WO2023152504A1 - An affinity-directed phosphatase system for targeted protein dephosphorylation - Google Patents

An affinity-directed phosphatase system for targeted protein dephosphorylation Download PDF

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Publication number
WO2023152504A1
WO2023152504A1 PCT/GB2023/050305 GB2023050305W WO2023152504A1 WO 2023152504 A1 WO2023152504 A1 WO 2023152504A1 GB 2023050305 W GB2023050305 W GB 2023050305W WO 2023152504 A1 WO2023152504 A1 WO 2023152504A1
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protein
gfp
flag
fusion protein
ppp2ca
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PCT/GB2023/050305
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French (fr)
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Gopal SAPKOTA
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University Of Dundee
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03016—Phosphoprotein phosphatase (3.1.3.16), i.e. calcineurin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present invention relates to a fusion protein comprising a phosphatase component linked to a protein binder of a target protein of interest.
  • the present invention relates to a fusion protein comprising a PP1 or a PP2A catalytic subunit linked to a polypeptide binder of a target protein of interest to dephosphorylate the target protein.
  • the present invention also includes associated methods and uses of the fusion protein in the treatment of neurodegenerative diseases and cancer.
  • Protein phosphorylation is a reversible post-translation modification (PTM) which involves the covalent addition of a phosphate group to primarily serine, threonine or tyrosine residues on the surface of a protein (Cohen, 2002, Ardito et al. , 2017). Protein phosphorylation is catalysed by protein kinases, while protein phosphatases mediate the reverse reaction by catalysing the hydrolysis of the phosphorylated amino acid residue (Ingebritsen and Cohen, 1983). The phosphorylation and dephosphorylation of a protein can alter protein function by potentially modulating its enzymatic activity, folding, stability, subcellular localisation and protein-protein interactions (Johnson and Lewis, 2001).
  • PTM post-translation modification
  • PP1 regulators There are many regulatory subunits, with at least 200 known PP1 regulators, and four classes of PP2A regulators (B/B55, B7B56, B”/PR72 and Striatin) that are each expressed as multiple different isoform variants (Brautigan, D. L., et al. (2016)).
  • PP1 regulators bind directly to the catalytic domain via short linear motifs (SLiMs), most commonly via an RVxF consensus sequence
  • the PP2A regulatory subunits assemble with invariable catalytic and scaffolding subunits into a heterotri meric holoenzyme fusion protein.
  • the B56 regulatory subunit then binds directly to SLiMs to target this holo-enzyme fusion protein to specific substrates (Brautigan, D. L., et al. (2018)).
  • phospho-POl phosphorylated proteins of interest
  • phosphatase catalytic subunits such as catalytic subunits of PP1 (PPP1CA) or PP2A (PPP2CA) (Hoermann et al., 2020, Ogris et al., 1999b, Casamayor and Ariho, 2020) are artificially recruited to different phospho-POIs via POI-specific polypeptide binders.
  • PPP1CA catalytic subunits of PP1
  • PPP2CA PP2A
  • This approach has been named an affinity-directed phosphatase (AdPhosphatase).
  • Heterobifunctional fusion proteins wherein a protein phosphatase is conjugated to a small molecule compound are known in the art (Chen et al., Yamazoe et al.). However, numerous disadvantages are associated with the existing approaches.
  • Yamazoe et al. have developed a heterobifunctional fusion protein wherein an AKT inhibitor or an EGDR inhibitor is conjugated to a phosphatase recruitment domain and a cell penetration domain.
  • Yamazoe et al. have shown this approach only results in dephosphorylation at very high concentrations due to poor intracellular penetration and instability toward cellular hydrolyases.
  • the present fusion protein has a number of advantages over these approaches. Such advantages include, but are not limited to, providing beautifully substrate- 1 eve I phospho-control without affecting phospho-status of other substrates of individual kinases, thus providing high target specificity and a demonstrated dependence on the phosphatase activity. Additionally, the fusion protein can be expressed directly in the cell or provided as a protein fusion protein, therefore does not require modifying an endogenous phosphatase or the POI, thereby simplifying the process of targeted dephosphorylation. The present fusion protein may also provide additional advantages for use as a research tool. Such examples include, but are not limited to, targeting a POI that is tagged with a marker protein e.g.
  • GFP, RFP, YFP etc. using a fusion protein of the present invention.
  • Such uses provide high specificity not observed with conventional approaches such as treatment with kinase inhibitors and is less technically challenging than the Halo tag approach as described in Chen et al.
  • the present fusion protein can employ an antigen-stabilised protein binder.
  • An antigen-stabilised protein binder is characterised in that the stability of the protein binder is dependent on the presence of its target protein. When the target protein is present, the protein binder is stable. However, when the target protein is absent the binder is unstable and degraded. This is particularly advantageous in the present fusion protein as the phosphatase component that is linked to the protein binder is also degraded in the absence of the target protein. This increases the specificity of the present invention and ultimately reduces off-target dephosphorylation.
  • the AdPhosphatase system can efficiently and selectively target specific proteins of interest for dephosphorylation.
  • the AdPhosphatase system is versatile and adaptable, where, in principle, any phosphatase can be redirected to dephosphorylate any phospho-POl in cells and tissues. This technology offers an excellent opportunity for dissecting the role of phosphorylation on potentially any POI due to its extraordinar specificity.
  • AdPhosphatase system is also suitable for therapeutic exploitation.
  • Substantial research has gone into developing specific protein kinase inhibitors in both therapeutic contexts and for studying cell signalling processes.
  • highly selective protein kinase inhibitors block phosphorylation of all downstream substrates and are known to elicit off-target effects.
  • the AdPhosphatase system has the potential to achieve extremely substrate- 1 eve I phospho-control and allow for dephosphorylation of phospho-substrates that may be phosphorylated by multiple upstream kinases, while not affecting the phospho-status of other substrates of individual kinases.
  • Targeted dephosphorylation is an exciting and promising new therapeutic modality and the AdPhosphatase system can be exploited to rapidly inform the utility of targeted POI dephosphorylation in any therapeutic application.
  • a fusion protein comprising a phosphatase component linked to at least one polypeptide protein binder.
  • the polypeptide protein binder of the fusion protein is an antigen-stabilised protein binder.
  • An antigen-stabilised protein binder refers to a protein that is unstable in the absence of its cognate antigen.
  • the antigen-stabilised protein binder as provided herein becomes stabilised when it binds its cognate antigen. It will be apparent to the skilled person that using an antigen- stabilised protein binder will increase the turnover rate of the fusion protein and reduce off- target effects.
  • the phosphatase component of the fusion protein selectively dephosphorylates a protein of interest (POI).
  • POI may be any phosphorylated protein.
  • the POI is phosphorylated at one or more serine, threonine and/or tyrosine residue.
  • dephosphorylation of the POI may modify the enzymatic activity, folding, conformation, stability, subcellular localisation and protein-protein interactions of the POI.
  • dephosphorylation of the POI by the fusion protein of the present invention may increase the enzymatic activity of the POI.
  • increasing the enzymatic activity it may be meant that the enzymatic activity of the dephosphorylated protein has increased by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 fold change compared to the phosphorylated POI.
  • dephosphorylation of the POI by the fusion protein of the present invention may decrease the enzymatic activity of the POI.
  • decreasing the enzymatic activity it may be meant that the enzymatic activity of the POI is reduced by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98 or 100% compared to the phosphorylated POI.
  • decreasing the enzymatic activity it may be meant that the enzymatic activity of the POI is reduced by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 fold change compared to the phosphorylated POI.
  • the fusion protein of the present invention regulates the activity of a POI by dephosphorylation of a POI that harbours a reversible post-translational phosphorylation.
  • the phosphatase component of the present invention can be essentially any phosphatase or binder of a phosphatase suitable to induce dephosphorylation of a target protein of interest.
  • the phosphatase component may be a phosphatase, a catalytic subunit of a phosphatase, a regulatory subunit of a phosphatase or any motif capable of recruiting a phosphatase.
  • PP1 and PP2A are ubiquitously expressed and highly conserved enzymes that are largely responsible for dephosphorylation of phospho-POIs.
  • less promiscuous phosphatases can also be employed as part of a fusion protein of the present invention, such examples include the metal-dependent protein phosphatase PPM1 H.
  • isoforms of the various phosphatases as described herein may be suitable for use in the present invention.
  • an isoform of PP1 may be PP1A, PP1 B or PP1y.
  • the phosphatase component is PP1A or a functional variant thereof.
  • the phosphatase component is PP2A or a functional variant thereof.
  • the phosphatase component is PPM1 H or a functional variant thereof.
  • the phosphatase component is any phosphatase selected from Table 1 or any isoforms thereof.
  • the phosphatase component is a phosphatase catalytic subunit.
  • the phosphatase catalytic subunit may be the catalytic subunit of any phosphatase selected from Table 1.
  • the phosphatase component of the fusion protein is a catalytic subunit of PP1.
  • the catalytic subunit may be PPP1CA or a functional variant thereof.
  • a functional variant comprises a sequence that is at least 60% identical to wild type PPP1CA (SEQ ID NO: 166; NCBI Gene ID: 5499), preferably at least 70, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to wild type PPP1CA (SEQ ID NO: 166; NCBI Gene ID: 5499).
  • a functional variant also comprises any variant of PPP1CA that retains the capacity to dephosphorylate the protein of interest.
  • the phosphatase component is the catalytic subunit of PP2A.
  • phosphatase component may be PPP2CA or a functional variant thereof.
  • a functional variant comprises a sequence that is at least 60% identical to wild type PPP2CA (SEQ ID NO: 169; NCBI GENE ID: 5515), preferably at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to wild type PPP2CA (SEQ ID NO: 169; NCBI GENE ID: 5515).
  • a functional variant also comprises any variant of PPP2CA that retains the capacity to dephosphorylate the protein of interest.
  • the phosphatase component is an apo-phosphatase, preferably PPM1 H.
  • the phosphatase component is PPM1 H or a functional variant thereof.
  • a functional variant comprises a sequence that is at least 60% identical to wild type PPM1 H (SEQ ID NO: 172; NCBI Gene ID: 57460), preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to wild type PPM1 H (SEQ ID NO: 172; NCBI Gene ID: 57460).
  • a functional variant also comprises any variant of PPM1 H that retains the capacity to dephosphorylate the protein of interest.
  • the phosphatase component may be the catalytic subunit of PPM1 H.
  • the phosphatase component is a binder of a phosphatase.
  • a binder of a phosphatase may be a regulatory phosphatase subunit or a short linear motif that can recruit a phosphatase.
  • Regulatory phosphatase subunits suitable for use in the present invention include, but are not limited to, PP2A family regulatory subunits including: B (commonly known as B55/PR55), B' (B56/PR61), B" (PR48/PR72/PR130), and B"' (PR93/PR110).
  • B55/PR55 may include any of the a, p, y, 5 isoforms.
  • B56/PR61 may include any of the a, p, y, 6, £ isoforms.
  • Regulatory phosphatase subunits suitable for use in the present invention may also include PP1A regulatory subunits.
  • Suitable PP1A regulatory subunits include any regulatory protein that contains a RVxF core PP1 binding motif.
  • PP1A regulatory subunits may include any of PPP1R1A, PPP1R1B, PPP1R1C, PPP1R2, PPP1R3A, PPP1R3B, PPP1R3C, PPP1R3D, PPP1R3E, PPP1R3F, PPP1R3G, PPP1R7, PPP1R8, PPP1R9A, PPP1R9B, PPP1R10, PPP1R11, PPP1R12A, PPP1R12B, PPP1R12C, TP53BP2, PPP1R13B, PPP1R14A, PPP1R14B, PPP1R14C, PPP1R14D, PPP1R15A, PPP1R15B, PPP1R16A, PPP1R16B, PPP1R17, PPP1R18, URI1, CCDC8, PPP1R21, CHCHD3, CHCHD6, CSMD1, DDX31,
  • PP1A and PP2A are multi-protein complexes that contain catalytic subunits and also contain regulatory proteins.
  • Polypeptide protein binders
  • polypeptide protein binders are stable regardless of antigen expression.
  • the fusion protein described herein may use traditional polypeptide protein binders such as, without limitation, antibodies, antibody fragments, monobodies, nanobodies, protein scaffolds to bind a protein of interest.
  • polypeptide protein binders such as, without limitation, antibodies, antibody fragments, monobodies, nanobodies, protein scaffolds to bind a protein of interest.
  • an antigen-stabilised polypeptide protein binder to bind a protein of interest.
  • an antigen- stabilised polypeptide protein binder relies on the presence its cognate antigen for stability, and as such provides conditional stability to the fusion protein. This allows fusion protein to be used in a cell-specific manner and limits non-specific dephosphorylation.
  • any polypeptide protein binder is suitable for use in the fusion protein of the present invention.
  • the ability of any putative polypeptide protein binder including antigen-stabilised protein binders to function in the context of the present invention can be assessed using the methodologies described herein or using techniques commonly known in the art (see e.g. Tang et al., 2016. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. eLife, 5.).
  • IP immunoprecipitation
  • binder performs as desired in IP
  • its ability to specifically bind to the POI in a fusion protein can also be assessed using IP.
  • the ability of the fusion protein to dephosphorylate a POI can be determined by assessing the phosphorylation status of the POI using an electrophoretic mobility shift assay (see e.g. Example 2), or using other techniques widely known in the art such as mass spectrometry.
  • the phosphorylation status of specific phosphorylation sites can also be used to assess the ability of the fusion protein to dephosphorylate a protein of interest using techniques commonly known in the art such as enzyme-linked immunosorbent assays, flow cytometry and phospho-IP.
  • the polypeptide protein binder is an antigen-stabilised protein binder.
  • the polypeptide protein binder is an antibody, an antibody fragment, a monobody and/or a nanobody.
  • any polypeptide protein binder may or may not be modified to be antigen-stabilised.
  • the polypeptide protein binder harbours at least one destabilising mutation.
  • a nanobody as used herein may refer to a single domain antibody derived from heavy-chain only (VHH) antibodies.
  • a nanobody includes any of monomeric, dimeric, bispecific or multivalent nanobodies that are specific for a POI and are capable of inducing dephosphorylation of the POI when part of the fusion protein of the present invention.
  • a nanobody harbours a destabilising mutation in the structurally conserved framework region of the nanobody.
  • Such nanobodies are referred to herein as destabilised nanobodies (dNb).
  • alpaca-derived dNbs can be generated by introducing 3 major transferable destabilising mutations (S73R, C/S98Y, S117F) into the structurally conserved scaffold regions of Nbs.
  • dNbs can also be generated by introducing A25V, E63V, S73R, C/S98Y, Q109H and S117F mutations into any target-specific nanobody.
  • screening assays can be performed to identify antigen- stabilised mutations. Such screening assays are known in the art, for example see Tang et al., 2016. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. Elife, 5.).
  • the polypeptide protein binder component of the fusion protein of the present invention is a destabilised nanobody.
  • the destabilised nanobody is aGFPeM or a variant thereof.
  • a functional variant of aGFPeM suitably comprises a sequence that is at least 60% identical to aGFPeM, more preferably at least 70, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to aGFP 6 M (SEQ ID NO: 164).
  • the functional variant of aGFPeM retains a similar or higher affinity for GFP compared to aGFPeM, e.g. at least 50%, 60%, 70%, 80%, 90% or 100% of the affinity for GFP as aGFPeM.
  • Nanobodies compared to conventional antibodies provides numerous advantages. Small polypeptide binders are ideal for intracellular expression as they do not require fusion protein folding or disulphide bridge formation. The small size of a nanobody also allows access to hidden and/or grooved epitopes. Nanobodies are particularly useful in CNS applications due to their ability to cross the blood brain barrier.
  • nanobodies may be of camelid origin (e.g. camel, alpaca and llama) and/or shark origin. Nanobodies are non-endogenous proteins but are considered non-immunogenic or of low immunogenicity due to their high similarity with human variable heavy (VH) sequences.
  • VH human variable heavy sequences.
  • the use of a nanobody as the protein binder of the present invention may have the additional advantage of reducing immunogenicity of the fusion protein.
  • the fusion protein comprises more than one polypeptide protein binder.
  • the fusion protein comprises a plurality of protein binders.
  • the fusion protein comprises at least two, three, four or five polypeptide protein binders.
  • the fusion protein comprises two polypeptide binders.
  • the fusion protein is a dual protein binder.
  • the more than one polypeptide protein binders recognise different proteins of interest.
  • the two polypeptide protein binders recognise different proteins of interest.
  • the phosphatase component may dephosphorylate all or some of the bound proteins of interest.
  • the phosphatase component may dephosphorylate at least 2, at least 3, at least 4 or at least 5 bound proteins of interest.
  • the skilled person will understand that the optimum orientation of the polypeptide binders can be readily identified using the methodologies described in the art and using the methodologies as described herein.
  • the fusion protein of the present invention is suitable for the targeted dephosphorylation of any phosphorylated protein of interest.
  • the phosphorylated protein may be selected from the group consisting of: transcription factors, cell cycle proteins, growth factors, immune signalling proteins, autophagy proteins and any kinase that is activated by T-loop phosphorylation.
  • the polypeptide protein binder recognises any protein of interest selected from the list consisting of: FAM83D, unc-51-like kinase (LILK1), LILK2, LC3, ATG12, ATG13, ATG7, GFP, YFP, RFP, TAU, SMAD1 , SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD8, FOXO1.
  • the fusion protein of the present invention dephosphorylates any protein of the autophagy pathway, such as LILK1 , LILK2, ATG7, ATG12 and LC3. In one embodiment, the fusion protein of the present invention dephosphorylates LILK1.
  • the fusion protein of the present invention dephosphorylates one or more transcription factors.
  • the fusion protein of the present invention dephosphorylates SMAD3 transcription factors.
  • the fusion protein of the present invention dephosphorylates TFEB.
  • the fusion protein of the present invention dephosphorylates TAU, preferably in a neuronal cell.
  • any target POI of interest may be dephosphorylated by a fusion protein of the present invention. It may be desirable in some embodiments that the target POI is modified to express a tag. Suitably, in some embodiments, the target POI is tagged with GFP, RFP or YFP. Suitably, when the polypeptide binder of the present invention is anti-GFPemthe target POI expresses a GFP tag.
  • the fusion protein dephosphorylates GFP-tagged TAU.
  • the fusion protein dephosphorylates GFP-tagged SMAD3.
  • the fusion protein dephosphorylates GFP-tagged TFEB.
  • the fusion protein dephosphorylates GFP-tagged LILK1.
  • any polypeptide protein binder that selectively binds the target POI and positions the POI for dephosphorylation by the phosphatase component is suitable for use in the fusion protein.
  • the phosphatase component may dephosphorylate the POI on its own or recruit regulatory subunits to allow for dephosphorylation of the POI.
  • Suitable combinations of polypeptide protein binders and phosphatase components can be readily identified using the methodologies described herein.
  • the fusion protein as described herein may be comprised of a phosphatase component and a polypeptide protein binder that are directly conjugated or joined via a linker.
  • linker refers to any means, entity or moiety used to join two or more entities.
  • a linker can be a covalent linker or non-covalent linker.
  • covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked.
  • the linker can also be a non-covalent bond, e.g., an organometallic bond through a metal centre such as a platinum atom.
  • various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea and the like.
  • Linker moieties include, but are not limited to, chemical linker moieties, or for example a peptide linker moiety (a linker sequence).
  • linker moieties include, but are not limited to, chemical linker moieties, or for example a peptide linker moiety (a linker sequence).
  • conjugate or “conjugation” or “linked” refers to the attachment of two or more entities to form one entity.
  • a conjugate encompasses both peptide-small molecule conjugates as well as peptide-protein/peptide conjugates.
  • the fusion protein comprises a phosphatase component linked to a polypeptide protein binder via a linker, preferably a flexible linker.
  • the linker is a poly-Gly or a poly-Gly-Ala or a Poly-Gly-Ser chain.
  • the poly-Gly or poly-Gly-Ala or Poly-Gly-Ser chain may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least
  • the linker protein comprises a poly-Gly or a poly-Gly-Ala or a Poly-Gly-Ser chain ranging from 7-21 residues.
  • the linker is a valine-aspartate linker (VD linker).
  • linker provides additional control over the fusion protein.
  • the linker may alter the structure and reach of the phosphatase component of the fusion protein described herein.
  • altering the length or nature of the linker can change what phospho-residues can be dephosphorylated by the fusion protein described herein.
  • the skilled person would understand the nature of the linker to mean any of altering the length, charge, folding pattern, conformation, amino acid composition and any other property that effects the structure and/or function of a polypeptide chain.
  • nucleic acid encoding a fusion protein of the first aspect.
  • the nucleic acid encodes a phosphatase component linked to a nucleic acid encoding a polypeptide protein binder.
  • the nucleic acid encoding the fusion protein comprises a nucleic acid encoding a phosphatase component and a nucleic acid encoding a polypeptide protein binder.
  • the nucleic acid encoding the fusion protein comprises a nucleic acid encoding a phosphatase component, a nucleic acid encoding a linker protein and a nucleic acid encoding a phosphatase component.
  • the nucleic acid encoding a polypeptide protein binder may encode an antigen- stabilised protein binder.
  • the nucleic acid encoding a phosphatase component may encode PP1 or PP2A.
  • the nucleic acid encoding a phosphatase component encodes PPP1CA or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder.
  • the nucleic acid encoding PPP1CA and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein.
  • the nucleic acid encoding the phosphatase component encodes PPP1CA according to SEQ ID NO: 165 (NCBI Gene ID: 5499) or a functional variant thereof.
  • the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 165 (NCBI Gene ID: 5499).
  • the nucleic acid encoding a phosphatase component encodes PPP2CA or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder.
  • the nucleic acid encoding PPP2CA and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein.
  • the nucleic acid encoding the phosphatase component encodes PPP2CA according to SEQ ID NO: 168 (NCBI GENE ID: 5515) or a functional variant thereof.
  • the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 168 (NCBI GENE ID: 5515).
  • nucleic acid encoding a phosphatase component encodes PPM1 H or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder.
  • nucleic acid encoding PPM1 H and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein.
  • nucleic acid encoding the phosphatase component encodes PPM1 H according to SEQ ID NO: 171 (NCBI Gene ID: 57460) or a functional variant thereof.
  • the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 171 (NCBI Gene ID: 57460).
  • the nucleic acid encoding a phosphatase component encodes a catalytic component of PPM1 H or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder.
  • the nucleic acid encoding the catalytic component of PPM1 H and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein.
  • the nucleic acid encodes a nanobody, preferably a destabilised nanobody.
  • the nucleic acid encodes aGFPeM or a functional variant or equivalent thereof.
  • the nucleic acid encoding the nanobody encodes aGFPeM according to SEQ ID NO: 163 or a functional variant thereof.
  • the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 163.
  • Nucleic acids which encode a fusion protein of the invention may be wholly or partially synthetic and may include, but are not limited to, DNA, cDNA and RNA.
  • Nucleic acid sequences encoding the fusion protein of the invention can be readily prepared by the skilled person using techniques which are well known to those skilled in the art, such as those described in Sambrook et al. "Molecular Cloning", A laboratory manual, Cold Spring Harbor Laboratory Press, Volumes 1-3, 2001 (ISBN-0879695773), and Ausubel et al. Short Protocols in Molecular Biology. John Wiley and Sons, 4th Edition, 1999 (ISBN - 0471250929). Said techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of nucleic acid, (ii) chemical synthesis, or (iii) preparation of cDNA sequences.
  • PCR polymerase chain reaction
  • DNA encoding the fusion protein of the invention may be generated and used in any suitable way known to those skilled in the art, including taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA.
  • the excised portion may then be operably linked to a suitable promoter and expressed in a suitable expression system, such as a commercially available expression system.
  • a suitable expression system such as a commercially available expression system.
  • the relevant portions of DNA can be amplified by using suitable PCR primers. Modifications to the DNA sequences can be made by using site directed mutagenesis.
  • Nucleic acid sequences encoding a fusion protein of the invention may be provided as expression constructs in the form of a plasmid, vector, transcription or expression cassette.
  • Expression constructs may comprise at least one nucleic acid as described above operably linked to one or more expression control sequences, e.g. a promoter, an enhancer, a poly-A sequence, an intron or suchlike.
  • expression control sequences are sufficient to provide expression of the fusion protein in a target cell.
  • the expression may be constitutive or regulatable.
  • an expression construct comprising a nucleic acid as set out above.
  • the expression construct is a vector, e.g. an expression vector adapted for expression in a eukaryotic or prokaryotic cell.
  • the expression construct comprises a nucleic acid of the second aspect operably linked to an expression control sequence.
  • the nucleic acid may be operably linked to a promoter, an enhancer, a poly-A sequences and/or an intron.
  • the promoter is a constitutive promoter. In an alternative embodiment, the promoter is an inducible promoter.
  • protein phosphorylation is important for regulating the activity of proteins. Therefore, the skilled person will understand that it may be desirable in some instances to control or regulate when a target protein of interest is dephosphorylated by a fusion protein of the present invention. Such control may be achieved, amongst other methods, using inducible gene expression systems.
  • inducible gene expression systems are available from a variety of commercial sources, including, without limitation, Takara, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art.
  • inducible systems include inducible promoters such as the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci.
  • an expression construct as described herein may be induced to express the nucleic acid encoding the fusion protein of the present invention by the presence or absence of a system-specific inducer.
  • Suitable inducers are widely known in the art and include, but are not limited to, antibiotics (e.g. tetracycline, doxycycline, etc.), alcohols (e.g. ethanol), hormones (e.g. steroid hormones) and environmental stresses (e.g. osmolality, heat, light etc.).
  • an expression construct of the present invention may be induced to express the nucleic acid encoding the fusion protein in the presence or absence of tetracycline or derivatives thereof.
  • an expression construct as described herein may be induced to express the nucleic acid encoding the fusion protein in the presence or absence of doxycycline or derivatives thereof.
  • the expression construct comprises a nucleic acid of the second aspect operably linked to a tetracycline inducible expression control system.
  • the tetracycline control system is the tet-ON system.
  • a reverse tetracycline-controlled transactivator rtTA
  • tTA reverse tetracycline-controlled transactivator
  • Gossen et al Science 23 Jun 1995: Vol. 268, Issue 5218, pp. 1766-1769 DOI: 10.1126/science.7792603
  • tTA can no longer bind its target sequence within the tTA-dependent promoter and there is no expression from the tTA-dependent promoter.
  • the tet-ON system may include the retro-X Tet- One Inducible Expression System whereby both the tetracycline-responsive transactivator and the nucleic acid of the second aspect are combined in the same expression vector.
  • the vector expresses the Tet-On transactivator from the constitutive human PGK promoter in the forward orientation, and the nucleic acid of the second aspect from the PTRESGS promoter in the reverse orientation.
  • the Tet-transactivator specifically binds and activates transcription from the inducible promoter that controls expression of the nucleic acid of the second aspect.
  • the tetracycline control system is the tet-OFF system.
  • tetracycline-controlled transactivator tTA
  • tTA allows transcriptional activation of a tTA-dependent promoter in the absence of tetracycline or its derivatives.
  • tTA and the tTA-dependent promoter were initially created by Gossen and Bujard, 1992 (Gossen, M. and Bujard, H. (1992)‘Tight control of gene expression in mammalian cells by tetracyclineresponsive promoters’, Proceedings of the National Academy of Sciences of the United States of America, 89(12), pp. 5547-5551.
  • tTA was created by fusion of the tetracycline resistance operon (tet repressor) encoded in TnlO of Escherichia coli with the activating cycline-controlled transactivator (tTA) and the tTA-dependent promoter was created by combining the tet operator sequence and a minimal promoter from the human cytomegalovirus promoter IE (hCMV-IE).
  • tet repressor tetracycline resistance operon
  • tTA-dependent promoter was created by combining the tet operator sequence and a minimal promoter from the human cytomegalovirus promoter IE (hCMV-IE).
  • the vector is a viral vector, such as a retroviral, lentiviral, adenoviral, or adeno-associated viral (AAV) vector.
  • the vector is an AAV vector.
  • the vector is a gene therapy vector, suitably an AAV vector, an adenoviral vector, a retroviral vector, a herpes simplex vector or a lentiviral vector.
  • Lentiviral vectors have been extensively used as a gene transfer tool in the CNS and are known to be able to successfully transduce neurones, astrocytes and oligodendrocytes. They are beneficial as they have relatively large cloning capacity and because viral genes are not expressed.
  • a particularly preferred lentiviral vector system is based on HIV-1.
  • Herpes simplex viral vectors and adenoviral vectors also show potential for use in as a gene transfer tool in CNS as they show successful transduction of CNS cells but are less preferred as due to their toxicity.
  • AAV vectors have been extensively discussed in the art. AAV vectors are of particular interest as AAV vectors do not typically integrate into the genome and do not elicit immune response.
  • AAV serotypes 1 , 2, 4, 5, 8, 9 and 2g9 (AAV1 , AAV2, AAV4, AAV5, AAV8, AAV9 and AAV2g9) have been noted to achieve efficient transduction in the CNS. Therefore, AAV1 , AAV2, AAV4, AAV5, AAV8, AAV9 and derivatives thereof are particularly preferred AAV serotypes.
  • AAV9 is particularly preferred AAV vector.
  • AAV2g9 is a particularly preferred AAV vector (WO2014/144229).
  • a particularly preferred AAV vector is AAVDJ8 (Hammond et al., 2017).
  • an AAV vector comprises a viral genome which comprises a nucleic acid sequence of the present invention positioned between two inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • WO2019/028306 discloses various wild type and modified AAV vectors that can be used in the CNS.
  • the AAV vector is capable of penetrating the blood brain barrier following delivery of the AAV vector.
  • AAV vectors of the present invention are recombinant AAV viral vectors which are replication defective, lacking sequences encoding functional Rep and Cap proteins within their viral genome.
  • AAV vectors for use herein comprise a virus that has been reduced to the minimum components necessary for transduction of a nucleic acid payload or cargo of interest.
  • AAV vectors are engineered as vehicles for specific delivery while lacking the deleterious replication and/or integration features found in wild-type viruses.
  • the AAV particle of the present invention is an scAAV. In another embodiment, the AAV particle of the present invention is an ssAAV.
  • the AAV vector comprises a capsid that allows for blood brain barrier penetration following intravascular (e.g. intravenous or intraarterial) administration (see e.g. WO2014/144229, which discusses, for example, capsids engineered for efficient crossing of the blood brain barrier, e.g.
  • capsids or peptide inserts including VOY101 , VOY201 , AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, and variants thereof).
  • Viral replication cells commonly used for production of recombinant AAV viral particles include but are not limited to HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines.
  • the vector is a non-viral vector, for example using cationic polymers or cationic lipids, as is known in the art.
  • cationic polymers or cationic lipids as is known in the art.
  • Various non-viral vectors are discussed in Selene Ingusci et al. (Gene Therapy Tools for Brain Diseases. Front. Pharmacol. 10:724. doi: 10.3389)
  • a virion comprising a vector, suitably a viral vector, according to the present invention.
  • the virion is an AAV virion.
  • the invention thus further provides recombinant virions (viral particles) comprising a vector as described above.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein of the present invention as set out above.
  • a composition typically comprises at least one pharmaceutically acceptable diluent or carrier.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the pharmaceutical composition is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.
  • Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • a pharmaceutical composition comprising the fusion protein, nucleic acid, expression construct, vector, or virion as discussed above and a pharmaceutically acceptable carrier or diluent.
  • the composition is suitable to induce targeted dephosphorylation in target cells.
  • the composition is suitable to regulate levels of phosphorylation in target cells. Dephosphorylation may increase or decrease the enzymatic activity, folding, stability, subcellular localisation and protein-protein interactions of the target protein.
  • the pharmaceutical composition results in the dephosphorylation of a target protein.
  • the fusion protein or the pharmaceutical composition according to any of the aspects and embodiments provided herein dephosphorylates a phosphorylated target protein of interest in a target cell.
  • the protein of interest may be phosphorylated at any one or more serine, threonine and/or tyrosine residues.
  • Suitable target cells include any eukaryotic cell.
  • the target cell is mammalian, more preferably human, murine, rat, pig or non-human primate. In a most preferred embodiment, the target cell is a human cell.
  • the target cell is a neuronal cell, preferably a cell from the central nervous system.
  • the neuronal cell may be a primary neuronal cell or a cell of a neurone derived cell line, e.g. an immortalised cell line.
  • the target cell is a neurone, astrocytes, oligodendrocytes, microglial cells and/or ependymal cells.
  • the target cell is a cancer cell.
  • a cancer cell may be a primary cell or an immortalised cancer cell line.
  • the cancer cell may be any transformed cell, pre-cancerous cell or metastatic cell.
  • the target cell is a lymphoma, leukaemic, gastric, pancreatic, bone, breast, liver, kidney, colon, nasopharyngeal, osteosarcoma, neuroblastoma or skin cancer cell.
  • the target cell is a human bone osteosarcoma epithelial cell line, preferably LI20S osteosarcoma cells.
  • the target cell is a human retinal pigment epithelial cell line, preferably ARPE-19 cells.
  • the target cell is a murine myoblast cell, preferably C2C12 cells.
  • the target cell is a human neuroblastoma cell, preferably SK-N-MC neuroepithelioma cells.
  • the target cell is in vitro, ex vivo or in vivo.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the present invention provides a method of treatment or prevention of a disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a fusion protein, expression construct, vector, virion or pharmaceutical composition as described herein.
  • the method comprises introducing into cells of the subject a fusion protein of the present invention, expression construct, vector, virion or pharmaceutical composition as discussed above. Suitable target cells are discussed above.
  • the method comprises administering a vector or virion according to the present invention to the subject.
  • the vector is a viral gene therapy vector, for example an AAV vector.
  • a suitable inducer may also be administered.
  • the method may also comprise administering a suitable gene expression inducer.
  • the inducer may be administered concurrently with the gene therapy vector.
  • the inducer may be administered after administration of the gene therapy vector.
  • the present invention also provides a fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition as described herein for use in a method of treatment or prevention of a disease in a subject.
  • the method suitably comprises administering to said subject a therapeutically effective amount of a fusion protein or pharmaceutical composition of the present invention.
  • the fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition as discussed above is used for the treatment, prophylaxis, palliation or amelioration of a neurological disease and/or disorder.
  • the fusion protein, expression construct, vector, virion or pharmaceutical composition is for use in the treatment of a subject with a neurodegenerative disorder.
  • the neurodegenerative disorder is a tauopathy.
  • a tauopathy may include any neurodegenerative disorder with abnormal tau inclusions.
  • the neurodegenerative disorder is any of Alzheimer’s disease, frontotemporal dementia with parkinsonism-17 (FTDP-17), Pick disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Parkinson’s disease, dementia and/or multiple system atrophy.
  • the subject in need thereof has a neurodegenerative disorder or cancer.
  • the neurodegenerative disorder is a tauopathy, optionally wherein the tauopathy is Alzheimer’s disease.
  • the fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition as discussed above is used for the treatment, prophylaxis, palliation or amelioration of a cancer.
  • the fusion protein, expression construct, vector, virion or pharmaceutical composition is for use in the treatment of a subject with cancer.
  • the cancer is any one of, but not limited to, neuroblastoma, glioblastoma, lymphoma, leukaemia, hepatocellular carcinoma, myeloma, breast cancer, ovarian cancer, pancreatic cancer, testicular cancer, throat cancer, skin cancer, colorectal cancer, stomach cancer, liver cancer, lung cancer, gallbladder cancer, prostate cancer, cervical cancer, renal cell cancer and retinoblastoma.
  • the method comprises administering a fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition systemically.
  • Systemic administration may be enteral (e.g. oral, sublingual, and rectal) or parenteral (e.g. injection).
  • Suitable methods of administration may be enteral (e.g. oral, sublingual, and rectal) or parenteral (e.g. injection) including intravenous, intraarterial, intracranial, intramuscular, subcutaneous, intra-articular, intrathecal, and intradermal injections.
  • Preferred administration methods are intravenous, intraarterial, intracranial and intrathecal injection.
  • the method comprises introducing into the CNS of the subject a pharmaceutical composition as described herein.
  • a particular difficulty with introducing a vector, virion or a pharmaceutical composition in the CNS is the blood brain barrier.
  • the blood brain barrier is a semipermeable border of endothelial cells that prevents certain chemicals and molecules in the bloodstream from crossing into the extracellular fluid of the central nervous system.
  • this obstacle has been overcome by injection directly into the brain of the animal, such as intracranial injection, suitably intracerebroventricular (ICV) injection (see e.g. Keiser et al., Curr Protoc Mouse Biol. 2018 Dec;8(4):e57).
  • This method of administration can be disadvantageous for gene therapy in humans as it is difficult to perform and can be dangerous for the subject.
  • the expression cassette as described herein is introduced into the CNS by intravenous or intraarterial (e.g. intracarotid) administration of a viral vector comprising the expression cassette.
  • the viral vector is an AAV vector.
