multidimensional liquid chromatography (LC) followed by tandem mass spectrometry (MS / MS), and laser densitometry.

[0083] Beyond degrading a-Synuclein and treating synucleinopathies, the invention also encompasses methods for targeted degradation of other non-SNARE proteins via engineered BoNT proteases. In particular, engineered BoNT / A proteases can be produced in accordance with the stepwise evolution strategy described herein to specifically target any IDPs that are implicated in the development of other human diseases. In various embodiments, the methods employ engineered BoNT / A proteases that are capable of specifically degrading any of these IDPs, e.g., c-myc, p53, EWS-FLI1, P-amyloid, TDP43 and TMEM106B.VII. Pharmaceutical compositions and administrations

[0084] The engineered a-Synuclein-degrading BoNT proteases of the invention, including derivative proteins and related compositions or compounds (e.g., conjugates with a toxin heavy chain or an antibody, polynucleotides, viral expression vectors, or lipid nanoparticles), can be administered directly to subjects in need of treatment. However, these therapeutic proteins are preferably administered to the subjects in pharmaceutical compositions which contain the engineered proteases (or modified derivative proteins) along with a pharmaceutically acceptable carrier, diluent or excipient in unit dosage form.Accordingly, the invention provides pharmaceutical or therapeutic compositions comprising one or more of the engineered BoNT / A proteases disclosed herein. The invention also provides a use of these engineered BoNT / A proteases in the preparation of pharmaceutical compositions or medicaments for treating or preventing the above-described diseases or medical disorders that are mediated by or associated with an IDP (e.g., a-Synuclein). The pharmaceutical compositions may be used in combination with other therapeutic agents that are suitable for treating or preventing Parkinson’s disease and other synucleinopathies, e.g., mAb exidavnemab, a-Synuclein vaccines PD01 A and PD03A, and small molecules minzasolmin, emrusolmin, and neflamapimod.

[0085] Pharmaceutically acceptable carriers are agents which are not biologically or otherwise undesirable. These agents can be administered to a subject along with the engineered BoNT / A protease without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition. Pharmaceutically carriers enhance or stabilize the composition or facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents,dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutically acceptable carrier employed should be suitable for various routes of administration described herein. Additional guidance for selecting appropriate pharmaceutically acceptable carriers is provided in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20thed., 2000. In addition to the engineered BoNT / A protease and the pharmaceutically acceptable carriers, the pharmaceutical compositions of the invention can further contain other active or inactive agents for treating synucleinopathies. For example, the pharmaceutical compositions can include known antioxidants and natural compounds such as flavonoids, squalamine, querceti and cuminaldehyde.

[0086] A pharmaceutical composition containing an engineered BoNT / A protease or derivative described herein and / or other therapeutic agents can be administered by a variety of methods known in the art, e.g., oral administration or intravenous administration. The routes and / or modes of administration vary depending upon the desired results.Depending on the route of administration, the active therapeutic agent may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the agent. Conventional pharmaceutical practice may be employed to provide suitable formulations to administer such compositions to subjects. Any appropriate route of administration may be employed. These include, but are not limited to, oral, intravenous, transcutaneous, subcutaneous, intranasal, intracranial, intraventricular, and intraspinal administration. Depending on the specific conditions of the subject to be treated, either systemic or localized delivery of the therapeutic agents may be used in the treatment.

[0087] When administered to a subject in vivo, the pharmaceutical compositions typically contain a therapeutically effective amount or dosage of the engineered BoNT / A protease or derivative (e.g., conjugate protein). A therapeutically effective amount is the total amount of the engineered BoNT / A protease that achieves the desired therapeutic effect. Effective dosages or doses vary depending upon many different factors, including means of administration, target site, physiological state of the patient, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. As a general guidance, the dosage ranges from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg, of the host body weight. For example dosages can be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg.

[0088] Exemplary treatment regimens entail administration once per day, once every other day, once every week, once every two weeks, once a month, once every 3 months, or once every 6 months or even longer. The pharmaceutical composition is usually administered on multiple occasions. As noted above, intervals between single dosages can be weekly, monthly, multiple-monthly, or even yearly. Intervals can also be irregular as indicated by measuring blood levels of the engineered BoNT / A protease. Alternatively, the engineered BoNT / A protease can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and administration frequency can also vary depending on the half-life of the administered enzyme in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

[0089] Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional (10thed., 2001); Remington: The Science and Practice of Pharmacy, Gennaro, ed., Lippincott Williams & Wilkins (20thed., 2003); Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7thed., 1999); and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions.EXAMPLES

[0090] The following examples are offered to illustrate, but not to limit the present invention.Example 1 q-Synuclein as a target

[0091] As a proof of concept, we chose a-Synuclein (a-Syn) as target due to its close association with Parkinson’s disease (PD) and its lack of any significant homology to SNAP25. In PD, loss of dopaminergic neurons is accompanied by the presence of Lewy bodies, abnormal plaques consisting mostly of precipitated a- Syn (11) - a 14 kDa protein that is highly expressed in neurons. Certain point mutations in the protein’s N-terminal region significantly increase the risk of developing PD and some are associated with early disease onset (12). Gene duplications (13) and triplications (14) also lead to early-onset autosomal dominant PD, indicating a correlation of dysregulated a-Syn expression levels to occurrence of PD. Furthermore, it has been observed that neuronal subpopulations with high relative a-Syn levels are more susceptible to Lewy body formation (15). Deletions of the gene cause minimal developmental changes in mouse studies (16, 17). Taken together, these data suggest that lowering intracellular Synuclein levels or degrading aggregates represents a promising strategy for improving disease outcome.

