Compositions and Methods for Controlled Proteolysis in Neurodegenerative Diseases

Abstract

Multifunctional polypeptides comprising an arbitrary signal peptide sequence, a cell-penetrating peptide, an antigen-binding domain (e.g., anti-synuclein, anti-tau, anti-huntingtin), and a programmable proteasome-targeting PEST motif, as well as methods of using these polypeptides in the treatment of protein aggregation diseases, such as neurodegenerative diseases, are disclosed herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 340,148, filed on May 10, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] The present disclosure relates to multifunctional polypeptides comprising a first domain comprising an antigen - binding domain (e.g., anti - α - synuclein, tau, or huntingtin) and a second domain comprising a programmable proteasome - targeting PEST motif, and methods of using these polypeptides in the treatment of protein aggregation diseases. These polypeptides can be used to treat neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, etc.).

Background Art

[0003] Neurodegenerative diseases such as synucleinopathies and tauopathies are associated with the accumulation of protein aggregates. These include α-synucleinopathies (e.g., Parkinson's disease, Lewy bodies, multiple system atrophy (MSA), etc.), and tauopathies (e.g., frontotemporal dementia (FTD), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia with parkinsonism on chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD-TAU), corticobasal degeneration (CBD), Alzheimer's disease, primary age-related tauopathy, Pick's disease, chronic traumatic encephalopathy (CTE) including boxer dementia, corticobasal body disease, ganglioglioma, gangliocytoma, meningovascular amyloidosis, posterior encephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), Hallervorden-Spatz disease, lead encephalopathy, tuberous sclerosis, lipofuscinosis, etc.). Neurodegenerative diseases can also result from glutamine repeats associated with the accumulation of protein aggregates. For example, Huntington's disease is a disorder caused by an expanded unstable trinucleotide repeat (CAG) in the huntingtin gene (HTT), which is interpreted as a polyglutamine repeat in the protein product. Currently, there is no well-established treatment to reduce the accumulation of protein aggregates to benefit patients with conditions such as tauopathies and synucleinopathies, and Huntington's disease, and such a treatment is highly desirable. Neural injuries such as traumatic brain injury (TBI) or spinal cord injury (SCI) are associated with the accumulation of protein aggregates including α-synuclein and abnormal tau deposition, which can cause neurodegeneration. Currently, there is no well-established neuroprotective treatment for TBI or SCI, and thus, a treatment that can delay, reduce the impact of, or prevent TBI- or SCI-induced neurodegeneration is also highly desirable. Summary of the Invention

[0004] The present disclosure relates to the characterization, delivery, and use of multifunctional polypeptides that target the degradation of antigens (e.g., α-synuclein, tau, or huntingtin), thereby altering the protein levels of these antigens. The present disclosure is based in part on the discovery that a cell-penetrating peptide can enhance the intracellular delivery of a bifunctional polypeptide that includes an intrabod y that binds to an epitope of an antigen (e.g., α-synuclein, tau, or huntingtin) and a programmable proteasome-targeting PEST motif. Further, certain modifications to the human PEST degron can alter the level of target antigen (e.g., synuclein, tau, huntingtin) degradation. There are various modifications (e.g., specific amino acid substitutions in the PEST degron) that can increase the degradation of a given target antigen or change the level of degradation such that a given antigen is not completely eliminated but is reduced to a pathogenicity-reducing extent. Such multifunctional polypeptides can be used to prevent the accumulation of disease-causing protein aggregates and thereby treat neurodegenerative conditions associated with such protein aggregation.

[0005] In some cases, the multifunctional polypeptides of the present disclosure include a first domain that includes an immunoglobulin heavy chain signal peptide sequence (SS), a second domain that includes a cell-penetrating peptide, a third domain that includes an antigen (e.g., anti-α-synuclein or huntingtin) binding domain, and a fourth domain that includes a programmable proteasome-targeting PEST motif, as well as methods for using these polypeptides in the treatment of protein aggregation neurodegenerative diseases.

[0006] In a first aspect, the present disclosure features a recombinant polypeptide comprising, from the N-terminus to the C-terminus, I. (a) an optional signal peptide domain; (b) a cell-penetrating peptide; (c) an antigen-binding domain that binds to α-synuclein; and (d) a programmable proteasome-targeting human or mouse PEST domain; or II. (a) an optional signal peptide domain; (b) a programmable proteasome-targeting human or mouse PEST domain; (c) an antigen-binding domain that binds to α-synuclein; and (d) a cell-penetrating peptide. In some embodiments, the programmable proteasome-targeting human PEST domain is at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and comprises a sequence having at least one amino acid substitution, wherein the polypeptide having at least one amino acid substitution increases or decreases the degradation of α-synuclein compared to an empty vector (EV) control.