  • Intravenous or intraarterial administration of some serotypes of AAV allows penetration of the AAV vectors into the brain.
  • Intravenous or intraarterial administration is safer and less invasive than intracranial administration, while still allowing penetration through the blood brain barrier.
  • a viral gene therapy vector may be administered concurrently or sequentially with one or more additional therapeutic agents or with one or more saturating agents designed to prevent clearance of the vectors by the reticular endothelial system.
  • the dosage of the vector may be from 1x10 10 gc/kg to 1x10 15 gc/kg or more, suitably from 1x10 12 gc/kg to 1x10 14 gc/kg, suitably from 5x10 12 gc/kg to 5x10 13 gc/kg.
  • the subject in need of treatment will be a mammal, and preferably a primate, more preferably a human.
  • the subject in need thereof will display symptoms characteristic of a disease, e.g. a disease discussed above, most preferably a neurodegenerative disorder or cancer.
  • the method typically comprises ameliorating the symptoms displayed by the subject in need thereof, by expressing the therapeutic amount of the therapeutic product of the invention.
  • Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo are well-known in the art and will not be discussed in detail here. Briefly, they include intramuscular injection, interstitial injection, instillation in airways, application to endothelium, intra-hepatic parenchyme, and intravenous or intra-arterial administration (e.g. intra-hepatic artery, intra- hepatic vein) of plasmid DNA vectors (naked or in liposomes) or viral vectors.
  • Various devices have been developed for enhancing the availability of DNA to the target cell. While a simple approach is to contact the target cell physically with catheters or implantable materials containing the relevant vector, more fusion protein approaches can use jet injection devices an suchlike.
  • a fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition for use as medicament, e.g. for treatment of a patient.
  • the patient is suffering from a neurodegenerative disorder or cancer.
  • a method of regulating protein activity comprising dephosphorylating a phosphorylated POI by administering a fusion protein of the present invention, a nucleic acid, an expression construct, a vector or a pharmaceutical composition to a cell or tissue containing the phosphorylated POI.
  • the cell or tissue is in vitro, ex vivo or in vivo.
  • a method for regulating protein activity comprising administering a fusion protein, a nucleic acid, an expression construct, a vector or the pharmaceutic composition of the present invention to a cell or a tissue in vitro, ex vivo or in vivo,
  • the polypeptide protein binder binds to the POI or when the POI target of the polypeptide protein binder component of the fusion protein is absent, the fusion protein destabilises;
  • the phosphatase component linked to the polypeptide protein binder selectively dephosphorylates the POI.
  • dephosphorylation of the POI increases the activity of the POI or decreases the activity of the POI.
  • Increased activity may refer to an increase of activity by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 fold change to that of the activity of the phosphorylated POI.
  • Decreased activity may refer to a decrease of activity by 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1 % compared to that of the activity of the phosphorylated POI.
  • the method may further comprise a step of inducing expression of the fusion protein by providing a suitable inducer.
  • a suitable inducer is tetracycline or derivatives thereof.
  • the fusion protein of the present invention is suitable for use as a tool to dissect the role of phosphorylation on any POI.
  • the fusion protein is applicable as a research tool.
  • a fusion protein of the present invention a nucleic acid, an expression construct, a vector, or a pharmaceutical composition as described herein for use as a research tool to regulate phosphorylation of a POI.
  • the fusion protein comprises a phosphatase component conjugated to at least one polypeptide protein binder.
  • the fusion protein dephosphorylates the POI.
  • the fusion protein regulates the activity of the POI.
  • the fusion protein is delivered to a cell, preferably a mammalian cell.
  • the cell is a central nervous system (CNS) cell.
  • the cell is a cancer cell.
  • the fusion protein of the present invention can be delivered to a cell in vitro, ex vivo or in vivo.
  • kit for use in any of the aspects and embodiments of the present invention comprises a fusion protein of the present invention, nucleic acid, expression construct, vector, virion or pharmaceutical composition as discussed above, and instructions for use.
  • Figure 1 Anti-GFP nanobody-directed recruitment of PPP1CA or PPP2CA mediates dephosphorylation of phospho-FAM83D-GFP.
  • FIG. 1 Schematic representation of anti-GFP nanobody (aGFPeM)-directed recruitment of either PPP1CA or PPP2CA to GFP-tagged FAM83D to mediate FAM83D-GFP dephosphorylation during mitosis.
  • B Wild-type (WT) and FA M83D GFP/GFP II 2 OS cells expressing FLAG-empty, FLAG-aGFPeM- PPP1CA or FLAG-aGFPeM-PPP2CA were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC) (5 pM) for 16 hr. Following incubation, mitotic (M) cells were isolated through shake-off.
  • STLC Eg5 inhibitor S-trityl-L-cysteine
  • Asynchronous (AS) cells were included as a control.
  • C U2OS FAM83D GFP/GFP FLAG-empty, FLAG-aGFP 6 M-PPP1CA or FLAG-aGFP 6 M-PPP2CA expressing cells were synchronised in mitosis using STLC and M cells were isolated through shake-off.
  • AS cells were included as a control. Cells were washed twice with ice-cold PBS, lysed and subjected to anti-GFP immunoprecipitation (IP).
  • IP anti-GFP immunoprecipitation
  • FIG. 3 PPP2CA AdPhosphatase targets phospho-GFP-ULK1 for dephosphorylation.
  • A Schematic representation of anti-GFP nanobody (aGFPeM)-directed recruitment of either PPP1CA or PPP2CA to GFP-tagged ULK1 to mediate phospho-GFP-ULK1 dephosphorylation.
  • B ARPE-19 wild-type (WT) and ULK1 GFP/GFP knock in cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA were lysed and subjected to immunoprecipitation (IP) with anti-FLAG M2 resin.
  • IP immunoprecipitation
  • ARPE-19 ULK1 GFP/GFP cells expressing FLAG-empty or FLAG-aGFPeM- PPP2CA were treated with the phosphatase inhibitors Calyculin A (Cal A, 20 nM) or Okadaic acid (OA, 1 M) for 20 min.
  • ARPE-19 ULK1 GFP/GFP cells expressing FLAG-empty, FLAG- aGFP6M-PPP2CA or FLAG- aGFPeM-PPP2CA H118Q were starved of amino acids with EBSS and treated with the lysosomal inhibitor Bafilomycin A1 (Baf-A1 , 50 nM) for 2 hr.
  • FIG. 5 FLAG-aGFP6M-PPP2CA expression mediates the recruitment of PP2A regulatory subunits.
  • U2OS FAM83D GFP/GFP cells expressing FLAG-empty, FLAG- aGFP6M-PPP2CA or FLAG-aGFPeM- PPP2CA H118Q were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC) (5 pM) for 16 hr. Following incubation, mitotic cells were isolated through shake-off and subjected to immunoprecipitation (IP) using anti-FLAG M2 resin.
  • STLC Eg5 inhibitor S-trityl-L-cysteine
  • Figure 6 TMT-labelled quantitative global phospho-proteomic analysis of FLAG- aGFP 6M -PPP2CA AdPhosphatase-mediated FAM83D-GFP and GFP-ULK1 dephosphorylation. Volcano plot following global (A) phospho- (11 ,821 unique phosphopeptides detected) and (B) total- (7,201 unique peptides detected) proteomic analysis of STLC-synchronised U2OS FAM83D GFP/GFP cells expressing FLAG-aGFP 6 M-PPP2CA compared with those expressing FLAG-aGFP6M-PPP2CA H118Q .
  • the top left quadrant indicates phosphopeptides/peptides that are downregulated in cells expressing FLAG-aGFPeM-PPP2CA over those expressing FLAG-aGFPeM-PPP2CA H118Q , coloured red, whilst the top right quadrant indicates phosphopeptides/peptides upregulated in cells expressing FLAG-aGFPeM-PPP2CA over those expressing FLAG-aGFP6M-PPP2CA H118Q , coloured blue.
  • Phospho-peptide detected corresponding to FAM83D is labelled in (A)
  • LILK1 is labelled in (C).
  • FIG. 7 Tet-inducible AdPhosphatase system for inducible dephosphorylation of endogenous TFEB-GFP: Tet-on system optimization.
  • C2C12 myoblast cells in which TFEB was homozygously tagged with a GFP tag were transduced with RetroX-Tet-One retroviruses encoding Flag-aGFP6M-PP1CA , Flag-aGFP6M-PP1CA-H125Q, Flag-aGFP6M-PP2CA, and Flag-aGFP6M-PP2CA-H118Q.
  • B A time course treatment (0-24 h) of cells with 100 ng/ml doxycycline, revealed a time-dependent dephosphorylation of TFEB-GFP, with the most robust dephosphorylation observed at 24 h.
  • the phosphatase dead mutants of both PP1 A and PP2A did not cause any dephosphorylation.
  • the expression of the AdPhosphatase constructs was detected with a Flag immunoblot, while GAPDH immunoblot served as a loading control.
  • Figure 8 Targeted dephosphorylation of endogenous Tau-GFP, knocked-in in SK-N-MC neuroepithelioma cells.
  • SK-N-MC cells in which TAU was tagged with a GFP tag were transduced with pBabeD (A) or RetroX-Tet-One (B) retroviruses encoding one or more of either Flag-aGFP6M, Flag-aGFP6M-PPP1CA, Flag-aGFP6M-PPP1CA-H125Q, Flag- aGFP6M-PPP2CA, or Flag-aGFP6M-PPP2CA-H118Q as shown.
  • A pBabeD
  • B RetroX-Tet-One
  • antibody as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site.
  • Antigen binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, I gG3, I gG4, I gA1 and lgA2.
  • the heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • nanobody as used herein may refer to single domain antibody derived from heavychain only (VHH) antibodies.
  • VHH antibodies may be of camelid origin including camels, alpacas and llamas.
  • VHH antibodies may be of shark origin.
  • Nanobody as used herein includes any of monomeric, dimeric, bispecific or multivalent nanobodies that are specific for a target protein.
  • the term ‘destabilised nanobody’ or ‘dNb’ may refer to a nanobody that naturally harbours a destabilising mutation or a destabilising mutation has been introduced into the nanobody.
  • a destabilising mutation may be in the structurally conserved framework region of the nanobody.
  • a destabilised nanobody may refer to a nanobody that is particularly susceptible to being broken down or degraded in living cells unless it is bound to its protein target inside the cell.
  • the term ‘monobody’ as used herein may refer to synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold. This class of binding proteins are built upon a diversified library of the 10th FN3 domain of human fibronectin. Various other scaffold protein-based synthetic binding proteins are known in the art, and can be used in the present invention.
  • FN3 fibronectin type III domain
  • a polypeptide protein binder as referred to herein refers to any polypeptide that recognises and binds to a target epitope.
  • the polypeptide protein binder can bind to its target when the binder is expressed in a cell or when introduced into a cell or binds to an isolated form of its target protein.
  • a polypeptide protein binder binder may include an antibody, an antibody fragment, a monobody, a nanobody and/or other types of binder based, e.g., on scaffold proteins, can be used.
  • antigen-stabilised refers to the stability of a protein when bound to its cognate antigen. Traditionally expressed protein binders are stable regardless of antigen binding. Antigen-stabilised protein binders as used herein rely on the presence of the protein binders cognate antigen for stability of the fusion protein. It will be apparent to the person skilled in the art that an antigen-stabilised protein binder does not refer to the antigen being bound to the protein when provided in the fusion protein of the present invention but refers to a protein that when it binds to its cognate antigen the protein binder becomes stabilised.
  • phosphatase component may refer to the whole or part of a phosphatase enzyme.
  • the phosphatase component may refer to any phosphatase or part of a phosphatase that is capable of inducing dephosphorylation of a protein.
  • phosphatase catalytic subunit may refer to a domain of a phosphatase protein that can form fusion proteins with other phosphatase domains such as a regulatory domains.
  • a phosphatase catalytic subunit in the context of the present invention may refer to any phosphatase domain that is capable of inducing dephosphorylation of a protein.
  • fusion protein refers to two or more associated polypeptide chains. Accordingly the two or more proteins of the present invention are ‘tethered’, ‘linked’ or ‘interlinked’. As such, these terms refer to joining the phosphatase component to the polypeptide protein binder. This may refer to the components of the fusion protein being directly conjugated or joined via a linker protein.
  • phosphorylation or ‘phosphorylated’ as used herein refer to the addition of a phosphate group to a protein.
  • Dephosphorylation or ‘dephosphorylated’ refer to the removal of a phosphate group from a protein. Proteins can be phosphorylated or dephosphorylated at more than one residue. Proteins are typically phosphorylated at serine, threonine and tyrosine residues.
  • ‘Selectivity’ as used herein refers to the binding preference of the polypeptide protein binder to target epitope. Higher selectivity may refer to a protein binder that exclusively or preferentially binds to the target epitope. In some embodiments, the more selective a protein binder, the less cross-reactive the polypeptide binder is with any protein present. Low selectivity may refer to a protein binder that binds an epitope that is shared with other proteins or is not unique to the target protein. A protein binder with high selectivity has reduced binding to a non-target proteins when compared to a protein binder with lower selectivity.
  • a ‘functional variant’ of a nucleic acid, or amino acid sequence or proteins in the context of the present invention is a variant of a reference sequence or protein that retains the ability to function in the same way as the reference sequence or protein.
  • Alternative terms for such functional variants include “biological equivalents” or “equivalents”.
  • Functional variants of nucleic acid or amino acid sequences that encode protein binders or protein binders per se refer to variants that selectively bind to the target POI.
  • suitable protein binders can be tested for their ability to bind the target POI using methods known in the art and those described herein e.g. see Example 2 either alone or in a fusion protein of the present invention.
  • nucleic acid or amino acid sequences that encode a phosphatase component or phosphatase components perse are those variants that retain the ability to dephosphorylate proteins. Suitable methods of testing the ability of a function variant, alone or in a fusion protein of the present invention are described above.
  • identity refers to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules. Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250). Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math.
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLASTTM Basic Local Alignment Search Tool
  • Bethesda, MD National Center for Biotechnology Information
  • Blastn the "Blast 2 sequences" function of the BLASTTM (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.
  • a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2.
  • the percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • vector refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention.
  • a vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell.
  • a vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide.
  • a vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated.
  • Vectors of the present invention can be episomal vectors (i.e. , that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome.
  • This definition includes both non-viral and viral vectors.
  • Non-viral vectors include but are not limited to plasmid vectors (e.g. pMA-RQ, plIC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc.
  • plasmid vectors e.g. pMA-RQ, plIC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)
  • transposons-based vectors e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vector
  • viral vectors such as artificial chromosomes (bacteria (BAG), yeast (YAC), or human (HAG)) may be used to accommodate larger inserts.
  • Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like.
  • viral vectors are replicationdeficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector.
  • some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis.
  • Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003).
  • Another example encompasses viral vectors mixed with cationic lipids.
  • expression construct may refer to a plasmid, vector, or expression cassette which comprises at least one nucleic acid .
  • An expression construct may comprise a nucleic acid operably linked to a sequence encoding an expression control sequence such as a promoter, an enhancer, polyadenylation sequence or an intron.
  • the sequence encoding an expression product e.g. gene
  • the gene typically encodes a polypeptide (protein) or RNA.
  • the gene may be a full- length cDNA or genomic DNA sequence, or any fragment, subunit or mutant thereof that has at least some desired biological activity.
  • the gene encodes a protein, it can be essentially any type of protein.
  • the protein can be an enzyme, an antibody or antibody fragment (e.g. a monoclonal antibody) or a fusion protein of the present invention.
  • ‘Expression control sequences’ as referred to herein may refer to promoters, enhancers, poly- A sequences and introns.
  • the expression control sequences in the present invention are sufficient to provide expression of the fusion protein in a target cell.
  • Expression control sequences may be constitutive or regulatable.
  • target cell refers to any cell that expresses a protein of interest.
  • a target cell can be, without limitation, a prokaryotic or eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
  • a target cell includes cancer cells and cells of the central nervous system (CNS cell).
  • CNS cell or “CNS cells” as used herein includes neurones, astrocytes, oligodendrocytes, microglial cells and/or ependymal cells.
  • cancer cell includes any cell that is transformed, harbours an oncogene, or displays uncontrolled proliferation.
  • pre-cancerous cell refers to any cell with abnormal growth characteristics.
  • metal-static cell refers to a cancer cell found at a location distant from the original tumour site.
  • compositions provided herein refers without limitation to an entity or ingredient is one that may be included in the compositions provided herein and that causes no significant adverse toxicological effects in the patient at specified levels, or if levels are not specified, in levels known to be acceptable by those skilled in the art. All ingredients in the compositions described herein are provided at levels that are pharmaceutically acceptable. For clarity, active ingredients may cause one or more side effects and inclusion of the ingredients with a side effect profile that is acceptable from a regulatory perspective for such ingredients will be deemed to be ‘pharmaceutically acceptable’ levels of those ingredients.
  • treatment may refer to reducing, ameliorating or eliminating one or more signs, symptoms, or effects of a disease or condition.
  • Treatment includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; (c) relieving the disease, i.e., causing regression of the disease; and (d) alleviating or reducing any symptoms of the disease.
  • the terms ‘inhibit’, ‘reduce’ and similar terms mean a decrease of at least about 5%, 10%, 15%; 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.
  • subject as used herein may be used interchangeably with ‘individual’ or ‘patient’, and refer to any individual subject with a disease or condition in need of prevention or treatment unless otherwise stated.
  • the subject may be a mammal, preferably a human.
  • Protein phosphorylation is a fundamental driver of all cell signalling processes and is therefore tightly regulated. Hyperphosphorylation of proteins is a known hallmark of many diseases, including cancer and neurodegenerative diseases.
  • the AdPhosphatase system can efficiently and selectively target specific proteins of interest (POIs) for dephosphorylation.
  • POIs proteins of interest
  • the AdPhosphatase system is versatile and adaptable, where, in principle, any phosphatase can be redirected to dephosphorylate any phospho-POl in cells and tissues.
  • the inventors have also demonstrated that inducible dephosphorylation is also possible, when an AdPhosphatase is placed under the expression of an inducible expression system. This technology offers an excellent opportunity to dissect the role of phosphorylation on potentially any POI.
  • Targeted dephosphorylation is also an exciting and promising new therapeutic modality and the AdPhosphatase system can be exploited to rapidly inform the utility of targeted POI dephosphorylation in any therapeutic
  • the inventors chose FAM83D, TAU, Transcription Factor-EB (TFEB) and the unc-51-like kinase 1 (LILK1) to demonstrate the ability of the fusion protein to dephosphorylate a protein of interest.
  • FAM83D Transcription Factor-EB
  • TFEB Transcription Factor-EB
  • LILK1 unc-51-like kinase 1
  • FAM83D is required for the recruitment of casein kinase 1a (CK1a) to the mitotic spindle to orchestrate proper spindle positioning and timely cell division (Fulcher et al., 2019).
  • CK1a casein kinase 1a
  • FAM83D is heavily phosphorylated at the mitotic spindle in a CK1a-dependent manner, causing a phospho-dependent electrophoretic mobility shift of approximately 25 kDa, and is subsequently degraded by the proteasome upon mitotic exit (Fulcher et al., 2019).
  • the precise role for CK1a-dependent hyperphosphorylation of FAM83D remains elusive.
  • the unc-51-like kinase 1 (LILK1) serine/threonine protein kinase functions in a fusion protein with both the autophagy-related protein 13 (ATG13) and the focal adhesion kinase family interacting protein of 200 kDa (FIP200) to regulate the initiation of autophagy (Ganley et al., 2009, Jung et al., 2009, Hosokawa et al., 2009).
  • LILK1 undergoes an intricate and diverse set of post-translational modifications (PTMs), including phosphorylation by different protein kinases at multiple residues, which can either activate or inhibit LILK1 and consequently initiation of autophagy (Zachari and Ganley, 2017).
  • PTMs post-translational modifications
  • Tau protein deposition in fibrillar lesions has been observed in numerous neurodegenerative diseases, most notably Alzheimer’s disease, and thus serves as a medically relevant phospho- POI demonstrating the potential therapeutic applicability of AdPhosphatases.
  • TFEB transcription factor EB
  • mTOR mechanistic target of rapamycin
  • FAM83D and LILK1 characterised cells harbouring endogenous GFP-tag knock ins, namely FAM83D GFP/GFP U2OS cells (Fulcher et al., 2019) and ULK1 GFP/GFP ARPE-19 cells (Simpson et al., 2020) were employed.
  • An AdPhosphatase construct was designed consisting of PPP1CA or PPP2CA conjugated to an antigen-stabilised anti-GFP nanobody (aGFPeM) (Tang et al., 2016) and a FLAG reporter ( Figure 1A).
  • aGFPeM is only stable when bound to GFP and destabilised and degraded when unbound, thereby maintaining homeostatic FLAG-aGFP6M-PPP1CA/- PPP2CA levels close to a 1 :1 ratio the target POI-GFP.
  • a key advantage of this approach would therefore be to limit the overexpression and potential off-target effects of the phosphatase (Hsu et al., 2006, Gergs et al., 2004).
  • ARPE-19 cells (ATCC, Cat# CRL-2302) are human retinal pigment epithelial cells derived from a 19-year-old male.
  • HEK293-FT cells (Invitrogen, Cat# R70007) are a clonal isolate of HEK293 cells transformed with the SV40 large T antigen.
  • U2OS cells (ATCC, Cat# HTB-96) are human epithelial bone osteosarcoma cells derived from a 15-year-old Caucasian female.
  • HEK293-FT and U2OS cells were maintained in DMEM (Life Technologies) containing 10% (v/v) foetal bovine serum (FBS, Thermo Fisher Scientific), 2 mM L-glutamine (Lonza), 100 U/ml penicillin (Lonza) and 0.1 mg/ml streptomycin (Lonza).
  • ARPE-19 cells were maintained in a 1 :1 mix of DMEM and Ham’s F-12 nutrient mix (Life Technologies) containing 15% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.
  • Cells were grown at 37°C with 5% CO2 in a water-saturated incubator. For passaging, cells were incubated with trypsin/EDTA at 37°C to detach cells.
  • the CRISPR/Cas9 genome editing system (Cong et al., 2013) was used to generate U2OS FAM83D homozygous C-terminal GFP knock in (KI) (FAM83D GFP/GFP ) cells (Fulcher et al., 2019), FAM83D knock-out (FAM83D KO) cells (Fulcher et al., 2019), and ARPE-19 ULK1 homozygous N-terminal GFP KI (ULK1 GFP/GFP ) cells (Simpson et al., 2020).
  • Retroviral pBABED-puromycin vectors encoding the desired construct (6 pg) were cotransfected with pCMV5-gag-pol (3.2 pg) and pCMV5-VSV-G (2.8 pg) (Cell Biolabs) into a 10 cm diameter dish of -70% confluent HEK293-FT cells. Briefly, plasmids were added to 1 ml Opti-MEM medium to which 24 pl of 1 mg/ml PEI was added. Following a gentle mix and incubation at room temperature for 20 min, the transfection mix was added dropwise to HEK293-FT cells. 16 hr post-transfection, fresh medium was added to the cells.
  • the retroviral medium was collected and passed through 0.45 pm sterile syringe filters.
  • Target cells -60% confluent
  • the retroviral medium was then replaced with fresh medium, and 24 hr later, the medium was again replaced with fresh medium containing 2 pg/ml puromycin for selection of cells which had integrated the constructs.
  • a pool of transduced cells were utilised for subsequent experiments following complete death of non-transduced cells placed under selection in parallel.
  • MG132 Abeam
  • CST Calyculin A
  • CST Okadaic acid
  • Bafilomycin-A1 Enzo Life Sciences
  • MRT68921 MRC PPU Reagents and Services.
  • Cells were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC, Sigma-Aldrich, 5 pM, 16 hr) (Fulcher et al., 2019, Simpson et al., 2020).
  • STLC Eg5 inhibitor S-trityl-L-cysteine
  • 5 pM 16 hr
  • mitotic cells were lysed after isolation through shake-off or after release into fresh media containing stated compounds for indicated times.
  • EBSS Earle’s balanced salt solution
  • Cells were harvested by washing twice with phosphate-buffered saline (PBS) and scraping into ice-cold lysis buffer (50 mM Tris-HCI pH 7.5, 0.27 M sucrose, 150 mM NaCI, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium p-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate and 1 % NP-40) supplemented with 1x completeTM protease inhibitor cocktail (Roche). After incubation for 10 min on ice, lysates were clarified by centrifugation at 20,000 xg for 20 min at 4°C.
  • PBS phosphate-buffered saline
  • IP immunoprecipitation
  • Cell lysates containing equal amounts of protein (10-20 pg) were resolved by SDS-PAGE and transferred to PVDF membrane.
  • Membranes were blocked in 5% (w/v) non-fat milk (Marvel) in TBS-T (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.2% Tween-20) and incubated overnight at 4°C in 5% (w/v) BSA/TBS-T or 5% (w/v) milk/TBS-T with the appropriate primary antibodies.
  • Primary antibodies used at indicated dilutions include: anti-Akt (9272S, CST, 1 :1 ,000), anti-Akt p-S473 (12694, CST, 1 :1 ,000), anti-ATG13 (SAB4200100, Sigma-Aldrich, 1 :1 ,000), anti- ATG13 p-S318 (NBP2-19127, Novus, 1 :1 ,000), anti-CK1a (A301-991A, Bethyl, 1 :1 ,000; SA527, MRC PPU Reagents & Services, 1 :1 ,000), anti-Cyclin B1 (4138, CST, 1 :1 ,000), anti- FAM83D (SA102, MRC PPU Reagents & Services, 1 :1 ,000), anti-FIP200 (17250-1-AP, Proteintech, 1 :1 ,000), anti-FLAG (A8592, Sigma-Aldrich, 1 :2,500), anti
  • HRP-coupled secondary antibodies used at indicated dilutions include: goat anti-rabbit-IgG (7074, CST, 1 :2,500), rabbit anti-sheep-IgG (31480, Thermo Fisher Scientific, 1 :5,000), goat anti-rat IgG (62-9520, Thermo Fisher Scientific, 1 :5,000), goat anti-mouse-IgG (31430, Thermo Fisher Scientific, 1 :5,000).
  • signal detection was performed using ECL (Merck) and ChemiDoc MP System (Bio-Rad). Imaged v1.49 (National Institutes of Health) was used to analyse protein bands by densitometry (Schneider et al., 2012).
  • Mouse anti-FLAG monoclonal F1804, Sigma-Aldrich
  • rabbit anti-FLAG monoclonal 14793, CST
  • sheep anti-CK1a polyclonal SA527, MRC PPU Reagents & Services
  • rabbit anti-ULK1 8054, CST
  • Donkey anti-rabbit IgG Alexa-Fluor 488 (A21206, Thermo Fisher Scientific), goat anti-mouse IgG Alexa-Fluor 594 (A11005, Thermo Fisher Scientific), goat anti-rabbit IgG Alexa-Fluor 594 (A11012, Thermo Fisher Scientific) and donkey anti-sheep IgG Alexa-Fluor 647 (A21448, Thermo Fisher Scientific) secondary antibodies were used at a 1 :500 dilution.
  • NP-40 lysis buffer Clarified lysates were incubated with Protein A agarose beads for 1 hr on a rotating wheel at 4°C to pre-clear nonspecific binding proteins and filtered through Spin-X columns by centrifugation for 5 min at 13,000 xg. Filtered extracts (5-10 mg protein) were incubated with 10-20 pl of appropriate beads for specified IP for 4 hr at 4°C on a rotating wheel. Following incubation, beads were washed 3x with standard lysis buffer. Bead-bound proteins were denatured and eluted in 2x LDS for 5 min at 95°C. Samples were then filtered through Spin-X columns to remove the beads from the eluate.
  • the filtered eluate was loaded onto a 4-12% Bis-Tris gradient gel and proteins were separated by SDS-PAGE. Gels were stained with InstantBlue and subsequently de-stained in deionised water. A small portion of the eluate was retained for analysis and validation by Western blotting. To minimise potential protein contaminants, all steps from this point were performed under a laminar flow hood. Disposable scalpels were used to cut protein bands of interest from the InstantBlue stained gels into 1-2 cm cubes, which were subsequently transferred into LoBind 1 .5 ml Eppendorf tubes. Gel pieces were washed once in HPLC grade water, and then shrank in anhydrous acetonitrile (ACN) for 5 min with gentle shaking.
  • ACN anhydrous acetonitrile
  • the ACN was aspirated, and gel pieces were re-swollen with 50 mM Tris-HCI pH 8.0 for 5 min with shaking. The shrinking-swelling process was repeated once more, and the proteins within the gel pieces were reduced with 5 mM DTT in 50 mM Tris-HCI pH 8.0 for 20 min at 65°C. Next, the proteins within the gel pieces were alkylated with 20 mM iodoacetamide (IAA) in 50 mM Tris-HCI pH 8.0 for 20 min at room temperature.
  • IAA iodoacetamide
  • Peptides were trapped on a nanoViper Trap column (2 cm x 100 pM, C185 pM, 100 A, Thermo Fisher Scientific) and subsequently separated on a 15 cm EasySpray column (Thermo Fisher Scientific) equilibrated with a flow rate of 300 nl/min. Data was acquired in the data-dependent mode, automatically switching between MS1 and MS2 acquisition. Full scan spectra (m/z 400- 1 ,600) were acquired in the orbitrap with resolution set to 60,000 at m/z 400.
  • urea lysis buffer 8 M urea, 20 mM HEPES pH 8.0, supplemented with 1 tablet of complete protease inhibitors per 25 ml lysis buffer and 1 tablet of PhosSTOP phosphatase inhibitors per 10 ml lysis buffer
  • Bioruptor® sonication 15 cycles at 30 sec intervals in LoBind Eppendorf tubes. Lysates were clarified by centrifugation at 13,000 xg for 20 min at 4°C and were then transferred to fresh LoBind Eppendorf tubes. Protein concentration was estimated using the PierceTM BCA method.
  • Equal protein from each condition were reduced with 5 mM DTT at room temperature for 30 min and alkylated with 20 mM IAA in the dark at room temperature for 15 min.
  • Samples were then digested with Lys-C (1 :100) at 30°C for 4 hr.
  • Samples were then diluted with 50 mM TEAB to a urea concentration of 1 .5 M and were then digested with trypsin (1 :20) at 30°C for 16 hr.
  • the digest was quenched with the addition of 1% FA (v/v) and samples were desalted on 200 mg SepPak C18 cartridges (Waters) and dried by SpeedVac.
  • TMT labels were resuspended in ACN, added to assigned samples and incubated for 1 hr at room temperature. Following label check by LC- MS/MS, the labelling reaction was quenched with 5% hydroxylamine for 15 min at room temperature. Labelled peptides from each condition were pooled together and dried.
  • IMAC beads were prepared from Ni-NTA superflow agarose beads that were stripped of Nickel with 100 mM EDTA and incubated in an aqueous solution of 10 mM iron(lll) chloride (FeCI3). Dried peptide fractions were reconstituted to a concentration of 0.5 pg/pl in 80% ACN/0.1% TFA. Peptide mixtures were enriched for phosphorylated peptides with 10 pl IMAC beads for 40 min. Enriched IMAC beads were loaded on Empore C18 silica packed stage tips. Stage tips were equilibrated with methanol followed by 50% ACN/0.1% FA then 1% FA.
  • FeCI3 iron(lll) chloride
  • the beads with enriched peptide were loaded onto C18 stage tips and washed with 80% ACN/0.1 % TFA.
  • Phosphorylated peptides were eluted from IMAC beads with 500 mM dibasic sodium phosphate, pH 7.0. Enriched phospho-peptides and peptides were analysed on an Orbitrap Fusion Tribrid mass spectrometer interfaced with Easy-nLC II nanoflow liquid chromatography system. Peptides were separated on an analytical column (75 pm x 50 cm, RSLC C18) at a flow rate of 280 nl/min using a step gradient of 8-22% solvent B (90% ACN/0.1 % FA) for the first 70 min, followed by 22-35% up to 103 min.
  • the total run time was set to 120 min.
  • the mass spectrometer was operated in a data-dependent acquisition mode.
  • a survey full scan MS (from m/z 350-1600) was acquired in the Orbitrap at a resolution of 120,000 at 200 m/z.
  • the AGC target for MS1 was set as 4 x 10 5 and ion filling time set at 50 ms.
  • the most intense ions with charge state >2 were isolated and fragmented using higher collision dissociation (HCD) fragmentation with 34% normalised collision energy and detected at a mass resolution of 50,000 at 200 m/z.
  • the AGC target for MS2 was set as 1 x 10 5 and ion filling time set at 100 ms, while dynamic exclusion was set for 30 s.
  • the mass spectrometry raw data were searched using Sequest HT search engines with Proteome Discoverer 2.1 (Thermo Fisher Scientific). Phosphopeptide-enriched fractions from each replicate were searched against the RefSeq protein database (version 89).
  • the search parameter used as carbamidomethylation of cysteine residues as a fixed modification. Oxidation of methionine, the phosphorylations of serine, threonine and tyrosine, were selected as dynamic modifications. Trypsin was set as the protease and a maximum of two missed cleavages were allowed. Precursor mass tolerance was set to 10 ppm, and a fragment mass tolerance of 0.05 Da was allowed.
  • Example 1 Anti-GFP nanobody-directed recruitment of PPP1CA or PPP2CA mediates dephosphorylation of mitotic phospho-FAM83D-GFP
  • AdPhosphatase fusion proteins were generated and expressed in FAM83D GFP/GFP knock in IISOS cells. The effect of the expression AdPhosphatase fusion proteins on FAM83D phosphorylation was then assessed.
  • FAM83D directs CK1a to the mitotic spindle to ensure proper spindle positioning and timely cell division (Fulcher et al., 2019). During mitosis, FAM83D also undergoes a CK1a-dependent hyperphosphorylation, causing ⁇ 25 kDa phospho-dependent electrophoretic mobility shift (Fulcher et al., 2019).
  • CK1a and the microtubule-associated protein hyaluronan- mediated motility receptor (HMMR, also referred to as RHAMM or CD168), which are validated endogenous FAM83D mitotic interactors (Fulcher et al., 2019), co-precipitated with FAM83D- GFP from mitotic extracts from cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA, suggesting that FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA do not interfere with the endogenous FAM83D-CK1a-HMMR mitotic interactions.
  • HMMR microtubule-associated protein hyaluronan- mediated motility receptor
  • Example 2 - Dephosphorylation is dependent on the catalytic activity of PPP1CA or PPP2CA
  • aGFPeM fused to catalytically dead PPP1CA FLAG-aGFP 6 M-PPP1CA H125Q
  • PPP2CA FLAG-aGFP 6 M- PPP2CA H118Q mutants
  • FAM83D is subsequently degraded by the proteasome following mitotic exit (Fulcher et al., 2019), although whether phosphorylation of FAM83D is required for its subsequent degradation is not known.
  • LI2OS FAM83D GFP/GFP cells expressing FLAG-empty, FLAG-aGFP 6 M-PPP1CA ( Figure 2B), FLAG- aGFP 6 M-PPP1CA H125Q ( Figure 2B), FLAG-aGFP 6 M-PPP2CA ( Figure 2C) or FLAG-aGFP 6 M- PPP2CA H118Q ( Figure 2C) were synchronised in mitosis using STLC and lysed at various time points following STLC washout.
  • FLAG-aGFPeM- PPP1CA or FLAG- aGFPeM-PPP2CA was expressed by retroviral transduction in ULK1 GFP/GFP knock in ARPE-19 cells (Simpson et al., 2020) to direct PPP1CA or PPP2CA to GFP-ULK1 for targeted dephosphorylation of phospho-GFP-ULK1 and to assess the impact on downstream autophagy signalling (Figure 3A).
  • GFP-ULK1 co-precipitated with IPs only from ULK1 GFP/GFP cell extracts expressing FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM- PPP2CA, but not from WT cells or ULK1 GFP/GFP cells expressing FLAG-empty control, confirming that FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA can interact only with GFP-ULK1 , but not with untagged LILK1.
  • both ATG13 and FIP200 co-precipitated in extracts from ULK1 GFP/GFP cells expressing FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA, suggesting that the expression of either FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM- PPP2CA does not interfere with the formation of the ULK1-ATG13-FIP200 complex.
  • constructs containing the catalytically dead PPP1CA (FLAG- aGFP 6M -PPP1CA H125Q ) or PPP2CA (FLAG-aGFP 6M -PPP2CA H118Q ) mutants were expressed by retroviral transduction in ARPE-19 ULK1 GFP/GFP cells ( Figure 3C).
  • GFP-ULK1 , ATG13 and FIP200 co-precipitated with IPs from ULK1 GFP/GFP cells expressing FLAG-aGFP 6M -PPP1CA, FLAG-aGFP 6M -PPP1CA H125Q , FLAG-aGFP 6M -PPP2CA or FLAG- aGFP 6M -PPP2CA H118Q , indicating that the catalytically dead AdPhosphatase constructs interact with GFP-ULK1 and do not interfere with the formation of the LILK1 complex.