[0092] The first step in designing a protease to degrade a-Syn was to identify a target cleavage site, which ideally corresponds to a conformationally flexible loop or disordered region of the protein required for activity. To this end, we aligned the amino acid sequence from SNAP25 cleaved by BoNT / A (24) with the central “non-amyloid core” region (NAC) (25) of a-Syn (Fig. 1, Panel A), a region that is considered responsible for the aggregation properties of the synucleins. Cleavage within this region would likely prevent the formation of aggregates, although it is unclear whether this region would be susceptible to cleavage in existing aggregates. We identified a potential a-Syn cleavage site between residues Q79 and K80 that corresponds to SNAP25’s Q197 / R198 amide bond hydrolytically cleaved by wild-type BoNT / A. Additionally, a-Syn residues T72, Q79, G86, and S87 align with SNAP25 residues T190, QI 97, G204, and S205, respectively. Attempting to proteolyze a-Syn at this site using an evolved BoNT / A therefore likely requires changing the recognition sequence of BoNT / A for many of the key residues in the putative 30 amino acid substrate binding site. However, even a single projected mutation in SNAP25 (R198K) has been shown to decrease the proteolytic activity of wild-type BoNT / A below detectable levels, (26) indicating a significant effort is required to alter the selectivity of this protease to target a-Syn.Example 2 Screening and Mutagenesis Strategy

[0093] To evolve the sequence specificity of BoNT / A, we required means to efficiently screen large numbers of diverse protease clones. To this end, we constructed the plasmid SS-SNAP harboring a fusion of a mutant lysozyme from coliphage T7 linked to T7 RNA polymerase (Fig. 1, B) that has been previously employed in PACE evolution campaigns (10, 27). The linker connecting the two proteins initially consisted of a peptide sequence containing 20 amino acids from SNAP25 surrounding the cleavage site, which likely plays the dominant role in determining the BoNT / A substrate Michaelis constant (KM). Additionally, our screening system harbors an sfGFP gene under control of a T7 promoter. In this construct, T7 lysozyme (T7LZ) acts as the natural inhibitor of T7 RNA polymerase (T7RNAP). Accordingly, with T7LZ translationally fused to T7RNAP only minimal background sfGFP production is observed by fluorescence measurement. (Fig. 1, B and 1, C). When E. coli DH10B (naturally araD139) are co-transformed with the plasmid SS-SNAP and a second plasmid harboring BoNT / A protease under control of the pBAD promoter, we measured trace production of sfGFP in the presence of 1 wt.% glucose (Fig. 1, C), comparable to the fluorescence observed in the absence of the protease plasmid. A strongly increased fluorescence signal was observed when BoNT / A expression was induced in the presence of 0.01 wt.% arabinose, indicating a correlation between proteolytic activity and sfGFP production. Consequently, cells expressing proteolytically active BoNT clones can be readily isolated based on their relative sfGFP expression levels using this system and fluorescence-activated cell sorting (FACS). Additionally, cells producing sfGFP independent of proteolytic activity, e.g., through mutations in the T7LZ or T7RNAP, can be distinguished due to their unchanged sfGFP levels when inducing or inhibiting protease expression. By varying the linker sequence connecting T7LZ to T7RNAP, protease activity against various targets can be probed. Moreover, nonspecific proteases are likely to be toxic if they act on the essential bacterial proteome, providing a simple negative selection.

[0094] We next sought to identify key residues in BoNT / A involved in proteasesubstrate interaction by examination of the co-crystal structure of an inactive BoNT / A mutant (E224Q, Y366F) bound to a SNAP25 peptide fragment.(5) We identified three important regions mediating interactions between SNAP25 and BoNT / A proximal to the Zn2+ catalytic site (site-saturation mutagenesis 1-3; SSM1-3, Fig. 2, Panels A and C) and four more remote sites that were judged to be of secondary importance (SSM4a-d, Fig. 2A). The design of our first (Fig. 2, B) targeted mutation library SSM1 was based on the hypothesis that the high selectivity of BoNT / A for residues proximal to the cleavage site between QI 97 and R198 ismediated in part by an unstructured loop flanking Y366, because the Y366F mutation severely impairs its catalytic activity(5). Additionally, we postulated that the interaction of BoNT / A D370 located in this loop is responsible for the observed substrate selectivity at SNAP25 sites R198 and A199(26). SSM1 is an NNK library derived from WT-BoNT / A with five randomized amino acids flanking Y366 but conserving the tyrosine itself (Fig. 2, A). For the corresponding substrate, we constructed a selection plasmid bearing a quadruple-mutant SNAP25 target sequence (R198K / A199T / T200V / L203A, SNAP25 numbering, Fig. 1, A and D) as a first step in transforming the wildtype BoNT / A cleavage site sequentially to that of a-Syn.Example 3 Subsite Mutation and Screening

[0095] Bacteria transformed with this protease library SSM1 and quadruple-mutant screening plasmid SSI (Fig. 1, D and Fig. 2, B) were then sorted by FACS. Throughout this study, -300 million cells were sorted per round and -40000 cells with the highest sfGFP levels were collected. Sequencing of green colonies from this initial selection revealed a strong mutational bias, with BoNT / A mutations F369L and D370P being frequently observed in selected clones (Fig. 2, C, Tier 1 mutations). To further investigate the evolved proteolytic activity of these new proteases, we selected clone 5 from this round (“Protease 1”, Fig. 2, D) and observed a significant signal increase in our fluorescence assay using SSI (Fig. 3, Panel A). Additionally, when Protease 1 was co- expressed with a Flag-tagged T7LZ-T7RNAP harboring the R198K / A199T / T200V / L203A quadruple mutant SNAP25 linker sequence (FLAG-SS1), the substrate could be clearly detected by Western blot even when inhibiting protease expression (Fig. 3, B). When protease expression was induced, complete disappearance of the substrate band was observed, indicating complete proteolytic cleavage of the SNAP25 quadruple mutant sequence.

[0096] We chose Protease 1 (cf. Fig. 2, C, clone 5 from SSM1) to continue the next round of our stepwise evolution (Fig. 2, B) campaign and focused the design of SSM2 on the BoNT region recognizing the C-terminus of SNAP25 comprising residues K201, M202, L203, and G204 (Fig. 1, A and Fig. 2A). SNAP25 residue K201 C-terminal to the cleavage site binds to a BoNT P-sheet near E257, which in turn interacts with BoNT’s K244, located in a second, antiparallel P-strand. This round of mutagenesis focused on generating clones that cleave a-Syn E83, reversing the substrate polarity of SNAP 25, which has a lysine at the corresponding position (Fig. 1, A). Consequently, we prepared protease library SSM2 fromProtease 1 with randomized BoNT / A residues K244 and E257 along with 4 proximal sites F243, V245, G255, and L256 (Fig. 2). These BoNT residues are all part of the same P-sheet binding the substrate C-terminal region. We constructed the corresponding screening substrate, SS2, additionally harboring mutations K201E and M202G compared to SSI (Fig.1, D). Cells harboring this quintuple mutant SS2 and second- tier protease library SSM2 were again sorted for the highest levels of sfGFP after inducing protease expression. Sequencing of active clones revealed a strong bias at select sites (Fig. 2, C, Tier 2 mutations). Notably, position E257 was mutated from glutamate to lysine or arginine, an observation in line with our expectation to reverse the polarity of SS2 at this site as compared to SS-SNAP. BoNT mutation K244Y was also prominently featured, perhaps interacting with the newly introduced cationic residue at position 257 within the BoNT P-sheet structure. The observation of aromatic or branched aliphatic amino acids overrepresented at BoNT positions 243, 245, 255, and 256 may also contribute to P-sheet stability. A strong increase in fluorescence was observed when expressing the protease clone 8 from this selection (“Protease 2”, Fig. 2, D) in the presence of SS2 (Fig. 3, A). We further validated the proteolytic activity by Western blot analysis, using Flag-tagged plasmid FLAG-SS2 (Fig. 3, B).