[0007] In some embodiments, at least one amino acid substitution is selected from alanine, glycine, valine, leucine, or isoleucine. In some embodiments, the programmable proteasome-targeting human PEST domain has the sequence NPDFX1X2X3VX4X5QX6AX7X8LX9VX 10 X 11 AWX 12 X 13 GMX 14 RHRAACASASINV (SEQ ID NO: 109), where X1 is (P / A), X2 is (P / A), X3 is (E / A), X4 is (E / A), X5 is (E / A), X6 is (D / A), X7 is (S / A), X8 is (T / A), X9 is (P / A), X 10 is (S / A), X 11 is (C / A), X 12 is (E / A), X 13 is (S / A), X 14comprises (K / A)), wherein the sequence is not NPDFPPEVEEQDASTLPVSCAWESGMKRHRAACASASINV (SEQ ID NO: 3), and the polypeptide increases the degradation of α-synuclein compared to an empty vector (EV) control.

[0008] In some embodiments, the programmable proteasome targeting human PEST domain has X1 being (P), X2 being (A), X3 being (E), X4 being (E), X5 being (E), X6 being (D), X7 being (S), X8 being (T), X9 being (P), X 10 being (S), X 11 being (C), X 12 being (E), X 13 being (S), X 14 being (K) (amino acids 164 - 191 in SEQ ID NO: 7), or X1 being (P), X2 being (P), X3 being (E), X4 being (E), X5 being (E), X6 being (A), X7 being (S), X8 being (T), X9 being (P), X 10 being (S), X 11 being (C), X 12 being (E), X 13 being (S), X 14 being (K) (amino acids 164 - 191 in SEQ ID NO: 8), or X1 being (P), X2 being (P), X3 being (E), X4 being (E), X5 being (E), X6 being (D), X7 being (S), X8 being (T), X9 being (P), X 10 being (S), X 11 being (C), X 12 being (E), X 13 being (A), X 14 comprises a sequence being (K) (amino acids 164 - 191 in SEQ ID NO: 10). In some embodiments, the programmable proteasome targeting human PEST domain comprises amino acids 164-191 of the amino acid sequence set forth in SEQ ID NO: 9, wherein the polypeptide reduces the degradation of α-synuclein compared to an empty vector (EV) control.

[0009] In some embodiments, the antigen-binding domain is an intrabody. In some embodiments, the intrabody is a single-chain variable fragment (scFv) or a single-domain antibody that binds to α-synuclein. In some embodiments, the single-domain antibody comprises an α-synuclein-specific VL domain (VL α-synuclein), an α-synuclein-specific VH domain (VH α-synuclein), or an α-synuclein-specific VHH domain. In some embodiments, the single-domain antibody comprises a VHH antibody having the amino acid sequence set forth in any one of SEQ ID NOs: 16-17, or a VH domain having the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the domains are arranged in the order of VL[α-synuclein]-VH[α-synuclein]-PEST motif. In some embodiments, the domains are arranged in the order of VH[α-synuclein]-VL[α-synuclein]-PEST motif. In some embodiments, the antigen-binding domain is selected from an antibody or a functional fragment thereof, an antibody heavy chain, an antibody light chain, a single-domain antibody, and an scFv. In some embodiments, the α-synuclein-specific VL domain (VL α-synuclein) and the α-synuclein-specific VH domain (VH α-synuclein) are linked by a polypeptide linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0010] In some embodiments, the antigen-binding domain is derived from a monoclonal antibody, a synthetic antibody, a human antibody, or a humanized antibody. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequences set forth in SEQ ID NO: 149 and SEQ ID NOs: 236-473. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequences set forth in any one of SEQ ID NOs: 150-152 and SEQ ID NOs: 170-235. In some embodiments, the cell-penetrating peptide enhances intracellular delivery of the recombinant polypeptide into cells as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide. In some embodiments, the cell-penetrating peptide enhances intracellular delivery of the recombinant polypeptide by an amount of (a) about 10% to about 30%, (b) about 30% to about 50%, (c) about 50% to about 70%, or (d) about 70% to about 90% as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide.

[0011] In a second aspect, the disclosure features a recombinant polypeptide that binds to α-synuclein and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequences set forth in any one of SEQ ID NOs: 154-158, SEQ ID NO: 161, SEQ ID NO: 163, and SEQ ID NO: 164.

[0012] In some embodiments, the disclosure features a method of treating a protein aggregation disease in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the recombinant polypeptide of the first aspect. In some embodiments, the protein aggregation disease is selected from the group consisting of Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia, Alzheimer's disease (AD), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), spinal cord injury (SCI), traumatic brain injury (TBI), and other synucleinopathies.

[0013] In some embodiments, the recombinant polypeptide is delivered to the midbrain dopaminergic neurons of a patient having PD or is expressed in midbrain dopaminergic neurons. In some embodiments, the recombinant polypeptide is delivered to the oligodendrocytes of a patient having MSA or is expressed in oligodendrocytes. In some embodiments, the recombinant polypeptide is delivered to the glutamatergic neurons of a patient having a synucleinopathy such as Lewy body disease or is expressed in glutamatergic neurons. In some embodiments, the method further comprises providing the recombinant polypeptide to the patient by gene therapy. In some embodiments, the degradation rate of α-synuclein is altered in a specified neuronal subtype. In some embodiments, the neuronal subtype is selected from neurons including but not limited to dopaminergic neurons, glutamatergic neurons, GABAergic neurons, cholinergic neurons, astrocytes, oligodendrocytes, and microglia. In some embodiments, the neuronal subtype is selected from neuron-specific promoters such as synapsin I, and cell-type specific promoters such as promoters in VGLUTI or tyrosine hydroxylase, or glia-specific promoters such as myelin basic protein or GFAP.