  • ARPE-19 ULK1 GFP/GFP cells expressing FLAG- empty, FLAG- aGFP 6M -PPP2CA or FLAG-aGFP 6M -PPP2CA H118Q were starved of amino acids with Earle’s balanced salt solution (EBSS) and treated with the lysosomal inhibitor Bafilomycin-A1 (Baf- A1 , 50 nM) to prevent autophagosome clearance (Yoshimori et al. , 1991 , Mauvezin et al., 2015, Klionsky et al., 2016) for 2 hr, fixed and analysed using anti-ULK1 and anti-FLAG immunostaining (Figure 3D).
  • EBSS Earle’s balanced salt solution
  • ULK1 punctate structures were observed in ARPE-19 ULK1 GFP/GFP cells expressing FLAG-empty, FLAG-aGFPeM- PPP2CA or FLAG-aGFP6M-PPP2CA H118Q .
  • FLAG punctate structures some of which overlapped with LILK1 puncta, were also observed in ULK1 GFP/GFP cells expressing FLAG-aGFP 6 M-PPP2CA or FLAG-aGFP 6 M PP2CA H118Q
  • ULK1 is phosphorylated at multiple sites, including S757 by mTORCI , to inhibit autophagy (Kim et al., 2011).
  • mTORCI is inactivated and the inhibitory phospho-sites on ULK1 are removed, resulting in increased ULK1 kinase activity (Kim et al., 2011).
  • Example 4 - FLAG-aGFP6M-PPP2CA expression mediates the recruitment of PP2A regulatory subunits
  • PP2A catalytic C subunits typically only exist in a fusion protein with a scaffold/structural A subunit (PP2A A) and regulatory B subunits, of which there are 26 (Janssens and Goris, 2001 , Mumby,2007, Seshacharyulu et al., 2013).
  • the nature of the PP2A holoenzyme fusion protein determines substrate specificity, subcellular localisation and catalytic activity (Janssens and Goris, 2001 , Mumby, 2007, Seshacharyulu et al., 2013, Lambrecht et al., 2013).
  • FLAG-aGFP 6M -PPP2CA interactors identified exclusively from mitotic U2OS FAM83D GFP/GFP cells included FAM83D, CK1a and HMMR ( Figure 5C), which were validated by Western blot ( Figure 5A), whilst those identified exclusively from EBSS- treated ARPE-19 ULK1 GFP/GFP cells included ULK1 , ATG13 and FIP200 ( Figure 5C), which were also validated by Western blot ( Figure 5B).
  • FLAG-aGFP 6M -PPP2CA interactors identified that were common to both mitotic LI2OS FAM83D GFP/GFP and EBSS- treated ARPE-19 ULK1 GFP/GFP cells included PPP2CA (bait), the PP2A 65 kDa regulatory subunit A alpha (PPP2R1A) and beta (PPP2R1 B) isoforms, the PP2A 55 kDa regulatory subunit B alpha isoform (PPP2R2A), and the PP2A 56 kDa regulatory subunit delta (PPP2R5D) and epsilon (PPP2R5E) isoforms (Figure 5C).
  • Example 5 Global phospho-proteomics demonstrates remarkable specificity of the FLAG-aGFP 6M -PPP2CA AdPhosphatase system
  • ARPE-19 ULK1 GFP/GFP cells expressing FLAG-aGFP 6M -PPP2CA or FLAG-aGFP 6M - PPP2CA H118Q were starved of amino acids with EBSS for 2 hr and subjected to quantitative phospho- and total-proteomic analyses (Figure 6C & D).
  • Example 6 Inducible dephosphorylation of a phospho-POl using AdPhosphatases.
  • AdPhosphatases investigated the ability of AdPhosphatases to dephosphorylate a phospho- POIs under the control of an inducible system.
  • AdPhosphatase was placed under the control of a tetracycline-inducible expression system, namely the retro-X Tet-One Inducible Expression System whereby both the tetracyclineresponsive transactivator and the nucleic acid encoding the fusion protein are combined in the same expression vector.
  • C2C12 myoblast cells in which TFEB is tagged with a GFP tag were employed.
  • Flag-aGFP6M-PP1CA (SEQ ID NO: 185), Flag-aGFP6M- PP1CA-H125Q (SEQ ID NO: 188) , Flag-aGFP6M-PP2CA (SEQ ID NO: 186), and Flag- aGFP6M-PP2CA-H118Q (SEQ ID NO: 187) were expressed by retroviral transduction using RetroX-Tet-One retroviruses.
  • AdPhosphatase expression was induced by treating the cells harbouring the AdPhosphatase constructs with doxycycline.
  • Tauopathies are neurodegenerative disorders characterised by the deposition of abnormal tau protein in the brain. Accumulation of phosphorylated tau is a key pathological feature of Alzheimer's disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. Therefore, the inventors sought to demonstrate the utility of AdPhosphatases on the dephosphorylation of TAU.
  • the inventors employed SK-N-MC cells in which TAU was tagged with GFP. This allows the aforementioned anti-GFP AdPhosphatases constructs to be used, which target GFP and by spatial proximity can target TAU for dephosphorylation in these cells.
  • Anti-TAU immunostaining demonstrates that TAU proteins levels are similar across the various cell lines, ruling out the possibility that the phospho-stains are inadvertently confounded by TAU levels (Figure 8B, panel IB: Tau).
  • Anti-Flag immunostaining confirms that AdPhosphatase protein is only detected when doxycycline is administered to the cells, in line with expectations as these constructs being under tetracycline-inducible expression systems (Figure 8B).
  • AdPhosphatase of the present invention can induce targeted phosphorylation of therapeutically relevant targets such as TAU, and provide further evidence that inducible dephosphorylation is possible using AdPhosphatases.
  • FLAG-aGFP 6M -PPP2CA or FLAG-aGFP 6M -PPP2CA H118Q UniProt ID is indicated for the protein, as are corresponding fold changes and p-values.
  • the threshold parameters included a significance level of p ⁇ 0.05 and fold change >2. Proteins are listed in alphabetical order.
  • AdPhosphatase construct consisting of aGFP 6M conjugated to PPP2CA mediated the targeted dephosphorylation of phospho-GFP-ULK1 when expressed in ULK1 GFP/GFP cells.
  • AdPhosphatase-mediated GFP-ULK1 dephosphorylation attenuated starvation-induced autophagy to the same extent as chemical inhibition of ULK1.
  • ULK1 undergoes phosphorylation at multiple residues that orchestrate either an activating or inhibitory role on its activity and subsequently its role in autophagy initiation (Zachari and Ganley, 2017).
  • FLAG-aGFP 6M -PPP2CA AdPhosphatase system could be further employed to determine whether the phosphorylation of LILK1 is involved in the regulation of additional selective forms of autophagy, such as mitophagy, ER-phagy/reticulophagy, pexophagy, ferritinophagy, nucleophagy, lysophagy, lipophagy, glycophagy, aggrephagy and xenophagy (Gatica et al., 2018).
  • a less promiscuous phosphatase such as the metal-dependent protein phosphatase PPM1 H, which is highly selective towards a subset of Rab GTPase proteins (Berndsen et al., 2019, Khan et al., 2021 , Waschbusch et al., 2021 , Malik et al., 2021), could be employed.
  • PPM1 H is likely to dimerise, PPM1 H does not require additional scaffold or regulatory subunits to facilitate target protein dephosphorylation (Waschbusch et al., 2021), which may prove beneficial for AdPhosphatase system applications.
  • the next stage for targeted dephosphorylation as a potential therapeutic approach would be to develop heterobifunctional small-molecules that directly bind the endogenous, untagged POI and recruit the phosphatase of interest.
  • the dephosphorylation-inducing heterobifunctional smallmolecules targeting Akt and EGFR have been reported albeit with poor efficiency of targeted dephosphorylation (Yamazoe et al., 2020).
  • PhosTACs phosphorylation targeting chimeras
  • FKBP12 F36V and Halo would need to be knocked in on to the respective phosphatase and POIs using, for example, CRISPR/Cas9 genome editing technology.
  • AdPhosphatase system can be exploited not only to explore the biological role of specific phospho-POIs, but also to rapidly inform the utility of phosphatase-mediated POI dephosphorylation before investing in the resource- and time-intensive development of POI-specific dephosphorylationinducing heterobifunctional small-molecules.
  • AdPhosphatase system Protein phosphorylation is a fundamental driver of all cell signalling processes and is therefore tightly regulated. Hyperphosphorylation of proteins is a known hallmark of many diseases, including cancer and neurodegenerative diseases. This new approach termed the AdPhosphatase system, can efficiently and selectively target specific proteins of interest (POIs) for dephosphorylation.
  • the AdPhosphatase system is versatile and adaptable, where, in principle, any promiscuous phosphatase can be redirected to dephosphorylate any phospho-POI.
  • this technology allows researchers to dissect the role of phosphorylation on potentially any POI and is a promising new therapeutic modality.
  • the AdPhosphatase system can be exploited to rapidly inform the utility of targeted POI dephosphorylation in any therapeutic application.
  • I-PPP1CA Cassette DNA atggactacaaggacgatgacgataagggatccgccatggccgatgtgcagctggttgaatctggcggtgcactggtgcagcc gggcggtagcctgcgtcttgcgtggccagtggctttccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaa agaacgtgaatgggtggcaggcatgagctctgcgggtgatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtc gtc gtgatgatgcgcaacacggtgtacctgcagatgaatagtctgaaaccggaagataccgccgttattactacaacgtgtgaatgt
  • P_ashed _ un_d_eriine_ represents > _F_LAG__re_ ion; bold sequence represents aGFP(6M) sequence; double underline represents PPP1CA sequence: VD is the linker sequence.
  • ADAMS R. R. et al. 2001.
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  • Calyculin A and okadaic acid inhibitors of protein phosphatase activity. Biochem Biophys Res Commun, 159, 871-7.
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Abstract

The present invention relates to a fusion protein that comprises a phosphatase component linked to a polypeptide protein binder that is suitable for inducible targeted dephosphorylation of a phosphorylated proteins of interest. The present invention also comprises methods of regulating protein activity, compositions and methods of treatment of neurodegenerative disorders and cancer.

Description

AN AFFINITY-DIRECTED PHOSPHATASE SYSTEM FOR TARGETED PROTEIN DEPHOSPHORYLATION
Field of the Invention
The present invention relates to a fusion protein comprising a phosphatase component linked to a protein binder of a target protein of interest. In some aspects, the present invention relates to a fusion protein comprising a PP1 or a PP2A catalytic subunit linked to a polypeptide binder of a target protein of interest to dephosphorylate the target protein. The present invention also includes associated methods and uses of the fusion protein in the treatment of neurodegenerative diseases and cancer.
Background of the Invention
The following discussion is provided to aid the reader in understanding the disclosure and does not constitute any admission as to the contents or relevance of the prior art.
Protein phosphorylation is a reversible post-translation modification (PTM) which involves the covalent addition of a phosphate group to primarily serine, threonine or tyrosine residues on the surface of a protein (Cohen, 2002, Ardito et al. , 2017). Protein phosphorylation is catalysed by protein kinases, while protein phosphatases mediate the reverse reaction by catalysing the hydrolysis of the phosphorylated amino acid residue (Ingebritsen and Cohen, 1983). The phosphorylation and dephosphorylation of a protein can alter protein function by potentially modulating its enzymatic activity, folding, stability, subcellular localisation and protein-protein interactions (Johnson and Lewis, 2001). Almost all aspects of cell biology are regulated by reversible protein phosphorylation, with abnormal phosphorylation being identified as the cause of a wide range of human diseases, including many cancers and neurodegenerative diseases (Cohen, 2001 , Ardito et al., 2017). Therefore, a lot of effort has gone in to developing specific protein kinase and phosphatase inhibitors and activators in both therapeutics and for studying cell signalling processes. Nevertheless, even highly selective protein kinase inhibitors block phosphorylation of all downstream substrates, and often both kinase and phosphatase inhibitors are known to elicit off-target effects (Fabian et al., 2005, Fedorov et al., 2007, Karaman et al., 2008, Elkins et al., 2016, Tsutsumi et al., 2018, Ferguson and Gray, 2018, Smyth and Collins, 2009, Munoz, 2017, Lanning et al., 2014).
It is estimated that 98% of phosphorylated residues in the human proteome are serine or threonine and the majority of these sites are dephosphorylated by just two ubiquitously expressed enzymes: protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). These enzymes, which are two of the most highly conserved proteins in eukaryotes, function within holo-enzyme fusion proteins that contain regulatory subunits, which direct the catalytic subunits towards specific substrates. There are many regulatory subunits, with at least 200 known PP1 regulators, and four classes of PP2A regulators (B/B55, B7B56, B”/PR72 and Striatin) that are each expressed as multiple different isoform variants (Brautigan, D. L., et al. (2018)). Whereas PP1 regulators bind directly to the catalytic domain via short linear motifs (SLiMs), most commonly via an RVxF consensus sequence, the PP2A regulatory subunits assemble with invariable catalytic and scaffolding subunits into a heterotri meric holoenzyme fusion protein. For the PP2A-B56 class, the B56 regulatory subunit then binds directly to SLiMs to target this holo-enzyme fusion protein to specific substrates (Brautigan, D. L., et al. (2018)).
One approach developed by the inventors to overcome the existing problems associated with kinase inhibitors is targeted dephosphorylation of specific phosphorylated proteins of interest (phospho-POl) by redirecting a catalytically active phosphatase to a desired neo-substrate. This approach has the potential to achieve exquisite substrate-level phosphorylation control and also allows for dephosphorylation of phospho-substrates that may be phosphorylated by multiple upstream kinases, while not affecting the phospho-status of other substrates of individual kinases.
To develop a system for targeted dephosphorylation, the inventors conceptualised an affinity- directed system whereby phosphatase catalytic subunits such as catalytic subunits of PP1 (PPP1CA) or PP2A (PPP2CA) (Hoermann et al., 2020, Ogris et al., 1999b, Casamayor and Ariho, 2020) are artificially recruited to different phospho-POIs via POI-specific polypeptide binders. This approach has been named an affinity-directed phosphatase (AdPhosphatase).
Heterobifunctional fusion proteins wherein a protein phosphatase is conjugated to a small molecule compound are known in the art (Chen et al., Yamazoe et al.). However, numerous disadvantages are associated with the existing approaches.
Yamazoe et al., have developed a heterobifunctional fusion protein wherein an AKT inhibitor or an EGDR inhibitor is conjugated to a phosphatase recruitment domain and a cell penetration domain. However, Yamazoe et al., have shown this approach only results in dephosphorylation at very high concentrations due to poor intracellular penetration and instability toward cellular hydrolyases.
Chen et al., have developed a chimeric fusion protein wherein a Halo-tag ligand is linked to a small molecule such as FKBP12 (F36V). This approach mediates fusion protein formation between a phosphatase and a targeted phosphoprotein for dephosphorylation. However, for this approach to be applied to endogenously regulated POIs, both FKBP12 (F36V) and Halo need to be knocked in to the respective phosphatase and POI, which presents a technical challenge to use both as a research tool and in therapeutic applications.
The present fusion protein has a number of advantages over these approaches. Such advantages include, but are not limited to, providing exquisite substrate- 1 eve I phospho-control without affecting phospho-status of other substrates of individual kinases, thus providing high target specificity and a demonstrated dependence on the phosphatase activity. Additionally, the fusion protein can be expressed directly in the cell or provided as a protein fusion protein, therefore does not require modifying an endogenous phosphatase or the POI, thereby simplifying the process of targeted dephosphorylation. The present fusion protein may also provide additional advantages for use as a research tool. Such examples include, but are not limited to, targeting a POI that is tagged with a marker protein e.g. GFP, RFP, YFP etc., using a fusion protein of the present invention. Such uses provide high specificity not observed with conventional approaches such as treatment with kinase inhibitors and is less technically challenging than the Halo tag approach as described in Chen et al.
Furthermore, the present fusion protein can employ an antigen-stabilised protein binder. An antigen-stabilised protein binder is characterised in that the stability of the protein binder is dependent on the presence of its target protein. When the target protein is present, the protein binder is stable. However, when the target protein is absent the binder is unstable and degraded. This is particularly advantageous in the present fusion protein as the phosphatase component that is linked to the protein binder is also degraded in the absence of the target protein. This increases the specificity of the present invention and ultimately reduces off-target dephosphorylation.
The AdPhosphatase system can efficiently and selectively target specific proteins of interest for dephosphorylation. The AdPhosphatase system is versatile and adaptable, where, in principle, any phosphatase can be redirected to dephosphorylate any phospho-POl in cells and tissues. This technology offers an excellent opportunity for dissecting the role of phosphorylation on potentially any POI due to its exquisite specificity.
However, as almost all aspects of cell biology are regulated by reversible protein phosphorylation, and abnormal phosphorylation has been identified as the cause of a wide range of human diseases, including many cancers and neurodegenerative diseases, the AdPhosphatase system is also suitable for therapeutic exploitation. Substantial research has gone into developing specific protein kinase inhibitors in both therapeutic contexts and for studying cell signalling processes. However, as discussed above, even highly selective protein kinase inhibitors block phosphorylation of all downstream substrates and are known to elicit off-target effects. The AdPhosphatase system has the potential to achieve exquisite substrate- 1 eve I phospho-control and allow for dephosphorylation of phospho-substrates that may be phosphorylated by multiple upstream kinases, while not affecting the phospho-status of other substrates of individual kinases.
Finally, there would be no need to mutate phospho-sites, which may lead to unintended conformational changes to the target proteins, to understand the role of phosphorylation of the target protein.
Targeted dephosphorylation is an exciting and promising new therapeutic modality and the AdPhosphatase system can be exploited to rapidly inform the utility of targeted POI dephosphorylation in any therapeutic application.
Summary of the Invention
In a first aspect of the present invention, there is provided a fusion protein comprising a phosphatase component linked to at least one polypeptide protein binder. In a preferred embodiment, the polypeptide protein binder of the fusion protein is an antigen-stabilised protein binder.
An antigen-stabilised protein binder refers to a protein that is unstable in the absence of its cognate antigen. The antigen-stabilised protein binder as provided herein becomes stabilised when it binds its cognate antigen. It will be apparent to the skilled person that using an antigen- stabilised protein binder will increase the turnover rate of the fusion protein and reduce off- target effects.
Suitably, in some embodiments the phosphatase component of the fusion protein selectively dephosphorylates a protein of interest (POI). A POI may be any phosphorylated protein. Suitably, in some embodiments the POI is phosphorylated at one or more serine, threonine and/or tyrosine residue.
Suitably, dephosphorylation of the POI may modify the enzymatic activity, folding, conformation, stability, subcellular localisation and protein-protein interactions of the POI. In one embodiment, dephosphorylation of the POI by the fusion protein of the present invention may increase the enzymatic activity of the POI. By increasing the enzymatic activity, it may be meant that the enzymatic activity of the dephosphorylated protein has increased by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 fold change compared to the phosphorylated POI. By increasing the enzymatic activity, it may be meant that the enzymatic activity of the dephosphorylated protein has increased by at least at least 50, 60, 70, 80, 90, 100, 200, 500, 1000 or 10,000% compared to the phosphorylated POI. In another embodiment, dephosphorylation of the POI by the fusion protein of the present invention may decrease the enzymatic activity of the POI. By decreasing the enzymatic activity, it may be meant that the enzymatic activity of the POI is reduced by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98 or 100% compared to the phosphorylated POI. By decreasing the enzymatic activity, it may be meant that the enzymatic activity of the POI is reduced by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 fold change compared to the phosphorylated POI.
The skilled person will appreciate that the fusion protein of the present invention regulates the activity of a POI by dephosphorylation of a POI that harbours a reversible post-translational phosphorylation.
Phosphatases:
The phosphatase component of the present invention can be essentially any phosphatase or binder of a phosphatase suitable to induce dephosphorylation of a target protein of interest. The phosphatase component may be a phosphatase, a catalytic subunit of a phosphatase, a regulatory subunit of a phosphatase or any motif capable of recruiting a phosphatase.
PP1 and PP2A are ubiquitously expressed and highly conserved enzymes that are largely responsible for dephosphorylation of phospho-POIs. However, less promiscuous phosphatases can also be employed as part of a fusion protein of the present invention, such examples include the metal-dependent protein phosphatase PPM1 H. The skilled person will understand that isoforms of the various phosphatases as described herein may be suitable for use in the present invention. For example, an isoform of PP1 may be PP1A, PP1 B or PP1y.
Suitably, in some embodiments of the present invention, the phosphatase component is PP1A or a functional variant thereof. In another embodiment, the phosphatase component is PP2A or a functional variant thereof. In yet another embodiment, the phosphatase component is PPM1 H or a functional variant thereof.
In some embodiments of the present invention, the phosphatase component is any phosphatase selected from Table 1 or any isoforms thereof. In some embodiments of the present invention, the phosphatase component is a phosphatase catalytic subunit. Suitably, the phosphatase catalytic subunit may be the catalytic subunit of any phosphatase selected from Table 1.
Table 1 :
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
In one embodiment, the phosphatase component of the fusion protein is a catalytic subunit of PP1. In one embodiment, the catalytic subunit may be PPP1CA or a functional variant thereof. A functional variant comprises a sequence that is at least 60% identical to wild type PPP1CA (SEQ ID NO: 166; NCBI Gene ID: 5499), preferably at least 70, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to wild type PPP1CA (SEQ ID NO: 166; NCBI Gene ID: 5499). A functional variant also comprises any variant of PPP1CA that retains the capacity to dephosphorylate the protein of interest.
In another embodiment, the phosphatase component is the catalytic subunit of PP2A. In one embodiment phosphatase component may be PPP2CA or a functional variant thereof. A functional variant comprises a sequence that is at least 60% identical to wild type PPP2CA (SEQ ID NO: 169; NCBI GENE ID: 5515), preferably at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to wild type PPP2CA (SEQ ID NO: 169; NCBI GENE ID: 5515). A functional variant also comprises any variant of PPP2CA that retains the capacity to dephosphorylate the protein of interest.
In a further embodiment, the phosphatase component is an apo-phosphatase, preferably PPM1 H. Suitably, in some embodiments, the phosphatase component is PPM1 H or a functional variant thereof. A functional variant comprises a sequence that is at least 60% identical to wild type PPM1 H (SEQ ID NO: 172; NCBI Gene ID: 57460), preferably at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to wild type PPM1 H (SEQ ID NO: 172; NCBI Gene ID: 57460). A functional variant also comprises any variant of PPM1 H that retains the capacity to dephosphorylate the protein of interest. Suitably, the phosphatase component may be the catalytic subunit of PPM1 H.
In some embodiments, the phosphatase component is a binder of a phosphatase. Suitably, a binder of a phosphatase may be a regulatory phosphatase subunit or a short linear motif that can recruit a phosphatase.
Regulatory phosphatase subunits suitable for use in the present invention include, but are not limited to, PP2A family regulatory subunits including: B (commonly known as B55/PR55), B' (B56/PR61), B" (PR48/PR72/PR130), and B"' (PR93/PR110). B55/PR55 may include any of the a, p, y, 5 isoforms. B56/PR61 may include any of the a, p, y, 6, £ isoforms. Regulatory phosphatase subunits suitable for use in the present invention may also include PP1A regulatory subunits. Suitable PP1A regulatory subunits include any regulatory protein that contains a RVxF core PP1 binding motif. PP1A regulatory subunits may include any of PPP1R1A, PPP1R1B, PPP1R1C, PPP1R2, PPP1R3A, PPP1R3B, PPP1R3C, PPP1R3D, PPP1R3E, PPP1R3F, PPP1R3G, PPP1R7, PPP1R8, PPP1R9A, PPP1R9B, PPP1R10, PPP1R11, PPP1R12A, PPP1R12B, PPP1R12C, TP53BP2, PPP1R13B, PPP1R14A, PPP1R14B, PPP1R14C, PPP1R14D, PPP1R15A, PPP1R15B, PPP1R16A, PPP1R16B, PPP1R17, PPP1R18, URI1, CCDC8, PPP1R21, CHCHD3, CHCHD6, CSMD1, DDX31, PPP1R26, PPP1R27, ELFN1, ELFN2, GPATCH2, HYDIN, PPP1R32, ARFGEF3, MARF1, PPP1R35, PPP1R36, PPP1R37, SH2D4A, SH3RF2, TRIM42, ZCCHC9, PPP1R42, AKAP1, AKAP11, AKAP9, APC, AURKA, AURKB, AXIN1, BCL2, BCL2L1, BCL2L2, BRCA1, DNAI7, KNL1, CASP9, CASP2, DLG2, ,CD2BP2, CDC25C, CENPE, CEP192, CLCN7, CNST, ANKRD28, DZIP3, EIF2S2, ELL, ZFYVE16, SH3GLB1, PTK2, CSRNP2, CSRNP3, FER, FARP1, FKBP15, AATK, AHCYL1, ANKRD42, CAMSAP3, CDCA2, DLG3, EIF2AK2, GPR12, GRM1, GRM5, GRM7, GRXCR1, HCFC1, HDAC6, HSPB6, IKZF1, ITGA2B, ITPR1, ITPR3, KCNA6, KCNK10, KIF18A, LMTK2, LMTK3, MAP1B, MAPT, MCM7, MKI67, MPHOSPH10, MYO16, MYO1D, NCOR1, NEFL, NEK2, NOC2L, NOM1, NONO, OCLN, OPN3, ORC5, PARD3, PCDH11X, PCDH7, PCIF1, PFKM, PHACTR3, PHACTR4, PHRF1, PKMYT1, PLCL1, POLD3, PREX2, RB1CC1, RBM26, RIMBP2, RPGRIP1L, RPL5, RRP1B, RYR1, SACS, SFI1, SFPQ, SLC12A2, SLC7A14, SLC9A1, SMARCB1, SPATA2, SPOCD1, SPRED1, SPZ1, SRSF10, STAU1, SYTL2, TMEM132C, TMEM132D, TMEM225, TNS1, TRA2B, TRIM28, TRPC4AP, TRPC5, TSC2, TSKS, UBN1, VDR, VPS54, WBP11, WDR81, WNK1, VWVC1, YLPM1.
The skilled person will understand that PP1A and PP2A are multi-protein complexes that contain catalytic subunits and also contain regulatory proteins.
It should be noted that, while the present invention has been demonstrated in the specific examples below using PPP1CA and PPP2CA as the phosphatase component, it will be apparent to the skilled person that other phosphatase components can be used. The suitability of any putative phosphatases, phosphatase catalytic subunits, phosphatase regulatory subunits, or short linear motifs that serve to recruit a phosphatase for use in the present invention can readily be assessed by substituting the candidate phosphatase components for PPP1CA or PPP2CA in the examples described below.
Polypeptide protein binders:
Conventional polypeptide protein binders are stable regardless of antigen expression. The fusion protein described herein may use traditional polypeptide protein binders such as, without limitation, antibodies, antibody fragments, monobodies, nanobodies, protein scaffolds to bind a protein of interest. Likewise, it is also suitable to use an antigen-stabilised polypeptide protein binder to bind a protein of interest. Without wishing to be bound by theory, an antigen- stabilised polypeptide protein binder relies on the presence its cognate antigen for stability, and as such provides conditional stability to the fusion protein. This allows fusion protein to be used in a cell-specific manner and limits non-specific dephosphorylation.
While in the examples below nanobodies have been shown to be particularly useful polypeptide protein binders, the skilled person will appreciate that any polypeptide protein binder is suitable for use in the fusion protein of the present invention. The ability of any putative polypeptide protein binder including antigen-stabilised protein binders to function in the context of the present invention can be assessed using the methodologies described herein or using techniques commonly known in the art (see e.g. Tang et al., 2016. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. eLife, 5.).
As an initial screen to test candidate polypeptide protein binders, the interaction between any binder and its cognate antigen can be carried out by immunoprecipitation (IP). Thereafter, assuming binder performs as desired in IP, its ability to specifically bind to the POI in a fusion protein can also be assessed using IP. Finally, the ability of the fusion protein to dephosphorylate a POI can be determined by assessing the phosphorylation status of the POI using an electrophoretic mobility shift assay (see e.g. Example 2), or using other techniques widely known in the art such as mass spectrometry. The phosphorylation status of specific phosphorylation sites can also be used to assess the ability of the fusion protein to dephosphorylate a protein of interest using techniques commonly known in the art such as enzyme-linked immunosorbent assays, flow cytometry and phospho-IP.
In some embodiments, the polypeptide protein binder is an antigen-stabilised protein binder. In one embodiment the polypeptide protein binder is an antibody, an antibody fragment, a monobody and/or a nanobody. Suitably, any polypeptide protein binder may or may not be modified to be antigen-stabilised. In some embodiments, the polypeptide protein binder harbours at least one destabilising mutation.
A nanobody as used herein may refer to a single domain antibody derived from heavy-chain only (VHH) antibodies. In some embodiments, a nanobody includes any of monomeric, dimeric, bispecific or multivalent nanobodies that are specific for a POI and are capable of inducing dephosphorylation of the POI when part of the fusion protein of the present invention.
In one embodiment, a nanobody harbours a destabilising mutation in the structurally conserved framework region of the nanobody. Such nanobodies are referred to herein as destabilised nanobodies (dNb). It is known in the art that alpaca-derived dNbs can be generated by introducing 3 major transferable destabilising mutations (S73R, C/S98Y, S117F) into the structurally conserved scaffold regions of Nbs. dNbs can also be generated by introducing A25V, E63V, S73R, C/S98Y, Q109H and S117F mutations into any target-specific nanobody. For other protein binders, screening assays can be performed to identify antigen- stabilised mutations. Such screening assays are known in the art, for example see Tang et al., 2016. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. Elife, 5.).
In some embodiments, the polypeptide protein binder component of the fusion protein of the present invention is a destabilised nanobody. In one embodiment, the destabilised nanobody is aGFPeM or a variant thereof. A functional variant of aGFPeM suitably comprises a sequence that is at least 60% identical to aGFPeM, more preferably at least 70, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to aGFP6M (SEQ ID NO: 164). Preferably the functional variant of aGFPeM retains a similar or higher affinity for GFP compared to aGFPeM, e.g. at least 50%, 60%, 70%, 80%, 90% or 100% of the affinity for GFP as aGFPeM.
The small size of nanobodies compared to conventional antibodies provides numerous advantages. Small polypeptide binders are ideal for intracellular expression as they do not require fusion protein folding or disulphide bridge formation. The small size of a nanobody also allows access to hidden and/or grooved epitopes. Nanobodies are particularly useful in CNS applications due to their ability to cross the blood brain barrier.
In some embodiments of the present invention, nanobodies may be of camelid origin (e.g. camel, alpaca and llama) and/or shark origin. Nanobodies are non-endogenous proteins but are considered non-immunogenic or of low immunogenicity due to their high similarity with human variable heavy (VH) sequences. Suitably, therefore, the use of a nanobody as the protein binder of the present invention may have the additional advantage of reducing immunogenicity of the fusion protein.
It is also apparent to the skilled person that in some instances it may be desirable to dephosphorylate more than one protein of interest simultaneously. Suitably, in some embodiments the fusion protein comprises more than one polypeptide protein binder. In some embodiments, the fusion protein comprises a plurality of protein binders. Suitably, in some embodiments the fusion protein comprises at least two, three, four or five polypeptide protein binders. In one embodiment, the fusion protein comprises two polypeptide binders. Suitably, in such embodiments the fusion protein is a dual protein binder. In some embodiments, the more than one polypeptide protein binders recognise different proteins of interest. In one embodiment, the two polypeptide protein binders recognise different proteins of interest. Suitably, the phosphatase component may dephosphorylate all or some of the bound proteins of interest. Suitably, in one embodiment, the phosphatase component may dephosphorylate at least 2, at least 3, at least 4 or at least 5 bound proteins of interest. The skilled person will understand that the optimum orientation of the polypeptide binders can be readily identified using the methodologies described in the art and using the methodologies as described herein.
The fusion protein of the present invention is suitable for the targeted dephosphorylation of any phosphorylated protein of interest. Suitably, the phosphorylated protein may be selected from the group consisting of: transcription factors, cell cycle proteins, growth factors, immune signalling proteins, autophagy proteins and any kinase that is activated by T-loop phosphorylation.
In some embodiments, the polypeptide protein binder recognises any protein of interest selected from the list consisting of: FAM83D, unc-51-like kinase (LILK1), LILK2, LC3, ATG12, ATG13, ATG7, GFP, YFP, RFP, TAU, SMAD1 , SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD8, FOXO1. FOXO3, FOXO4, FOXO6, FOXP3, NFKB, AHR, TFEB, CDK1 , CDK4, CDK5, PLK, AURK, NRK, GATA1 , GATA2, GATA3, GATA4, GATA5, GATA6, VEGRK1. VEGRK2, VEGRK3, EGFR, MAPK, ERK1 , ERK2, Rab GTPases, PKC, BTK, JAK1 , JAK2, JAK3, SYK, AKT, STAT1 , STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6.
In some embodiments, the fusion protein of the present invention dephosphorylates any protein of the autophagy pathway, such as LILK1 , LILK2, ATG7, ATG12 and LC3. In one embodiment, the fusion protein of the present invention dephosphorylates LILK1.
In some embodiments, the fusion protein of the present invention dephosphorylates one or more transcription factors. Suitably, in one embodiment, the fusion protein of the present invention dephosphorylates SMAD3 transcription factors. In another embodiment, the fusion protein of the present invention dephosphorylates TFEB.
In one embodiment, the fusion protein of the present invention dephosphorylates TAU, preferably in a neuronal cell.
The skilled person will understand that any target POI of interest may be dephosphorylated by a fusion protein of the present invention. It may be desirable in some embodiments that the target POI is modified to express a tag. Suitably, in some embodiments, the target POI is tagged with GFP, RFP or YFP. Suitably, when the polypeptide binder of the present invention is anti-GFPemthe target POI expresses a GFP tag.
In one embodiment of the present invention, the fusion protein dephosphorylates GFP-tagged TAU.
In one embodiment, the fusion protein dephosphorylates GFP-tagged SMAD3.
In another embodiment, the fusion protein dephosphorylates GFP-tagged TFEB.
In yet another embodiment, the fusion protein dephosphorylates GFP-tagged LILK1.
It is believed that any polypeptide protein binder that selectively binds the target POI and positions the POI for dephosphorylation by the phosphatase component is suitable for use in the fusion protein. The phosphatase component may dephosphorylate the POI on its own or recruit regulatory subunits to allow for dephosphorylation of the POI. Suitable combinations of polypeptide protein binders and phosphatase components can be readily identified using the methodologies described herein.
Linkers
The fusion protein as described herein may be comprised of a phosphatase component and a polypeptide protein binder that are directly conjugated or joined via a linker.
The term “linker” refers to any means, entity or moiety used to join two or more entities. A linker can be a covalent linker or non-covalent linker. Examples of covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked. The linker can also be a non-covalent bond, e.g., an organometallic bond through a metal centre such as a platinum atom. For covalent linkages, various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea and the like. To provide for linking, the domains can be modified by oxidation, hydroxylation, substitution, reduction etc. to provide a site for coupling. Methods for conjugation are well known by the person skilled in the art and are encompassed for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or for example a peptide linker moiety (a linker sequence). As used herein, the term “conjugate” or “conjugation” or “linked” refers to the attachment of two or more entities to form one entity. A conjugate encompasses both peptide-small molecule conjugates as well as peptide-protein/peptide conjugates. Suitably, in some embodiments of the present invention the fusion protein comprises a phosphatase component linked to a polypeptide protein binder via a linker, preferably a flexible linker.
In some embodiments, the linker is a poly-Gly or a poly-Gly-Ala or a Poly-Gly-Ser chain. Suitably, the poly-Gly or poly-Gly-Ala or Poly-Gly-Ser chain may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least
11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 30, at least
50, at least 100 residues. Preferably, the linker protein comprises a poly-Gly or a poly-Gly-Ala or a Poly-Gly-Ser chain ranging from 7-21 residues.
In an alternative embodiment, the linker is a valine-aspartate linker (VD linker).
Altering the nature of the linker provides additional control over the fusion protein. The linker may alter the structure and reach of the phosphatase component of the fusion protein described herein. The skilled person would understand that altering the length or nature of the linker can change what phospho-residues can be dephosphorylated by the fusion protein described herein. The skilled person would understand the nature of the linker to mean any of altering the length, charge, folding pattern, conformation, amino acid composition and any other property that effects the structure and/or function of a polypeptide chain.
Nucleic Acids and Vectors:
In a second aspect of the present invention, there is provided a nucleic acid encoding a fusion protein of the first aspect.
In some embodiments the nucleic acid encodes a phosphatase component linked to a nucleic acid encoding a polypeptide protein binder. In some embodiments, the nucleic acid encoding the fusion protein comprises a nucleic acid encoding a phosphatase component and a nucleic acid encoding a polypeptide protein binder. In some embodiments, the nucleic acid encoding the fusion protein comprises a nucleic acid encoding a phosphatase component, a nucleic acid encoding a linker protein and a nucleic acid encoding a phosphatase component.