[0097] With an evolved protease readily cleaving SNAP25 quintuple mutant SS2 in hand, we turned to the N-terminal region of the substrate encompassing SNAP25 residues T190-Q197 as a next step (Fig. 2, B). This substrate region binds to another P-strand in BoNT / A, itself deeply nested in the protease core as part of an 8-strand P-sheet motif.BoNT / A residues C165, K166, S167, F168 Q184, and R231 appear involved in interactions with SNAP25 in this region, and we accordingly prepared library SSM3 containing proteases randomized at these positions (Fig. 2, A). For screening we devised and created the corresponding substrate SS3 (Fig. 1, D), which has an additional six substrate residues that bind to this BoNT / A P-strand altered relative to SS2: R191G, I192V, D193T, E194A, Al 95V, and N196A (SNAP25#, Fig. 1, A). Overall, the substrate sequence in SS3 differs at 12 sites from the original SNAP25 sequence and already closely resembles that of a-Syn (Fig. 1, A). Despite this large change from the previous substrate sequence SS2, we were able to isolate active protease clones against SS3 when sorting cells expressing this round 3 library by FACS. Strong mutational biases were again observed, with R23 IF being the only functional mutation isolated at this position. Clones with conserved Cl 65 appeared to require an additional cysteine at BoNT position 184, presumably forming a new disulfide bond.K166R was another frequently isolated mutation, with lysine at this site being notably absent. Otherwise, native amino acid identities were frequently observed, perhaps indicating the importance of this deeply embedded core region of the enzyme. We investigated clone 1 from this selection (“Protease 3”, Fig. 2, D) in more detail. In our fluorescence assay using SS3, an increase in fluorescence was observed in the presence of this Protease 3 (Fig. 3, A). However, the increase was significantly smaller than in previous iterations. We also constructed Flag-tagged plasmid Flag-SS3, and cleavage of this mutant substrate was visualized by Western blot, which showed significant but incomplete degradation of Flag-SS3 by Protease 3 (Fig.3, B).

[0098] Before optimizing for increased proteolytic activity, we first sought to investigate whether selectivity is retained on a larger a-Syn fragment. Consequently, we increased the substrate length with screening system SS-aSyn containing a 60 amino acid linker sequence derived exclusively from a-Syn connecting T7LZ and T7RNAP (Fig. 1, D). The increased substrate size also served the dual purpose of identifying possible remote binding sites for increased substrate affinity and minimizing the possibility of selecting mutants that bind to either T7LZ or T7RNAP. Notably, we observed that the longer linker sequence leads to slightly less efficient inhibition of T7RNAP activity and higher background fluorescence in the absence of protease (Fig. 3, A). Despite Protease 3 cleaving highly homologous SS3, no significant activity of this protease was detected on SS-aSyn either by fluorescence assay or by Western blot (Fig. 3). We thus attempted to further increase both activity and selectivity of Protease 3 by mutating distal SNAP25 binding sites in the protease. Consequently, four libraries were separately constructed from Protease 3, each randomizing residues in separate unstructured loops at BoNT sites K23, 124, P25, A27, G28, and Q29 (SSM4a, Fig. 2, A and B), G169, H170, E171, V172, N174, and T176 (SSM4c), and V304, G305, T306, T307, A308, and Y312 (SSM4d), as well as in P-sheet residues D131, T132, C134, N136, Y144, and S146 (SSM4b). From these four libraries, protease clones active against SS-aSyn were identified by FACS. The mutated regions isolated in each round of selection were then permutated. First, a separate PCR for each of the pooled active protease libraries SSM1-4 was used to produce linear gene fragments; pooling of the resulting PCR products and reassembly using Gibson assembly yielded plasmids encoding full length proteases with interchanged gene cassettes. We again tested the proteolytic activity of Protease 4 containing permutated sequences from previous selections (Fig. 2, D) using SS-aSyn and observed a significant increase in fluorescence in the presence of protease (Fig.3, A). Importantly no fluorescence increase was detected in the presence of SS-SNAP, SSI or SS2 (Fig. 3, A). However, further analysis using our fluorescence assay showed remaining activity against SS3 and a Western Blot revealed that more, but still only partial proteolysis of substrate SS-aSyn was achieved (Fig. 3, B). At the same time, Protease 4 retained significant activity on previous substrate SS3, suggesting that selectivity also required additional improvement.Example 4 Random Mutagenesis and Recombination

[0099] To further optimize the catalytic activity and specificity of our evolved proteases for a-Syn, we conducted random, error-prone mutagenesis using Protease 4 as the template, reasoning mutations distal to the binding site might optimize the geometry of key interactions. Clones were sorted for activity, and a library of 400 active clones was then subjected to NeXT DNA shuffling (28) along with clones from previous screens. Cells were again challenged against SS-aSyn and sorted for high fluorescence intensity. The active protease clones were then counter-screened for minimal activity against SNAP25 using SS-SNAP and this complete protocol was repeated to yield Protease 5. Interestingly, Q363R reverted to its original wild-type amino acid (Fig. 2, D), and we also observed additional mutations E368L and A372L in this unstructured loop. In addition the P-sheet evolved in SSM2 contained the adjacent mutation V242A and proximal mutations N248D and S254R. All point mutations identified during random mutagenesis appeared to mediate interactions with the C-terminal region of the substrate except for T109M. Additionally, some sites mutated in previous rounds showed variability, suggesting that these regions play a minor role in controlling overall activity and selectivity.