[0014] In a third aspect, the present disclosure features a recombinant polypeptide that, from the N-terminus to the C-terminus, comprises: I. (a) an optional signal peptide domain; (b) a cell-permeable peptide; (c) an antigen-binding domain that binds to tau; and (d) a programmable proteasome-targeting human or mouse PEST domain; or II. (a) an optional signal peptide domain; (b) a programmable proteasome-targeting human or mouse PEST domain; (c) an antigen-binding domain that binds to tau; and (d) a cell-permeable peptide.

[0015] In some embodiments, the programmable proteasome-targeting human PEST domain is at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and comprises a sequence having at least one amino acid substitution, wherein the polypeptide increases or decreases the degradation of tau as compared to an empty vector (EV) control. In some embodiments, the at least one amino acid substitution is selected from alanine, glycine, valine, leucine, or isoleucine. In some embodiments, the programmable proteasome-targeting human PEST domain has the sequence NPDFX1X2X3VX4X5QX6AX7X8LX9VX 10 X 11 AWX 12 X 13 GMX 14 RHRAACASASINV (SEQ ID NO: 109), wherein X1 is (P / A), X2 is (P / A), X3 is (E / A), X4 is (E / A), X5 is (E / A), X6 is (D / A), X7 is (S / A), X8 is (T / A), X9 is (P / A), X 10 is (S / A), X 11 is (C / A), X 12 is (E / A), X 13 is (S / A), X 14It includes (K / A), where the sequence is not NPDFPPEVEEQDASTLPVSCAWESGMKRHRAACASASINV (SEQ ID NO: 3), and the polypeptide increases tau degradation compared to the empty vector (EV) control.

[0016] In some embodiments, the programmable proteasome targeting human PEST domain has X1 being (P), X2 being (A), X3 being (E), X4 being (E), X5 being (E), X6 being (D), X7 being (S), X8 being (T), X9 being (P), X 10 being (S), X 11 being (C), X 12 being (E), X 13 being (S), X 14 being (K) (amino acids 164 - 191 in SEQ ID NO: 7), or X1 being (P), X2 being (P), X3 being (E), X4 being (E), X5 being (E), X6 being (A), X7 being (S), X8 being (T), X9 being (P), X 10 being (S), X 11 being (C), X 12 being (E), X 13 being (S), X 14 being (K) (amino acids 164 - 191 in SEQ ID NO: 8), or X1 being (P), X2 being (P), X3 being (E), X4 being (E), X5 being (E), X6 being (D), X7 being (S), X8 being (T), X9 being (P), X 10 being (S), X 11 being (C), X 12 being (E), X 13 being (A), X 14 and includes a sequence being (K) (amino acids 164 - 191 in SEQ ID NO: 10).

[0017] In some embodiments, the programmable proteasome targeting human PEST domain comprises amino acids 164 to 191 of the amino acid sequence set forth in SEQ ID NO: 9, where the polypeptide reduces the degradation of tau compared to an empty vector (EV) control. In some embodiments, the antigen-binding domain is an intrabody. In some embodiments, the intrabody is a single-chain variable fragment (scFv) or a single-domain antibody that binds to tau. In some embodiments, the single-domain antibody comprises a tau-specific VL domain (VLtau), a tau-specific VH domain (VHtau), or a tau-specific VHH domain. In some embodiments, the recombinant polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 162.

[0018] In some embodiments, the single-domain antibody comprises a VH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 65 to 81, or a VL domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 82 to 98. In some embodiments, the domains are arranged in the order of VL[tau]-VH[tau]-PEST motif. In some embodiments, the domains are arranged in the order of VH[tau]-VL[tau]-PEST motif. In some embodiments, the antigen-binding domain is selected from an antibody or a functional fragment thereof, an antibody heavy chain, an antibody light chain, a single-domain antibody, and an scFv. In some embodiments, a tau-specific VL domain (VLtau) and a tau-specific VH domain (VHtau) are linked by a polypeptide linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the antigen-binding domain is derived from a monoclonal antibody, a synthetic antibody, a human antibody, or a humanized antibody.

[0019] In some embodiments, the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequences set forth in SEQ ID NO: 149 and SEQ ID NOs: 236 to 473. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 150 to 152 and SEQ ID NOs: 170 to 235.