Suitably, the nucleic acid encoding a polypeptide protein binder may encode an antigen- stabilised protein binder. Suitably, the nucleic acid encoding a phosphatase component may encode PP1 or PP2A. In one embodiment, the nucleic acid encoding a phosphatase component encodes PPP1CA or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder. Suitably, the nucleic acid encoding PPP1CA and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein. In one embodiment, the nucleic acid encoding the phosphatase component encodes PPP1CA according to SEQ ID NO: 165 (NCBI Gene ID: 5499) or a functional variant thereof. Preferably, the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 165 (NCBI Gene ID: 5499).
In one embodiment, the nucleic acid encoding a phosphatase component encodes PPP2CA or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder. Suitably, the nucleic acid encoding PPP2CA and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein. In one embodiment, the nucleic acid encoding the phosphatase component encodes PPP2CA according to SEQ ID NO: 168 (NCBI GENE ID: 5515) or a functional variant thereof. Preferably, the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 168 (NCBI GENE ID: 5515).
In a further embodiment, the nucleic acid encoding a phosphatase component encodes PPM1 H or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder. Suitably, the nucleic acid encoding PPM1 H and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein. In one embodiment, the nucleic acid encoding the phosphatase component encodes PPM1 H according to SEQ ID NO: 171 (NCBI Gene ID: 57460) or a functional variant thereof. Preferably, the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 171 (NCBI Gene ID: 57460). In one embodiment, the nucleic acid encoding a phosphatase component encodes a catalytic component of PPM1 H or a functional variant thereof linked to a nucleic acid encoding an polypeptide protein binder. Suitably, the nucleic acid encoding the catalytic component of PPM1 H and the nucleic acid encoding the polypeptide protein binder may be linked via a nucleic acid encoding a linker protein.
Suitably, the nucleic acid encodes a nanobody, preferably a destabilised nanobody. In some embodiments the nucleic acid encodes aGFPeM or a functional variant or equivalent thereof. In one embodiment, the nucleic acid encoding the nanobody encodes aGFPeM according to SEQ ID NO: 163 or a functional variant thereof. Preferably, the functional variant is at least 60, 70, 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 163. Nucleic acids which encode a fusion protein of the invention may be wholly or partially synthetic and may include, but are not limited to, DNA, cDNA and RNA. Nucleic acid sequences encoding the fusion protein of the invention can be readily prepared by the skilled person using techniques which are well known to those skilled in the art, such as those described in Sambrook et al. "Molecular Cloning", A laboratory manual, Cold Spring Harbor Laboratory Press, Volumes 1-3, 2001 (ISBN-0879695773), and Ausubel et al. Short Protocols in Molecular Biology. John Wiley and Sons, 4th Edition, 1999 (ISBN - 0471250929). Said techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of nucleic acid, (ii) chemical synthesis, or (iii) preparation of cDNA sequences. DNA encoding the fusion protein of the invention may be generated and used in any suitable way known to those skilled in the art, including taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The excised portion may then be operably linked to a suitable promoter and expressed in a suitable expression system, such as a commercially available expression system. Alternatively, the relevant portions of DNA can be amplified by using suitable PCR primers. Modifications to the DNA sequences can be made by using site directed mutagenesis.
Nucleic acid sequences encoding a fusion protein of the invention may be provided as expression constructs in the form of a plasmid, vector, transcription or expression cassette. Expression constructs may comprise at least one nucleic acid as described above operably linked to one or more expression control sequences, e.g. a promoter, an enhancer, a poly-A sequence, an intron or suchlike. Suitably the expression control sequences are sufficient to provide expression of the fusion protein in a target cell. The expression may be constitutive or regulatable.
Accordingly, in a third aspect of the present invention, there is provided an expression construct comprising a nucleic acid as set out above. Suitably the expression construct is a vector, e.g. an expression vector adapted for expression in a eukaryotic or prokaryotic cell.
In one embodiment, the expression construct comprises a nucleic acid of the second aspect operably linked to an expression control sequence. Suitably, the nucleic acid may be operably linked to a promoter, an enhancer, a poly-A sequences and/or an intron. Suitably, in some embodiments the promoter is a constitutive promoter. In an alternative embodiment, the promoter is an inducible promoter.
As described above, protein phosphorylation is important for regulating the activity of proteins. Therefore, the skilled person will understand that it may be desirable in some instances to control or regulate when a target protein of interest is dephosphorylated by a fusion protein of the present invention. Such control may be achieved, amongst other methods, using inducible gene expression systems.
Inducible gene expression systems are available from a variety of commercial sources, including, without limitation, Takara, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible systems include inducible promoters such as the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracyclineinducible system (Gossen et al., Science, 268: 1766- 1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the Retro-X Tet-One Inducible Expression System (Heinz, N. et al. Retroviral and transposon-based tet-regulated all-in-one vectors with reduced background expression and improved dynamic range. Hum. Gene Ther. 22, 166-76 (2011)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Then, 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)).
Suitably, in some embodiments of the present invention, an expression construct as described herein may be induced to express the nucleic acid encoding the fusion protein of the present invention by the presence or absence of a system-specific inducer. Suitable inducers are widely known in the art and include, but are not limited to, antibiotics (e.g. tetracycline, doxycycline, etc.), alcohols (e.g. ethanol), hormones (e.g. steroid hormones) and environmental stresses (e.g. osmolality, heat, light etc.).
In some embodiments, an expression construct of the present invention may be induced to express the nucleic acid encoding the fusion protein in the presence or absence of tetracycline or derivatives thereof. Alternatively, in some embodiments of the present invention, an expression construct as described herein may be induced to express the nucleic acid encoding the fusion protein in the presence or absence of doxycycline or derivatives thereof.
In one preferred embodiment, the expression construct comprises a nucleic acid of the second aspect operably linked to a tetracycline inducible expression control system.
Suitably, in some embodiments, the tetracycline control system is the tet-ON system. In the tet-ON system, a reverse tetracycline-controlled transactivator (rtTA) allows transcriptional activation of a tTA-dependent promoter in the presence of tetracycline or its derivatives as described in Gossen et al (Science 23 Jun 1995: Vol. 268, Issue 5218, pp. 1766-1769 DOI: 10.1126/science.7792603). In the absence of tetracycline or its derivatives, tTA can no longer bind its target sequence within the tTA-dependent promoter and there is no expression from the tTA-dependent promoter. Alternatively, the tet-ON system may include the retro-X Tet- One Inducible Expression System whereby both the tetracycline-responsive transactivator and the nucleic acid of the second aspect are combined in the same expression vector. Suitably, in some embodiments, the vector expresses the Tet-On transactivator from the constitutive human PGK promoter in the forward orientation, and the nucleic acid of the second aspect from the PTRESGS promoter in the reverse orientation. Suitably, in the presence of tetracycline or doxycycline, the Tet-transactivator specifically binds and activates transcription from the inducible promoter that controls expression of the nucleic acid of the second aspect.
Alternatively, in some embodiments, the tetracycline control system is the tet-OFF system. In the tet-OFF system, tetracycline-controlled transactivator (tTA) allows transcriptional activation of a tTA-dependent promoter in the absence of tetracycline or its derivatives. tTA and the tTA-dependent promoter were initially created by Gossen and Bujard, 1992 (Gossen, M. and Bujard, H. (1992)‘Tight control of gene expression in mammalian cells by tetracyclineresponsive promoters’, Proceedings of the National Academy of Sciences of the United States of America, 89(12), pp. 5547-5551. doi: 10.1073/pnas.89.12.5547), which is incorporated herein by reference. tTA was created by fusion of the tetracycline resistance operon (tet repressor) encoded in TnlO of Escherichia coli with the activating cycline-controlled transactivator (tTA) and the tTA-dependent promoter was created by combining the tet operator sequence and a minimal promoter from the human cytomegalovirus promoter IE (hCMV-IE). When tetracycline or its derivatives are added, tTA can no longer bind its target sequence within the tTA-dependent promoter and there is no expression from the tTA- dependent promoter. The mechanism of the conformational change brought by binding of tetracycline or its derivatives to tTA is described in Orth et al., 2000 (Orth, P. et al. (2000) ‘Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system’, Nature Structural Biology, 7(3), pp. 215-219. doi: 10.1038/73324), which is incorporated herein by reference. Binding of tetracycline to TetR increases the separation of the attached DNA binding domains which abolishes the affinity of TetR for its operator DNA.
The skilled person will understand that any suitable expression control system may be used with the present invention to control the expression of the fusion protein. In some embodiments of the present invention the vector is a viral vector, such as a retroviral, lentiviral, adenoviral, or adeno-associated viral (AAV) vector. In some preferred embodiments the vector is an AAV vector.
In some embodiments, the vector is a gene therapy vector, suitably an AAV vector, an adenoviral vector, a retroviral vector, a herpes simplex vector or a lentiviral vector. Lentiviral vectors have been extensively used as a gene transfer tool in the CNS and are known to be able to successfully transduce neurones, astrocytes and oligodendrocytes. They are beneficial as they have relatively large cloning capacity and because viral genes are not expressed. A particularly preferred lentiviral vector system is based on HIV-1. Herpes simplex viral vectors and adenoviral vectors also show potential for use in as a gene transfer tool in CNS as they show successful transduction of CNS cells but are less preferred as due to their toxicity.
AAV vectors have been extensively discussed in the art. AAV vectors are of particular interest as AAV vectors do not typically integrate into the genome and do not elicit immune response. AAV serotypes 1 , 2, 4, 5, 8, 9 and 2g9 (AAV1 , AAV2, AAV4, AAV5, AAV8, AAV9 and AAV2g9) have been noted to achieve efficient transduction in the CNS. Therefore, AAV1 , AAV2, AAV4, AAV5, AAV8, AAV9 and derivatives thereof are particularly preferred AAV serotypes. In some embodiments, AAV9 is particularly preferred AAV vector. In other embodiments, AAV2g9 is a particularly preferred AAV vector (WO2014/144229). In yet other embodiments, a particularly preferred AAV vector is AAVDJ8 (Hammond et al., 2017). Suitably an AAV vector comprises a viral genome which comprises a nucleic acid sequence of the present invention positioned between two inverted terminal repeats (ITRs). WO2019/028306, for example discloses various wild type and modified AAV vectors that can be used in the CNS. In one embodiment, the AAV vector is capable of penetrating the blood brain barrier following delivery of the AAV vector. In one embodiment, AAV vectors of the present invention are recombinant AAV viral vectors which are replication defective, lacking sequences encoding functional Rep and Cap proteins within their viral genome. These defective AAV vectors may lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and the nucleic acid of interest for delivery to a cell, a tissue, an organ or an organism. Suitably AAV vectors for use herein comprise a virus that has been reduced to the minimum components necessary for transduction of a nucleic acid payload or cargo of interest. In this manner, AAV vectors are engineered as vehicles for specific delivery while lacking the deleterious replication and/or integration features found in wild-type viruses. In one embodiment, the AAV particle of the present invention is an scAAV. In another embodiment, the AAV particle of the present invention is an ssAAV. Methods for producing and/or modifying AAV particles are disclosed extensively in the art (see e.g. W02000/28004; W02001/23001 ; W02004/112727; WO 2005/005610 and WO 2005/072364, which are incorporated herein by reference). In one embodiment the AAV vector comprises a capsid that allows for blood brain barrier penetration following intravascular (e.g. intravenous or intraarterial) administration (see e.g. WO2014/144229, which discusses, for example, capsids engineered for efficient crossing of the blood brain barrier, e.g. capsids or peptide inserts including VOY101 , VOY201 , AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, and variants thereof).
Methods of making AAV vectors are well known in the art and are described in e.g., U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551 , US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191 , US6566118, US8137948; or International Publication Nos. WO1996039530, WG1998010088, WO 1999014354, WO1999/015685, W01999/047691 , W02000/055342, W02000/075353 and WO2001/023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al, Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J Fir.63:3822-8 (1989); Kajigaya et al, Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al, Vir., 219:37-44 (1996); Zhao et al, Vir.272: 382-93 (2000); the contents of each of which are herein incorporated by reference. Viral replication cells commonly used for production of recombinant AAV viral particles include but are not limited to HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines.
In some embodiments the vector is a non-viral vector, for example using cationic polymers or cationic lipids, as is known in the art. Various non-viral vectors are discussed in Selene Ingusci et al. (Gene Therapy Tools for Brain Diseases. Front. Pharmacol. 10:724. doi: 10.3389)
In a further aspect, there is provided a virion (viral particle) comprising a vector, suitably a viral vector, according to the present invention. In some embodiments the virion is an AAV virion.
The invention thus further provides recombinant virions (viral particles) comprising a vector as described above.
Pharmaceutical Compositions
In another aspect the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a fusion protein of the present invention as set out above. Such a composition typically comprises at least one pharmaceutically acceptable diluent or carrier. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the pharmaceutical composition is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure. Various other conventional pharmaceutical ingredients may be provided in the pharmaceutical composition, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.
Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
Suitably in one aspect of the invention, there is provided a pharmaceutical composition comprising the fusion protein, nucleic acid, expression construct, vector, or virion as discussed above and a pharmaceutically acceptable carrier or diluent. Suitably, in one embodiment, the composition is suitable to induce targeted dephosphorylation in target cells. In a further embodiment, the composition is suitable to regulate levels of phosphorylation in target cells. Dephosphorylation may increase or decrease the enzymatic activity, folding, stability, subcellular localisation and protein-protein interactions of the target protein. Suitably, the pharmaceutical composition results in the dephosphorylation of a target protein.
Suitably, in one embodiment the fusion protein or the pharmaceutical composition according to any of the aspects and embodiments provided herein, dephosphorylates a phosphorylated target protein of interest in a target cell. The protein of interest may be phosphorylated at any one or more serine, threonine and/or tyrosine residues. Suitable target cells include any eukaryotic cell. Preferably, the target cell is mammalian, more preferably human, murine, rat, pig or non-human primate. In a most preferred embodiment, the target cell is a human cell.
In some embodiments the target cell is a neuronal cell, preferably a cell from the central nervous system. The neuronal cell may be a primary neuronal cell or a cell of a neurone derived cell line, e.g. an immortalised cell line. In some embodiments, the target cell is a neurone, astrocytes, oligodendrocytes, microglial cells and/or ependymal cells.
In some embodiments the target cell is a cancer cell. A cancer cell may be a primary cell or an immortalised cancer cell line. The cancer cell may be any transformed cell, pre-cancerous cell or metastatic cell. In some embodiments, the target cell is a lymphoma, leukaemic, gastric, pancreatic, bone, breast, liver, kidney, colon, nasopharyngeal, osteosarcoma, neuroblastoma or skin cancer cell.
In one embodiment, the target cell is a human bone osteosarcoma epithelial cell line, preferably LI20S osteosarcoma cells.
In one embodiment, the target cell is a human retinal pigment epithelial cell line, preferably ARPE-19 cells.
In one embodiment, the target cell is a murine myoblast cell, preferably C2C12 cells.
In one embodiment, the target cell is a human neuroblastoma cell, preferably SK-N-MC neuroepithelioma cells.
In some embodiments, the target cell is in vitro, ex vivo or in vivo.
A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
Therapy and Related Methods In a further aspect the present invention provides a method of treatment or prevention of a disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a fusion protein, expression construct, vector, virion or pharmaceutical composition as described herein.
Suitably the method comprises introducing into cells of the subject a fusion protein of the present invention, expression construct, vector, virion or pharmaceutical composition as discussed above. Suitable target cells are discussed above.
In some embodiments the method comprises administering a vector or virion according to the present invention to the subject. Suitably the vector is a viral gene therapy vector, for example an AAV vector.
The skilled person will understand that in embodiments where a regulatable gene expression construct is used in any of the above methods, a suitable inducer may also be administered. Suitably, in some embodiments, the method may also comprise administering a suitable gene expression inducer. Suitably, the inducer may be administered concurrently with the gene therapy vector. Alternatively, and more preferably, the inducer may be administered after administration of the gene therapy vector.
The present invention also provides a fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition as described herein for use in a method of treatment or prevention of a disease in a subject. The method suitably comprises administering to said subject a therapeutically effective amount of a fusion protein or pharmaceutical composition of the present invention.
Suitably the fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition as discussed above is used for the treatment, prophylaxis, palliation or amelioration of a neurological disease and/or disorder. In one embodiment, the fusion protein, expression construct, vector, virion or pharmaceutical composition is for use in the treatment of a subject with a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is a tauopathy. A tauopathy may include any neurodegenerative disorder with abnormal tau inclusions. In some embodiments, the neurodegenerative disorder is any of Alzheimer’s disease, frontotemporal dementia with parkinsonism-17 (FTDP-17), Pick disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Parkinson’s disease, dementia and/or multiple system atrophy. In some embodiments, the subject in need thereof has a neurodegenerative disorder or cancer. Suitably, in one embodiment, the neurodegenerative disorder is a tauopathy, optionally wherein the tauopathy is Alzheimer’s disease.
Suitably the fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition as discussed above is used for the treatment, prophylaxis, palliation or amelioration of a cancer. In one embodiment, the fusion protein, expression construct, vector, virion or pharmaceutical composition is for use in the treatment of a subject with cancer. In some embodiments, the cancer is any one of, but not limited to, neuroblastoma, glioblastoma, lymphoma, leukaemia, hepatocellular carcinoma, myeloma, breast cancer, ovarian cancer, pancreatic cancer, testicular cancer, throat cancer, skin cancer, colorectal cancer, stomach cancer, liver cancer, lung cancer, gallbladder cancer, prostate cancer, cervical cancer, renal cell cancer and retinoblastoma.
In some embodiments, the method comprises administering a fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition systemically. Systemic administration may be enteral (e.g. oral, sublingual, and rectal) or parenteral (e.g. injection). Suitable methods of administration may be enteral (e.g. oral, sublingual, and rectal) or parenteral (e.g. injection) including intravenous, intraarterial, intracranial, intramuscular, subcutaneous, intra-articular, intrathecal, and intradermal injections. Preferred administration methods are intravenous, intraarterial, intracranial and intrathecal injection.
In some embodiments the method comprises introducing into the CNS of the subject a pharmaceutical composition as described herein. A particular difficulty with introducing a vector, virion or a pharmaceutical composition in the CNS is the blood brain barrier. The blood brain barrier is a semipermeable border of endothelial cells that prevents certain chemicals and molecules in the bloodstream from crossing into the extracellular fluid of the central nervous system. In animal studies, this obstacle has been overcome by injection directly into the brain of the animal, such as intracranial injection, suitably intracerebroventricular (ICV) injection (see e.g. Keiser et al., Curr Protoc Mouse Biol. 2018 Dec;8(4):e57). This method of administration can be disadvantageous for gene therapy in humans as it is difficult to perform and can be dangerous for the subject.
Instead, in a gene therapy setting in human, it is preferred that the expression cassette as described herein is introduced into the CNS by intravenous or intraarterial (e.g. intracarotid) administration of a viral vector comprising the expression cassette. Suitably, the viral vector is an AAV vector. Intravenous or intraarterial administration of some serotypes of AAV allows penetration of the AAV vectors into the brain. Intravenous or intraarterial administration is safer and less invasive than intracranial administration, while still allowing penetration through the blood brain barrier.
In some embodiments, a viral gene therapy vector may be administered concurrently or sequentially with one or more additional therapeutic agents or with one or more saturating agents designed to prevent clearance of the vectors by the reticular endothelial system.
Where the vector is an AAV vector, the dosage of the vector may be from 1x1010 gc/kg to 1x1015 gc/kg or more, suitably from 1x1012 gc/kg to 1x1014 gc/kg, suitably from 5x1012 gc/kg to 5x1013 gc/kg.
In general, the subject in need of treatment will be a mammal, and preferably a primate, more preferably a human. Typically, the subject in need thereof will display symptoms characteristic of a disease, e.g. a disease discussed above, most preferably a neurodegenerative disorder or cancer. The method typically comprises ameliorating the symptoms displayed by the subject in need thereof, by expressing the therapeutic amount of the therapeutic product of the invention.
Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo, are well-known in the art and will not be discussed in detail here. Briefly, they include intramuscular injection, interstitial injection, instillation in airways, application to endothelium, intra-hepatic parenchyme, and intravenous or intra-arterial administration (e.g. intra-hepatic artery, intra- hepatic vein) of plasmid DNA vectors (naked or in liposomes) or viral vectors. Various devices have been developed for enhancing the availability of DNA to the target cell. While a simple approach is to contact the target cell physically with catheters or implantable materials containing the relevant vector, more fusion protein approaches can use jet injection devices an suchlike. Gene transfer into mammalian cells has been performed using both ex vivo and in vivo procedures. The ex vivo approach typically requires harvesting of the cells, in vitro transduction with suitable expression vectors, followed by reintroduction of the transduced hepatocytes the liver. In vivo gene transfer has been achieved by injecting DNA or viral vectors.
In a further aspect of the invention there is provided a fusion protein, nucleic acid, expression construct, vector, virion or pharmaceutical composition according to any aspect of the invention for use as medicament, e.g. for treatment of a patient. Suitably the patient is suffering from a neurodegenerative disorder or cancer.
Methods of Regulating Protein Activity In a further aspect, there is provided herein a method of regulating protein activity wherein the method comprises dephosphorylating a phosphorylated POI by administering a fusion protein of the present invention, a nucleic acid, an expression construct, a vector or a pharmaceutical composition to a cell or tissue containing the phosphorylated POI. In some embodiments, the cell or tissue is in vitro, ex vivo or in vivo.
In a further aspect, there is provided a method for regulating protein activity, wherein the method comprises administering a fusion protein, a nucleic acid, an expression construct, a vector or the pharmaceutic composition of the present invention to a cell or a tissue in vitro, ex vivo or in vivo,
- wherein when a POI target of the polypeptide protein binder component of fusion protein is present, the polypeptide protein binder binds to the POI or when the POI target of the polypeptide protein binder component of the fusion protein is absent, the fusion protein destabilises; and
- wherein when the polypeptide protein binder is bound to the POI, the phosphatase component linked to the polypeptide protein binder selectively dephosphorylates the POI.
Suitably, dephosphorylation of the POI increases the activity of the POI or decreases the activity of the POI. Increased activity may refer to an increase of activity by at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 fold change to that of the activity of the phosphorylated POI. Decreased activity may refer to a decrease of activity by 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1 % compared to that of the activity of the phosphorylated POI.
In some embodiments of any of the methods as described herein, the method may further comprise a step of inducing expression of the fusion protein by providing a suitable inducer. Suitably, in some embodiments the inducer is tetracycline or derivatives thereof.
Research Tools
The fusion protein of the present invention is suitable for use as a tool to dissect the role of phosphorylation on any POI. Suitably, the fusion protein is applicable as a research tool. In one aspect, there is provided a fusion protein of the present invention, a nucleic acid, an expression construct, a vector, or a pharmaceutical composition as described herein for use as a research tool to regulate phosphorylation of a POI.
In some embodiments the fusion protein comprises a phosphatase component conjugated to at least one polypeptide protein binder. Suitably, the fusion protein dephosphorylates the POI. In some embodiments, the fusion protein regulates the activity of the POI. Suitably, the fusion protein is delivered to a cell, preferably a mammalian cell. In some embodiments the cell is a central nervous system (CNS) cell. In another embodiment the cell is a cancer cell.
In some embodiments, where the fusion protein of the present invention is used as a research tool, the fusion protein can be delivered to a cell in vitro, ex vivo or in vivo.
Kits:
In one aspect, there is provided a kit for use in any of the aspects and embodiments of the present invention, wherein the kit comprises a fusion protein of the present invention, nucleic acid, expression construct, vector, virion or pharmaceutical composition as discussed above, and instructions for use.
Brief Description of the Figures
Figure 1 : Anti-GFP nanobody-directed recruitment of PPP1CA or PPP2CA mediates dephosphorylation of phospho-FAM83D-GFP.
(A) Schematic representation of anti-GFP nanobody (aGFPeM)-directed recruitment of either PPP1CA or PPP2CA to GFP-tagged FAM83D to mediate FAM83D-GFP dephosphorylation during mitosis. (B) Wild-type (WT) and FA M83DGFP/GFP II 2 OS cells expressing FLAG-empty, FLAG-aGFPeM- PPP1CA or FLAG-aGFPeM-PPP2CA were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC) (5 pM) for 16 hr. Following incubation, mitotic (M) cells were isolated through shake-off. Asynchronous (AS) cells were included as a control. (C) U2OS FAM83DGFP/GFP FLAG-empty, FLAG-aGFP6M-PPP1CA or FLAG-aGFP6M-PPP2CA expressing cells were synchronised in mitosis using STLC and M cells were isolated through shake-off. AS cells were included as a control. Cells were washed twice with ice-cold PBS, lysed and subjected to anti-GFP immunoprecipitation (IP). (D) AS and M U2OS FAM83DGFP/GFP FLAG-empty, FLAG-aGFP6M-PPP1CA and FLAG- aGFP6M-PPP2CA expressing cells were treated with the phosphatase inhibitor Calyculin A(Cal A, 20 nM) for 20 min prior to lysis. For (B,C & D), extracts and IPs were resolved by SDS-PAGE and transferred on to PVDF membranes, which were subjected to immunoblotting with indicated antibodies. Figure 2: AdPhosphatase-mediated dephosphorylation of FAM83D-GFP in mitosis inhibits FAM83D-GFP proteasomal degradation. (A) LI2OS FAM83DGPP/GPP FLAG-empty, FLAG-aGFP6M-PPP1CA, FLAG-aGFP6M-PPP1CAH125Q, FLAG-aGFP6M-PPP2CA or FLAG- aGFP6M-PPP2CAH1180 expressing cells were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC) (5 M) for 16 hr. Following incubation, mitotic (M) cells were isolated through shake-off. Asynchronous (AS) cells were included as a control. (B & C) LI2OS FAM83DGFP/GFP FLAG-empty, FLAG-aGFP6M-PPP1CA, FLAG-aGFP6M- PPP1CAH125Q, FLAG- aGFP6M- 2CA or FLAG-aGFP6M-PPP2CAH118Q expressing cells were synchronised in mitosis using STLC and lysed at indicated time points following STLC washout (w/o) and release into medium ± the proteasomal inhibitor MG132 (20 pM), where stated. AS cells were included as a control. For (A, B & C), extracts were resolved by SDS-PAGE and transferred on to PVDF membranes, which were subjected to immunoblotting with indicated antibodies. (D) U2OS FAM83D knockout (KO) (n=38) and FAM83DGFP/GFP FLAG-empty (n=55), FLAG-aGFP6M-PPP1CA (n=61), FLAG-aGFP6M-PPP1CAH125Q (n=56), FLAG-aGFP6M- PPP2CA (n=59) or FLAG-aGFP6M-PPP2CAH118Q (n=56) expressing cells were synchronised in mitosis using STLC and subjected to GFP fluorescence and anti-FLAG and anti-CK1a immunofluorescence microscopy. DNA is stained with DAPI. Scale bars, 10 pm. (E) Quantification of CK1a spindle localisation for the cells described in (D) ± SD. Statistical analysis was carried out on indicated number of cells by one-way analysis of variance (ANOVA) using Tukey’s post-test, n=2 independent experiments.
Figure 3: PPP2CA AdPhosphatase targets phospho-GFP-ULK1 for dephosphorylation. (A) Schematic representation of anti-GFP nanobody (aGFPeM)-directed recruitment of either PPP1CA or PPP2CA to GFP-tagged ULK1 to mediate phospho-GFP-ULK1 dephosphorylation. (B) ARPE-19 wild-type (WT) and ULK1GFP/GFP knock in cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA were lysed and subjected to immunoprecipitation (IP) with anti-FLAG M2 resin. (C) ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA, FLAG-aGFP6M-PPP1CAH125Q, FLAG- aGFP6M-PPP2CA or FLAG-aGFP6M-PPP2CAH118Q were lysed and subjected to IP using anti- FLAG M2 resin. For (B & C), extracts and IPs were resolved by SDS-PAGE and transferred on to PVDF membranes, which were subjected to immunoblotting with indicated antibodies. (D) ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty, FLAG-aGFP6M-PPP2CA or FLAG- aGFP6M-PPP2CAH118Q were starved of amino acids with EBSS and treated with the lysosomal inhibitor Bafilomycin A1 (Baf-A1 , 50 nM) for 2 hr and subjected to anti-ULK1 and anti-FLAG immunofluorescence microscopy. DNA is stained with DAPI. Scale bars, 10pm. Figure 4: Targeted GFP-ULK1 dephosphorylation inhibits starvation-induced autophagy. (A) ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty or FLAG-aGFPeM- PPP2CA were treated with the phosphatase inhibitors Calyculin A (Cal A, 20 nM) or Okadaic acid (OA, 1 M) for 20 min. (B) ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty, FLAG- aGFP6M-PPP2CA or FLAG- aGFPeM-PPP2CAH118Q were starved of amino acids with EBSS and treated with the lysosomal inhibitor Bafilomycin A1 (Baf-A1 , 50 nM) for 2 hr. Quantification of (C) p-S318 ATG13 normalised to total ATG13 protein levels and (D) LC3-II protein levels normalised to GAPDH from (B) ± SD of n=3 independent experiments. (E) ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty or FLAG-aGFPeM-PPP2CA were pre- treated with the ULK1 inhibitor MRT68921 (2 M, 2 hr), where indicated, and amino acid starved with EBSS and treated with Baf-A1 (50 nM) for 2 hr. Quantification of (F) p-S318 ATG13 normalised to total ATG13 protein levels and (G) LC3-II protein levels normalised to GAPDH from (E) ± SD of n=3 independent experiments. For (A, B & E), extracts were resolved by SDS-PAGE and transferred on to PVDF membranes, which were subjected to immunoblotting with indicated antibodies. +AA = amino acid-rich conditions, n.s. = not significant. Statistical analyses were carried out by one-way analysis of variance (ANOVA) using Tukey’s post-test.
Figure 5: FLAG-aGFP6M-PPP2CA expression mediates the recruitment of PP2A regulatory subunits. (A) U2OS FAM83DGFP/GFP cells expressing FLAG-empty, FLAG- aGFP6M-PPP2CA or FLAG-aGFPeM- PPP2CAH118Q were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC) (5 pM) for 16 hr. Following incubation, mitotic cells were isolated through shake-off and subjected to immunoprecipitation (IP) using anti-FLAG M2 resin. (B) ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty, FLAG-aGFP6M-PPP2CA or FLAG-aGFPeM- PPP2CAH118Q were starved of amino acids with EBSS for 2 hr, lysed and subjected to IP using anti-FLAG M2 resin. For (A & B), extracts and IPs were resolved by SDS-PAGE and transferred on to PVDF membranes, which were subjected to immunoblotting with indicated antibodies. (C) Anti-FLAG IPs from FLAG-aGFPeM-PPP2CA expressing cell extracts from (A & B) were subjected to SDS-PAGE, followed by in-gel trypsin digest of proteins and protein identification by LC-MS/MS. Venn diagram depicts FLAG-aGFPeM- PPP2CA interactors exclusive to and common between mitotic U2OS FAM83DGFP/GFP cells and EBSS-starved ARPE-19 ULK1GFP/GFP cells.
Figure 6: TMT-labelled quantitative global phospho-proteomic analysis of FLAG- aGFP6M-PPP2CA AdPhosphatase-mediated FAM83D-GFP and GFP-ULK1 dephosphorylation. Volcano plot following global (A) phospho- (11 ,821 unique phosphopeptides detected) and (B) total- (7,201 unique peptides detected) proteomic analysis of STLC-synchronised U2OS FAM83DGFP/GFP cells expressing FLAG-aGFP6M-PPP2CA compared with those expressing FLAG-aGFP6M-PPP2CAH118Q. Volcano plot following global (C) phospho- (22,574 unique phospho-peptides detected) and (D) total- (8,153 unique peptides detected) proteomic analysis of EBSS-starved ARPE-19 ULK1GFP/GFP cells expressing FLAG-aGFPeM-PPP2CA compared with those expressing FLAG- aGFP6M- PPP2CAH1180 Grey horizontal threshold indicates significance level of p=0.05. Grey vertical threshold indicates 2-fold change. The top left quadrant indicates phosphopeptides/peptides that are downregulated in cells expressing FLAG-aGFPeM-PPP2CA over those expressing FLAG-aGFPeM-PPP2CAH118Q, coloured red, whilst the top right quadrant indicates phosphopeptides/peptides upregulated in cells expressing FLAG-aGFPeM-PPP2CA over those expressing FLAG-aGFP6M-PPP2CAH118Q, coloured blue. Phospho-peptide detected corresponding to FAM83D is labelled in (A), and LILK1 is labelled in (C).
Figure 7: Tet-inducible AdPhosphatase system for inducible dephosphorylation of endogenous TFEB-GFP: Tet-on system optimization. C2C12 myoblast cells in which TFEB was homozygously tagged with a GFP tag were transduced with RetroX-Tet-One retroviruses encoding Flag-aGFP6M-PP1CA , Flag-aGFP6M-PP1CA-H125Q, Flag-aGFP6M-PP2CA, and Flag-aGFP6M-PP2CA-H118Q. (A) A range of doxycycline concentrations (0-200 ng/ml), were administered to the cells for 24 h and it was determined that 100 ng/ml was the optimal dose to cause targeted dephosphorylation of TFEB-GFP, as observed through a downward mobility shift evident for GFP in immunoblots. (B) A time course treatment (0-24 h) of cells with 100 ng/ml doxycycline, revealed a time-dependent dephosphorylation of TFEB-GFP, with the most robust dephosphorylation observed at 24 h. The phosphatase dead mutants of both PP1 A and PP2A (PP1AH125Q and PP2AH118Q, respectively) did not cause any dephosphorylation. The expression of the AdPhosphatase constructs was detected with a Flag immunoblot, while GAPDH immunoblot served as a loading control.
Figure 8: Targeted dephosphorylation of endogenous Tau-GFP, knocked-in in SK-N-MC neuroepithelioma cells. SK-N-MC cells in which TAU was tagged with a GFP tag were transduced with pBabeD (A) or RetroX-Tet-One (B) retroviruses encoding one or more of either Flag-aGFP6M, Flag-aGFP6M-PPP1CA, Flag-aGFP6M-PPP1CA-H125Q, Flag- aGFP6M-PPP2CA, or Flag-aGFP6M-PPP2CA-H118Q as shown. Targeted dephosphorylation of Tau on several known phospho-sites were tested using immunoblotting (IB) with corresponding phospho-specific antibodies. Both catalytically active PP1A and PP2A AdPhosphatases appear to cause a robust dephosphorylation of TAU at multiple sites. The expression of the AdPhosphatase constructs were detected with a Flag immunoblot, while GAPDH immunoblot served as a loading control.
Detailed Description of Embodiments of the Invention and Examples While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in- chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.
Definitions
The term ‘antibody’ as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising an antigen binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, I gG3, I gG4, I gA1 and lgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The term ‘nanobody’ as used herein may refer to single domain antibody derived from heavychain only (VHH) antibodies. VHH antibodies may be of camelid origin including camels, alpacas and llamas. VHH antibodies may be of shark origin. ‘Nanobody’ as used herein includes any of monomeric, dimeric, bispecific or multivalent nanobodies that are specific for a target protein. The term ‘destabilised nanobody’ or ‘dNb’ may refer to a nanobody that naturally harbours a destabilising mutation or a destabilising mutation has been introduced into the nanobody. A destabilising mutation may be in the structurally conserved framework region of the nanobody. A destabilised nanobody may refer to a nanobody that is particularly susceptible to being broken down or degraded in living cells unless it is bound to its protein target inside the cell.
The term ‘monobody’ as used herein may refer to synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold. This class of binding proteins are built upon a diversified library of the 10th FN3 domain of human fibronectin. Various other scaffold protein-based synthetic binding proteins are known in the art, and can be used in the present invention.
A polypeptide protein binder as referred to herein refers to any polypeptide that recognises and binds to a target epitope. Suitably, the polypeptide protein binder can bind to its target when the binder is expressed in a cell or when introduced into a cell or binds to an isolated form of its target protein. A polypeptide protein binder binder may include an antibody, an antibody fragment, a monobody, a nanobody and/or other types of binder based, e.g., on scaffold proteins, can be used.
The term ‘antigen-stabilised’ refers to the stability of a protein when bound to its cognate antigen. Traditionally expressed protein binders are stable regardless of antigen binding. Antigen-stabilised protein binders as used herein rely on the presence of the protein binders cognate antigen for stability of the fusion protein. It will be apparent to the person skilled in the art that an antigen-stabilised protein binder does not refer to the antigen being bound to the protein when provided in the fusion protein of the present invention but refers to a protein that when it binds to its cognate antigen the protein binder becomes stabilised.
The term ‘phosphatase component’ may refer to the whole or part of a phosphatase enzyme. The phosphatase component may refer to any phosphatase or part of a phosphatase that is capable of inducing dephosphorylation of a protein.