[0100] Protease 5 displayed significantly increased activity in our fluorescencebased activity assay against SS-aSyn, and a strong increase in sfGFP production was observed even when low protease expression in the presence of glucose (Fig. 4, Panel A). To evaluate its selectivity, we co-transformed cells with Protease 5 plasmid and separately one of our four screening plasmids SS-SNAP, SSI, SS2 or SS3. As a control, we also collected data using cells containing no protease, but only the respective screening plasmid. In the absence of protease, cells showed only a marginal increase in fluorescence in the presence of arabinose over those with glucose (see Figure 5). This small difference is likely due to differences in metabolism and growth behavior. In the presence of our evolved protease, none of the earlier screening systems showed significant proteolytic activity as judged by ourfluorescence assay either in the presence of glucose or arabinose (Fig. 4, A). Selection system SS-aSyn bearing the 60 residue a-Syn linker was the only substrate exhibiting a significant increase in fluorescence in the presence of Protease 5 over the no protease control, independent of whether expression was induced, highlighting the activity and fidelity of this evolved protease. We further confirmed these data by Western Blot, where induction of protease expression led to complete disappearance of the substrate, and partial proteolysis was observed even when protease expression was inhibited (Fig. 4, A). The selectivity of this protease for aSyn may result in part from degradation of bacterial proteins by lower fidelity proteases with broader substrate specificity, resulting in slower growth rates and subsequently exponential underrepresentation during selection.Example 5 Proteolytic Activity of Evolved Protease 5

[0101] The time course of substrate cleavage in bacteria was assessed by transforming bacteria with SS-SNAP and WT-BoNT protease or SS-aSyn and evolved Protease 5. Saturated cultures were diluted into multiple aliquots and grown for a total of 3h at 37 °C. At specific timepoints arabinose protease expression was induced. For WT-BoNT, partial substrate cleavage was observed when protease expression was induced for 30 minutes (Fig. 4, C). In contrast, no substrate band could be observed at 30 minutes with evolved Protease 5. Furthermore, only trace amounts of substrate could be observed at high exposure even when no arabinose was added at all (timepoint 0 min), indicating highly efficient degradation by our evolved protease at low uninduced protease expression levels. This is in accordance with our previous finding that notable SS-aSyn substrate degradation was observed by Western blot when using Protease 5, even under conditions that repress protease expression with glucose (Fig. 4, A). The presence of bands for the substrate Flag-SS-aSyn in the absence of protease plasmid additionally showcases that Protease 5 is responsible for degradation of this construct. We then purified Protease 5 with an N-terminal GB1 solubility tag and subsequently incubated it with a substrate consisting of full-length a-Synuclein with N-terminal YFP and C-terminal CFP fusions. Analysis by LCMS revealed that this substrate was cleaved between a-Synuclein residues T92 and G93 (Fig. 5). This data further confirms that only two fragments are produced by evolved Protease 5. It proved difficult to directly quantify the specific activities for the evolved protease by FRET with this system due to aggregation of the various Synuclein containing fusion proteins. Both variation of the fluorescent proteins or Synuclein-derived linker did not yield any monomeric substrateamenable to in vitro measurements. These difficulties are in line with previous reports highlighting the complex and disputed aggregation status of a-Synuclein.(29, 30) A short peptide consisting of 17 amino acids derived from Synuclein amino acids G68-G84 for kinetic measurements proved to be insoluble.

[0102] To begin to investigate whether our findings extend to the degradation of full-length, native Synuclein in mammalian cells, we constructed two CMV vectors expressing Protease 5 and human a-Syn. When HEK293T cells were transfected with only the a-Syn expression plasmid, a strong a-Syn band was observed by Western blot using antibody Syn211 (Fig. 4, C). Under these imaging conditions, none of the endogenous a-Syn produced by HEK293T was detectable, underpinning the high level of a-Syn overexpression achieved by transient transfection. In contrast, co-transfection of both protease and Syn plasmids (1:5 ratio, respectively) led to significant degradation of a-Syn when probed after 24 h. Co-transfected cells were investigated for viability to determine any detrimental effects of either proteolytic fragments or off-target protease activity, but no significant decrease in viability was observed (Fig. 4).Example 6 General procedures for stepwise evolving BoNT / A and substrate sequence

[0103] We have developed a generalizable method for evolving BoNT / A proteolytic cleavage against any target protein of interest. The method relies on an iterative evolution strategy in which specific regions of the BoNT / A light chain are randomized to identify BoNT / A mutants that cleave a substrate that has been changed from the wild-type target (SNAP25) to the target sequence at defined positions relative to the BoNT / A cleavage site in SNAP25. Specifically, 7 libraries site saturation libraries of BoNT / A have been designed, here referred to as SSM1 (residues R363X, L367X, N368X, F369X, D370X, A372X), SSM2 (F243X, K244X, V245X, G255X, L256X, E257X), SSM3 (C165X, K166X, S167X, F168X, Q184X, R231X), and SSM4a (K23X, I24X, P25X, A27X, G28X, Q29X), SSM4b (G169X, H170X, E171X, V172X, N174X, T176X), SSM4c (D131X, T132X, C134X, N136X, Y144X, S146X), SSM4d (V304X, G305X, T306X, T307X, A308X, Y312X). These libraries are screened to affect changes in BoNT / A cleavage specificity for defined residues in the wild-type SNAP25 target sequence to the desired new recognition sequence. Specifically, SSM1 libraries are screened for the identification of BoNT / A mutants for which the sequence specificity of residues at -1,+1, +2, +3, and +6, relative to the original cut site in SNAP25 has been changed; SSM2 libraries are screened for the identification ofBoNT / A mutants for which the sequence specificity of residues at +4 and +5 relative to the original cut site in SNAP25 has been changed, SSM3 libraries are screened for the identification of BoNT / A mutants for which the sequence specificity of residues at -7, -6, -5, -4, -3, and -2 relative to the original cut site in SNAP25 has been changed. Finally, libraries SSM4a, SSM4b, SSM4c, and SSM4d are screened for mutations in BoNT / A for which the sequence specificity of residues at -11, -10, -9, and -8 relative to the original cut site in SNAP25 have been changed. The process of changing the cleavage site from the original SNAP25 to the desired target sequence using these 7 libraries (SSM1, SSM2, SSM3, SSM4a, SSM4b, SSM4c, SSM4d) is carried out in an iterative manner in no defined order. Both BoNT / A mutations identified in any step of this process, as well as changes to the recognition sequence that have been carried out are carried over to screening of consecutive libraries. Screening of any individual- or combinations of the outlined BoNT / A libraries (SSM1, SSM2, SSM3, SSM4a, SSM4b, SSM4c, SSM4d) is used to identify BoNT / A mutants that can cleave any target sequence of interest.Example 7 Some exemplified materials and methods