[0020] In some embodiments, the cell-penetrating peptide enhances the intracellular delivery of the recombinant polypeptide into cells as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide. In some embodiments, the cell-penetrating peptide enhances the intracellular delivery of the recombinant polypeptide by an amount of (a) about 10% to about 30%, (b) about 30% to about 50%, (c) about 50% to about 70%, or (d) about 70% to about 90% as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide. In some embodiments, the recombinant polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence set forth in SEQ ID NO: 162.

[0021] In some embodiments, the present disclosure features a method of treating a protein aggregation disease in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the recombinant polypeptide of the third aspect. In some embodiments, the protein aggregation disease is selected from frontotemporal dementia (FTD), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia with parkinsonism on chromosome 17 (FTDP-17), frontotemporal lobar degeneration (FTLD-TAU), corticobasal degeneration (CBD), primary age-related tauopathy, Pick's disease, chronic traumatic encephalopathy (CTE), Lewy body dementia, vascular dementia, tuberous sclerosis, spinal cord injury (SCI), traumatic brain injury (TBI), or other tauopathies. In some embodiments, the recombinant polypeptide is delivered to the midbrain dopaminergic neurons of a patient having PD or is expressed in midbrain dopaminergic neurons. In some embodiments, the recombinant polypeptide is delivered to oligodendrocytes in a patient having MSA or is expressed in oligodendrocytes. In some embodiments, the recombinant polypeptide is delivered to glutamatergic neurons of a patient having a tauopathy or is expressed in glutamatergic neurons. In some embodiments, the method further comprises providing the recombinant polypeptide to a patient by gene therapy.

[0022] In a fourth aspect, the disclosure features a recombinant polypeptide comprising, from the N-terminus to the C-terminus, I. (a) an optional signal peptide domain; (b) a cell-penetrating peptide; (c) an antigen-binding domain that binds to huntingtin; and (d) a programmable proteasome-targeting human or mouse PEST domain; or II. (a) an optional signal peptide domain; (b) a programmable proteasome-targeting human or mouse PEST domain; (c) an antigen-binding domain that binds to huntingtin; and (d) a cell-penetrating peptide.

[0023] In some embodiments, the programmable proteasome targeting human PEST domain is at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and includes a sequence having at least one amino acid substitution, where the polypeptide increases or decreases the degradation of huntingtin compared to an empty vector (EV) control. In some embodiments, the at least one amino acid substitution is selected from alanine, glycine, valine, leucine, or isoleucine. In some embodiments, the programmable proteasome targeting human PEST domain has the sequence NPDFX1X2X3VX4X5QX6AX7X8LX9VX 10 X 11 AWX 12 X 13 GMX 14 RHRAACASASINV (SEQ ID NO: 109), wherein X1 is (P / A), X2 is (P / A), X3 is (E / A), X4 is (E / A), X5 is (E / A), X6 is (D / A), X7 is (S / A), X8 is (T / A), X9 is (P / A), X 10 is (S / A), X 11 is (C / A), X 12 is (E / A), X 13 is (S / A), X 14 is (K / A)), where the sequence is not NPDFPPEVEEQDASTLPVSCAWESGMKRHRAACASASINV (SEQ ID NO: 3) and the polypeptide increases the degradation of the protein compared to an empty vector (EV) control.

[0024] In some embodiments, for the programmable proteasome targeting human PEST domain, X1 is (P), X2 is (A), X3 is (E), X4 is (E), X5 is (E), X6 is (D), X7 is (S), X8 is (T), X9 is (P), X 10 is (S), X 11 is (C), X 12 is (E), X13 is (S), X 14 is (K) (amino acids 164 - 191 in SEQ ID NO: 7), or X1 is (P), X2 is (P), X3 is (E), X4 is (E), X5 is (E), X6 is (A), X7 is (S), X8 is (T), X9 is (P), X 10 is (S), X 11 is (C), X 12 is (E), X 13 is (S), X 14 is (K) (amino acids 164 - 191 in SEQ ID NO: 8), or X1 is (P), X2 is (P), X3 is (E), X4 is (E), X5 is (E), X6 is (D), X7 is (S), X8 is (T), X9 is (P), X 10 is (S), X 11 is (C), X 12 is (E), X 13 is (A), X 14 includes a sequence that is (K) (amino acids 164 - 191 in SEQ ID NO: 10). In some embodiments, the programmable proteasome targeting human PEST domain comprises amino acids 164 - 191 of the amino acid sequence set forth in SEQ ID NO: 9, where the polypeptide reduces the degradation of huntingtin compared to an empty vector (EV) control.

[0025] In some embodiments, the antigen-binding domain is an intrabody. In some embodiments, the intrabody is a single-chain variable fragment (scFv) or a single-domain antibody that binds to huntingtin. In some embodiments, the single-domain antibody comprises a huntingtin-specific VL domain (VL huntingtin), a huntingtin-specific VH domain (VH huntingtin), or a huntingtin-specific VHH domain. In some embodiments, the scFV comprises a VH domain described herein as SEQ ID NO: 106 and a VL domain described herein as SEQ ID NO: 107. In some embodiments, the domains are arranged in the order of VL[huntingtin]-VH[huntingtin]-PEST motif or VH[huntingtin]-VL[huntingtin]-PEST motif. In some embodiments, the huntingtin-specific VL domain comprises the amino acid sequence described herein as SEQ ID NO: 108. In some embodiments, the recombinant polypeptide comprises the amino acid sequence described in SEQ ID NO: 160. In some embodiments, the antigen-binding domain is selected from an antibody or a functional fragment thereof, an antibody heavy chain, an antibody light chain, a single-domain antibody, and an scFv.