The term ‘phosphatase catalytic subunit’ may refer to a domain of a phosphatase protein that can form fusion proteins with other phosphatase domains such as a regulatory domains. A phosphatase catalytic subunit in the context of the present invention may refer to any phosphatase domain that is capable of inducing dephosphorylation of a protein.
The term ‘fusion protein’ as used herein refers to two or more associated polypeptide chains. Accordingly the two or more proteins of the present invention are ‘tethered’, ‘linked’ or ‘interlinked’. As such, these terms refer to joining the phosphatase component to the polypeptide protein binder. This may refer to the components of the fusion protein being directly conjugated or joined via a linker protein.
The terms ‘phosphorylation’ or ‘phosphorylated’ as used herein refer to the addition of a phosphate group to a protein. ‘Dephosphorylation’ or ‘dephosphorylated’ refer to the removal of a phosphate group from a protein. Proteins can be phosphorylated or dephosphorylated at more than one residue. Proteins are typically phosphorylated at serine, threonine and tyrosine residues.
‘Selectivity’ as used herein refers to the binding preference of the polypeptide protein binder to target epitope. Higher selectivity may refer to a protein binder that exclusively or preferentially binds to the target epitope. In some embodiments, the more selective a protein binder, the less cross-reactive the polypeptide binder is with any protein present. Low selectivity may refer to a protein binder that binds an epitope that is shared with other proteins or is not unique to the target protein. A protein binder with high selectivity has reduced binding to a non-target proteins when compared to a protein binder with lower selectivity.
A ‘functional variant’ of a nucleic acid, or amino acid sequence or proteins in the context of the present invention is a variant of a reference sequence or protein that retains the ability to function in the same way as the reference sequence or protein. Alternative terms for such functional variants include “biological equivalents” or “equivalents”. Functional variants of nucleic acid or amino acid sequences that encode protein binders or protein binders per se refer to variants that selectively bind to the target POI. As discussed, suitable protein binders can be tested for their ability to bind the target POI using methods known in the art and those described herein e.g. see Example 2 either alone or in a fusion protein of the present invention. Likewise, functional variants of nucleic acid or amino acid sequences that encode a phosphatase component or phosphatase components perse are those variants that retain the ability to dephosphorylate proteins. Suitable methods of testing the ability of a function variant, alone or in a fusion protein of the present invention are described above.
The terms ‘identity’ and ‘identical’ and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules. Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250). Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881- 90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31 ; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.
The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the "help" section for BLAST™. For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.
For example, a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2. The percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.
The terms ‘peptide’, ‘polypeptide’, and ‘protein’ are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
The term ‘vector’ is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated. Vectors of the present invention can be episomal vectors (i.e. , that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to plasmid vectors (e.g. pMA-RQ, plIC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAG), yeast (YAC), or human (HAG)) may be used to accommodate larger inserts. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replicationdeficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.
The term ‘expression construct’ may refer to a plasmid, vector, or expression cassette which comprises at least one nucleic acid . An expression construct may comprise a nucleic acid operably linked to a sequence encoding an expression control sequence such as a promoter, an enhancer, polyadenylation sequence or an intron. The sequence encoding an expression product (e.g. gene) typically encodes a polypeptide (protein) or RNA. The gene may be a full- length cDNA or genomic DNA sequence, or any fragment, subunit or mutant thereof that has at least some desired biological activity. Where the gene encodes a protein, it can be essentially any type of protein. By way of non-limiting example, the protein can be an enzyme, an antibody or antibody fragment (e.g. a monoclonal antibody) or a fusion protein of the present invention.
‘Expression control sequences’ as referred to herein may refer to promoters, enhancers, poly- A sequences and introns. Suitably the expression control sequences in the present invention are sufficient to provide expression of the fusion protein in a target cell. Expression control sequences may be constitutive or regulatable.
The term ‘target cell’ as used herein refers to any cell that expresses a protein of interest. A target cell can be, without limitation, a prokaryotic or eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell. A target cell includes cancer cells and cells of the central nervous system (CNS cell).
The term “CNS cell” or “CNS cells” as used herein includes neurones, astrocytes, oligodendrocytes, microglial cells and/or ependymal cells.
The term ‘cancer cell’ includes any cell that is transformed, harbours an oncogene, or displays uncontrolled proliferation. A ‘pre-cancerous cell’ as used herein refers to any cell with abnormal growth characteristics. A ‘metastatic cell’ refers to a cancer cell found at a location distant from the original tumour site.
The term ‘pharmaceutically acceptable’ as used herein refers without limitation to an entity or ingredient is one that may be included in the compositions provided herein and that causes no significant adverse toxicological effects in the patient at specified levels, or if levels are not specified, in levels known to be acceptable by those skilled in the art. All ingredients in the compositions described herein are provided at levels that are pharmaceutically acceptable. For clarity, active ingredients may cause one or more side effects and inclusion of the ingredients with a side effect profile that is acceptable from a regulatory perspective for such ingredients will be deemed to be ‘pharmaceutically acceptable’ levels of those ingredients.
The term ‘treatment’ or ‘treating’ as used herein may refer to reducing, ameliorating or eliminating one or more signs, symptoms, or effects of a disease or condition. ‘Treatment’ as used herein includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; (c) relieving the disease, i.e., causing regression of the disease; and (d) alleviating or reducing any symptoms of the disease. As used herein, the terms ‘inhibit’, ‘reduce’ and similar terms mean a decrease of at least about 5%, 10%, 15%; 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.
The term ‘subject’ as used herein may be used interchangeably with ‘individual’ or ‘patient’, and refer to any individual subject with a disease or condition in need of prevention or treatment unless otherwise stated. For the purposes of the present disclosure, the subject may be a mammal, preferably a human.
Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings.
Examples
Introduction:
Protein phosphorylation is a fundamental driver of all cell signalling processes and is therefore tightly regulated. Hyperphosphorylation of proteins is a known hallmark of many diseases, including cancer and neurodegenerative diseases. The AdPhosphatase system can efficiently and selectively target specific proteins of interest (POIs) for dephosphorylation. The AdPhosphatase system is versatile and adaptable, where, in principle, any phosphatase can be redirected to dephosphorylate any phospho-POl in cells and tissues. The inventors have also demonstrated that inducible dephosphorylation is also possible, when an AdPhosphatase is placed under the expression of an inducible expression system. This technology offers an excellent opportunity to dissect the role of phosphorylation on potentially any POI. Targeted dephosphorylation is also an exciting and promising new therapeutic modality and the AdPhosphatase system can be exploited to rapidly inform the utility of targeted POI dephosphorylation in any therapeutic application.
For phospho-POIs, the inventors chose FAM83D, TAU, Transcription Factor-EB (TFEB) and the unc-51-like kinase 1 (LILK1) to demonstrate the ability of the fusion protein to dephosphorylate a protein of interest.
FAM83D is required for the recruitment of casein kinase 1a (CK1a) to the mitotic spindle to orchestrate proper spindle positioning and timely cell division (Fulcher et al., 2019). During mitosis, FAM83D is heavily phosphorylated at the mitotic spindle in a CK1a-dependent manner, causing a phospho-dependent electrophoretic mobility shift of approximately 25 kDa, and is subsequently degraded by the proteasome upon mitotic exit (Fulcher et al., 2019). The precise role for CK1a-dependent hyperphosphorylation of FAM83D remains elusive. The unc-51-like kinase 1 (LILK1) serine/threonine protein kinase functions in a fusion protein with both the autophagy-related protein 13 (ATG13) and the focal adhesion kinase family interacting protein of 200 kDa (FIP200) to regulate the initiation of autophagy (Ganley et al., 2009, Jung et al., 2009, Hosokawa et al., 2009). To orchestrate and regulate the initiation of autophagy, LILK1 undergoes an intricate and diverse set of post-translational modifications (PTMs), including phosphorylation by different protein kinases at multiple residues, which can either activate or inhibit LILK1 and consequently initiation of autophagy (Zachari and Ganley, 2017).
Tau protein deposition in fibrillar lesions has been observed in numerous neurodegenerative diseases, most notably Alzheimer’s disease, and thus serves as a medically relevant phospho- POI demonstrating the potential therapeutic applicability of AdPhosphatases.
The transcription factor EB (TFEB) plays a pivotal role in the regulation of basic cellular processes, such as lysosomal biogenesis and autophagy. The subcellular localization and activity of TFEB are regulated by mechanistic target of rapamycin (mTOR)-mediated phosphorylation, which occurs at the lysosomal surface. Phosphorylated TFEB is retained in the cytoplasm, whereas dephosphorylated TFEB translocates to the nucleus to induce the transcription of target genes.
For FAM83D and LILK1 , characterised cells harbouring endogenous GFP-tag knock ins, namely FAM83DGFP/GFP U2OS cells (Fulcher et al., 2019) and ULK1GFP/GFP ARPE-19 cells (Simpson et al., 2020) were employed. An AdPhosphatase construct was designed consisting of PPP1CA or PPP2CA conjugated to an antigen-stabilised anti-GFP nanobody (aGFPeM) (Tang et al., 2016) and a FLAG reporter (Figure 1A). In this case, aGFPeM is only stable when bound to GFP and destabilised and degraded when unbound, thereby maintaining homeostatic FLAG-aGFP6M-PPP1CA/- PPP2CA levels close to a 1 :1 ratio the target POI-GFP. A key advantage of this approach would therefore be to limit the overexpression and potential off-target effects of the phosphatase (Hsu et al., 2006, Gergs et al., 2004). This is evident upon the expression of FLAG-aGFP6M-PPP2CA in FAM83DGFP/GFP U2OS and ULK1GFP/GFP ARPE-19 cells, where the levels of FLAG-aGFPeM-PPP2CA are much lower than endogenous PPP2CA when detected using anti-PPP2CA antibody (Figure 6A&B). The ability of these AdPhosphatases to target the dephosphorylation of phospho-FAM83D-GFP and phospho- ULK1-GFP and investigate downstream biology was then tested. For TFEB, C2C12 myoblast cells in which TFEB was tagged with a GFP tag were employed. For TAU, SK-N-MC cells were employed in which TAU has been tagged with GFP. In both instances, aGFPeM as described above was used to target GFP-POI for dephosphorylation. Materials and Methods:
Cell Lines
All procedures were carried out under aseptic conditions meeting biological safety requirements. ARPE-19 cells (ATCC, Cat# CRL-2302) are human retinal pigment epithelial cells derived from a 19-year-old male. HEK293-FT cells (Invitrogen, Cat# R70007) are a clonal isolate of HEK293 cells transformed with the SV40 large T antigen. U2OS cells (ATCC, Cat# HTB-96) are human epithelial bone osteosarcoma cells derived from a 15-year-old Caucasian female. For growth, HEK293-FT and U2OS cells were maintained in DMEM (Life Technologies) containing 10% (v/v) foetal bovine serum (FBS, Thermo Fisher Scientific), 2 mM L-glutamine (Lonza), 100 U/ml penicillin (Lonza) and 0.1 mg/ml streptomycin (Lonza). ARPE-19 cells were maintained in a 1 :1 mix of DMEM and Ham’s F-12 nutrient mix (Life Technologies) containing 15% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were grown at 37°C with 5% CO2 in a water-saturated incubator. For passaging, cells were incubated with trypsin/EDTA at 37°C to detach cells.
Plasmids
For production of retroviral vectors, the following were cloned into pBABED-puromycin plasmids: FLAG-aGFP6M-PPP1CA (DU62917), FLAG-aGFP6M-PPP1CAH125Q (DU62964), FLAG-aGFP6M-PPP2CA (DU62902), FLAG-aGFP6M-PPP2CAH118Q (DU62960). All constructs were sequence-verified by the DNA Sequencing Service, University of Dundee.
Generation of cell lines using CRISPR/Cas9
The CRISPR/Cas9 genome editing system (Cong et al., 2013) was used to generate U2OS FAM83D homozygous C-terminal GFP knock in (KI) (FAM83DGFP/GFP) cells (Fulcher et al., 2019), FAM83D knock-out (FAM83D KO) cells (Fulcher et al., 2019), and ARPE-19 ULK1 homozygous N-terminal GFP KI (ULK1GFP/GFP) cells (Simpson et al., 2020).
Retroviral generation of stable cell lines
Retroviral pBABED-puromycin vectors encoding the desired construct (6 pg) were cotransfected with pCMV5-gag-pol (3.2 pg) and pCMV5-VSV-G (2.8 pg) (Cell Biolabs) into a 10 cm diameter dish of -70% confluent HEK293-FT cells. Briefly, plasmids were added to 1 ml Opti-MEM medium to which 24 pl of 1 mg/ml PEI was added. Following a gentle mix and incubation at room temperature for 20 min, the transfection mix was added dropwise to HEK293-FT cells. 16 hr post-transfection, fresh medium was added to the cells. 24 hr later, the retroviral medium was collected and passed through 0.45 pm sterile syringe filters. Target cells (-60% confluent) were transduced with the optimised titre of the retroviral medium diluted in fresh medium (typically 1 :1-1 :10) containing 8 pg/ml polybrene (Sigma-Aldrich) for 24 hr. The retroviral medium was then replaced with fresh medium, and 24 hr later, the medium was again replaced with fresh medium containing 2 pg/ml puromycin for selection of cells which had integrated the constructs. A pool of transduced cells were utilised for subsequent experiments following complete death of non-transduced cells placed under selection in parallel.
Treatment of cells with compounds
The following chemicals were added to cell media at indicated concentrations and times: MG132 (Abeam), Calyculin A (CST), Okadaic acid (CST), Bafilomycin-A1 (Enzo Life Sciences), MRT68921 (MRC PPU Reagents and Services). Cells were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC, Sigma-Aldrich, 5 pM, 16 hr) (Fulcher et al., 2019, Simpson et al., 2020). Following incubation, mitotic cells were lysed after isolation through shake-off or after release into fresh media containing stated compounds for indicated times. For amino acid starvation, cells were washed twice in Earle’s balanced salt solution (EBSS, Gibco) and incubated in EBSS for 2 hr.
Cell lysis and immunoprecipitation
Cells were harvested by washing twice with phosphate-buffered saline (PBS) and scraping into ice-cold lysis buffer (50 mM Tris-HCI pH 7.5, 0.27 M sucrose, 150 mM NaCI, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium p-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate and 1 % NP-40) supplemented with 1x complete™ protease inhibitor cocktail (Roche). After incubation for 10 min on ice, lysates were clarified by centrifugation at 20,000 xg for 20 min at 4°C. Protein concentration was determined according to the Bradford assay to enable normalisation between samples. Following determination of protein concentration by Bradford assay, immunoprecipitation (IP) was utilised to isolate a particular protein of interest. For anti-FLAG IPs, anti-FLAG M2 resin (Sigma-Aldrich) was used; for anti-GFP IPs, GFP-TRAP beads (ChromoTek) were used. Before an IP was performed, an input from each lysate was retained to compare and determine IP efficiency. Samples were incubated for 4 hr at 4°C on a rotating wheel. Beads were collected by centrifugation at 1000 xg for 1 min at 4°C and a sample of the supernatant was retained (flow-through). IPs were subsequently washed three times with lysis buffer. Input, IP and flow-through samples were reduced in LDS sample buffer (Invitrogen). SDS-PAGE and Western blotting
Cell lysates containing equal amounts of protein (10-20 pg) were resolved by SDS-PAGE and transferred to PVDF membrane. Membranes were blocked in 5% (w/v) non-fat milk (Marvel) in TBS-T (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 0.2% Tween-20) and incubated overnight at 4°C in 5% (w/v) BSA/TBS-T or 5% (w/v) milk/TBS-T with the appropriate primary antibodies. Primary antibodies used at indicated dilutions include: anti-Akt (9272S, CST, 1 :1 ,000), anti- Akt p-S473 (12694, CST, 1 :1 ,000), anti-ATG13 (SAB4200100, Sigma-Aldrich, 1 :1 ,000), anti- ATG13 p-S318 (NBP2-19127, Novus, 1 :1 ,000), anti-CK1a (A301-991A, Bethyl, 1 :1 ,000; SA527, MRC PPU Reagents & Services, 1 :1 ,000), anti-Cyclin B1 (4138, CST, 1 :1 ,000), anti- FAM83D (SA102, MRC PPU Reagents & Services, 1 :1 ,000), anti-FIP200 (17250-1-AP, Proteintech, 1 :1 ,000), anti-FLAG (A8592, Sigma-Aldrich, 1 :2,500), anti-GAPDH (2118, CST, 1 :5,000), anti-GFP (S268B, MRC PPU Reagents & Services, 1 :2,000), anti-HMMR (124729, Abeam), anti-LC3 (S400D, MRC PPU Reagents & Services, 1 :200), anti-mono- and poly- ubiquitinylated conjugates (BML-PW8810, Enzo, 1 :2,000), anti-PP2A (S274B, MRC PPU Reagents & Services, 1 :1 ,000), anti-a-tubulin (MA1-80189, Thermo Fisher Scientific, 1 :5,000), anti-ULK1 (8054, CST, 1 :1 ,000), anti-ULK1 p-S757 (6888, CST, 1 :1 ,000). Membranes were subsequently washed with TBS-T and incubated with HRP-conjugated secondary antibody for 1 hr at room temperature. HRP-coupled secondary antibodies used at indicated dilutions include: goat anti-rabbit-IgG (7074, CST, 1 :2,500), rabbit anti-sheep-IgG (31480, Thermo Fisher Scientific, 1 :5,000), goat anti-rat IgG (62-9520, Thermo Fisher Scientific, 1 :5,000), goat anti-mouse-IgG (31430, Thermo Fisher Scientific, 1 :5,000). After further washing, signal detection was performed using ECL (Merck) and ChemiDoc MP System (Bio-Rad). Imaged v1.49 (National Institutes of Health) was used to analyse protein bands by densitometry (Schneider et al., 2012).
Immunofluorescence microscopy
Cells were seeded onto sterile glass coverslips in 6-well dishes. Coverslips were washed twice with PBS, fixed with 4% (w/v) paraformaldehyde (Thermo Fisher Scientific) for 10 min, washed twice with and incubated for 10 min in DMEM/10 mM HEPES pH 7.4. After one wash in PBS, cell permeabilisation was carried out using 0.2% NP-40 in PBS for 4 min. Samples were blocked by washing twice and incubation for 15 min in blocking buffer (1 % (w/v) BSA/PBS). Coverslips were incubated for 1 hr at 37°C with primary antibodies in blocking buffer and washed three times in blocking buffer. Mouse anti-FLAG monoclonal (F1804, Sigma-Aldrich), rabbit anti-FLAG monoclonal (14793, CST), sheep anti-CK1a polyclonal (SA527, MRC PPU Reagents & Services) and rabbit anti-ULK1 (8054, CST) primary antibodies were used at a 1 :500 dilution. Coverslips were then incubated for 30 min at room temperature with Alexafluor coupled secondary antibodies in blocking buffer and washed an additional three times in blocking buffer. Donkey anti-rabbit IgG Alexa-Fluor 488 (A21206, Thermo Fisher Scientific), goat anti-mouse IgG Alexa-Fluor 594 (A11005, Thermo Fisher Scientific), goat anti-rabbit IgG Alexa-Fluor 594 (A11012, Thermo Fisher Scientific) and donkey anti-sheep IgG Alexa-Fluor 647 (A21448, Thermo Fisher Scientific) secondary antibodies were used at a 1 :500 dilution. After submerging in ddH2O, cells were mounted onto glass slides using Prolong gold antifade mountant with DAPI (Life Technologies) and visualised using a DeltaVision system (Applied Precision) and deconvolved using SoftWoRx (Applied Precision). Images were processed using Imaged and OMERO 5.4.10 software (Allan et al., 2012). Imaged macro quantification of CK1a spindle localisation was performed as previously described (Fulcher et al., 2019, Simpson et al., 2020).
Mass spectrometry
Interactome analysis
For interactome analysis, cells were first lysed in NP-40 lysis buffer. Clarified lysates were incubated with Protein A agarose beads for 1 hr on a rotating wheel at 4°C to pre-clear nonspecific binding proteins and filtered through Spin-X columns by centrifugation for 5 min at 13,000 xg. Filtered extracts (5-10 mg protein) were incubated with 10-20 pl of appropriate beads for specified IP for 4 hr at 4°C on a rotating wheel. Following incubation, beads were washed 3x with standard lysis buffer. Bead-bound proteins were denatured and eluted in 2x LDS for 5 min at 95°C. Samples were then filtered through Spin-X columns to remove the beads from the eluate. The filtered eluate was loaded onto a 4-12% Bis-Tris gradient gel and proteins were separated by SDS-PAGE. Gels were stained with InstantBlue and subsequently de-stained in deionised water. A small portion of the eluate was retained for analysis and validation by Western blotting. To minimise potential protein contaminants, all steps from this point were performed under a laminar flow hood. Disposable scalpels were used to cut protein bands of interest from the InstantBlue stained gels into 1-2 cm cubes, which were subsequently transferred into LoBind 1 .5 ml Eppendorf tubes. Gel pieces were washed once in HPLC grade water, and then shrank in anhydrous acetonitrile (ACN) for 5 min with gentle shaking. The ACN was aspirated, and gel pieces were re-swollen with 50 mM Tris-HCI pH 8.0 for 5 min with shaking. The shrinking-swelling process was repeated once more, and the proteins within the gel pieces were reduced with 5 mM DTT in 50 mM Tris-HCI pH 8.0 for 20 min at 65°C. Next, the proteins within the gel pieces were alkylated with 20 mM iodoacetamide (IAA) in 50 mM Tris-HCI pH 8.0 for 20 min at room temperature. Gel pieces were then shrunk again in ACN for 5 min and re-swollen in 50 mM triethylammonium bicarbonate (TEAB) pH 8.0 containing 5 pg/ml trypsin for 16 hr at 30°C for digestion. An equivalent volume of ACN was added to the digest for 15 min with shaking and the supernatant was collected into a fresh LoBind 1 .5 ml Eppendorf tube. Gel pieces were then re-swollen with 0.1% (v/v) trifluoroacetic acid (TFA) for 5 min with shaking, and peptides were extracted twice with ACN for 5 min each with shaking. After each extraction, the supernatant was removed and combined with the previous supernatant. The supernatants were then dried by vacuum centrifugation using a SpeedVac. Digested peptides were reconstituted in HPLC-grade 5% (v/v) ACN containing 0.1 % (v/v) formic acid (FA) and injected into a LI3000 RSLC (rapid separation liquid chromatography) HPLC chromatography system (Thermo Fisher Scientific) coupled to a linear ion trap-orbitrap hybrid mass spectrometer (Orbitrap Velos Pro, Thermo Fisher Scientific). Peptides were trapped on a nanoViper Trap column (2 cm x 100 pM, C185 pM, 100 A, Thermo Fisher Scientific) and subsequently separated on a 15 cm EasySpray column (Thermo Fisher Scientific) equilibrated with a flow rate of 300 nl/min. Data was acquired in the data-dependent mode, automatically switching between MS1 and MS2 acquisition. Full scan spectra (m/z 400- 1 ,600) were acquired in the orbitrap with resolution set to 60,000 at m/z 400. The 20 most intense ions, above a specified minimum signal threshold of 2,000, were fragmented by collision induced dissociation and recorded in the linear ion trap (full automatic gain control (AGC) target; 30,000, Msn AGC target; 5,000). Raw files were subsequently converted into a list of identified peptides, along with the precursor intensity of the identified peptides, and submitted to the in-house Mascot server (MRC PPU, University of Dundee). Data was searched against the SwissProt human database with variable modifications allowing for oxidation of Met, phosphorylation of Ser/Thr or Tyr residues, along with oxidation or dioxidation modifications. Carbamidomethylation of Cys was set as a fixed modification. Error tolerances were set to 10 ppm (parts per million) for MS1 and 0.6 Da for MS2. Data analysis was performed using Scaffold v 4.4.6 (Proteome Software).
Global proteome and phospho-proteome analysis
Cells were lysed in urea lysis buffer (8 M urea, 20 mM HEPES pH 8.0, supplemented with 1 tablet of complete protease inhibitors per 25 ml lysis buffer and 1 tablet of PhosSTOP phosphatase inhibitors per 10 ml lysis buffer) by Bioruptor® sonication for 15 cycles at 30 sec intervals in LoBind Eppendorf tubes. Lysates were clarified by centrifugation at 13,000 xg for 20 min at 4°C and were then transferred to fresh LoBind Eppendorf tubes. Protein concentration was estimated using the Pierce™ BCA method. Equal protein from each condition were reduced with 5 mM DTT at room temperature for 30 min and alkylated with 20 mM IAA in the dark at room temperature for 15 min. Samples were then digested with Lys-C (1 :100) at 30°C for 4 hr. Samples were then diluted with 50 mM TEAB to a urea concentration of 1 .5 M and were then digested with trypsin (1 :20) at 30°C for 16 hr. The digest was quenched with the addition of 1% FA (v/v) and samples were desalted on 200 mg SepPak C18 cartridges (Waters) and dried by SpeedVac. Peptides were resuspended in 50 mM TEAB and labelled using tandem mass tag (TMT) labels as per the manufacturer’s instructions. TMT labels were resuspended in ACN, added to assigned samples and incubated for 1 hr at room temperature. Following label check by LC- MS/MS, the labelling reaction was quenched with 5% hydroxylamine for 15 min at room temperature. Labelled peptides from each condition were pooled together and dried. Pooled peptides were separated by basic reversed phase (bRP) chromatography fractionation with Ultimate 3000 HPLC system (Dionex) operating at 569 pl/min with two buffers: buffer A (10 mM ammonium formate, pH 10) and buffer B (80% ACN, 10 mM ammonium formate, pH 10). Peptides were resuspended in 100 pl of buffer A (10 mM ammonium formate, pH 10) and separated on a C18 reverse phase column. A total of 96 fractions were collected. 10% of each fraction were concentrated into 24 fractions for proteome analysis, whilst the remaining 90% were concentrated into 12 fractions for IMAC-based phospho-peptide enrichment. Each concentrated fraction was then dried by SpeedVac.
IMAC beads were prepared from Ni-NTA superflow agarose beads that were stripped of Nickel with 100 mM EDTA and incubated in an aqueous solution of 10 mM iron(lll) chloride (FeCI3). Dried peptide fractions were reconstituted to a concentration of 0.5 pg/pl in 80% ACN/0.1% TFA. Peptide mixtures were enriched for phosphorylated peptides with 10 pl IMAC beads for 40 min. Enriched IMAC beads were loaded on Empore C18 silica packed stage tips. Stage tips were equilibrated with methanol followed by 50% ACN/0.1% FA then 1% FA. The beads with enriched peptide were loaded onto C18 stage tips and washed with 80% ACN/0.1 % TFA. Phosphorylated peptides were eluted from IMAC beads with 500 mM dibasic sodium phosphate, pH 7.0. Enriched phospho-peptides and peptides were analysed on an Orbitrap Fusion Tribrid mass spectrometer interfaced with Easy-nLC II nanoflow liquid chromatography system. Peptides were separated on an analytical column (75 pm x 50 cm, RSLC C18) at a flow rate of 280 nl/min using a step gradient of 8-22% solvent B (90% ACN/0.1 % FA) for the first 70 min, followed by 22-35% up to 103 min. The total run time was set to 120 min. The mass spectrometer was operated in a data-dependent acquisition mode. A survey full scan MS (from m/z 350-1600) was acquired in the Orbitrap at a resolution of 120,000 at 200 m/z. The AGC target for MS1 was set as 4 x 105 and ion filling time set at 50 ms. The most intense ions with charge state >2 were isolated and fragmented using higher collision dissociation (HCD) fragmentation with 34% normalised collision energy and detected at a mass resolution of 50,000 at 200 m/z. The AGC target for MS2 was set as 1 x 105 and ion filling time set at 100 ms, while dynamic exclusion was set for 30 s. The mass spectrometry raw data were searched using Sequest HT search engines with Proteome Discoverer 2.1 (Thermo Fisher Scientific). Phosphopeptide-enriched fractions from each replicate were searched against the RefSeq protein database (version 89). The search parameter used as carbamidomethylation of cysteine residues as a fixed modification. Oxidation of methionine, the phosphorylations of serine, threonine and tyrosine, were selected as dynamic modifications. Trypsin was set as the protease and a maximum of two missed cleavages were allowed. Precursor mass tolerance was set to 10 ppm, and a fragment mass tolerance of 0.05 Da was allowed. All peptide-spectrum matches (PSM) were identified at a 1 % false-discovery rate (FDR). The probability of phosphorylation for each site was calculated by the phosphoRS node in Proteome Discoverer. Only phospho-peptides with >75% site localisation probability were considered for further analysis.
Quantification and Statistical Analysis
Statistical analysis was determined using unpaired Student’s t-test for single comparisons and for multiple treatments analysis of variance was performed followed by the post-hoc tests described in figure legends using Prism® Version 8.0.
Example 1 - Anti-GFP nanobody-directed recruitment of PPP1CA or PPP2CA mediates dephosphorylation of mitotic phospho-FAM83D-GFP
To evaluate whether target-specific nanobody-directed recruitment of the phosphatase catalytic subunits PPP1CA or PPP2CA mediates dephosphorylation of a protein of interest, FLAG-aGFPeM-PPP1CA or FI_AG-aGFPeM-PPP2CA AdPhosphatase fusion proteins were generated and expressed in FAM83DGFP/GFP knock in IISOS cells. The effect of the expression AdPhosphatase fusion proteins on FAM83D phosphorylation was then assessed.
FAM83D directs CK1a to the mitotic spindle to ensure proper spindle positioning and timely cell division (Fulcher et al., 2019). During mitosis, FAM83D also undergoes a CK1a-dependent hyperphosphorylation, causing ~25 kDa phospho-dependent electrophoretic mobility shift (Fulcher et al., 2019). Therefore, the inventors postulated that upon expression of the FLAG- aGFP6M-PPP1CA or -PPP2CA AdPhosphatases in FAM83DGFP/GFP knock in U2OS cells (Fulcher et al., 2019) but not in wild type (WT) cells, the phosphatase activity would be directed to FAM83D-GFP and mediate the dephosphorylation of hyperphosphorylated FAM83D-GFP during mitosis (Figure 1A).
U2OS wild-type (WT) and FAM83DGFP/GFP cells transduced with retroviruses encoding FLAG- empty control, FLAG-aGFP6M-PPP1CA or FI_AG-aGFPeM-PPP2CA were synchronised in mitosis using the Eg5 inhibitor S-trityl-L-cysteine (STLC) (Fulcher et al., 2019) and FAM83D protein levels were analysed (Figure 1 B). In mitotic LI2OS WT cells expressing FLAG-empty control, FLAG-aGFP6M-PPP1CA or FI_AG-aGFPeM-PPP2CA, FAM83D phosphorylation was observed by the striking ~25 kDa electrophoretic mobility shift compared to asynchronous controls. This data suggests that the low levels of expression of FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA alone in cells does not interfere with endogenous FAM83D phosphorylation during mitosis (Figure 1 B). In FAM83DGFP/GFP cel Is expressing FLAG-aGFPeM- PPP1CA or FLAG-aGFPeM-PPP2CA, but not those expressing FLAG-empty control, the mitotic phospho-FAM83D-GFP electrophoretic mobility shift collapsed, suggesting that targeted dephosphorylation of mitotic phospho-FAM83D-GFP was potentially achieved by both FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA (Figure 1 B). To determine that phospho-FAM83D-GFP dephosphorylation is mediated through an interaction with FLAG- aGFPeM-PPPICA or FLAG-aGFPeM-PPP2CA, extracts from both asynchronous and mitotic U2OS FAM83DGFP/GFP cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA or FLAG- aGFP6M-PPP2CA were subjected to anti-GFP IP (Figure 1C). In both asynchronous and mitotic extracts, FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA co-precipitated with FAM83D-GFP. In addition, both CK1a and the microtubule-associated protein hyaluronan- mediated motility receptor (HMMR, also referred to as RHAMM or CD168), which are validated endogenous FAM83D mitotic interactors (Fulcher et al., 2019), co-precipitated with FAM83D- GFP from mitotic extracts from cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA, suggesting that FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA do not interfere with the endogenous FAM83D-CK1a-HMMR mitotic interactions.
These data suggest that FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA interact with FAM83D-GFP to mediate the reduction in mitotic phosphorylation of phospho-FAM83D-GFP.
Example 2 - Dephosphorylation is dependent on the catalytic activity of PPP1CA or PPP2CA
To establish that the reduction in mitotic phosphorylation of phospho-FAM83D-GFP is reliant on the catalytic activity of PPP1CA or PPP2CA, asynchronous and mitotic U2OS FAM83DGFP/GFP cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA or FLAG-aGFP6M- PPP2CA were treated with the protein phosphatase inhibitor Calyculin A (Cal A) (Resjo et al., 1999, Ishihara et al., 1989) (Figure 1 D). In cells expressing FLAG-empty, FLAG-aGFPeM- PPP1CA or FLAG-aGFPeM-PPP2CA, an increase in Akt S473 phosphorylation was observed upon Cal A treatment compared to DMSO-treated controls, suggesting effective protein phosphatase inhibition. The phospho-FAM83D-GFP electrophoretic mobility shift observed in mitotic DMSO-treated FLAG-empty control cells was shifted upwards further by Cal A treatment. Moreover, the mitotic phospho-FAM83D-GFP mobility shift in FAM83DGFP/GFP cells that collapsed upon the expression of FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA was partially restored with Cal A treatment, suggesting that the dephosphorylation of phospho- FAM83D-GFP in mitosis is reliant on the phosphatase activity of FLAG-aGFP6M-PPP1CA or FLAG-aGFP6M-PPP2CA. To further ascertain that phospho-FAM83D-GFP dephosphorylation is reliant on the catalytic activity of the aGFPeM-directed recruitment of PPP1CA or PPP2CA, aGFPeM fused to catalytically dead PPP1CA (FLAG-aGFP6M-PPP1CAH125Q) and PPP2CA (FLAG-aGFP6M- PPP2CAH118Q) mutants (Egloff et al., 1995, Ogris et al., 1999a, Ogris et al., 1999b) were expressed by retroviral transduction in LI2OS FAM83DGFP/GFP cells (Figure 2A). Whilst a robust collapse of the mitotic phospho-FAM83D-GFP electrophoretic mobility shift was observed in mitotic extracts from FAM83DGFP/GFP cells expressing FLAG-aGFPeM-PPP1CA or FLAG- aGFP6M-PPP2CA compared to FLAG-empty controls, substantial mitotic phospho-FAM83D- GFP electrophoretic mobility shift was still evident in mitotic extracts expressing FLAG- aGFP6M-PPP1CAH125Q or FLAG-aGFP6M-PPP2CAH118Q catalytically dead mutants.
This data demonstrates that mitotic phospho-FAM83D-GFP dephosphorylation is reliant on the catalytic activity of the aGFPeM-directed recruitment of PPP1CA or PPP2CA.
Previously, it has been shown that following CK1a-dependent phosphorylation of FAM83D at the mitotic spindle, FAM83D is subsequently degraded by the proteasome following mitotic exit (Fulcher et al., 2019), although whether phosphorylation of FAM83D is required for its subsequent degradation is not known.
To determine the effects of AdPhosphatase-mediated dephosphorylation of mitotic phospho- FAM83D-GFP on FAM83D-GFP protein stability following STLC release, LI2OS FAM83DGFP/GFP cells expressing FLAG-empty, FLAG-aGFP6M-PPP1CA (Figure 2B), FLAG- aGFP6M-PPP1CAH125Q (Figure 2B), FLAG-aGFP6M-PPP2CA (Figure 2C) or FLAG-aGFP6M- PPP2CAH118Q (Figure 2C) were synchronised in mitosis using STLC and lysed at various time points following STLC washout. Following STLC release, as expected, a reduction in Cyclin B1 (Chang et al., 2003), HMMR and phosphorylated FAM83D-GFP (Fulcher et al., 2019) levels can be observed 3-6 hr after STLC washout in DMSO-treated FLAG-empty expressing control cells. In MG132-treated FLAG-empty control cells, an increase in polyubiquitylated substrates (Ub) was observed 3-6 hr after STLC wash-out compared with DMSO-treated controls, suggesting successful inhibition of the proteasome. Under these conditions, the reduction in Cyclin B1 , HMMR and phosphorylated FAM83D-GFP protein levels was not observed, suggesting they are degraded by the proteasome following STLC release. In cells expressing FLAG-aGFP6M-PPP1CA (Figure 2B), FLAG-aGFP6M-PPP1CAH125Q (Figure 2B), FLAG-aGFP6M-PPP2CA (Figure 2C) or FLAG-aGFP6M-PPP2CAH118Q (Figure 2C), a reduction in Cyclin B1 and HMMR levels was observed 3-6 hr after STLC washout, similar to that seen in FLAG-empty control cells, suggesting that the expression of FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM-PPP2CA does not negatively impact the cell cycle-dependent regulation of Cyclin B1 or HMMR. Interestingly, there was no reduction in FAM83D-GFP protein abundance following STLC washout in cells expressing FLAG-aGFP6M-PPP1CA (Figure 2B) or FLAG- aGFP6M-PPP2CA (Figure 2C), which was still evident in cells expressing FLAG-aGFP6M- PPP1CAH125Q (Figure 2B) or FLAG-aGFP6M-ppp2CAH118Q (Figure 2C). This suggests that following FAM83D-GFP phosphorylation in mitosis, phospho-FAM83D-GFP is degraded by the proteasome following STLC release, and that AdPhosphatase-mediated phospho- FAM83D-GFP dephosphorylation during mitosis inhibits phosphorylation-dependent proteasomal degradation of FAM83D-GFP.