[0104] Site-Saturation Library Assembly: The respective protease plasmid was amplified with primers containing degenerate NNK codons at the indicated sites using Q5 DNA Polymerase (New England Biolabs). PCR fragments were assembled using Gibson assembly. (39) Assembly mixtures were purified using QIAquick PCR Purification Kit (Qiagen). Electrocompetent NEB 10-beta cells (New England Biolabs) were mixed with precooled, purified DNA and electroporated and recovered using the manufacturer’s protocol. Pooled cells from multiple electroporations were then plated on LB agar and grown at 37 °C overnight. Small aliquots were diluted and plated on separate plates to estimate library diversity. The next day, cells were resuspended in PBS, pelleted and plasmid DNA was isolated using ZymoPURE II Plasmid Midiprep Kit (Zymo Research).

[0105] Error Prone PCR: Gene fragments were amplified using Taq polymerase rendered more error prone by addition of 0.3 mM MnCh and an uneven mix of dNTPs. In short, primers (each 0.2 pM). 400 ng plasmid DNA template, 4 pL Taq DNA polymerase, “error-prone” dNTPs (350 pM dATP, 400 pM dCTP, 200 pM dGTP, 1350 pM dTTP), MgCh (2.95 mM) and molecular biology grade water to 388pL were mixed and MnCh (12 pL, 0.3 mM) was added. The mixture was distributed into a PCR strip with 8 wells containing each 50 pL each. Molarities indicate final concentrations. A PCR protocolconsisted of: Initial Denaturation: 95 °C for 1:00 min, cycle 34x: 95°C for 30 sec, 60 °C for 45 sec, 68 °C 1:30 then one final amplification at 68 °C for 5:00 minutes. PCR mixtures were then pooled into 200 pL batches, purified using QIAquick PCR Purification Kit (Qiagen) and assembled into circular plasmids using Gibson assembly. Plasmid backbones for Gibson assembly were amplified under non-error prone conditions using Q5 DNA polymerase.Isolation of library plasmids was executed as described above for site-saturation libraries.

[0106] Random DNA Shuffling: Mutagenized clones were pooled and 100 firnol were amplified using Taq polymerase in ThermoPol Buffer following the manufacturer’s protocol with 30% of dTTP substituted with dUTP. PCR products were isolated using QIAquick PCR Purification Kit (Qiagen) and digested using 2 units USER enzyme (NEB) for 50pL reactions in lx rCutSmart (NEB) for 2 h at 37 °C. Complete digestion was validated by agarose gel. To the digested mixtures was added 1 pL Dpnl and incubated for 15 minutes at 37 °C. DNA fragments were isolated using the QIAex II Kit (Qiagen). Digested fragments were reassembled into full length genes as previously reported(40) and cloned into linear plasmids using Gibson assembly and electrocompetent NEB 10-beta cells (New England Biolabs).

[0107] Protease Activity Selection by FACS and Fluorescence Measurements: NEB 10-beta cells (New England Biolabs) competent cells containing respective selection plasmids SS-SNAP, SSI, SS2, SS3, or SS-aSyn were made electrocompetent(41), mixed with the respective protease plasmid library and transformed by electroporation. Recovered cells were plated on LB agar containing maintenance antibiotics and 0.01 wt.% arabinose and grown overnight at 37 °C. Cells were harvested in PBS and sorted by fluorescence-activated cells sorting (FACS) using a Sony MA 900 cell sorter until >300million events were reached and ~40000 cells with the highest sfGFP levels had been collected. Collected cells were plated on LB agar containing maintenance antibiotics and 0.01 wt.% arabinose and grown overnight at 37 °C. The next morning, single green colonies were picked into a 96 well plate containing 150 pL / well 2xYT media with maintenance antibiotics. Cultures were grown to saturation and diluted 30-fold into 96 well plates containing 2xYT media with maintenance antibiotics and either 1 wt.% glucose or 0.01 wt.% arabinose and shaken for 6h at 37 °C in a Logphase 600 microbiology reader (BioTek) at 800 rpm in a black-walled, clear bottom 96-well plate. Cells were pelleted, resuspended in PBS and fluorescence was measured at 470 nm excitation / 510 nm emission using a SpectraMax iD3 plate reader (Molecular Devices). Clones with higher fluorescence in the presence of glucose were excluded as falsepositives and the remaining clones were ranked and selected according to their relative fluorescence in the presence of arabinose.

[0108] Western Blots: NEB 10-beta cells containing the respective FLAG-tagged selection system plasmid and protease plasmid were grown overnight. Then cultures were diluted 30-fold into 96 well plates containing 2xYT media with maintenance antibiotics and either 1% glucose or 0.01% arabinose and shaken for 6h at 37 °C in a Logphase 600 microbiology reader (BioTek) at 800 rpm in a black-walled, clear bottom 96-well plate. 50 pL of culture were pelleted and resuspended in 50 pL lx NuPage LDS sample buffer (Life Technologies). Proteins were separated by electrophoresis on Bolt 4-12% Bis-Tris Plus WedgeWell gels (Invitrogen) at 200V for 30 minutes using submerged in MOPS SDS Running Buffer (Invitrogen). Proteins were blotted onto 0.45 um PVDF membrane (Immobilon) at 20V for 60 minutes using Bolt Transfer Buffer (Invitrogen) containing 10% MeOH. Membranes were blocked for 1 h using 5% non-fat dry milk in TBST (lx TBS+0.1% Tween80 (Millipore). Membranes were then incubated overnight with the primary antibody, then washed 6x with TBST over 30 minutes. Membranes were incubated with secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature and washed 12x with TBST over Ih followed by a final wash with lx TBS. Horseradish peroxidase conjugates were visualized using ProSignal Dura ECL Reagent (Prometheus) on a ChemiDoc Touch Imaging System (Bio-Rad).