[0026] In some embodiments, the huntingtin-specific VL domain (VL huntingtin) and the huntingtin-specific VH domain (VH huntingtin) are linked by a polypeptide linker. In some embodiments, the linker comprises the amino acid sequence described in SEQ ID NO: 14. In some embodiments, the antigen-binding domain is derived from a monoclonal antibody, a synthetic antibody, a human antibody, or a humanized antibody. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequences described in SEQ ID NO: 149 and SEQ ID NOs: 236-473.

[0027] In some embodiments, the cell-penetrating peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 150 to 152 and SEQ ID NOs: 170 to 235. In some embodiments, the cell-penetrating peptide enhances the intracellular delivery of the recombinant polypeptide to cells as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide. In some embodiments, the cell-penetrating peptide enhances the intracellular delivery of the recombinant polypeptide by an amount of (a) about 10% to about 30%, (b) about 30% to about 50%, (c) about 50% to about 70%, or (d) about 70% to about 90% as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide. In a fifth aspect, the present disclosure features a recombinant polypeptide that binds to huntingtin and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence set forth in SEQ ID NO: 160.

[0028] In some embodiments, the present disclosure features a method of treating a protein aggregation disease in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the recombinant polypeptide of the fourth or fifth aspect. In some embodiments, the protein aggregation disease is selected from Huntington's disease, or other protein aggregation neurodegenerative diseases including Parkinson's disease (PD), multiple system atrophy (MSA), and Lewy body dementia, Alzheimer's disease (AD), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), spinal cord injury (SCI), and traumatic brain injury (TBI). In some embodiments, the protein aggregation disease is Huntington's disease.

[0029] In some embodiments, the recombinant polypeptide is delivered to the midbrain dopaminergic neurons of a patient with PD or expressed in the midbrain dopaminergic neurons. In some embodiments, the recombinant polypeptide is delivered to the oligodendrocytes in a patient with MSA or expressed in the oligodendrocytes. In some embodiments, the recombinant polypeptide is delivered to the glutamatergic neurons of a patient with Huntington's disease or expressed in the glutamatergic neurons. In some embodiments, the method further comprises providing the recombinant polypeptide to the patient by gene therapy.

[0030] In some embodiments, the present disclosure features a polynucleotide encoding a recombinant polypeptide of any of the above aspects. In some embodiments, the present disclosure features a vector comprising the polynucleotide of the present disclosure. In some embodiments, the present disclosure features an isolated host cell transfected with the polynucleotide of the present disclosure. In some embodiments, the present disclosure features an isolated host cell transfected with the vector of the present disclosure. In some embodiments, the present disclosure features a pharmaceutical composition comprising a human gene therapy vector comprising the polynucleotide of the present disclosure. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the present disclosure features a method for the preparation of a recombinant polypeptide, the method comprising culturing a host cell transfected with the polynucleotide of the present disclosure and expressing the polynucleotide, and isolating the polypeptide from the cells.

[0031] In another aspect, the present disclosure features a recombinant polypeptide comprising, from the N-terminus to the C-terminus, I. (a) an optional signal peptide domain; (b) a cell-penetrating peptide; (c) an antigen-binding domain that binds to a protein; and (d) a programmable proteasome-targeting human or mouse PEST domain; or II. (a) an optional signal peptide domain; (b) a programmable proteasome-targeting human or mouse PEST domain; (c) an antigen-binding domain that binds to a protein; and (d) a cell-penetrating peptide. In some embodiments, the programmable proteasome-targeting human PEST domain is at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and comprises a sequence having at least one amino acid substitution, wherein the polypeptide having at least one amino acid substitution increases or decreases protein degradation compared to an empty vector (EV) control. In some embodiments, at least one amino acid substitution is selected from alanine, glycine, valine, leucine, or isoleucine. In some embodiments, the programmable proteasome-targeting human PEST domain has the sequence NPDFX1X2X3VX4X5QX6AX7X8LX9VX 10 X 11 AWX 12 X 13 GMX 14 RHRAACASASINV (SEQ ID NO: 109), wherein X1 is (P / A), X2 is (P / A), X3 is (E / A), X4 is (E / A), X5 is (E / A), X6 is (D / A), X7 is (S / A), X8 is (T / A), X9 is (P / A), X 10 is (S / A), X 11 is (C / A), X 12 is (E / A), X 13 is (S / A), X 14comprises (K / A)), wherein said sequence is not NPDFPPEVEEQDASTLPVSCAWESGMKRHRAACASASINV (SEQ ID NO: 3), and the polypeptide increases protein degradation compared to the empty vector (EV) control.