Finally, to investigate the localisation of FAM83D-GFP and CK1a at the mitotic spindle following AdPhosphatase-mediated phospho-FAM83D-GFP dephosphorylation, LI2OS FAM83D KO (Fulcher et al., 2019) and FAM83DGFP/GFP cells expressing FLAG-empty, FLAG- aGFP6M-PPP1CA, FLAG-aGFP6M-PPP1CAH125Q, FLAG-aGFP6M-PPP2CA or FLAG-aGFP6M- PPP2CAH118Q were synchronised using STLC, fixed, and analysed by anti-CK1a and anti- FLAG immunostaining as well as GFP fluorescence microscopy (Figure 2D), and relative CK1a spindle fluorescence intensity was quantified (Figure 2E). As expected, a significant reduction in CK1a levels at the mitotic spindle was observed in FAM83D KO cells compared to FAM83DGFP/GFP cells (Figure 2D & E). Overlapping FAM83D-GFP, CK1a and FLAG localisation signals were observed at the mitotic spindle in FAM83DGFP/GFP cells expressing FLAG-aGFP6M-PPP1CA, FLAG-aGFP6M-PPP1CAH125Q, FLAG-aGFP6M-PPP2CA or FLAG- aGFP6M-PPP2CAH118Q (Figure 2D & E). These observations suggest that the AdPhosphatase- mediated dephosphorylation of phospho-FAM83D-GFP does not interfere with the mitotic localisation of either FAM83D-GFP or CK1a. Interestingly, a significant increase in CK1a levels at the mitotic spindle was observed in cells expressing FLAG-aGFP6M-PPP2CA compared to FLAG-empty controls (Figure 2D & E), potentially due to stabilisation of FAM83D- GFP upon targeted dephosphorylation.
Example 3: PPP2CA AdPhosphatase targets phospho-GFP-ULK1 for dephosphorylation
To investigate the versatility and broader applicability of the AdPhosphatase system, the inventors next targeted phospho-ULK1 for targeted dephosphorylation. FLAG-aGFPeM- PPP1CA or FLAG- aGFPeM-PPP2CA was expressed by retroviral transduction in ULK1GFP/GFP knock in ARPE-19 cells (Simpson et al., 2020) to direct PPP1CA or PPP2CA to GFP-ULK1 for targeted dephosphorylation of phospho-GFP-ULK1 and to assess the impact on downstream autophagy signalling (Figure 3A).
First, to determine the interaction between GFP-ULK1 and FLAG-aGFPeM-PPP1CA or FLAG- aGFP6M-PPP2CA, extracts from ARPE-19 WT control or ULK1GFP/GFP cells expressing FLAG- empty, FLAG-aGFP6M-PPP1CA or FLAG-aGFPeM- PPP2CA were subjected to anti-FLAG IP (Figure 3B). GFP-ULK1 co-precipitated with IPs only from ULK1GFP/GFP cell extracts expressing FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM- PPP2CA, but not from WT cells or ULK1GFP/GFP cells expressing FLAG-empty control, confirming that FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA can interact only with GFP-ULK1 , but not with untagged LILK1. In addition, both ATG13 and FIP200 co-precipitated in extracts from ULK1GFP/GFP cells expressing FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM-PPP2CA, suggesting that the expression of either FLAG-aGFPeM-PPP1CA or FLAG-aGFPeM- PPP2CA does not interfere with the formation of the ULK1-ATG13-FIP200 complex. Interestingly, a striking downward electrophoretic mobility shift of GFP-ULK1 and ATG13 was observed in extracts from ULK1GFP/GFP cells expressing FLAG-aGFP6M-PPP2CA, but not FLAG-aGFP6M-PPP1CA, suggesting potential dephosphorylation of both phospho-GFP-ULK1 and phospho-ATG13 by FLAG-aGFP6M-PPP2CA.
To confirm that the observed GFP-ULK1 dephosphorylation was reliant on the catalytic activity of the AdPhosphatase system, constructs containing the catalytically dead PPP1CA (FLAG- aGFP6M-PPP1CAH125Q) or PPP2CA (FLAG-aGFP6M-PPP2CAH118Q) mutants were expressed by retroviral transduction in ARPE-19 ULK1GFP/GFP cells (Figure 3C). Following anti-FLAG IP, GFP-ULK1 , ATG13 and FIP200 co-precipitated with IPs from ULK1GFP/GFP cells expressing FLAG-aGFP6M-PPP1CA, FLAG-aGFP6M-PPP1CAH125Q, FLAG-aGFP6M-PPP2CA or FLAG- aGFP6M-PPP2CAH118Q, indicating that the catalytically dead AdPhosphatase constructs interact with GFP-ULK1 and do not interfere with the formation of the LILK1 complex. Under these conditions, neither FLAG-aGFP6M-PPP1CA nor FLAG-aGFP6M-PPP1CAH125Q caused any detectable GFP-ULK1 or ATG13 mobility shift. Furthermore, the downward GFP-ULK1 and ATG13 electrophoretic mobility shift evident in FLAG-aGFP6M-PPP2CA expressing cells was not observed in FLAG-aGFP6M-PPP2CAH118Q expressing cells, suggesting that this potential dephosphorylation-induced mobility shift is reliant on PPP2CA catalytic activity.
Next, the localisation of GFP-ULK1 following AdPhosphatase- mediated GFP-ULK1 dephosphorylation was investigated. Upon starvation, ULK1 forms punctate structures that co-localise with omegasomes, thereby supporting autophagosome biogenesis (Karanasios et al., 2013, Zachari et al., 2020). ARPE-19 ULK1GFP/GFP cells expressing FLAG- empty, FLAG- aGFP6M-PPP2CA or FLAG-aGFP6M-PPP2CAH118Q were starved of amino acids with Earle’s balanced salt solution (EBSS) and treated with the lysosomal inhibitor Bafilomycin-A1 (Baf- A1 , 50 nM) to prevent autophagosome clearance (Yoshimori et al. , 1991 , Mauvezin et al., 2015, Klionsky et al., 2016) for 2 hr, fixed and analysed using anti-ULK1 and anti-FLAG immunostaining (Figure 3D). Under these conditions, ULK1 punctate structures were observed in ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty, FLAG-aGFPeM- PPP2CA or FLAG-aGFP6M-PPP2CAH118Q. In addition, FLAG punctate structures, some of which overlapped with LILK1 puncta, were also observed in ULK1GFP/GFP cells expressing FLAG-aGFP6M-PPP2CA or FLAG-aGFP6M PP2CAH118Q
To further ascertain that AdPhosphatase-mediated GFP-ULK1 dephosphorylation was reliant on the catalytic activity of the AdPhosphatase system, ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty control or FLAG-aGFP6M-PPP2CA were treated with the protein phosphatase inhibitors Cal A or Okadaic acid (OA) (Cohen et al., 1989, Fernandez et al., 2002, Resjo et al., 1999, Ishihara et al., 1989) (Figure 4A). In FLAG-empty or FLAG-aGFP6M-PPP2CA expressing cells, an increase in Akt-S473 phosphorylation levels was observed upon Cal A and OA treatment compared to DM SO-treated controls, suggesting effective protein phosphatase inhibition. No changes in GFP-ULK1 or ATG13 mobility shift or abundance were apparent in cells expressing FLAG-empty control upon Cal A or OA treatment compared to DMSO-treated controls. In contrast, in cells expressing FLAG-aGFP6M-PPP2CA, the downward GFP-ULK1 and ATG13 electrophoretic mobility shift observed in DMSO-treated cells was restored back to the levels seen in FLAG-empty cells upon treatment with either Cal A or OA, suggesting that the AdPhosphatase-mediated dephosphorylation of phospho-GFP- ULK1 and phospho-ATG13 is dependent on the phosphatase activity of FLAG-aGFP6M- PPP2CA.
Under nutrient-rich conditions, ULK1 is phosphorylated at multiple sites, including S757 by mTORCI , to inhibit autophagy (Kim et al., 2011). During periods of starvation, mTORCI is inactivated and the inhibitory phospho-sites on ULK1 are removed, resulting in increased ULK1 kinase activity (Kim et al., 2011). This leads to downstream autophagy signalling, including phosphorylation of ATG13 at S318 by activated ULK1 (Joo et al., 2011), expansion of the autophagosome, marked by lipidated microtubule-associated protein 1A/1 B-light chain 3 (LC3-II), which engulfs cargo and then fuses with the lysosome for cargo degradation (Zachari and Ganley, 2017). To investigate the effects of aGFP6M-PPP2CA-directed dephosphorylation of GFP-ULK1 on downstream starvation-induced autophagy signalling, ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty, FLAG-aGFP6M-PPP2CA or FLAG- aGFP6M-PPP2CAH118Q were starved of amino acids for 2 hr with EBSS (Figure 4B, C & D). During this period, cells were also treated with Baf-A1 to inhibit lysosomal degradation and monitor autophagic flux (Yoshimori et al., 1991 , Mauvezin et al., 2015, Klionsky et al., 2016). As expected, in EBSS-treated ULK1 GFP/GFP cells expressing FLAG-empty control, a reduction in GFP-ULK1 phosphorylation at S757 with a concomitant increase in ATG13 phosphorylation at S318 was observed compared to amino acid-rich controls, indicating ULK1 activation. Interestingly, in FLAG-aGFP6M-PPP2CA expressing cells under basal conditions, a substantial reduction in basal phosphorylation of LILK1 at S757 were observed compared to FLAG-empty control cells but with no concomitant phosphorylation of ATG13 at S318.
Furthermore, the EBSS-induced phosphorylation of ATG13 at S318 evident in FLAG-empty control cells were absent in cells expressing FLAG-aGFP6M-PPP2CA (Figure 4B & C). Moreover, inhibition of both basal and starvation-induced autophagy signalling was observed in cells expressing FLAG-aGFP6M-PPP2CA, as indicated by the large reduction in LC3-II flux, compared to FLAG-empty control cells (Figure 4B & D). These results suggest that one or more phospho-residues on ULK1 , other than S757, that are critical for ULK1 activation are also targeted for dephosphorylation by the AdPhosphatase leading to the inhibition of ULK1. In EBSS-stimulated cells expressing the FLAG-aGFP6M-PPP2CAH118Q catalytically dead mutant, robust ATG13 phosphorylation at S318 was observed, comparable to that observed in cells expressing FLAG-empty control (Figure 4B & C). Furthermore, in cells expressing FLAG-aGFP6M-PPP2CAH118Q, both basal and starvation-induced LC3-II flux were comparable to cells expressing FLAG-empty control (Figure 4B & D). These data serve to demonstrate that the reduction in ATG13 S318 phosphorylation and basal and starvation-induced LC3-II flux observed in cells expressing FLAG-aGFP6M-PPP2CA is dependent on FLAG-aGFP6M- PPP2CA catalytic activity.
Next, we sought to compare the efficacy of autophagy inhibition by AdPhosphatase-directed dephosphorylation of GFP-ULK1 to that of GFP-ULK1 inhibition using the small-molecule ULK1 inhibitor MRT68921 (Petherick et al., 2015, Zachari et al., 2020). ARPE-19 ULK1GFP/GFP cells expressing FLAG-empty control were pre-treated with or without MRT68921 (2 pM, 2 hr) and, along with cells expressing FLAG-aGFP6M-PPP2CA that were not treated with MRT68921 , were starved of amino acids with EBSS and treated with or without Baf-A1 (50 nM) for 2 hr (Figure 4E, F & G). Under starvation conditions, the reduction in ATG13 phosphorylation at S318 relative to amino acid-rich controls was comparable between MRT68921 -treated FLAG-empty control cells and those expressing FLAG-aGFP6M-PPP2CA (Figure 4E & F). In addition, under starvation conditions, LC3-II levels were comparable between MRT68921 -treated FLAG-empty control cells and those expressing FLAG-aGFP6M- PPP2CA (Figure 4E & G). These data suggest that the attenuation of starvation-induced autophagy observed following AdPhosphatase-mediated GFP-ULK1 dephosphorylation reflects that observed when cells were treated with a small-molecule inhibitor of ULK1 .
Example 4 - FLAG-aGFP6M-PPP2CA expression mediates the recruitment of PP2A regulatory subunits PP2A catalytic C subunits typically only exist in a fusion protein with a scaffold/structural A subunit (PP2A A) and regulatory B subunits, of which there are 26 (Janssens and Goris, 2001 , Mumby,2007, Seshacharyulu et al., 2013). The nature of the PP2A holoenzyme fusion protein determines substrate specificity, subcellular localisation and catalytic activity (Janssens and Goris, 2001 , Mumby, 2007, Seshacharyulu et al., 2013, Lambrecht et al., 2013). To determine whether FLAG-aGFP6M-PPP2CA exists by itself, or recruits additional scaffold and regulatory subunits, to mediate POI dephosphorylation, STLC-synchronised LI2OS FAM83DGFP/GFP cells (Figure 5A) or EBSS-treated ARPE-19 ULK1GFP/GFP cells (Figure 5B) expressing FLAG- aGFP6M- PPP2CA extracts were subjected to anti-FLAG IP. The resultant anti-FLAG IPs were resolved by SDS-PAGE, subjected to in-gel trypsin digestion and the resulting peptides were analysed by LC-MS/MS (Figure 5C). FLAG-aGFP6M-PPP2CA interactors identified exclusively from mitotic U2OS FAM83DGFP/GFP cells included FAM83D, CK1a and HMMR (Figure 5C), which were validated by Western blot (Figure 5A), whilst those identified exclusively from EBSS- treated ARPE-19 ULK1GFP/GFP cells included ULK1 , ATG13 and FIP200 (Figure 5C), which were also validated by Western blot (Figure 5B). Interestingly, FLAG-aGFP6M-PPP2CA interactors identified that were common to both mitotic LI2OS FAM83DGFP/GFP and EBSS- treated ARPE-19 ULK1GFP/GFP cells included PPP2CA (bait), the PP2A 65 kDa regulatory subunit A alpha (PPP2R1A) and beta (PPP2R1 B) isoforms, the PP2A 55 kDa regulatory subunit B alpha isoform (PPP2R2A), and the PP2A 56 kDa regulatory subunit delta (PPP2R5D) and epsilon (PPP2R5E) isoforms (Figure 5C). This data suggests that, FLAG- aGFP6M-PPP2CA does not exist by itself, but rather recruits additional regulatory subunits to mediate targeted dephosphorylation of phospho-FAM83D-GFP and phospho-GFP-ULK1.
Example 5 - Global phospho-proteomics demonstrates remarkable specificity of the FLAG-aGFP6M-PPP2CA AdPhosphatase system
To determine the specificity of the FLAG-aGFP6M-PPP2CA AdPhosphatase-mediated dephosphorylation of mitotic phospho-FAM83D-GFP or phospho-GFP-ULK1 following amino acid starvation, an unbiased global phospho-proteomic approach was employed (Figure 6). U2OS FAM83DGFP/GFP cells expressing FLAG-aGFP6M-PPP2CA or FLAG-aGFP6M- PPP2CAH118Q were synchronised in mitosis with STLC and subjected to quantitative phospho- and total-proteomic analyses (Figure 6A & B). Following total protein analysis of STLC- synchronised U2OS FAM83DGFP/GFP cells expressing FLAG-aGFP6M-PPP2CA compared with those expressing FLAG-aGFP6M-PPP2CAH118Q (Figure 6B), a total of 7,201 unique peptides were identified and of those, no proteins were observed to significantly change in abundance more than two-fold between the two conditions. This suggests that the expression of FLAG- aGFP6M-PPP2CA, compared to FLAG-aGFP6M-PPP2CAH118Q, in U2OS FAM83DGFP/GFP cells does not cause any substantial changes to total protein levels. Following phospho-peptide enrichment and analysis of STLC-synchronised LI2OS FAM83DGFP/GFP cells expressing FLAG- aGFP6M-PPP2CA compared with those expressing FLAG-aGFP6M-PPP2CAH118Q (Figure 6A and Table 1), 11 ,821 unique phospho-peptides were identified. Of these, phospho-peptides corresponding to 21 proteins were significantly downregulated by more than two-fold, and 6 were significantly upregulated by more than two-fold, in LI2OS FAM83DGFP/GFP cells expressing FLAG-aGFP6M-PPP2CA compared to those expressing FLAG-aGFP6M-PPP2CAH118Q (Figure 6A and Table 1). Of the significantly downregulated phospho-peptides detected in FLAG- aGFP6M-PPP2CA expressing cells corresponding to the 21 proteins listed, 2 FAM83D phospho-peptides were detected (pS493 and pS462). Further work is needed to determine whether phosphorylation of FAM83D at pS493 or pS462 mediates FAM83D proteasomal degradation following mitotic exit. Of the other phospho- peptides identified, some corresponding to mitosis-regulating proteins, including INCENP (inner centromere protein) (Adams et al., 2001 , Li et al., 2004, Honda et al., 2003, Sasai et al., 2016, Kang et al., 2011 , Qi et al., 2006), were found to be downregulated. Whether the down/upregulation observed in the phospho-peptides corresponding to these other proteins is a direct consequence of FAM83D-GFP dephosphorylation or rather due to being in proximity of FLAG-aGFP6M-PPP2CA requires further investigation.
Next, ARPE-19 ULK1GFP/GFP cells expressing FLAG-aGFP6M-PPP2CA or FLAG-aGFP6M- PPP2CAH118Q were starved of amino acids with EBSS for 2 hr and subjected to quantitative phospho- and total-proteomic analyses (Figure 6C & D). Following total protein analysis of EBSS-starved ARPE-19 ULK1GFP/GFP cells expressing FLAG-aGFP6M-PPP2CA compared with those expressing FLAG-aGFP6M-PPP2CAH118Q (Figure 6D and Table 2), a total of 8,153 unique peptides were identified and of those, those corresponding to 3 proteins were observed to be significantly downregulated more than two-fold between the two conditions, whilst those corresponding to 3 other proteins were observed to be significantly upregulated. Whether the observed changes in the abundance of these 6 proteins (Figure 6D and Table 2) is a direct consequence of GFP-ULK1 dephosphorylation needs to be further investigated. Following phospho-peptide enrichment and analysis of EBSS- starved ARPE-19 ULK1GFP/GFP cells expressing FLAG-aGFP6M-PPP2CA compared with those expressing FLAG-aGFP6M- PPP2CAH118Q (Figure 6C and Table 3), 22,574 unique phospho-peptides were identified. Of these, phospho-peptides corresponding to 43 proteins were significantly downregulated by more than two-fold, and 63 were significantly upregulated by more than two-fold, in ARPE-19 ULK1GFP/GFP cells expressing FLAG-aGFP6M- PPP2CA compared to those expressing FLAG- aGFP6M-PPP2CAH118Q (Figure 6C and Table 3). Of the significantly downregulated phospho- peptides detected in FLAG-aGFP6M-PPP2CA expressing cells corresponding to the 43 proteins listed, 3 LILK1 phospho-residues were detected (S539, S544 and S694). Further work is needed to determine whether phosphorylation of LILK1 at S539, S544 or S694 impacts its role in starvation-induced autophagy. Of the other phospho-peptides identified, those corresponding to autophagy-regulating proteins, including Beclin-1 (Russell et al., 2013, Qian et al., 2017), AP-3 fusion protein subunit beta-1 (AP3B1) (Kannangara et al., 2021) and La-related protein 1 (LARP1) (McKnight et al., 2012), were found to be downregulated. Whether the down/upregulation observed in the phospho-peptides corresponding to these other proteins is a direct consequence of GFP-ULK1 dephosphorylation and inactivation or rather due to being in proximity of FLAG-aGFP6M-PPP2CA needs to be further investigated.
Overall, these results indicate that the AdPhosphatase system is highly specific and a change in the phosphorylation status of relatively few off-target proteins is observed. This provides a significant advantage over existing kinase inhibitors that target significantly more off-target proteins than observed with fusion proteins of the present invention.
Example 6 - Inducible dephosphorylation of a phospho-POl using AdPhosphatases.
To further investigate the versatility and broader applicability of the AdPhosphatase system, the inventors investigated the ability of AdPhosphatases to dephosphorylate a phospho- POIs under the control of an inducible system.
AdPhosphatase was placed under the control of a tetracycline-inducible expression system, namely the retro-X Tet-One Inducible Expression System whereby both the tetracyclineresponsive transactivator and the nucleic acid encoding the fusion protein are combined in the same expression vector. C2C12 myoblast cells in which TFEB is tagged with a GFP tag were employed. Constructs Flag-aGFP6M-PP1CA (SEQ ID NO: 185), Flag-aGFP6M- PP1CA-H125Q (SEQ ID NO: 188) , Flag-aGFP6M-PP2CA (SEQ ID NO: 186), and Flag- aGFP6M-PP2CA-H118Q (SEQ ID NO: 187) were expressed by retroviral transduction using RetroX-Tet-One retroviruses. AdPhosphatase expression was induced by treating the cells harbouring the AdPhosphatase constructs with doxycycline. In cells expressing PP1A and PP2A, but not in cells expressing the catalytically dead variants PP1CAH125Q or PP2CAH118Q, dephosphorylation was detectable through an observable electrophoretic mobility shift (Figure 7A). The observable dose-dependence of GFP-TFEB dephosphorylation in correlation with the concentration of doxycycline administered suggests that dephosphorylation is achieved specifically through the induction of the AdPhosphatases (Figure 7A). This is further demonstrated by the Flag immunoblots, which confirm that AdPhosphatase protein levels correlate with the doxycycline concentrations administered. Furthermore, time-course immunoblots demonstrate that in cells expressing PP1A and PP2A, but not in cells expressing PP1CAH125Q or PP2CAH118Q, GFP-TFEB dephosphorylation gradually increases up to 24 hours after doxycycline-mediated AdPhosphatase induction, and this also correlates with the levels of AdPhosphatase protein measured by anti-Flag immunostaining (Figure 7B). 100 ng/mL was identified as being an optimum induction concentration, but this does not restrict the concentration that may be useful in other examples of the invention.
Taken together, this data demonstrates inducible targeted dephosphorylation of phospho- POIs.
Example 7 - AdPhosphatase-mediated dephosphorylation of TAU
Tauopathies are neurodegenerative disorders characterised by the deposition of abnormal tau protein in the brain. Accumulation of phosphorylated tau is a key pathological feature of Alzheimer's disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. Therefore, the inventors sought to demonstrate the utility of AdPhosphatases on the dephosphorylation of TAU.
To demonstrate TAU dephosphorylation, the inventors employed SK-N-MC cells in which TAU was tagged with GFP. This allows the aforementioned anti-GFP AdPhosphatases constructs to be used, which target GFP and by spatial proximity can target TAU for dephosphorylation in these cells.
Cells harbouring GFP-tagged TAU were transformed with either constitutively expressed AdPhosphatase constructs, encoded in pBabeD vectors (Figure 8A), or inducible AdPhosphatases, encoded in RetroX-Tet-One vectors (Figure 8B). Dephosphorylation on TAU was tested on several known phospho-sites (T181 , S393, S404, S202/205) by immunostaining. The immunoblots show that catalytically active PPP2CA, but not the catalytically inactive PPP2CAH118Q, induce dephosphorylation on all the tested phospho-sites when expressed constitutively (Figure 8A). This demonstrates the utility of AdPhosphatases in being able to dephosphorylate therapeutically relevant phospho-POIs such as TAU.
This conclusion is further supported by experiments where PPP1CA and PPP2CA, and catalytically dead versions PPP1CAH125Q and PPP2CAH118Q, are under the inducible expression of doxycycline. In absence of doxycycline, there is no observable difference in TAU phospho-site phosphorylation levels across the transformed cells regardless of which AdPhosphatase they harbour (Figure 7B, lanes under ‘- Dox’). However, when doxycycline is administered in the optimal concentration and duration of time as established in Example 6 (100 ng/mL, 24 h), the expression of catalytically active PPP1CA and PPPC2A, but not the catalytically dead PPP1CAH125Q and PPP2CAH118Q, correlate with dephosphorylation of TAU (Figure 8B, lanes under ‘+ Dox (100ng/ml, 24 h)’). The most notable dephosphorylation is observable for phospho-site T181 , which appears almost completely dephosphorylated (Figure 8B). Anti-TAU immunostaining demonstrates that TAU proteins levels are similar across the various cell lines, ruling out the possibility that the phospho-stains are inadvertently confounded by TAU levels (Figure 8B, panel IB: Tau). Anti-Flag immunostaining confirms that AdPhosphatase protein is only detected when doxycycline is administered to the cells, in line with expectations as these constructs being under tetracycline-inducible expression systems (Figure 8B).
These results demonstrate that the AdPhosphatase of the present invention can induce targeted phosphorylation of therapeutically relevant targets such as TAU, and provide further evidence that inducible dephosphorylation is possible using AdPhosphatases.
Table 1. Down/upregulated phospho-peptides identified following global phospho- proteomic analysis of AdPhosphatase-mediated FAM83D-GFP dephosphorylation.
Values of down/upregulated phospho-peptides identified (out of a total of 11 ,821 unique phospho- peptides detected) from global analysis of STLC-synchronised LI2OS FAM83DGFP/GFP cells expressing either FLAG-aGFP6M-PPP2CA or FLAG-aGFP6M- PPP2CAH118Q. UniProt ID is indicated for the protein, as are corresponding peptides detected, fold changes and p-values. The threshold parameters included a significance level of p<0.05 and fold change >2. Phospho-peptides detected corresponding to FAM83D are highlighted in green, including phospho-residues S493 and S462 that were detected. Proteins are listed in alphabetical order.
Figure imgf000062_0001
Figure imgf000063_0001
Table 2. Down/upregulated peptides identified following global total proteome analysis of AdPhosphatase-mediated GFP-ULK1 dephosphorylation.
Values of down/upregulated peptides identified (out of a total of 8,153 unique peptides detected) from global analysis of EBSS-starved ARPE-19 ULKGFP/GFP cells expressing either
FLAG-aGFP6M-PPP2CA or FLAG-aGFP6M-PPP2CAH118Q UniProt ID is indicated for the protein, as are corresponding fold changes and p-values. The threshold parameters included a significance level of p<0.05 and fold change >2. Proteins are listed in alphabetical order.
Figure imgf000063_0002
Table 3. Down/upregulated phospho-peptides identified following global phospho- proteomic analysis of AdPhosphatase-mediated GFP-ULK1 dephosphorylation.
Values of down/upregulated phospho-peptides identified (out of a total of 22,574 unique phospho- peptides detected) from global analysis of EBSS-starved ARPE-19 ULKGFP/GFP cells expressing either FLAG-aGFP6M-PPP2CA or FLAG-aGFP6M-PPP2CAH118Q. UniProt ID is indicated for the protein, as are corresponding peptides detected, fold changes and p-values. The threshold parameters included a significance level of p<0.05 and fold change >2. Phospho-peptides detected corresponding to LILK1 are highlighted in green, including phospho-residues S694, S539 and S544 that were detected. Proteins are listed in alphabetical order.
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Discussion
Through the conjugation of high-affinity polypeptide binders of specific POIs to a catalytic subunit of a phosphatase (AdPhosphatase), the inventors have demonstrated that the phosphatase catalytic activity can be redirected to the POI to mediate targeted phospho-POl dephosphorylation with exquisite selectivity.
By directing an AdPhosphatase construct consisting of aGFP6M conjugated to PPP1CA or PPP2CA to FAM83D-GFP in FAM83DGFP/GFP cells, dephosphorylation of phospho-FAM83D- GFP was promoted in mitosis to prevent phosphorylation-triggered FAM83D-GFP proteasomal degradation. Using the AdPhosphatase system, the role of CK1a-mediated phosphorylation of FAM83D in mitosis was established to lead to its proteasomal degradation, highlighting the applicability of the AdPhosphatase system in understanding the function of phospho-modifications on POIs. The PPP2CA AdPhosphatase-mediated dephosphorylation of phospho-FAM83D-GFP in mitotic FAM 83 DGFP/GFP cells was remarkably specific, with only a short list of other phospho-peptides shown to be significantly downregulated in an unbiased global phospho-proteomic screen.
Furthermore, an AdPhosphatase construct consisting of aGFP6M conjugated to PPP2CA mediated the targeted dephosphorylation of phospho-GFP-ULK1 when expressed in ULK1GFP/GFP cells. AdPhosphatase-mediated GFP-ULK1 dephosphorylation attenuated starvation-induced autophagy to the same extent as chemical inhibition of ULK1. ULK1 undergoes phosphorylation at multiple residues that orchestrate either an activating or inhibitory role on its activity and subsequently its role in autophagy initiation (Zachari and Ganley, 2017). The PPP2CA AdPhosphatase system resulted in a profound dephosphorylation of phospho-GFP-ULK1 and resulted in the inhibition of starvation-induced autophagy, suggesting that this system targeted the dephosphorylation of dominant ULK1 phosphoresidues that potentiate ULK1 activity. Following global phospho-proteomic analysis, three ULK1 phospho-residues, S539, S544 and S694, were found to be significantly downregulated more than two-fold in EBSS-starved cells expressing FLAG-aGFP6M-PPP2CA compared to FLAG-aGFP6M-PPP2CAH118Q. Further work is needed to determine whether phosphorylation of LILK1 at S539, S544 orS694 are necessary for initiating starvation-induced autophagy. In addition, the FLAG-aGFP6M-PPP2CA AdPhosphatase system could be further employed to determine whether the phosphorylation of LILK1 is involved in the regulation of additional selective forms of autophagy, such as mitophagy, ER-phagy/reticulophagy, pexophagy, ferritinophagy, nucleophagy, lysophagy, lipophagy, glycophagy, aggrephagy and xenophagy (Gatica et al., 2018). Also of interest was the observation that the LILK1 -mediated phosphorylation of ATG13 at S318 following amino acid starvation was attenuated by the FLAG-aGFP6M-PPP2CA AdPhosphatase system. However, as ATG13 is in complex with LILK1 , further work needs to be undertaken to determine whether ATG13 dephosphorylation was a direct consequence of GFP-ULK1 inactivation following AdPhosphatase-mediated dephosphorylation or rather due to FLAG-aGFP6M-PPP2CA directly dephosphorylating ATG13 due to its proximity.
Interestingly, the AdPhosphatase system consisting of aGFPeM conjugated to PPP1CA was unable to dephosphorylate LILK1 , although it acted to dephosphorylate FAM83D-GFP, suggesting a possibility that PPP1CA may not have accessed the same phospho-residues on LILK1 that PPP2CA did. Further investigations are needed to ascertain the range of dephosphorylation activity of the different phosphatases when used with the AdPhosphatase system. To expand upon the phosphatase candidates that can be exploited with the AdPhosphatase system and potentially provide further specificity, a less promiscuous phosphatase, such as the metal-dependent protein phosphatase PPM1 H, which is highly selective towards a subset of Rab GTPase proteins (Berndsen et al., 2019, Khan et al., 2021 , Waschbusch et al., 2021 , Malik et al., 2021), could be employed. Although PPM1 H is likely to dimerise, PPM1 H does not require additional scaffold or regulatory subunits to facilitate target protein dephosphorylation (Waschbusch et al., 2021), which may prove beneficial for AdPhosphatase system applications.
As the AdPhosphatase system demonstrates efficient targeted dephosphorylation, the next stage for targeted dephosphorylation as a potential therapeutic approach would be to develop heterobifunctional small-molecules that directly bind the endogenous, untagged POI and recruit the phosphatase of interest. The dephosphorylation-inducing heterobifunctional smallmolecules targeting Akt and EGFR have been reported albeit with poor efficiency of targeted dephosphorylation (Yamazoe et al., 2020). Tag-based phosphorylation targeting chimeras (PhosTACs) involving dTAG/Halo-recruiting heterobifunctional small-molecules have also been designed to recruit overexpressed FKBP12F36V-tagged PP2A to an overexpressed Halo- tagged POI to mediate POI dephosphorylation (Chen et al., 2021) for a chemical proof-of- concept. To transfer this dTAG/Halo-recruiting PhosTAC approach to endogenously regulated POIs, FKBP12F36V and Halo would need to be knocked in on to the respective phosphatase and POIs using, for example, CRISPR/Cas9 genome editing technology. The AdPhosphatase system can be exploited not only to explore the biological role of specific phospho-POIs, but also to rapidly inform the utility of phosphatase-mediated POI dephosphorylation before investing in the resource- and time-intensive development of POI-specific dephosphorylationinducing heterobifunctional small-molecules.
Protein phosphorylation is a fundamental driver of all cell signalling processes and is therefore tightly regulated. Hyperphosphorylation of proteins is a known hallmark of many diseases, including cancer and neurodegenerative diseases. This new approach termed the AdPhosphatase system, can efficiently and selectively target specific proteins of interest (POIs) for dephosphorylation. The AdPhosphatase system is versatile and adaptable, where, in principle, any promiscuous phosphatase can be redirected to dephosphorylate any phospho-POI.
Without wishing to be bound by theory, this technology allows researchers to dissect the role of phosphorylation on potentially any POI and is a promising new therapeutic modality. The AdPhosphatase system can be exploited to rapidly inform the utility of targeted POI dephosphorylation in any therapeutic application.