[0109] Western Blots visualizing native a-Synuclein were conducted with an adjusted method to avoid detachment of a-Synuclein from the PVDF membrane after transfer. In short, after transfer the membrane was incubated in PBS containing 0.4% paraformaldehyde for 30 minutes, then blocked for Ih with 5% non-fat dry milk in TBST. Afterwards, the membrane was incubated with antibody Syn211 (Invitrogen) in 5% non-fat dry milk in TBST overnight, followed by washing 6x with TBST over 30min, incubation with HRP conjugated secondary goat anti-mouse HRP conjugated antibody (Invitrogen) in 5% non-fat dry milk in TBST for Ih and washing with TBST 12x over Ih. After a final wash with lx TBS, horseradish peroxidase conjugates were visualized using ProSignal Dura ECL Reagent (Prometheus) on a ChemiDoc Touch Imaging System (Bio-Rad).

[0110] Tissue Culture: HEK293T cells were cultured as adherent cells in tissue culture treated petri dishes in a humidified incubator in a 5% CO2 atmosphere at 37 °C using Dulbecco’s modified Eagle’s medium with 4.5g / L glucose, L-glutamine and sodium pyruvate (Corning) supplemented with 10% FBS (Gibco). Cells were passaged 1:10 at -80%confluency using TrypLE Express (Gibco) to detach cells. For Western Blots 0.2* 106cells were seeded into clear 6-well dishes, allowed to attach and transfected using FugeneHD (Promega) following the manufacturers protocol. In short, 2 pg DNA was topped up to 92 L total volume using OptiMEM reduced serum medium (Gibco). Then, 8 pL FugeneHD reagent was slowly added without touching the plastic walls, pipetted up and down to mix the reagents and incubated for 10 minutes and room temperature. The complete mixture was then added dropwise to the culture dish and incubated for further 24 h. For viability assays 0.01 *106cells were seeded into a white-walled 96 well plate (Corning Costar) and transfected with 10 pL of FugeneHD transfection mixture prepared as above and again incubated for 24 h afterwards. Measurements were recorded in triplicates.

[0111] Protein Expression: BL21(DE3) chemically competent cells (New England Biolabs) were transformed with a pET21 vector encoding evolved Protease 5 preceded by an N-terminal His-tag and a GB1 solubility tag. The substrate was expressed using the same cell line with a pET21 plasmid encoding a fusion of an N-terminal His-tag, YFP, full-length human a-Synuclein, and CFP. To express either protein, a single colony was picked and used to inoculate an overnight culture of 2xYT with maintenance antibiotic. The next day, the culture was diluted 1: 100 into a 4 L baffled flask containing 1 L of 2xYT with maintenance antibiotic, grown to OD=1 and then cooled on ice for 1 h. IPTG was added to a final concentration of 0.2 mM and the flask was shaken at 200 rpm at 18 °C overnight. Cells were pelleted, resuspended in 100 mL ice-cold lysis buffer containing cOmplete EDTA free protease inhibitor (Roche), 300 mM NaCl and 20mM HEPES and lysed by sonication using a Q700 (Qsonica) using the following parameters: Amplitude 15, 1 second on time, 2 second off time and for a total processing time of 15 minutes. The sample was kept on ice during the sonication. Cell debris was removed by centrifugation at 29000 rpm, the supernatant was further cleared by filtration, 5 M imidazole (neutralized by HC1, pH=7) was added to a final concentration of 10 mM and a pre-equilibrated Ni-NTA agarose resin (Qiagen) column was then incubated with this cell lysate for Ih at 4 °C. The lysate was allowed to exit the column by gravity flow, the remaining resin was washed with lysis buffer containing 25 mM imidazole followed by elution of the desired protein using lysis buffer containing 250 mM imidazole. The imidazole was removed from the eluted protein using a PD10 column (Cytiva).Some cited references:1. J. Jankovic, Botulinum toxin in clinical practice. Journal of Neurology, Neurosurgery & Psychiatry 75, 951-957 (2004).2. D. D. Truong, W. H. Jost, Botulinum toxin: Clinical use. Parkinsonism & Related Disorders 12, 331-355 (2006).3. M. R. Popoff, Overview of Bacterial Protein Toxins from Pathogenic Bacteria: Mode of Action and Insights into Evolution. Toxins 16, 182 (2024).4. T. C. Siidhof, J. E. Rothman, Membrane Fusion: Grappling with SNARE and SM Proteins. Science 323, 474-477 (2009).5. M. A. Breidenbach, A. T. Brunger, Substrate recognition strategy for botulinum neurotoxin serotype A. Nature 432, 925-929 (2004).6. T. Binz, et al., Mutations in light chain of botulinum neurotoxin a enable cleavage of human SNAP-23. Toxicon 156, S10 (2018).7. S. Sikorra, et al., Engineering an Effective Human SNAP-23 Cleaving Botulinum Neurotoxin A Variant. Toxins 12, 804 (2020).8. R. P. Dyer, et al., Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution. Sci Rep 12, 9956 (2022).9. S. Chen, J. T. Barbieri, Engineering botulinum neurotoxin to extend therapeutic intervention. Proceedings of the National Academy of Sciences 106, 9180-9184 (2009). 10. T. R. Blum, et al., Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity. Science 371, 803-810 (2021).11. M. G. Spillantini, et al., a-Synuclein in Lewy bodies. Nature 388, 839-840 (1997).12. P. Flagmeier, et al., Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of a-Synuclein aggregation. Proceedings of the National Academy of Sciences 113, 10328-10333 (2016).13. M.-C. Chartier-Harlin, et al., a-Synuclein locus duplication as a cause of familial Parkinson’s disease. The Lancet 364, 1167-1169 (2004).14. A. B. Singleton, et al., a-Synuclein Locus Triplication Causes Parkinson’s Disease. Science 302, 841-841 (2003).15. J. Courte, et al., The expression level of a-Synuclein in different neuronal populations is the primary determinant of its prion-like seeding. Sci Rep 10, 4895 (2020).16. A. Abeliovich, et al., Mice Lacking a-Synuclein Display Functional Deficits in the Nigrostriatal Dopamine System. Neuron 25, 239-252 (2000).17. aPy-Synuclein triple knockout mice reveal age-dependent neuronal dysfunction.Available at: https: / / www.pnas.org / doi / 10.1073 / pnas.1005005107 [Accessed 2 December 2024],18. S. K. Madden, A. D. de Araujo, M. Gerhardt, D. P. Fairlie, J. M. Mason, Taking the Myc out of cancer: toward therapeutic strategies to directly inhibit c-Myc. Molecular Cancer 20, 3 (2021).19. R. Dhanasekaran, et al., The MYC oncogene — the grand orchestrator of cancer growth and immune evasion. Nat Rev Clin Oncol 19, 23-36 (2022).20. V. N. Uversky, p53 Proteoforms and Intrinsic Disorder: An Illustration of the Protein Structure-Function Continuum Concept. International Journal of Molecular Sciences 17, 1874 (2016).21. H. V. Erkizan, V. N. Uversky, J. A. Toretsky, Oncogenic Partnerships: EWS-FLI1 Protein Interactions Initiate Key Pathways of Ewing’s Sarcoma. Clin Cancer Res 16, 4077-4083 (2010).22. M. Neumann, et al., Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis. Science 314, 130-133 (2006).23. Y. X. Jiang, et al., Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43. Nature 605, 304-309 (2022).24. L. von Berg, et al., Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology. SciRep 9, 5531 (2019).25. J. A. Rodriguez, et al., Structure of the toxic core of a-Synuclein from invisible crystals. Nature 525, 486-490 (2015).26. J. J. Schmidt, K. A. Bostian, Endoproteinase Activity of Type A Botulinum Neurotoxin: Substrate Requirements and Activation by Serum Albumin. J Protein Chem 16, 19-26 (1997).27. M. S. Packer, H. A. Rees, D. R. Liu, Phage-assisted continuous evolution of proteases with altered substrate specificity. Nat Commun 8, 956 (2017).28. K. M. Muller, et al., Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution. Nucleic Acids Research 33, el 17 (2005).29. T. Bartels, J. G. Choi, D. J. Selkoe, a-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature Ml, 107-110 (2011).30. J. Burre, et al., Properties of native brain a-Synuclein. Nature 498, E4-E6 (2013).31. Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates.32. S.-I. Miyashita, J. Zhang, S. Zhang, C. B. Shoemaker, M. Dong, Delivery of singledomain antibodies into neurons using a chimeric toxin-based platform is therapeutic in mouse models of botulism. Science Translational Medicine 13, eaaz4197 (2021).33. P. M. McNutt, et al., Neuronal delivery of antibodies has therapeutic effects in animal models of botulism. Science Translational Medicine 13, eabd7789 (2021).34. S. Tian, et al., Targeted intracellular delivery of Casl3 and Cas9 nucleases using bacterial toxin-based platforms. Cell Reports 38 (2022).35. H. Roh, B. G. Dorner, A. Y. Ting, Cell-Type-Specific Intracellular Protein Delivery with Inactivated Botulinum Neurotoxin. J. Am. Chem. Soc. 145, 10220-10226 (2023).36. M. F. Naso, B. Tomkowicz, W. L. Perry, W. R. Strohl, Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31, 317-334 (2017).37. D. Wang, P. W. L. Tai, G. Gao, Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 18, 358-378 (2019).38. J.-H. Wang, D. J. Gessler, W. Zhan, T. L. Gallagher, G. Gao, Adeno-associated virus as a delivery vector for gene therapy of human diseases. Sig Transduct Target Ther 9, 1-33 (2024).39. D. G. Gibson, et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343-345 (2009).40. N. M. Gaudelli, et al., Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage. Nature 551, 464-471 (2017).41. Q. Tu, et al., Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency. Sci Rep 6, 24648 (2016).***