[0032] In some embodiments, the programmable proteasome targeting human PEST domain has X1 is (P), X2 is (A), X3 is (E), X4 is (E), X5 is (E), X6 is (D), X7 is (S), X8 is (T), X9 is (P), X 10 is (S), X 11 is (C), X 12 is (E), X 13 is (S), X 14 is (K) (amino acids 164-191 in SEQ ID NO: 7), or X1 is (P), X2 is (P), X3 is (E), X4 is (E), X5 is (E), X6 is (A), X7 is (S), X8 is (T), X9 is (P), X 10 is (S), X 11 is (C), X 12 is (E), X 13 is (S), X 14 is (K) (amino acids 164-191 in SEQ ID NO: 8), or X1 is (P), X2 is (P), X3 is (E), X4 is (E), X5 is (E), X6 is (D), X7 is (S), X8 is (T), X9 is (P), X 10 is (S), X 11 is (C), X 12 is (E), X 13 is (A), X 14 comprises a sequence that is (K) (amino acids 164-191 in SEQ ID NO: 10).

[0033] In some embodiments, the programmable proteasome targeting human PEST domain comprises amino acids 164 to 191 of the amino acid sequence set forth in SEQ ID NO: 9, wherein the polypeptide reduces protein degradation compared to an empty vector (EV) control. In some embodiments, the antigen-binding domain is an intrabody. In some embodiments, the intrabody is a single-chain variable fragment (scFv) or a single-domain antibody that binds to a protein. In some embodiments, the single-domain antibody comprises a VL domain specific for the protein, a VH domain specific for the protein, or a VHH domain specific for the protein. In some embodiments, the antigen-binding domain is selected from an antibody or a functional fragment thereof, an antibody heavy chain, an antibody light chain, a single-domain antibody, and an scFv. In some embodiments, the VL domain specific for the protein and the VH domain specific for the protein are linked by a polypeptide linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0034] In some embodiments, the antigen-binding domain is derived from a monoclonal antibody, a synthetic antibody, a human antibody, or a humanized antibody. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequences set forth in SEQ ID NO: 149 and SEQ ID NOs: 236-473. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150-152 and SEQ ID NOs: 170-235. In some embodiments, the cell-penetrating peptide enhances the intracellular delivery of the recombinant polypeptide into cells as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide. In some embodiments, the cell-penetrating peptide enhances the intracellular delivery of the recombinant polypeptide by an amount of (a) about 10% to about 30%, (b) about 30% to about 50%, (c) about 50% to about 70%, or (d) about 70% to about 90% as compared to an equivalent recombinant polypeptide that does not contain the cell-penetrating peptide.

[0035] In some embodiments, the protein is α-synuclein. In some embodiments, the single-domain antibody is a VHH antibody having an amino acid sequence set forth in any one of SEQ ID NOs: 16-17, or comprises a VH domain having the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the domains are arranged in the order of VL[α-synuclein]-VH[α-synuclein]-PEST motif. In some embodiments, the domains are arranged in the order of VH[α-synuclein]-VL[α-synuclein]-PEST motif.

[0036] In some embodiments, the protein is tau. In some embodiments, the single domain antibody comprises a VH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 65 to 81, or a VL domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 82 to 98. In some embodiments, the domains are arranged in the order of VL[tau]-VH[tau]-PEST motif. In some embodiments, the domains are arranged in the order of VH[tau]-VL[tau]-PEST motif. In some embodiments, the protein is huntingtin. In some embodiments, the scFv comprises a VH domain described herein as SEQ ID NO: 106 and a VL domain described herein as SEQ ID NO: 107. In some embodiments, the domains are arranged in the order of VL[huntingtin]-VH[huntingtin]-PEST motif or VH[huntingtin]-VL[huntingtin]-PEST motif. In some embodiments, the huntingtin-specific VL domain comprises the amino acid sequence described herein as SEQ ID NO: 108.

[0037] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0038] To avoid any doubt, in "some embodiments", "in certain embodiments", "in certain cases", "in some cases", "in further embodiments", "in one embodiment" and "in further embodiments" and the like, the expressions are to be read such that any of the embodiments described therein are to be read in view of combining the respective features of those embodiments, and it is emphasized that they are used and intended such that the present disclosure must be treated in the same manner as if a combination of the features of those embodiments were detailed in one embodiment. The same applies to any combination of embodiments and features of the appended claims, as illustrated in the examples, which are also intended to be combined with features from the corresponding embodiments disclosed in the description, where, for the sake of consistency and brevity only, the embodiments are characterized by dependencies, while in fact, each combination of embodiments and features that can be construed as resulting from (multiple) dependencies must be recognized as being literally disclosed and not regarded as a choice among alternatives. In this context, those skilled in the art will understand that the embodiments and features disclosed in the examples are intended to be generalized to equivalents having the same functions as those illustrated therein. Other features and advantages of the present disclosure will be apparent from the following detailed description and the appended claims.