Sequences aGFP DNA Sequence atggccgatgtgcagctggttgaatctggcggtgcactggtgcagccgggcggtagcctgcgtctgtcttgcgtggccagtggcttt ccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaaagaacgtgaatgggtggcaggcatgagctctgcgggt gatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtcgtgatgatgcgcgcaacacggtgtacctgcagatgaat agtctgaaaccggaagataccgccgtttattactacaacgtgaatgttggcttcgaatattggggccatggtacccaggtgacggtt tctttttga (SEQ ID NO: 163) aGFP(6M) Amino Acid Sequence
MADVQLVESGGALVQPGGSLRLSCVASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGD RSSYVDSVKGRFTIRRDDARNTVYLQMNSLKPEDTAVYYYNVNVGFEYWGHGTQVTVSF (SEQ ID NO: 164)
PPP1CA DNA Sequence aggccggaaggaggctgccggagggcgggaggcaggagcgggccaggagctgctgggctggagcggcggcgccgccat gtccgacagcgagaagctcaacctggactcgatcatcgggcgcctgctggaagtgcagggctcgcggcctggcaagaatgta cagctgacagagaacgagatccgcggtctgtgcctgaaatcccgggagatttttctgagccagcccattcttctggagctggagg cacccctcaagatctgcggtgacatacacggccagtactacgaccttctgcgactatttgagtatggcggtttccctcccgagagc aactacctctttctgggggactatgtggacaggggcaagcagtccttggagaccatctgcctgctgctggcctataagatcaagta ccccgagaacttcttcctgctccgtgggaaccacgagtgtgccagcatcaaccgcatctatggtttctacgatgagtgcaagaga cgctacaacatcaaactgtggaaaaccttcactgactgcttcaactgcctgcccatcgcggccatagtggacgaaaagatcttct gctgccacggaggcctgtccccggacctgcagtctatggagcagattcggcggatcatgcggcccacagatgtgcctgaccag ggcctgctgtgtgacctgctgtggtctgaccctgacaaggacgtgcagggctggggcgagaacgaccgtggcgtctcttttaccttt ggagccgaggtggtggccaagttcctccacaagcacgacttggacctcatctgccgagcacaccaggtggtagaagacggct acgagttctttgccaagcggcagctggtgacacttttctcagctcccaactactgtggcgagtttgacaatgctggcgccatgatga gtgtggacgagaccctcatgtgctctttccagatcctcaagcccgccgacaagaacaaggggaagtacgggcagttcagtggc ctgaaccctggaggccgacccatcaccccaccccgcaattccgccaaagccaagaaatagcccccgcacaccaccctgtgc cccagatgatggattgattgtacagaaatcatgctgccatgctgggggggggtcaccccgacccctcaggcccacctgtcacgg ggaacatggagccttggtgtatttttcttttctttttttaatgaatcaatagcagcgtccagtcccccagggctgcttcctgcctgcacctg cggtgactgtgagcaggatcctggggccgaggctgcagctcagggcaacggcaggccaggtcgtgggtctccagccgtgcttg gcctcagggctggcagccggatcctggggcaacccatctggtctcttgaataaaggtcaaagctggattctc (SEQ ID NO: 165)
PPP1CA Amino Acid Sequence
/WSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFLSQPILLELEAPLKICGDI HGQYYDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLRGNHEC ASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIVDEKIFCCHGGLSPDLQSMEQIRRIMR PTDVPDQGLLCDLLWSDPDKDVQGWGENDRGVSFTFGAEWAKFLHKHDLDLICRAHQW EDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPADKNKGKYGQFSG LNPGGRPITPPRNSAKAKK (SEQ ID NO: 166)
PPP1CA H125Q
MSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFLSQPILLELEAPLKICGDI HGQYYDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYKIKYPENFFLLRGNQEC ASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIVDEKIFCCHGGLSPDLQSMEQIRRIMR PTDVPDQGLLCDLLWSDPDKDVQGWGENDRGVSFTFGAEWAKFLHKHDLDLICRAHQVV EDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQILKPADKNKGKYGQFSG LNPGGRPITPPRNSAKAKK (SEQ ID NO: 167)
PPP2CA DNA Sequence acagagagccgagctctggagcctcagcgagcggaggaggaggcgcagcggccgacggccgagtactgcggtgagagc cagcgggccagcgccagcctcaacagccgccagaagtacacgaggaaccggcggcggcgtgtgcgtgtaggcccgtgtgc gggcggcggcgcgggagcagcgcggagcggcagccggctggggcgggtggcatcatggacgagaaggtgttcaccaagg agctggaccagtggatcgagcagctgaacgagtgcaagcagctgtccgagtcccaggtcaagagcctctgcgagaaggcta aagaaatcctgacaaaagaatccaacgtgcaagaggttcgatgtccagttactgtctgtggagatgtgcatgggcaatttcatgat ctcatggaactgtttagaattggtggcaaatcaccagatacaaattacttgtttatgggagattatgttgacagaggatattattcagtt gaaacagttacactgcttgtagctcttaaggttcgttaccgtgaacgcatcaccattcttcgagggaatcatgagagcagacagat cacacaagtttatggtttctatgatgaatgtttaagaaaatatggaaatgcaaatgtttggaaatattttacagatctttttgactatcttc ctctcactgccttggtggatgggcagatcttctgtctacatggtggtctctcgccatctatagatacactggatcatatcagagcacttg atcgcctacaagaagttccccatgagggtccaatgtgtgacttgctgtggtcagatccagatgaccgtggtggttggggtatatctc ctcgaggagctggttacacctttgggcaagatatttctgagacatttaatcatgccaatggcctcacgttggtgtctagagctcacca gctagtgatggagggatataactggtgccatgaccggaatgtagtaacgattttcagtgctccaaactattgttatcgttgtggtaac caagctgcaatcatggaacttgacgatactctaaaatactctttcttgcagtttgacccagcacctcgtagaggcgagccacatgtt actcgtcgtaccccagactacttcctgtaatgaaattttaaacttgtacagtattgccatgaaccatatatcgacctaatggaaatgg gaagagcaacagtaactccaaagtgtcagaaaatagttaacattcaaaaaacttgttttcacatggaccaaaagatgtgccatat aaaaatacaaagcctcttgtcatcaacagccgtgaccactttagaatgaaccagttcattgcatgctgaagcgacattgttggtca agaaaccagtttctggcatagcgctatttgtagttacttttgctttctctgagagactgcagataataagatgtaaacattaacacctc gtgaatacaatttaacttccatttagctatagctttactcagcatgactgtagataaggatagcagcaaacaatcattggagcttaat gaacatttttaaaaataattaccaaggcctcccttctacttgtgagttttgaaattgttctttttattttcagggataccgtttaatttaattata tgatttgtctgcactcagtttattccctactcaaatctcagccccatgttgttctttgttattgtcagaacctggtgagttgttttgaacagaa ctgttttttccccttcctgtaagacgatgtgactgcacaagagcactgcagtgtttttcataataaacttgtgaactaagaactgagaa ggtcaaattttaattgtatcaatgggcaagactggtgctgtttattaaaaaagttaaatcaattgagtaaattttagaatttgtagacttg taggtaaaataaaaatcaagggcactacataacctctctggtaactccttgacattcttcagattaacttcaggatttatttgtatttcac atattacaatttgtcacattgttggtgtgcactttgtgggttcttcctgcatattaacttgtttgtaagaaaggaaatctgtgctgcttcagta agacttaattgtaaaaccatataacttgagatttaagtctttgggttttgttttaataaaacagcatgttttcaggtagagcttaaactaa atgatgtgtttacttagtgcagtttctggttatgaatattatattgctatgtgtatattatatggactctttaaaatgattgacagattggcaa attcttaaatctttgtacattgttgagtcatatgttcttagagttaaatttgtctcagataagaaagtgttaaagcattagcctgtgtcaagt tctttgagtgatactagtgaaaccaaatagaaaactattgttggatcatgatttagtcttatgtacattcacccgaagacaaaaatggt acttaaagtggcagtgttcaacatttaatgagtttttccccttttatccttcgaataggattagatgtttaaaaaaaagttcttctgtggga actaatatttgatattttaacctaccagagtaaacaggaacacttaatcatacttgtgagtgtagtaaataaaagttttcttgctttgtgct gtgttgaatctggaaccaacagggaagttatagcatatcccctttctaaaatgcttgaggaacacatacataccgaatgtcttttctg atctaattgatagtatttttagtggcttgtggagttaattttccaaagcaaaaggccattagggtttctacatttcatttcatttcattcttttctt tcacaagaaatacattctctgtgtgtctttttgttgctctgtcactctatgccctttctctccgactgaacaaatagcttatccatgtgcagt ggttttaatacccaaacaatctagacaccaagcagctattttttccggtcctgtgatatcagaattgaccaaggaatacgtatattgta attgacacgtggtggtatcttccaggtacaaattctctaaattttgtggttagcagaatgggacttgtgataagaatagcttggttttag cataactagggtttaaaataattgtttaattatagagactgaccgggctttggccatctaaactggaaagtgttagtaccctaccttctt ttgaaaatggctatggtaaggaaaatgtgttagtaaattatgtattttcttgaaaaatacataattatggttggatgggaatcactaagt tgggtgttaactgatgtctcaattagtaacattaggattttcattaataaacctaaaaagctttccctaagaacaggcctggcacagt ggctcatgcctgtaatcccagcactttgggagaccagggtgggtggatcccttgaggtcaggagttgaagagcagcctggccaa catggcaaaaccccatctctacaaaaaatatgaaaatcagccgggcttggtggcataccatagtcccagctacttgggaggctg aggtgagaggatcgcctgagcccaggagatggaggttgcagtgggctgagattgtgccactgtactccagtctgggtgacaga gccagaccctgtctcaaaaataaagaggattctgagtttgtatagtgagggcttgcagaaattttgaaacttattttgtaagtttacaa tgaatttgtacatgatgtgctcatgtcttgggttgagtatcctagacatgattttttcatttgctgcatattaaacatttgttggttgtagtcgg tatttcttaaatagaagtttgtcaatattagattagtttcaagaaggacttagctcaggaaaaggatagttatttctgtggttctcagcttt gatgcctacagagattctgatttaaattgctggaggagggcccaggcatacattttttaagtttcccaagtgattctaatttacatctag ggttaaaaacagccaggcgcggtggctcatgcctgtaatcccagcacttggggaggccggggtgggtggatcacctgatgtca ggagatcgagaccagcctggccagcgtggtgaaaccccatctctattaaaaatacaaaactgaccggtgtggtggcaggagc ctgtaatcccagctacttgggaggctgaggcaggagaatcacttgaactcaggaggaggaggttacagtgagcccagatcgtg ccattgcactccagcctgggcaaaaagagcaaaactccatctcaaaaaacaaaaacacctcactgctgtttcctaagtacatac ttaagaaaattgggatacatggtggtggttcatggatgttgataaggaattaaaatgtaccgtgcgactctctgtttcagtggtgacttt tacctgtttagtataaatattcctttgcttccaaccataaatgtgttcttagaaatgggcctatagtttagtaacctatagtttggtaatagg cttgtttgttttcagatggattttggttctgtgagctaaagctattttgcattaaagccttcgtcctcaca (SEQ ID NO: 168)
PPP2CA Amino Acid
Figure imgf000072_0001
M DEKVFTKELDQWI EQLN ECKQLSESQVKSLCEKAKEI LTKESNVQEVRCPVTVCGDVHG
QFHDLMELFRIGGKSPDTNYLFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNHESRQ
ITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVDGQIFCLHGGLSPSIDTLDHIRALDR
LQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETFNHANGLTLVSRAHQLV
MEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQFDPAPRRGEPHVTRRT
PDYFL (SEQ ID NO: 169)
PPP2CA H118Q
M DEKVFTKELDQWI EQLN ECKQLSESQVKSLCEKAKEI LTKESNVQEVRCPVTVCGDVHG
QFHDLMELFRIGGKSPDTNYLFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNQESRQ
ITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVDGQIFCLHGGLSPSIDTLDHIRALDR
LQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETFNHANGLTLVSRAHQLV
MEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQFDPAPRRGEPHVTRRT
PDYFL (SEQ ID NO: 170)
Figure imgf000072_0002
acatgacccggcggcagtagccgtggcagcagccgcggcggctccgcgagctcgccgggtgggctcagttcagcgcacgcc ggagccgagcgcagggggcggggaagggacctgctgcagctgcagccgcctgggcgctcctggagcgcgcggtgactccc ccggtcggcccgctccatgcagctccgttgcggaagtgtagcggggggaggcggcggccaccgcggcactaagcacgaga ggccggggctcggccccctgcagcactaggctctgggagccgcgcgcggcgcgtcccagtggcccgactcgccgtgcgccc ggcgcccaccgcagcctgcatgccccgcgctgcgccttgcccggcccccgccgcctcctgctcgcaccgctgcagccgggcg ccggagtaatatgctcactcgagtgaaatctgccgtggccaatttcatgggcggcatcatggctggcagctcaggctccgagcac ggcggcggcagctgcggaggctcggacctgcccctgcgtttcccctacgggcggccagagttcctggggctgtctcaggacga ggtggagtgcagcgccgaccacatcgcccgccccatcctcatcctcaaggagactcggcggctgccctgggccactggctacg cagaggttatcaatgccgggaagagcacacacaatgaagaccaagccagctgtgaggtgctcactgtgaagaagaaggca ggggccgtgacctcaaccccaaacaggaactcatccaagagacggtcctcccttcccaatggggaagggctgcagctgaag gagaactcggaatccgagggtgtttcctgccactattggtcgctgtttgacgggcacgcggggtccggggccgcggtggtggcgt cacgcctgctgcagcaccacatcacggagcagctgcaggacatcgtggacatcctgaagaactccgccgtcctgccccctacc tgcctgggggaggagcctgagaacacgcccgccaacagccggactctgacccgggcagcctccctgcgcggaggggtggg ggccccgggctcccccagcacgccccccacacgcttctttaccgagaagaagattccccatgagtgcctggtcatcggagcgct tgaaagtgcattcaaggaaatggacctacagatagaacgagagaggagttcatataatatatctggtggctgcacggccctcatt gtgatttgccttttggggaagctgtatgttgcaaatgctggggatagcagggccataatcatcagaaatggagaaattatccccatg tcttcagaatttacccccgagacggagcgccagcgacttcagtacctggcattcatgcagcctcacttgctgggaaatgagttcac acatttggagtttccaaggagagtacagagaaaggagcttggaaagaagatgctctacagggactttaatatgacaggctggg catacaaaaccattgaggatgaggacttgaagttcccccttatatatggagaaggcaagaaggcccgggtaatggcaactattg gagtgaccaggggacttggggaccatgacctgaaggtgcatgactccaacatctacattaaaccattcctgtcttcagctccaga ggtaagaatctacgatctttcaaaatatgatcatggatcagatgatgtgctgatcttggccactgatggactctgggacgttttatcaa atgaagaagtagcagaagcaatcactcagtttcttcctaactgtgatccagatgatcctcacaggtacacactggcagctcagga cctggtgatgcgtgcccggggtgtgctgaaggacagaggatggcggatatctaatgaccgactgggctcaggagacgacatttc tgtatatgtcattcctttaatacatggaaacaagctgtcatgaaaatggcccaggggattgggaggacagaggggaagaaagct gggatgcctcttggcaggacggaactgggaagtgccccagctgagttccaagtgatgcagtctcttcccagcccaagcgggga gttcatggccaaaagactatgcttcaagatgaccctttggtttccatttcttctttagtaacaggtcaactcaacaagagcaaaacac aaaggctgctaccaagtgttgttgtatttcagttcctttcataggcctccgaggtggccattgactatttggggtatatatgtcatatttatt ttatctagagtagctggggcagccattttcaggtgtaaatggcagaggactcttcagcctgtcaagctgccagcttatctacgggtta aaaagtgctgcattggaaagtagggggtcatgcctcaaaatgtaagtaagtgcccaccttctaggaagcctgaggtttatttcagg gattgccgtctgccccccgccccccttctctttttttcttctctgtttctattcttttatggcagtggtggagtgaggcagggatttttttttttttttt tcgtgtttttgacattccttgaatctgttttttattccccttccacagaacaggcctgggactttccaacaccctgctaaggaagttctgtgt ccaagtcccacccaggctgggttgtccccacctcctccagcccacacagcccaggcagcatccgggccagtgccctgcatgac agagggtctttgttgtgtaatgtttgttcccaagttgcattttctaaccgaatcagtgtgttttcatgaaactgagtgtttctgtggaccagt agttcctctgttgtcttcagtggtcttcctgtgtggctcaagggttctctgtgagagtctggattttcatttctggaatggctggccccatcc cacttttctgtatcatggggacacatataaagcagtgtttaatagagcagtttaagaagttgcttgcatctgttggttcaccatggctca tctggggaccattttggattcatgtttcatggcttgtgactgtccccaagcccactccaaacaaagtgtaaggatcagagttctgtca aggagcagcagttctgctctccccatcatctttgtgcaaggcccctcggggggcactttaataaaagaatttgaaatgggttgactg gccattctcatgctgtgctccctgtctcttctcttctctaaagaatcatgtcccagctcctcaaggtccctctatggttccacatctgagtg ttcgccacaagagcagcagcagcaggcacagtgcatgccatatctacctgctgcttctctgctgggaggaatggccaagtagatt ataaaactcacttctgtctcttaggcagacttgtacggccacaaaattacctagtcttcttcctgctgagctactgaggtattgccacc attttgacaactttgagtaattaaaacactcttctgacccaaaaaggaaaaaaggtcactgacgtgacccccccagcatgctaga gagctaattccagttctcatatttgtttgaatttcttcccagaggagaggataggaacctctcctccagggcagtaaatcacctgcatt tctggagttgtcggtattgtattcgaaaaggcctggagcccctcctgctcaggaaagaactcattccagggtgtggagacagtgcc gtctggcaggtgaaatactgtgggaattcacgccaccaggtgtttgtgcaagtgttggcctgggaagaatgggacttcggccttgt caggagttgtcttcatctgcagcacgtttcttcctcctgcagtagatcttagctaccccagatatctctatggagagaagtttgtggaa aatgctttgcttcgtggcagagtctgatgctgtaggaaaaccttcgggcatgtgacagcagtgtggtccactccctgttctgccctgg cactcagagtcatgtgtaagtaggaaacctgagcaagtcttccgtggaggaccctgagctgccgtctttgggatccttcctgtgtcc ccaccgtctttcatttatttgctttcctgggcctctatctgggccctaccttgagcttctccagttttattcaagccaccagagtaagaattt gggtgtagatgtcacaactaccttctactcaattcaccaattcatttactgctatggcacgtctcaggaataactctagaaacctctaa atcgaaatattataaaatcttgagcacttagtcctgctggttttagttagaaaggcatccaggaattgttttcctacgcccccttgagtg gaaagatcttagttagaagataaagtcaagtttgtgttcaggggatgggaggaagactataaataagatgaagaaatcaaaagt aggaaacatgatgtaaacgaagcatggcagatctgtccagcactgatattgctctataaattgagcttactcagttttggccttattttt ttacccaggccccatgtcacccagtcctaaaacagtaaccgtgtctacataacgggttggcccctggtgcatccctggaaaagtc aaaggacgcacacttcgaaattctgcagaacgtatttatacatggttcagaaatcttgcgtatctgacttatagccaaatctgcttgct cgaatagcctcagaggaagtcttgtttaataaaaaccttttgatttcctagtcaagtctttatggttgtctcgaggggtgtgtggctacttt aatgaaaggctttcctgctctaaatctctttgctgggctgggcctcttcagactatctggtgaaactcctttccttagaacaaactcagt ccgtccatgctctgtggcattttgctagatgataaccaaagccttattcctgtagccagtgtcagcagtcagagaggtggagggtgt gttctgctgtggttatgcatacctatctgctgttcttgaggtgtaaaaggaaaggtgaaaatcgggccaggccaagtactcagctgt cttaataggatgaagccttaagcagtggaaatttcagttattttccacagtattccattttggaggatttggggtgtttactttttaaattctt gaacaacttaacctccatgaggctttgtgaagtcagctgtgaccaccctcctcttactgtgttctcagtattcattcacttccagggaa gaatgacagccacagggagatggtggtgggcaagaatgagagtcccaggatccagatttagcctcagatcttccccattcagg aagggttttccatttaacaagagcactagtatgaaaacattagggacaaatctcccatgtctttgaaattcggattctcctcttgagat ccccttcctcacctgccaatcaactttataaggccacaagtggtcactggttttccttccacaggtttgaggttctcagctttccttaagc gacccagcagctccgctgttttcagagtgaatatgttaagctttgatgagattctattttcagtaagttagtgcttctgggacacttgga gaaagctgtgagagtcattgtctacgcaaagaacaacgaagctgatcctaaaagtgatccaatctaagaaaatggtaaaacga gctctggccacagcacagaattttatgtgaggaactcagatttttgaagacttaacaattgcagagaaaggttgcagcctgcacac catagcccacctctctgagcagactttggttttgtgtggtgacgtggcacatgtttgtacactgggatttttcaaaggacgctacgcga gcagactgacttgcctcttctgtgagcactgtggcttttgtcagatggagtgccggtctgcagaggactgctctttcgaatccacagtg ttatctgtgtaaatagctttaatttttcttctgtgtcttaggtgaagttttgttcatgtagcaaccaggtagacagtgaccaaataaggctgt aaatgtgctgtagttttctactgtgatgtacttgaaggagaacctgtgtcctctacttttctgatctcccacaagtattttgtgtttgtttcctg agtcctgaggttattattttactcctgttttgcccccagttttctttgttttttttctggagacccagggaggcccatggtggagatcatttgta aggaatggatcatggtctgggtttccaaaactaccctagtacagtgaatgagagaaatctgcctggaaattgtttcagaaccatgt acctttatgctttgtgattgtgaaacattgacttttttgtaaccccaaaatgaaaactgtttagtaaaggggatctattttgtgtgttttgaa acttaggtgcaatgtcccctggaaaaagctaaagaaatgtatatgttcaatgacattttaaaataaaatattatatatatgtatatacg ca (SEQ ID NO: 171)
PPM1 H Amino Acid Sequence
MLTRVKSAVANFMGGIMAGSSGSEHGGGSCGGSDLPLRFPYGRPEFLGLSQDEVECSAD HIARPILILKETRRLPWATGYAEVINAGKSTHNEDQASCEVLTVKKKAGAVTSTPNRNSSKR RSSLPNGEGLQLKENSESEGVSCHYWSLFDGHAGSGAAVVASRLLQHHITEQLQDIVDILK NSAVLPPTCLGEEPENTPANSRTLTRAASLRGGVGAPGSPSTPPTRFFTEKKIPHECLVIGA LESAFKEMDLQIERERSSYNISGGCTALIVICLLGKLYVANAGDSRAIIIRNGEIIPMSSEFTPE TERQRLQYLAFMQPHLLGNEFTHLEFPRRVQRKELGKKMLYRDFNMTGWAYKTIEDEDLK FPLIYGEGKKARVMATIGVTRGLGDHDLKVHDSNIYIKPFLSSAPEVRIYDLSKYDHGSDDVL ILATDGLWDVLSNEEVAEAITQFLPNCDPDDPHRYTLAAQDLVMRARGVLKDRGWRISNDR LGSGDDISVYVIPLIHGNKLS (SEQ ID NO: 172) aGFP(6M)-PPP1CA DNA Sequence gtcgacatgtccgacagcgagaagctcaacctggactcgatcatcgggcgcctgctggaagtgcagggctcgcggcctggca agaatgtacagctgacagagaacgagatccgcggtctgtgcctgaaatcccgggagatttttctgagccagcccattcttctgga gctggaggcacccctcaagatctgcggtgacatacacggccagtactacgaccttctgcgactatttgagtatggcggtttccctcc cgagagcaactacctctttctgggggactatgtggacaggggcaagcagtccttggagaccatctgcctgctgctggcctataag atcaagtaccccgagaacttcttcctgctccgtgggaaccacgagtgtgccagcatcaaccgcatctatggtttctacgatgagtg caagagacgctacaacatcaaactgtggaaaaccttcactgactgcttcaactgcctgcccatcgcggccatagtggacgaaa agatcttctgctgccacggaggcctgtccccggacctgcagtctatggagcagattcggcggatcatgcggcccacagatgtgcc tgaccagggcctgctgtgtgacctgctgtggtctgaccctgacaaggacgtgcagggctggggcgagaacgaccgtggcgtctc ttttacctttggagccgaggtggtggccaagttcctccacaagcacgacttggacctcatctgccgagcacaccaggtggtagaa gacggctacgagttctttgccaagcggcagctggtgacacttttctcagctcccaactactgtggcgagtttgacaatgctggcgcc atgatgagtgtggacgagaccctcatgtgctctttccagatcctcaagcccgccgacaagaacaaggggaagtacgggcagtt cagtggcctgaaccctggaggccgacccatcaccccaccccgcaattccgccaaagccaagaaataggcggccgc (SEQ ID NO: 173)
FLAG-aGFP(6M)-Linker-PPP1CA
MDYKDDDDKGSA/WADVQLVESGGALVQPGGSLRLSCVASGFPVNRYSMRWYRQAPGK
EREWVAGMSSAGDRSSYVDSVKGRFTIRRDDARNTVYLQMNSLKPEDTAVYYYNVNVGF
EYWGH GTQVTVSF V'D/WSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFL
IKYPENFFLLRGNHECASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIVDEKIFCCHGG
Figure imgf000075_0001
Dashed underline
Figure imgf000075_0002
FLAG region; bold sequence represents aGFP(6M) sequence; double underline represents PPP1CA sequence: VD is the linker sequence
I-PPP1CA Cassette DNA atggactacaaggacgatgacgataagggatccgccatggccgatgtgcagctggttgaatctggcggtgcactggtgcagcc gggcggtagcctgcgtctgtcttgcgtggccagtggctttccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaa agaacgtgaatgggtggcaggcatgagctctgcgggtgatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtc gtgatgatgcgcgcaacacggtgtacctgcagatgaatagtctgaaaccggaagataccgccgtttattactacaacgtgaatgtt ggcttcgaatattggggccatggtacccaggtgacggtttcttttgtcgacatgtccgacagcgagaagctcaacctggactcgatc atcgggcgcctgctggaagtgcagggctcgcggcctggcaagaatgtacagctgacagagaacgagatccgcggtctgtgcc tgaaatcccgggagatttttctgagccagcccattcttctggagctggaggcacccctcaagatctgcggtgacatacacggcca gtactacgaccttctgcgactatttgagtatggcggtttccctcccgagagcaactacctctttctgggggactatgtggacaggggc aagcagtccttggagaccatctgcctgctgctggcctataagatcaagtaccccgagaacttcttcctgctccgtgggaaccacga gtgtgccagcatcaaccgcatctatggtttctacgatgagtgcaagagacgctacaacatcaaactgtggaaaaccttcactgac tgcttcaactgcctgcccatcgcggccatagtggacgaaaagatcttctgctgccacggaggcctgtccccggacctgcagtctat ggagcagattcggcggatcatgcggcccacagatgtgcctgaccagggcctgctgtgtgacctgctgtggtctgaccctgacaa ggacgtgcagggctggggcgagaacgaccgtggcgtctcttttacctttggagccgaggtggtggccaagttcctccacaagca cgacttggacctcatctgccgagcacaccaggtggtagaagacggctacgagttctttgccaagcggcagctggtgacacttttct cagctcccaactactgtggcgagtttgacaatgctggcgccatgatgagtgtggacgagaccctcatgtgctctttccagatcctca agcccgccgacaagaacaaggggaagtacgggcagttcagtggcctgaaccctggaggccgacccatcaccccaccccgc aattccgccaaagccaagaaataggcggccgc (SEQ ID NO: 175) aGFP(6M)-PPP1CA H125Q DNA Sequence ggatccgccatggccgatgtgcagctggttgaatctggcggtgcactggtgcagccgggcggtagcctgcgtctgtcttgcgtggc cagtggctttccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaaagaacgtgaatgggtggcaggcatgag ctctgcgggtgatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtcgtgatgatgcgcgcaacacggtgtacct gcagatgaatagtctgaaaccggaagataccgccgtttattactacaacgtgaatgttggcttcgaatattggggccatggtaccc aggtgacggtttcttttgtcgacatgtccgacagcgagaagctcaacctggactcgatcatcgggcgcctgctggaagtgcaggg ctcgcggcctggcaagaatgtacagctgacagagaacgagatccgcggtctgtgcctgaaatcccgggagatttttctgagcca gcccattcttctggagctggaggcacccctcaagatctgcggtgacatacacggccagtactacgaccttctgcgactatttgagt atggcggtttccctcccgagagcaactacctctttctgggggactatgtggacaggggcaagcagtccttggagaccatctgcctg ctgctggcctataagatcaagtaccccgagaacttcttcctgctccgtgggaaccaagagtgtgccagcatcaaccgcatctatg gtttctacgatgagtgcaagagacgctacaacatcaaactgtggaaaaccttcactgactgcttcaactgcctgcccatcgcggc catagtggacgaaaagatcttctgctgccacggaggcctgtccccggacctgcagtctatggagcagattcggcggatcatgcg gcccacagatgtgcctgaccagggcctgctgtgtgacctgctgtggtctgaccctgacaaggacgtgcagggctggggcgaga acgaccgtggcgtctcttttacctttggagccgaggtggtggccaagttcctccacaagcacgacttggacctcatctgcc gagcacaccaggtggtagaagacggctacgagttctttgccaagcggcagctggtgacacttttctcagctcccaactactgtgg cgagtttgacaatgctggcgccatgatgagtgtggacgagaccctcatgtgctctttccagatcctcaagcccgccgacaagaac aaggggaagtacgggcagttcagtggcctgaaccctggaggccgacccatcaccccaccccgcaattccgccaaagccaag aaataggcggccgc (SEQ ID NO: 176)
FLAG-aGFP(6M)-Linker-PPP1CA H125Q Amino Acid Sequence
MDYKDPDDKGSAMADVQLVESGGALVQPGGSLRLSCVASGFPVNRYSMRWYRQAPGK EREWVAGMSSAGDRSSYVDSVKGRFTIRRDDARNTVYLQMNSLKPEDTAVYYYNVNVGF EYWGH GTQVTVSF V'DMSDSEKLNLDSIIGRLLEVQGSRPGKNVQLTENEIRGLCLKSREIFL SQPILLELEAPLKICGDIHGQYYDLLRLFEYGGFPPESNYLFLGDYVDRGKQSLETICLLLAYK IKYPENFFLLRGNQECASINRIYGFYDECKRRYNIKLWKTFTDCFNCLPIAAIVDEKIFCCHGG LSPDLQSMEQIRRIMRPTDVPDQGLLCDLLWSDPDKDVQGWGENDRGVSFTFGAEWAKF LHKHDLDLICRAHQWEDGYEFFAKRQLVTLFSAPNYCGEFDNAGAMMSVDETLMCSFQIL KPADKNKGKYGQFSGLNPGGRPITPPRNSAKAKK (SEQ ID NO: 177)
Amino acid substitution H125Q indicated in bold. Dashed underline FLAG region; bold sequence represents aGFP(6M) sequence; double underline represents PPP1CA sequence: VD is the linker sequence. i-PPPICA H125Q Cassette DNA atggactacaaggacgatgacgataagggatccgccatggccgatgtgcagctggttgaatctggcggtgcactggtgcagcc gggcggtagcctgcgtctgtcttgcgtggccagtggctttccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaa agaacgtgaatgggtggcaggcatgagctctgcgggtgatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtc gtgatgatgcgcgcaacacggtgtacctgcagatgaatagtctgaaaccggaagataccgccgtttattactacaacgtgaatgtt ggcttcgaatattggggccatggtacccaggtgacggtttcttttgtcgacatgtccgacagcgagaagctcaacctggactcgatc atcgggcgcctgctggaagtgcagggctcgcggcctggcaagaatgtacagctgacagagaacgagatccgcggtctgtgcc tgaaatcccgggagatttttctgagccagcccattcttctggagctggaggcacccctcaagatctgcggtgacatacacggcca gtactacgaccttctgcgactatttgagtatggcggtttccctcccgagagcaactacctctttctgggggactatgtggacaggggc aagcagtccttggagaccatctgcctgctgctggcctataagatcaagtaccccgagaacttcttcctgctccgtgggaaccaag agtgtgccagcatcaaccgcatctatggtttctacgatgagtgcaagagacgctacaacatcaaactgtggaaaaccttcactga ctgcttcaactgcctgcccatcgcggccatagtggacgaaaagatcttctgctgccacggaggcctgtccccggacctgcagtct atggagcagattcggcggatcatgcggcccacagatgtgcctgaccagggcctgctgtgtgacctgctgtggtctgaccctgaca aggacgtgcagggctggggcgagaacgaccgtggcgtctcttttacctttggagccgaggtggtggccaagttcctc cacaagcacgacttggacctcatctgccgagcacaccaggtggtagaagacggctacgagttctttgccaagcggcagctggt gacacttttctcagctcccaactactgtggcgagtttgacaatgctggcgccatgatgagtgtggacgagaccctcatgtgctctttc cagatcctcaagcccgccgacaagaacaaggggaagtacgggcagttcagtggcctgaaccctggaggccgacccatcac cccaccccgcaattccgccaaagccaagaaataggcggccgc (SEQ ID NO: 178)
Figure imgf000077_0001
gtcgacatggacgagaaggtgttcaccaaggagctggaccagtggatcgagcagctgaacgagtgcaagcagctgtccgagt cccaggtcaagagcctctgcgagaaggctaaagaaatcctgacaaaagaatccaacgtgcaagaggttcgatgtccagttact gtctgtggagatgtgcatgggcaatttcatgatctcatggaactgtttagaattggtggcaaatcaccagatacaaattacttgtttatg ggagattatgttgacagaggatattattcagttgaaacagttacactgcttgtagctcttaaggttcgttaccgtgaacgcatcaccat tcttcgagggaatcatgagagcagacagatcacacaagtttatggtttctatgatgaatgtttaagaaaatatggaaatgcaaatgt ttggaaatattttacagatctttttgactatcttcctctcactgccttggtggatgggcagatcttctgtctacatggtggtctctcgccatct atagatacactggatcatatcagagcacttgatcgcctacaagaagttccccatgagggtccaatgtgtgacttgctgtggtcagat ccagatgaccgtggtggttggggtatatctcctcgaggagctggttacacctttgggcaagatatttctgagacatttaatcatgcca atggcctcacgttggtgtctagagctcaccagctagtgatggagggatataactggtgccatgaccggaatgtagtaacgattttc agtgctccaaactattgttatcgttgtggtaaccaagctgcaatcatggaacttgacgatactctaaaatactctttcttgcagtttgac ccagcacctcgtagaggcgagccacatgttactcgtcgtaccccagactacttcctgtaagcggccgc (SEQ ID NO: 179)
FLAG-aGFP(6M)-Linker-PPP2CA Amino Acid
MDYKDDDDKGSAMADVQLVESGGALVQPGGSLRLSCVASGFPVNRYSMRWYRQAPGK
EREWVAGMSSAGDRSSYVDSVKGRFTIRRDDARNTVYLQMNSLKPEDTAVYYYNVNVGF
Figure imgf000078_0001
) sequence; double underline represents PPP1CA sequence; VD is the linker sequence. aGFP(6M)-PPP2CA Cassette DNA atggactacaaggacgatgacgataagggatccgccatggccgatgtgcagctggttgaatctggcggtgcactggtgcagcc gggcggtagcctgcgtctgtcttgcgtggccagtggctttccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaa agaacgtgaatgggtggcaggcatgagctctgcgggtgatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtc gtgatgatgcgcgcaacacggtgtacctgcagatgaatagtctgaaaccggaagataccgccgtttattactacaacgtgaatgtt ggcttcgaatattggggccatggtacccaggtgacggtttcttttgtcgacatggacgagaaggtgttcaccaaggagctggacca gtggatcgagcagctgaacgagtgcaagcagctgtccgagtcccaggtcaagagcctctgcgagaaggctaaagaaatcctg acaaaagaatccaacgtgcaagaggttcgatgtccagttactgtctgtggagatgtgcatgggcaatttcatgatctcatggaact gtttagaattggtggcaaatcaccagatacaaattacttgtttatgggagattatgttgacagaggatattattcagttgaaacagtta cactgcttgtagctcttaaggttcgttaccgtgaacgcatcaccattcttcgagggaatcatgagagcagacagatcacacaagttt atggtttctatgatgaatgtttaagaaaatatggaaatgcaaatgtttggaaatattttacagatctttttgactatcttcctctcactgcctt ggtggatgggcagatcttctgtctacatggtggtctctcgccatctatagatacactggatcatatcagagcacttgatcgcctacaa gaagttccccatgagggtccaatgtgtgacttgctgtggtcagatccagatgaccgtggtggttggggtatatctcctcgaggagct ggttacacctttgggcaagatatttctgagacatttaatcatgccaatggcctcacgttggtgtctagagctcaccagctagtgatgg agggatataactggtgccatgaccggaatgtagtaacgattttcagtgctccaaactattgttatcgttgtggtaaccaagctgcaat catggaacttgacgatactctaaaatactctttcttgcagtttgacccagcacctcgtagaggcgagccacatgttactcgtcgtacc ccagactacttcctgtaagcggccgc (SEQ ID NO: 181) aGFP(6M)-PPP2CA H118Q DNA Seguence ggatccgccatggccgatgtgcagctggttgaatctggcggtgcactggtgcagccgggcggtagcctgcgtctgtcttgcgtggc cagtggctttccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaaagaacgtgaatgggtggcaggcatgag ctctgcgggtgatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtcgtgatgatgcgcgcaacacggtgtacct gcagatgaatagtctgaaaccggaagataccgccgtttattactacaacgtgaatgttggcttcgaatattggggccatggtaccc aggtgacggtttcttttgtcgacatggacgagaaggtgttcaccaaggagctggaccagtggatcgagcagctgaacgagtgca agcagctgtccgagtcccaggtcaagagcctctgcgagaaggctaaagaaatcctgacaaaagaatccaacgtgcaagagg ttcgatgtccagttactgtctgtggagatgtgcatgggcaatttcatgatctcatggaactgtttagaattggtggcaaatcaccagat acaaattacttgtttatgggagattatgttgacagaggatattattcagttgaaacagttacactgcttgtagctcttaaggttcgttacc gtgaacgcatcaccattcttcgagggaatcaagagagcagacagatcacacaagtttatggtttctatgatgaatgtttaagaaaa tatggaaatgcaaatgtttggaaatattttacagatctttttgactatcttcctctcactgccttggtggatgggcagatcttctgtctacat ggtggtctctcgccatctatagatacactggatcatatcagagcacttgatcgcctacaagaagttccccatgagggtccaatgtgt gacttgctgtggtcagatccagatgaccgtggtggttggggtatatctcctcgaggagctggttacacctttgggcaagatatttctg agacatttaatcatgccaatggcctcacgttggtgtctagagctcaccagctagtgatggagggatataactggtgccatgaccgg aatgtagtaacgattttcagtgctccaaactattgttatcgttgtggtaaccaagctgcaatcatggaacttgacgatactctaaaata ctctttcttgcagtttgacccagcacctcgtagaggcgagccacatgttactcgtcgtaccccagactacttcctgtaagcggccgct (SEQ ID NO: 182)
Flag-aGFP(6M)-Linker-PPP2CA H118Q Amino Acid Sequence:
MDYKDPDDKGSAMADVQLVESGGALVQPGGSLRLSCVASGFPVNRYSMRWYRQAPGK EREWVAGMSSAGDRSSYVDSVKGRFTIRRDDARNTVYLQMNSLKPEDTAVYYYNVNVGF EYWGH GTQVTVSF V'DMDEKVFTKELDQWIEQLNECKQLSESQVKSLCEKAKEILTKESNV
QEVRCPVTVCGDVHGQFHDLMELFRIGGKSPDTNYLFMGDYVDRGYYSVETVTLLVALKV RYRERITILRGNQESRQITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVDGQIFCLH GGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETF NHANGLTLVSRAHQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQ FDPAPRRGEPHVTRRTPDYFL (SEQ ID NO: 183)
Amino acid substitution H118Q indicated in bold.