[0112] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0113] All patents, patent applications, published applications and publications, GenBank sequences, databases, ATCC deposits, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety and for all purposes as if each is individually so denoted.

Claims

TSRI 2277.1PC WE CLAIM:

1. A library of Botulinum neurotoxin A (BoNT / A) protease variants for stepwise evolution of the enzyme, comprising a plurality of BoNT / A variants that, relative to the wildtype BoNT / A sequence, contain randomized mutations at each residue selected from (1) R363, L367, N368, F369, D370, and A372, (2) F243, K244, V245, G255, L256, and E257, (3) C165, K166, S167, F168, Q184, and R231, (4) K23, 124, P25, A27, G28, and Q29, (5) G169, H170, E171, V172, N174, and T176, (6) D131, T132, C134, N136, Y144, and S146, or (7) V304, G305, T306, T307, A308, and Y312; wherein the wildtype BoNT / A sequence is based on UniProt ID Q7B8V4 (SEQ ID NO: 1).

2. A method for evolving Botulinum neurotoxin A (BoNT / A) protease to generate a variant BoNT / A protease that specifically cleaves a target protein at a selected substrate sequence, comprising:(1) selecting in the target protein a substrate sequence that comprises residues from position -7 to position +6 around a desired cleavage site;(2) preparing (i) a first library of BoNT / A variants that, relative to a first reference enzyme, contain randomized mutations at each residue selected from R363, L367, N368, F369, D370, and A372, and a first corresponding substrate sequence; wherein the first corresponding substrate sequence, relative to a first reference substrate sequence, has residues at -1, +1, +2, +3 and +6 positions being respectively identical to the corresponding residues in the selected substrate sequence in the target protein, (ii) a second library of BoNT / A variants that, relative to a second reference enzyme, contain randomized mutations at each residue selected from F243, K244, V245, G255, L256, and E257, and a second corresponding substrate sequence; wherein the second corresponding substrate sequence, relative to a second reference substrate sequence, has residues at +4 and +5 positions being respectively identical to the corresponding residues in the selected substrate sequence in the target protein, and (iii) a third library of BoNT / A variants that, relative to a third reference enzyme, contain randomized mutations at each residue selected from C165, K166, S167, F168, Q184, and R231, and a third corresponding substrate sequence; wherein the third corresponding substrate sequence, relative to a third reference substrate sequence, has residues at -7, -6, -5, -4, -3, and -2 positions being respectively identical to the corresponding residues in the selected substrate sequence in the target protein, andTSRI 2277.1PC (3) performing 3 rounds of primary screening iteratively without a defined order, each round employing a separate library of BoNT / A variants and the corresponding substrate sequence set forth in (2), to identify a BoNT / A variant that cleaves the corresponding substrate sequence in each round of screening; wherein (i) in the first round of primary screening, the reference enzyme for the employed library of BoNT / A variants is wildtype BoNT / A, and the reference substrate sequence for the employed corresponding substrate sequence comprises the native BoNT / A cleavage site in the SNAP25 protein; (ii) in the second round of primary screening, the reference enzyme for the employed library of BoNT / A variants is the BoNT / A variant identified from the first round of primary screening, and the reference substrate sequence for the employed corresponding substrate sequence is the corresponding substrate sequence used in the first round of primary screening, and (iii) in the third round of primary screening, the reference enzyme for the employed library of BoNT / A variants is the BoNT / A variant identified from the second round of primary screening, and the reference substrate sequence for the employed corresponding substrate sequence is the corresponding substrate sequence used in the second round of primary screening; whereby the BoNT / A variant identified from the third round of primary screening is a variant BoNT / A protease that specifically cleaves the target protein at the selected substrate sequence.