Brief Description of the Drawings

[0039]

FIG. 1A-C

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Mode for Carrying Out the Invention

[0040] Overview The present disclosure is based on the discovery that a cell-penetrating peptide can enhance the intracellular delivery of a bifunctional polypeptide comprising an intrabody that binds to an epitope of an antigen (e.g., α-synuclein, tau, or huntingtin) and a programmable proteasome-targeting PEST motif. The bifunctional polypeptide is useful for the treatment and prevention of protein aggregation diseases such as synucleinopathies and tauopathies, Huntington's disease, and spinal cord injury (SCI) and traumatic brain injury (TBI). The bifunctional polypeptides for use in the present disclosure are described in U.S. Provisional Application No. 63 / 112,381, filed November 11, 2020, U.S. Provisional Application No. 63 / 112,383, filed November 11, 2020, and U.S. Provisional Application No. 63 / 112,385, filed November 11, 2020, the contents of which are incorporated herein by reference in their entirety.

[0041] α-synuclein protein α-Synuclein is a molecule important for the function of the nervous system. α-Synuclein accumulates to toxic levels in many neurodegenerative diseases, as well as in traumatic injuries, and thus reducing the intracellular level of this protein is a beneficial therapeutic approach. Since α-synuclein is an essential protein, completely removing it is harmful to the cell. Thus, provided herein are methods where the therapeutic goal is to achieve a level of α-synuclein degradation that reduces the amount of intracellular α-synuclein to a level that is not toxic to the cell but does not completely eliminate the protein. More precisely, the level of α-synuclein is reduced to a desired level.

[0042] In synucleinopathies, α-synuclein undergoes intracellular cascades of pathogenic misfolding, abnormal accumulation, and transcellular propagation. This process induces synuclein aggregation and neurotoxicity, as observed in vertebrate models, suggesting this process as a novel therapeutic target. However, none of these events proceed in the absence of major intracellular α-synuclein misfolding events. Therefore, it is important to target α-synuclein to prevent this pathological cascade. This was addressed by developing bifunctional intrabodies that have the potential to eliminate synuclein accumulation using the cell's normal protein removal process. An anti-α-synuclein body intrabody that targets synuclein to the proteasome for degradation was identified. To avoid potential immunogenic responses, the proteasome targeting signal was optimized for human use by substitution of the mouse PEST degron with the human PEST (hPEST) degron derived from ornithine decarboxylase (ODC). One specific intrabody, designated VH14-hPEST herein, resulted in efficient degradation of endogenous α-synuclein in human induced pluripotent stem cell (iPSC)-derived neurons. Furthermore, the novel anti-synuclein bifunctional intrabodies N77D and DB1 are able to efficiently degrade synuclein from both human and squid.

[0043] Tau protein Tau is a molecule important for the function of the nervous system. Tau accumulates to toxic levels in many neurodegenerative diseases, as well as in traumatic injuries, and thus reducing the intracellular level of the protein is a beneficial therapeutic approach. Since Tau has important functions in the nervous system, it is harmful to remove it all. Therefore, provided herein is a method in which the therapeutic goal is to achieve a level of Tau degradation that reduces the amount of intracellular Tau to a level that is not toxic to the cell but does not completely eliminate the protein. More precisely, the level of Tau is reduced to a desirable level. As described herein, an intrabodymolecule that targets tau provides specificity to a protein, and a PEST degron provides a target to the proteasome. Modification of the PEST degron by specific alteration of a protein sequence provides the ability to regulate the level of degradation.

[0044] Tau is a microtubule-associated phosphorylated protein expressed in the central and peripheral nervous systems. Tau plays roles in many biological processes such as microtubule stabilization, neurite outgrowth, neuronal migration, signal transduction, and organelle transport. Under normal conditions, tau expression is abundant within the axons of neurons. Misfolding and aggregation of tau within neurons define the pathological features in various tauopathies. The incidence of tauopathies represents an urgent and unmet medical need.

[0045] In tauopathies, the tau protein may lose its ability to bind to microtubules, and as a result, tau is mislocalized to the neuronal cell body-dendritic compartment by mistake. During this process, tau is hyperphosphorylated and misfolds into insoluble aggregates of straight filaments and paired helical filaments (PHFs) including neurofibrillary tangles and threads (NFTs). Tau hyperphosphorylation is presumed to occur prior to NFT formation. Furthermore, abnormal tau can recruit properly folded isoforms into misfolded complexes, and the abnormal forms may be secreted from one cell and taken up by other cells, which can trigger a cascade of misfolded tau complexes and potentially spread the disease throughout the central nervous system. Immunotherapy for reducing the intracellular levels of tau available for misfolding and / or aggregation represents a potential therapeutic approach for the treatment of tauopathies. However, full-length antibodies that bind to tau have limited penetration into brain cells where tau protein aggregates are present.