P_ashed _ un_d_eriine_ represents > _F_LAG__re_ ion; bold sequence represents aGFP(6M) sequence; double underline represents PPP1CA sequence: VD is the linker sequence. aGFP(6M)-PPP2CA H118Q Cassette DNA Sequence: atggactacaaggacgatgacgataagggatccgccatggccgatgtgcagctggttgaatctggcggtgcactggtgcagcc gggcggtagcctgcgtctgtcttgcgtggccagtggctttccggttaaccgctatagtatgcgttggtaccgccaggcgccgggtaa agaacgtgaatgggtggcaggcatgagctctgcgggtgatcgtagtagctatgtagatagcgttaaaggccgctttaccattcgtc gtgatgatgcgcgcaacacggtgtacctgcagatgaatagtctgaaaccggaagataccgccgtttattactacaacgtgaatgtt ggcttcgaatattggggccatggtacccaggtgacggtttcttttgtcgacatggacgagaaggtgttcaccaaggagctggacca gtggatcgagcagctgaacgagtgcaagcagctgtccgagtcccaggtcaagagcctctgcgagaaggctaaagaaatcctg acaaaagaatccaacgtgcaagaggttcgatgtccagttactgtctgtggagatgtgcatgggcaatttcatgatctcatggaact gtttagaattggtggcaaatcaccagatacaaattacttgtttatgggagattatgttgacagaggatattattcagttgaaacagtta cactgcttgtagctcttaaggttcgttaccgtgaacgcatcaccattcttcgagggaatcaagagagcagacagatcacacaagtt tatggtttctatgatgaatgtttaagaaaatatggaaatgcaaatgtttggaaatattttacagatctttttgactatcttcctctcactgcct tggtggatgggcagatcttctgtctacatggtggtctctcgccatctatagatacactggatcatatcagagcacttgatcgcctaca agaagttccccatgagggtccaatgtgtgacttgctgtggtcagatccagatgaccgtggtggttggggtatatctcctcgaggagc tggttacacctttgggcaagatatttctgagacatttaatcatgccaatggcctcacgttggtgtctagagctcaccagctagtgatgg agggatataactggtgccatgaccggaatgtagtaacgattttcagtgctccaaactattgttatcgttgtggtaaccaagctgcaat catggaacttgacgatactctaaaatactctttcttgcagtttgacccagcacctcgtagaggcgagccacatgttactcgtcgtacc ccagactacttcctgtaagcggccgct (SEQ ID NO: 184) pRetroX-TetOne-Puro-Flag-aGFP6m-PP1CA aagctagctttgctcttaggagtttcctaatacatcccaaactcaaatatataaagcatttgacttgttctatgcgcctaggagcttggc ccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagt tattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctgg ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtc aatgggtggagtatttacgctaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatg acggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgct attaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattg acgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg catctagagcgcttctgctccccgagctctataaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtc gcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtga ttgactacccgtcagcgggggtctttcatttgggggctcgtccgggatcgggagacccctgcccagggaccaccgacccaccac cgggaggtaagctggccagcaacttatctgtgtctgtccgattgtctagtgtctatgactgattttatgcgcctgcgtcggtactagtta gctaactagctctgtatctggcggacccgtggtggaactgacgagttcggaacacccggccgcaaccctgggagacgtcccag ggacttcgggggccgtttttgtggcccgacctgagtcctaaaatcccgatcgtttaggactctttggtgcaccccccttagaggagg gatatgtggttctggtaggagacgagaacctaaaacagttcccgcctccgtctgaatttttgctttcggtttgggaccgaagccgcg ccgcgcgtcttgtctgctgcagcatcgttctgtgttgtctctgtctgactgtgtttctgtatttgtctgaaaatgagggcccgggctagcct gttaccactcccttaagtttgaccttaggtcactggaaagatgtcgagcggatcgctcacaaccagtcggtagatgtcaagaaga gacgttgggttaccttctgctctgcagaatggccaacctttaacgtcggatggccgcgagacggcacctttaaccgagacctcatc acccaggttaagatcaaggtcttttcacctggcccgcatggacacccagaccaggtggggtacatcgtgacctgggaagccttg g cttttg acccccctccctgg gtcaag ccctttgtacaccctaag cctccg cctcctcttcctccatccg ccccgtctctcccccttg aa cctcctcgttcgaccccgcctcgatcctccctttatccagccctcactccttctctaggcgcccccatatggccatatgagatcctatat ggggcacccccgccccttgtaaacttccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacagg ctctctacttagtccagcacgaagtctggagacctctggcggcagcctaccaagaacaactggaccgaccggtggtacctcacc cttaccgagtcggcgacacagtgtgggtccgccgacaccagactaagaacctagaacctcgctggaaaggaccttacacagt cctgctgaccacccccaccgccctcaaagtagacggcatcgcagcttggatacacgccgcccacgtgaaggctgccgacccc gggggtggaccatcctctagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccct gaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaggcaatagcatcaca aatttcacaaataaggcatttttttcactgcattctagttttggtttgtccaaactcatcaatgtatcttatcatgtctggatctcaaatccct cggaagctgcgcctgtcttaggttggagtgatacatttttatcacttttacccgtctttggattaggcagtagctctgacggccctcctgt cttaggttagtgaaaaatgtcactctcttacccgtcattggctgtccagcttagctcgcaggggaggtggtctggatccctatttcttgg ctttggcggaattgcggggtggggtgatgggtcggcctccagggttcaggccactgaactgcccgtacttccccttgttcttgtcggc gggcttgaggatctggaaagagcacatgagggtctcgtccacactcatcatggcgccagcattgtcaaactcgccacagtagtt gggagctgagaaaagtgtcaccagctgccgcttggcaaagaactcgtagccgtcttctaccacctggtgtgctcggcagatgag gtccaagtcgtgcttgtggaggaacttggccaccacctcggctccaaaggtaaaagagacgccacggtcgttctcgccccagcc ctgcacgtccttgtcagggtcagaccacagcaggtcacacagcaggccctggtcaggcacatctgtgggccgcatgatccgcc gaatctgctccatagactgcaggtccggggacaggcctccgtggcagcagaagatcttttcgtccactatggccgcgatgggca ggcagttgaagcagtcagtgaaggttttccacagtttgatgttgtagcgtctcttgcactcatcgtagaaaccatagatgcggttgat gctggcacactcgtggttcccacggagcaggaagaagttctcggggtacttgatcttataggccagcagcaggcagatggtctc caaggactgcttgcccctgtccacatagtcccccagaaagaggtagttgctctcgggagggaaaccgccatactcaaatagtcg cagaaggtcgtagtactggccgtgtatgtcaccgcagatcttgaggggtgcctccagctccagaagaatgggctggctcagaaa aatctcccgggatttcaggcacagaccgcggatctcgttctctgtcagctgtacattcttgccaggccgcgagccctgcacttccag caggcgcccgatgatcgagtccaggttgagcttctcgctgtcggacatgtcgacaaaagaaaccgtcacctgggtaccatggcc ccaatattcgaagccaacattcacgttgtagtaataaacggcggtatcttccggtttcagactattcatctgcaggtacaccgtgttgc gcgcatcatcacgacgaatggtaaagcggcctttaacgctatctacatagctactacgatcacccgcagagctcatgcctgccac ccattcacgttctttacccggcgcctggcggtaccaacgcatactatagcggttaaccggaaagccactggccacgcaagacag acgcaggctaccgcccggctgcaccagtgcaccgccagattcaaccagctgcacatcggccatcttatcgtcatcgtccttgtagt ccatggtggcgaattctttacgagggtaggaagtggtacggaaagttggtataagacaaaagtgttgtggaattgaagtttactca aaaaatcagcactcttttataggcgccctggtttacataagcaaagcttatacgttctctatcactgatagggagtaaactggatata cgttctctatcactgatagggagtaaactgtagatacgttctctatcactgatagggagtaaactggtcatacgttctctatcactgata gggagtaaactccttatacgttctctatcactgatagggagtaaagtctgcatacgttctctatcactgatagggagtaaactcttcat acgttctctatcactgatagggagtaaactcgaggtgataattccacggggttggggttgcgccttttccaaggcagccctgggtttg cgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttc gcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttg cggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagc aatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaa ggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccg gagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatcatcgaattaccatgtctagac tggacaagagcaaagtcataaactctgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaactcgctc aaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatg ctggacaggcatcatacccactcctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccg ctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaa atcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggct gcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgaa acaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaa cagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgac tttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaactaagtaagaattgaatgtgtgtc agttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgt ggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaac tccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgagg ccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaacgcgaccatgaccgagt acaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgg gctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgaacgcgtaattccctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaact atgttgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctccttgtataa atcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccact ggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcc tgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtcggggaagctgacgtcgattcctgcg gagatatcagtggtccaggctctagttttgactcaacaatatcaccagctgaagcctatagagtacgagccatagataaaataaa agattttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagcttctcgcttctgttcgcgcg cttctgctcccagacatcaacaaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtcgcccgggtac ccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtgattgactacccg tcagcgggggtctttcacgcgtatgccgcatagttaagccaccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgacgcccgccccacgacccgcagcgcccgaccgaaaggagcgcacgaccccatggctccgac cgaagccgacccgggcggccccgccgaccccgcacccgcccccgaggcccaccgactctagaggatcataatcagccata ccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgtta acttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgt ggtttgtccaaactcatcaatgtatcttatcatggcgcgtcgaattaattcactggccgtcgttttacaacgtcgtgactgggaaaacc ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatat ggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgac gggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcac cgaaacgcgcgatgacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtg gcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcctt cctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgg atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgc ggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagt cacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcca acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgtt gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccac ttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg gggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagat cgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcattttt aatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac cccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacca gcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtc cttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtgg ctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaa cggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaa gcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggg agcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct gcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccca ggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatt acgcc (SEQ ID NO: 185) pRetroX-TetOne-Puro-Flag-aGFP6m-PP2CA aagctagctttgctcttaggagtttcctaatacatcccaaactcaaatatataaagcatttgacttgttctatgcgcctaggagcttggc ccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagt tattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctgg ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtc aatgggtggagtatttacgctaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatg acggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgct attaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattg acgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg catctagagcgcttctgctccccgagctctataaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtc gcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtga ttgactacccgtcagcgggggtctttcatttgggggctcgtccgggatcgggagacccctgcccagggaccaccgacccaccac cgggaggtaagctggccagcaacttatctgtgtctgtccgattgtctagtgtctatgactgattttatgcgcctgcgtcggtactagtta gctaactagctctgtatctggcggacccgtggtggaactgacgagttcggaacacccggccgcaaccctgggagacgtcccag ggacttcgggggccgtttttgtggcccgacctgagtcctaaaatcccgatcgtttaggactctttggtgcaccccccttagaggagg gatatgtggttctggtaggagacgagaacctaaaacagttcccgcctccgtctgaatttttgctttcggtttgggaccgaagccgcg ccgcgcgtcttgtctgctgcagcatcgttctgtgttgtctctgtctgactgtgtttctgtatttgtctgaaaatgagggcccgggctagcct gttaccactcccttaagtttgaccttaggtcactggaaagatgtcgagcggatcgctcacaaccagtcggtagatgtcaagaaga gacgttgggttaccttctgctctgcagaatggccaacctttaacgtcggatggccgcgagacggcacctttaaccgagacctcatc acccaggttaagatcaaggtcttttcacctggcccgcatggacacccagaccaggtggggtacatcgtgacctgggaagccttg gcttttgacccccctccctgggtcaagccctttgtacaccctaagcctccgcctcctcttcctccatccgccccgtctctcccccttgaa cctcctcgttcgaccccgcctcgatcctccctttatccagccctcactccttctctaggcgcccccatatggccatatgagatcctatat ggggcacccccgccccttgtaaacttccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacagg ctctctacttagtccagcacgaagtctggagacctctggcggcagcctaccaagaacaactggaccgaccggtggtacctcacc cttaccgagtcggcgacacagtgtgggtccgccgacaccagactaagaacctagaacctcgctggaaaggaccttacacagt cctgctgaccacccccaccgccctcaaagtagacggcatcgcagcttggatacacgccgcccacgtgaaggctgccgacccc gggggtggaccatcctctagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccct gaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaggcaatagcatcaca aatttcacaaataaggcatttttttcactgcattctagttttggtttgtccaaactcatcaatgtatcttatcatgtctggatctcaaatccct cggaagctgcgcctgtcttaggttggagtgatacatttttatcacttttacccgtctttggattaggcagtagctctgacggccctcctgt cttaggttagtgaaaaatgtcactctcttacccgtcattggctgtccagcttagctcgcaggggaggtggtctggatccttacaggaa gtagtctggggtacgacgagtaacatgtggctcgcctctacgaggtgctgggtcaaactgcaagaaagagtattttagagtatcgt caagttccatgattgcagcttggttaccacaacgataacaatagtttggagcactgaaaatcgttactacattccggtcatggcacc agttatatccctccatcactagctggtgagctctagacaccaacgtgaggccattggcatgattaaatgtctcagaaatatcttgccc aaaggtgtaaccagctcctcgaggagatataccccaaccaccacggtcatctggatctgaccacagcaagtcacacattggac cctcatggggaacttcttgtaggcgatcaagtgctctgatatgatccagtgtatctatagatggcgagagaccaccatgtagacag aagatctgcccatccaccaaggcagtgagaggaagatagtcaaaaagatctgtaaaatatttccaaacatttgcatttccatatttt cttaaacattcatcatagaaaccataaacttgtgtgatctgtctgctctcatgattccctcgaagaatggtgatgcgttcacggtaacg aaccttaagagctacaagcagtgtaactgtttcaactgaataatatcctctgtcaacataatctcccataaacaagtaatttgtatctg gtgatttgccaccaattctaaacagttccatgagatcatgaaattgcccatgcacatctccacagacagtaactggacatcgaacc tcttgcacgttggattcttttgtcaggatttctttagccttctcgcagaggctcttgacctgggactcggacagctgcttgcactcgttcag ctgctcgatccactggtccagctccttggtgaacaccttctcgtccatgtcgacaaaagaaaccgtcacctgggtaccatggcccc aatattcgaagccaacattcacgttgtagtaataaacggcggtatcttccggtttcagactattcatctgcaggtacaccgtgttgcg cgcatcatcacgacgaatggtaaagcggcctttaacgctatctacatagctactacgatcacccgcagagctcatgcctgccacc cattcacgttctttacccggcgcctggcggtaccaacgcatactatagcggttaaccggaaagccactggccacgcaagacaga cgcaggctaccgcccggctgcaccagtgcaccgccagattcaaccagctgcacatcggccatcttatcgtcatcgtccttgtagtc catggtggcgaattctttacgagggtaggaagtggtacggaaagttggtataagacaaaagtgttgtggaattgaagtttactcaa aaaatcagcactcttttataggcgccctggtttacataagcaaagcttatacgttctctatcactgatagggagtaaactggatatac gttctctatcactgatagggagtaaactgtagatacgttctctatcactgatagggagtaaactggtcatacgttctctatcactgata gggagtaaactccttatacgttctctatcactgatagggagtaaagtctgcatacgttctctatcactgatagggagtaaactcttcat acgttctctatcactgatagggagtaaactcgaggtgataattccacggggttggggttgcgccttttccaaggcagccctgggtttg cgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttc gcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttg cggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagc aatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaa ggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccg gagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatcatcgaattaccatgtctagac tggacaagagcaaagtcataaactctgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaactcgctc aaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatg ctggacaggcatcatacccactcctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccg ctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaa atcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggct gcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgaa acaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaa cagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgac tttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaactaagtaagaattgaatgtgtgtc agttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgt ggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaac tccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgagg ccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaacgcgaccatgaccgagt acaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgg gctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgaacgcgtaattccctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaact atgttgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctccttgtataa atcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccact ggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcc tgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtcggggaagctgacgtcgattcctgcg gagatatcagtggtccaggctctagttttgactcaacaatatcaccagctgaagcctatagagtacgagccatagataaaataaa agattttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagcttctcgcttctgttcgcgcg cttctgctcccagacatcaacaaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtcgcccgggtac ccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtgattgactacccg tcagcgggggtctttcacgcgtatgccgcatagttaagccaccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgacgcccgccccacgacccgcagcgcccgaccgaaaggagcgcacgaccccatggctccgac cgaagccgacccgggcggccccgccgaccccgcacccgcccccgaggcccaccgactctagaggatcataatcagccata ccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgtta acttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgt ggtttgtccaaactcatcaatgtatcttatcatggcgcgtcgaattaattcactggccgtcgttttacaacgtcgtgactgggaaaacc ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatat ggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgac gggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcac cgaaacgcgcgatgacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtg gcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcctt cctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgg atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgc ggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagt cacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcca acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgtt gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccac ttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg gggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagat cgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcattttt aatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac cccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacca gcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtc cttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtgg ctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaa cggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaa gcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggg agcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct gcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccca ggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatt acgcc (SEQ ID NO: 186) pRetroX-TetOne-Puro-Flag-aGFP6m-PP2CA H118Q aagctagctttgctcttaggagtttcctaatacatcccaaactcaaatatataaagcatttgacttgttctatgcgcctaggagcttggc ccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagt tattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctgg ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtc aatgggtggagtatttacgctaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatg acggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgct attaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattg acgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg catctagagcgcttctgctccccgagctctataaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtc gcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtga ttgactacccgtcagcgggggtctttcatttgggggctcgtccgggatcgggagacccctgcccagggaccaccgacccaccac cgggaggtaagctggccagcaacttatctgtgtctgtccgattgtctagtgtctatgactgattttatgcgcctgcgtcggtactagtta gctaactagctctgtatctggcggacccgtggtggaactgacgagttcggaacacccggccgcaaccctgggagacgtcccag ggacttcgggggccgtttttgtggcccgacctgagtcctaaaatcccgatcgtttaggactctttggtgcaccccccttagaggagg gatatgtggttctggtaggagacgagaacctaaaacagttcccgcctccgtctgaatttttgctttcggtttgggaccgaagccgcg ccgcgcgtcttgtctgctgcagcatcgttctgtgttgtctctgtctgactgtgtttctgtatttgtctgaaaatgagggcccgggctagcct gttaccactcccttaagtttgaccttaggtcactggaaagatgtcgagcggatcgctcacaaccagtcggtagatgtcaagaaga gacgttgggttaccttctgctctgcagaatggccaacctttaacgtcggatggccgcgagacggcacctttaaccgagacctcatc acccaggttaagatcaaggtcttttcacctggcccgcatggacacccagaccaggtggggtacatcgtgacctgggaagccttg g cttttg acccccctccctgg gtcaag ccctttgtacaccctaag cctccg cctcctcttcctccatccg ccccgtctctcccccttg aa cctcctcgttcgaccccgcctcgatcctccctttatccagccctcactccttctctaggcgcccccatatggccatatgagatcctatat ggggcacccccgccccttgtaaacttccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacagg ctctctacttagtccagcacgaagtctggagacctctggcggcagcctaccaagaacaactggaccgaccggtggtacctcacc cttaccgagtcggcgacacagtgtgggtccgccgacaccagactaagaacctagaacctcgctggaaaggaccttacacagt cctgctgaccacccccaccgccctcaaagtagacggcatcgcagcttggatacacgccgcccacgtgaaggctgccgacccc gggggtggaccatcctctagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccct gaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaggcaatagcatcaca aatttcacaaataaggcatttttttcactgcattctagttttggtttgtccaaactcatcaatgtatcttatcatgtctggatctcaaatccct cggaagctgcgcctgtcttaggttggagtgatacatttttatcacttttacccgtctttggattaggcagtagctctgacggccctcctgt cttaggttagtgaaaaatgtcactctcttacccgtcattggctgtccagcttagctcgcaggggaggtggtctggatccttacaggaa gtagtctggggtacgacgagtaacatgtggctcgcctctacgaggtgctgggtcaaactgcaagaaagagtattttagagtatcgt caagttccatgattgcagcttggttaccacaacgataacaatagtttggagcactgaaaatcgttactacattccggtcatggcacc agttatatccctccatcactagctggtgagctctagacaccaacgtgaggccattggcatgattaaatgtctcagaaatatcttgccc aaaggtgtaaccagctcctcgaggagatataccccaaccaccacggtcatctggatctgaccacagcaagtcacacattggac cctcatggggaacttcttgtaggcgatcaagtgctctgatatgatccagtgtatctatagatggcgagagaccaccatgtagacag aagatctgcccatccaccaaggcagtgagaggaagatagtcaaaaagatctgtaaaatatttccaaacatttgcatttccatatttt cttaaacattcatcatagaaaccataaacttgtgtgatctgtctgctctcttgattccctcgaagaatggtgatgcgttcacggtaacg aaccttaagagctacaagcagtgtaactgtttcaactgaataatatcctctgtcaacataatctcccataaacaagtaatttgtatctg gtgatttgccaccaattctaaacagttccatgagatcatgaaattgcccatgcacatctccacagacagtaactggacatcgaacc tcttgcacgttggattcttttgtcaggatttctttagccttctcgcagaggctcttgacctgggactcggacagctgcttgcactcgttcag ctgctcgatccactggtccagctccttggtgaacaccttctcgtccatgtcgacaaaagaaaccgtcacctgggtaccatggcccc aatattcgaagccaacattcacgttgtagtaataaacggcggtatcttccggtttcagactattcatctgcaggtacaccgtgttgcg cgcatcatcacgacgaatggtaaagcggcctttaacgctatctacatagctactacgatcacccgcagagctcatgcctgccacc cattcacgttctttacccggcgcctggcggtaccaacgcatactatagcggttaaccggaaagccactggccacgcaagacaga cgcaggctaccgcccggctgcaccagtgcaccgccagattcaaccagctgcacatcggccatcttatcgtcatcgtccttgtagtc catggtggcgaattctttacgagggtaggaagtggtacggaaagttggtataagacaaaagtgttgtggaattgaagtttactcaa aaaatcagcactcttttataggcgccctggtttacataagcaaagcttatacgttctctatcactgatagggagtaaactggatatac gttctctatcactgatagggagtaaactgtagatacgttctctatcactgatagggagtaaactggtcatacgttctctatcactgata gggagtaaactccttatacgttctctatcactgatagggagtaaagtctgcatacgttctctatcactgatagggagtaaactcttcat acgttctctatcactgatagggagtaaactcgaggtgataattccacggggttggggttgcgccttttccaaggcagccctgggtttg cgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttc gcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttg cggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagc aatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaa ggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccg gagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatcatcgaattaccatgtctagac tggacaagagcaaagtcataaactctgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaactcgctc aaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatg ctggacaggcatcatacccactcctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccg ctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaa atcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggct gcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgaa acaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaa cagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgac tttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaactaagtaagaattgaatgtgtgtc agttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgt ggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaac tccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgagg ccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaacgcgaccatgaccgagt acaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgg gctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgaacgcgtaattccctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaact atgttgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctccttgtataa atcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccact ggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcc tgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtcggggaagctgacgtcgattcctgcg gagatatcagtggtccaggctctagttttgactcaacaatatcaccagctgaagcctatagagtacgagccatagataaaataaa agattttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagcttctcgcttctgttcgcgcg cttctgctcccagacatcaacaaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtcgcccgggtac ccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtgattgactacccg tcagcgggggtctttcacgcgtatgccgcatagttaagccaccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgacgcccgccccacgacccgcagcgcccgaccgaaaggagcgcacgaccccatggctccgac cgaagccgacccgggcggccccgccgaccccgcacccgcccccgaggcccaccgactctagaggatcataatcagccata ccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgtta acttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgt ggtttgtccaaactcatcaatgtatcttatcatggcgcgtcgaattaattcactggccgtcgttttacaacgtcgtgactgggaaaacc ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatat ggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgac gggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcac cgaaacgcgcgatgacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtg gcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcctt cctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgg atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgc ggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagt cacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcca acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgtt gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccac ttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg gggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagat cgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcattttt aatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac cccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacca gcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtc cttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtgg ctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaa cggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaa gcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggg agcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct gcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccca ggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatt acgcc (SEQ ID NO: 187) pRetroX-T etOne-Puro-Flag-aGFP6m-PP1 CA H 125Q aagctagctttgctcttaggagtttcctaatacatcccaaactcaaatatataaagcatttgacttgttctatgcgcctaggagcttggc ccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagt tattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctgg ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtc aatgggtggagtatttacgctaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatg acggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgct attaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattg acgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg catctagagcgcttctgctccccgagctctataaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtc gcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtga ttgactacccgtcagcgggggtctttcatttgggggctcgtccgggatcgggagacccctgcccagggaccaccgacccaccac cgggaggtaagctggccagcaacttatctgtgtctgtccgattgtctagtgtctatgactgattttatgcgcctgcgtcggtactagtta gctaactagctctgtatctggcggacccgtggtggaactgacgagttcggaacacccggccgcaaccctgggagacgtcccag ggacttcgggggccgtttttgtggcccgacctgagtcctaaaatcccgatcgtttaggactctttggtgcaccccccttagaggagg gatatgtggttctggtaggagacgagaacctaaaacagttcccgcctccgtctgaatttttgctttcggtttgggaccgaagccgcg ccgcgcgtcttgtctgctgcagcatcgttctgtgttgtctctgtctgactgtgtttctgtatttgtctgaaaatgagggcccgggctagcct gttaccactcccttaagtttgaccttaggtcactggaaagatgtcgagcggatcgctcacaaccagtcggtagatgtcaagaaga gacgttgggttaccttctgctctgcagaatggccaacctttaacgtcggatggccgcgagacggcacctttaaccgagacctcatc acccaggttaagatcaaggtcttttcacctggcccgcatggacacccagaccaggtggggtacatcgtgacctgggaagccttg gcttttgacccccctccctgggtcaagccctttgtacaccctaagcctccgcctcctcttcctccatccgccccgtctctcccccttgaa cctcctcgttcgaccccgcctcgatcctccctttatccagccctcactccttctctaggcgcccccatatggccatatgagatcctatat ggggcacccccgccccttgtaaacttccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacagg ctctctacttagtccagcacgaagtctggagacctctggcggcagcctaccaagaacaactggaccgaccggtggtacctcacc cttaccgagtcggcgacacagtgtgggtccgccgacaccagactaagaacctagaacctcgctggaaaggaccttacacagt cctgctgaccacccccaccgccctcaaagtagacggcatcgcagcttggatacacgccgcccacgtgaaggctgccgacccc gggggtggaccatcctctagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccct gaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaggcaatagcatcaca aatttcacaaataaggcatttttttcactgcattctagttttggtttgtccaaactcatcaatgtatcttatcatgtctggatctcaaatccct cggaagctgcgcctgtcttaggttggagtgatacatttttatcacttttacccgtctttggattaggcagtagctctgacggccctcctgt cttaggttagtgaaaaatgtcactctcttacccgtcattggctgtccagcttagctcgcaggggaggtggtctggatccctatttcttgg ctttggcggaattgcggggtggggtgatgggtcggcctccagggttcaggccactgaactgcccgtacttccccttgttcttgtcggc gggcttgaggatctggaaagagcacatgagggtctcgtccacactcatcatggcgccagcattgtcaaactcgccacagtagtt gggagctgagaaaagtgtcaccagctgccgcttggcaaagaactcgtagccgtcttctaccacctggtgtgctcggcagatgag gtccaagtcgtgcttgtggaggaacttggccaccacctcggctccaaaggtaaaagagacgccacggtcgttctcgccccagcc ctgcacgtccttgtcagggtcagaccacagcaggtcacacagcaggccctggtcaggcacatctgtgggccgcatgatccgcc gaatctgctccatagactgcaggtccggggacaggcctccgtggcagcagaagatcttttcgtccactatggccgcgatgggca ggcagttgaagcagtcagtgaaggttttccacagtttgatgttgtagcgtctcttgcactcatcgtagaaaccatagatgcggttgat gctggcacactcttggttcccacggagcaggaagaagttctcggggtacttgatcttataggccagcagcaggcagatggtctcc aaggactgcttgcccctgtccacatagtcccccagaaagaggtagttgctctcgggagggaaaccgccatactcaaatagtcgc agaaggtcgtagtactggccgtgtatgtcaccgcagatcttgaggggtgcctccagctccagaagaatgggctggctcagaaaa atctcccgggatttcaggcacagaccgcggatctcgttctctgtcagctgtacattcttgccaggccgcgagccctgcacttccagc aggcgcccgatgatcgagtccaggttgagcttctcgctgtcggacatgtcgacaaaagaaaccgtcacctgggtaccatggccc caatattcgaagccaacattcacgttgtagtaataaacggcggtatcttccggtttcagactattcatctgcaggtacaccgtgttgc gcgcatcatcacgacgaatggtaaagcggcctttaacgctatctacatagctactacgatcacccgcagagctcatgcctgccac ccattcacgttctttacccggcgcctggcggtaccaacgcatactatagcggttaaccggaaagccactggccacgcaagacag acgcaggctaccgcccggctgcaccagtgcaccgccagattcaaccagctgcacatcggccatcttatcgtcatcgtccttgtagt ccatggtggcgaattctttacgagggtaggaagtggtacggaaagttggtataagacaaaagtgttgtggaattgaagtttactca aaaaatcagcactcttttataggcgccctggtttacataagcaaagcttatacgttctctatcactgatagggagtaaactggatata cgttctctatcactgatagggagtaaactgtagatacgttctctatcactgatagggagtaaactggtcatacgttctctatcactgata gggagtaaactccttatacgttctctatcactgatagggagtaaagtctgcatacgttctctatcactgatagggagtaaactcttcat acgttctctatcactgatagggagtaaactcgaggtgataattccacggggttggggttgcgccttttccaaggcagccctgggtttg cgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttc gcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttg cggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagc aatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaa ggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccg gagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatcatcgaattaccatgtctagac tggacaagagcaaagtcataaactctgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaactcgctc aaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatg ctggacaggcatcatacccactcctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccg ctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaa atcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggct gcgtattggaggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgaa acaagcaattgagctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaa cagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgac tttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaactaagtaagaattgaatgtgtgtc agttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgt ggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaac tccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgagg ccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaacgcgaccatgaccgagt acaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgg gctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgaacgcgtaattccctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaact atgttgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctccttgtataa atcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccact ggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcc tgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtcggggaagctgacgtcgattcctgcg gagatatcagtggtccaggctctagttttgactcaacaatatcaccagctgaagcctatagagtacgagccatagataaaataaa agattttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagcttctcgcttctgttcgcgcg cttctgctcccagacatcaacaaaagagcccacaacccctcactcggggcgccagtcctccgattgactgagtcgcccgggtac ccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtgattgactacccg tcagcgggggtctttcacgcgtatgccgcatagttaagccaccgcggtggcggtctggaccacgccggagagcgtcgaagcgg gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcct cctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgc aacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgacgcccgccccacgacccgcagcgcccgaccgaaaggagcgcacgaccccatggctccgac cgaagccgacccgggcggccccgccgaccccgcacccgcccccgaggcccaccgactctagaggatcataatcagccata ccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgtta acttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgt ggtttgtccaaactcatcaatgtatcttatcatggcgcgtcgaattaattcactggccgtcgttttacaacgtcgtgactgggaaaacc ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatat ggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgac gggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcac cgaaacgcgcgatgacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtg gcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcctt cctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgg atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgc ggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagt cacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcca acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgtt gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccac ttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg gggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagat cgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcattttt aatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagac cccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctacca gcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtc cttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtgg ctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaa cggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaa gcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggg agcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct gcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagc tggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccca ggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatt acgcc (SEQ ID NO: 188) References:
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Claims

Claims:
1. A fusion protein comprising a phosphatase component linked to at least one polypeptide protein binder.
2. The fusion protein according to claim 1 , wherein the polypeptide protein binder is an antigen-stabilised protein binder.
3. The fusion protein according to claims 1 or 2, wherein the phosphatase component dephosphorylates a protein of interest (POI).
4. The fusion protein according to claim 3, wherein the phosphatase component is a phosphatase catalytic subunit.
5. The fusion protein according to claim 4, wherein the phosphatase component is PPP1CA or a functional variant thereof.
6. The fusion protein according to claim 4, wherein the phosphatase component is PPP2CA or a functional variant thereof.
7. The fusion protein according to any preceding claim, wherein the at least one polypeptide protein binder is an antibody, an antibody fragment, a monobody and/or a nanobody.
8. The fusion protein according to any preceding claim, wherein the phosphatase component is linked to two polypeptide protein binders.
9. The fusion protein according to claim 8, wherein the two polypeptide protein binders recognise different proteins of interest.
10. The fusion protein according to any preceding claim, wherein the at least one polypeptide protein binder recognises any POI from the list comprising: FAM83D, unc-51-like kinase (ULK1), ULK2, ATG13, ATG, GFP, YFP, Tau, SMAD1 , SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD, SMAD8, FOXO1. FOXO3, FOXO4, FOXO6, FOXP3, NFKB, AHR, CDK1 , CDK4, CDK5, PLK, AURK, NRK, GATA1 , GATA2, GATA3, GATA4, GATA5, GATA6, VEGRK1. VEGRK2, VEGRK3, EGFR, MAPK, ERK1 , ERK2, Rab GTPases, PKC, BTK, JAK1 , JAK2, JAK3, SYK, AKT, STAT1 , STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6.
11. The fusion protein according to any preceding claim, wherein the protein of interest is phosphorylated at serine, threonine and/or tyrosine residues.
12. The fusion protein according to any preceding claim, wherein the protein of interest is GFP- tagged.
13. A nucleic acid encoding a fusion protein of any preceding claim.
14. The nucleic acid according to claim 13, wherein the phosphatase component is PPP1CA or PPP2CA or functional variants thereof.
15. The nucleic acid according to claims 13 or 14, wherein the polypeptide protein binder is anti-GFPeM nanobody or a functional variant thereof.
16. A expression construct comprising the nucleic acid of any of claims 13-15 operably linked to one or more expression control sequences.
17. The expression construct of claim 16, wherein the expression control sequence is constitutive or inducible.
18. A vector comprising an expression construct according to claims 16 or 17.
19. The vector of claim 18, which is a gene therapy vector, suitably a viral vector, suitably an AAV vector, an adenoviral vector, a retroviral vector or a lentiviral vector.
20. A pharmaceutical composition comprising the fusion protein of any of claims 1-12, a nucleic acid construct according to any of claims 13-15, an expression construct according to claims 16 or 17 or a vector according to claims 18 or 19 and a pharmaceutically acceptable carrier or diluent.
21. The fusion protein of any of claims 1-12, a nucleic acid according to any of claims 13-15, an expression construct according to claims 16 or 17, a vector according to claims 18 or 19 or the pharmaceutical composition according to claim 20 for use in the treatment of a subject with cancer or a neurodegenerative disorder.
22. A method of regulating protein activity wherein the method comprises dephosphorylating a phosphorylated POI by administering the fusion protein of any of claims 1-12, a nucleic acid according to any of claims 13-15, an expression construct according to claims 16 or 17, a vector according to claims 18 or 19 or the pharmaceutical composition according to claim 20 to a cell or tissue containing the phosphorylated POI.
23. A method for regulating protein activity, wherein the method comprises administering a fusion protein, a nucleic acid, an expression construct, a vector or the pharmaceutic composition of any preceding claim to a cell or a tissue in vitro, ex vivo or in vivo,
- wherein when a POI target of the polypeptide protein binder component of fusion protein is present, the polypeptide protein binder binds to the POI or when the POI target of the polypeptide protein binder component of the fusion protein is absent, the fusion protein destabilises; and
- wherein when the polypeptide protein binder is bound to the POI, the phosphatase component linked to the polypeptide protein binder selectively dephosphorylates the POI.
24. A method of treatment of a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of the fusion protein of any of claims 1- 12, a nucleic acid according to any of claims 13-15, an expression construct according to claims 16 or 17, a vector according to claim 18 or 19, or the pharmaceutical composition according to claim 20.
25. The method of claim 24, wherein the subject has a neurodegenerative disorder or cancer.
26. The method of claim 25, wherein the neurodegenerative disorder is a tauopathy, optionally Alzheimer’s disease.
27. The fusion protein of any of claims 1-12, a nucleic acid according to any of claims 13-15, an expression construct according to claims 16 or 17, a vector according to claims 18 or 19, or the pharmaceutical composition according to claim 20 for use as a research tool to regulate phosphorylation of a POI.
28. A kit for use in a method of any of claims 22-26 or for use as a research tool according to claim 27, wherein the kit comprises the fusion protein of any of claims 1-12, a nucleic acid according to any of claims 13-15, an expression construct according to claims 16 or 17, a vector according to claims 18 or 19, or the pharmaceutical composition according to claim
20, and instructions for use.
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