3. The method of claim 2, further comprising one or more rounds of secondary iterative screening without a defined order to identify a final BoNT / A variant that specifically cleaves the target protein at a substrate sequence that additionally comprises residues at positions -8, -9, -10, and -11 around the cleavage site; wherein the one or more rounds of secondary screening comprise screening one or more of 4 additional libraries of BoNT / A variants that, relative to a reference enzyme, contain randomized mutations at each residue selected from (i) K23, 124, P25, A27, G28, and Q29, (ii) D131, T132, C134, N136, Y144, and S146, (iii) G169, H170, E171, V172, N174, and T176, or (iv) V304, G305, T306, T307, A308, and Y312, with a corresponding substrate sequence that, relative to the corresponding substrate sequence used in the third round of primary screening, has residue at positions -8, -9, -10 and -11 being respectively identical to the corresponding residues in the selected substrate sequence in the target protein; thereby identifying a final BoNT / A variant that cleaves the corresponding substrate sequence.

4. The method of claim 3, wherein the one or more rounds of secondary screening are performed sequentially, and wherein in each of the one or more rounds ofTSRI 2277.1PC secondary screening, the reference enzyme for the employed library of BoNT / A variants is the BoNT / A variant identified from the previous round of screening.

5. The method of claim 4, wherein the one or more rounds of secondary screening comprise screening of each of the 4 additional libraries of BoNT / A variants.

6. The method of claim 3, wherein the one or more rounds of secondary screening are performed in parallel, wherein in each of the one or more rounds of secondary screening, the reference enzyme for the employed library of BoNT / A variants is the BoNT / A variant identified from the third round of primary screening, and wherein the identified final BoNT / A variant comprises, in addition to mutations in the reference enzyme, all additional mutations relative to the reference enzyme that are present in the BoNT / A variants identified from each round of secondary screening.

7. The method of claim 2, wherein the target protein is an intrinsically disordered protein (IDP).

8. The method of claim 7, wherein the substrate sequence is located in an intrinsically disordered region of the protein.

9. The method of claim 7, wherein the IDP is a-Synuclein, c-myc, p53, EWS-FLI1, P-amyloid, TDP43, or TMEM106B.

10. An engineered Botulinum neurotoxin A (BoNT / A) protease that specifically degrade a-Synuclein, comprising the light chain sequence of a wildtype BoNT / A except for the following amino acid substitutions, K23I, I24L, A27N, G28M, Q29D, Q67K, T109M, D131S, N136V, Y144Q, S146M, K166R, F168R, G169E, H170F, E171S, V172M, N174P, Q184C, Fl 961, F213I, T220L, R231F, V242A, F243I, K244W, V245F, N248D, S254R, G255W, L256M, E257K, N280D, F282L, R363R, N368L, F369L, D370P, and A372L; wherein the amino acid numbering is based on the sequence of the wildtype BoNT / A protein under UniProt ID Q7B8V4 (SEQ ID NO: 1).

11. The engineered BoNT / A protease of claim 10, wherein the wildtype BoNT / A is of subtype Al.

12. The engineered BoNT / A protease of claim 10, wherein the wildtype BoNT / A comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-13.

13. The engineered BoNT / A protease of claim 10, comprising said amino acid substitutions that are introduced into a BoNT / A light chain sequence that is at least 95% or 99% identical to SEQ ID NO: 14.TSRI 2277.1PC 14. The engineered BoNT / A protease of claim 10, comprising an amino acid sequence set forth in SEQ ID NO: 15 or a conservatively modified variant thereof that does not substantially affect the catalytic activity, folding or stability of the protease.

15. The engineered BoNT / A protease of claim 10, further comprising a conjugated targeting moiety.

16. The engineered BoNT / A protease of claim 15, wherein the conjugated targeting moiety is a bacterial toxin heavy chain, an antibody or monobody.

17. The engineered BoNT / A protease of claim 15, wherein the conjugated targeting moiety is an engineered BoNT / A heavy chain that targets a specific cell type or tissue type.

18. The engineered BoNT / A protease of claim 17, wherein the specific cell type is neuron or glia.

19. The engineered BoNT / A protease of claim 17, wherein the engineered BoNT / A protease is conjugated to the engineered BoNT / A heavy chain via a disulfide bond.

20. A method of treating or preventing a synucleinopathy in a subject, comprising administering to the subject a pharmaceutical composition that contains a therapeutically effective amount of an engineered Botulinum neurotoxin A (BoNT / A) protease of claim 10, thereby treating or preventing the synucleinopathy in the subject.

21. The method of claim 20, wherein the engineered BoNT / A protease comprises an amino acid sequence set forth in SEQ ID NO: 15 or a conservatively modified variant thereof.

22. The method of claim 20, wherein the synucleinopathy is Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or pure autonomic failure (PAF).

23. The method of claim 20, wherein the engineered BoNT / A protease further comprises a conjugated BoNT / A heavy chain that targets a specific cell type or tissue type.

24. A polynucleotide that encodes the engineered Botulinum neurotoxin A (BoNT / A) protease of claim 10.

25. The polynucleotide of claim 24, wherein the engineered Botulinum neurotoxin A (BoNT / A) protease comprises an amino acid sequence set forth in SEQ ID NO: 15 or a conservatively modified variant thereof.TSRI 2277.1PC 26. An expression vector that expresses the engineered Botulinum neurotoxin A (BoNT / A) protease of claim 10.

27. The expression vector of claim 26, which is a viral vector.

28. The expression vector of claim 27, wherein the viral vector is an adeno-associated virus (AAV) vector or a retroviral vector.

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