[0046] Huntingtin protein Huntingtin is a molecule important for the function of the nervous system. Huntingtin accumulates to toxic levels in many neurodegenerative diseases, particularly Huntington's disease, and thus reducing the intracellular level of the protein is a beneficial therapeutic approach. Since huntingtin has important functions in the nervous system, completely removing it is harmful. Accordingly, this specification provides a method for achieving a level of huntingtin degradation at which the therapeutic goal is to reduce the amount of intracellular huntingtin to a level that is not toxic to the cell but does not completely eliminate the protein. More precisely, the level of huntingtin is reduced to a desired level.

[0047] Huntingtin is a protein present in many body tissues and is the causative gene / protein (HTT) of Huntington's disease. The genetic mutation that causes Huntington's disease is known as an expansion of the CAG trinucleotide repeat. This mutation increases the size of the CAG segment in the HTT gene. People with Huntington's disease have 36 to over 120 CAG repeats. People with 36 to 39 CAG repeats (SEQ ID NO: 135) may or may not develop the signs and symptoms of Huntington's disease, while people with 40 or more repeats almost always develop the disorder. The exact function of the huntingtin protein is unknown, but the huntingtin protein is thought to play an important role in nerve cells (neurons) in the brain and is essential for normal prenatal development. Huntingtin is found in many body tissues and is found at the highest levels of activity in the brain. Inside the cell, this protein may be involved in chemical signaling, transport of substances, attachment (binding) to proteins and other structures, and protection from cell self-destruction (apoptosis). Some studies have suggested that huntingtin plays a role in the repair of damaged DNA.

[0048] One region of the HTT gene contains a specific DNA segment known as a CAG trinucleotide repeat. This segment is composed of a series of three DNA building blocks (cytosine, adenine, and guanine) that appear multiple times in a row. Typically, the CAG segment is repeated 10 to 35 times within the gene (SEQ ID NO: 136). An expansion of the CAG segment leads to the production of an abnormally long version of the huntingtin protein. The expanded protein is cleaved into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal function of these cells. It has also been suggested that the loss of the DNA repair function of the huntingtin protein can lead to the accumulation of DNA damage in neurons, especially as molecular damage increases during aging. Brain regions that help coordinate movement and control thought and emotion (the striatum and cerebral cortex) are particularly affected. Dysfunction and eventual death of neurons in these regions of the brain underlie the signs and symptoms of Huntington's disease.

[0049] When the altered HTT gene is passed from one generation to the next, the size of the CAG trinucleotide repeat often increases. Typically, a greater number of repeats is associated with earlier onset of signs and symptoms. This phenomenon is called anticipation. People with adult-onset Huntington's disease (which appears in middle age) usually have 40 to 50 CAG repeats (SEQ ID NO: 137) in the HTT gene, while people with the less common juvenile form of the disorder (which appears in childhood or adolescence) tend to have more than 60 CAG repeats. Individuals with 27 to 35 CAG repeats (SEQ ID NO: 138) in the HTT gene do not develop Huntington's disease, but they are at risk of having children who develop the disorder. When the gene is passed from parent to child, the size of the CAG trinucleotide repeat can increase to a range associated with Huntington's disease (36 repeats or more).

[0050] Intrabodies targeting α-synuclein, tau, and huntingtin In some embodiments, the recombinant polypeptides of the present disclosure are intrabodies having several domains. In some embodiments, an intrabody useful for achieving an increase in the degradation of α-synuclein as described herein may have a structure as described herein (see FIGS. 1A and 1B for schematic diagrams). An intrabody targeting α-synuclein provides specificity to the protein, and the PEST degron provides the target to the proteasome. Modification of the PEST degron by specific alterations in the protein sequence provides the ability to regulate the level of degradation. In some embodiments, the intrabody is a cell-permeable intrabody having an immunoglobulin heavy chain signal peptide sequence (SS) added to the N-terminus to direct the complex to the secretory pathway and a cell-penetrating peptide derived from the Drosophila antennapedia homeodomain, termed penetratin (PEN), added to the N-terminus of the intrabody to facilitate translocation across the cell membrane. In some embodiments, the intrabody may have a first domain as an immunoglobulin heavy chain signal peptide sequence, a second domain as a cell-penetrating peptide (e.g., PEN), a third domain having an antigen-binding domain of an antibody or a functional fragment thereof that binds to an epitope of an antigen of the present disclosure (e.g., α-synuclein, tau, or huntingtin), and a fourth domain having a programmable proteasome-targeting PEST motif.

[0051] In some embodiments, the recombinant polypeptide of the present disclosure contains a methionine (M) amino acid representing the translation start codon (ATG) at position 1. Methionine is required for protein expression. In some embodiments, the recombinant polypeptide is structured from the N-terminus to the C-terminus as follows: (start codon) leader sequence; cell-penetrating peptide; antigen-binding domain that binds to α-synuclein; programmable proteasome-targeting human or mouse PEST domain. In other embodiments, the recombinant polypeptide is structured from the N-terminus to the C-terminus as follows: (start codon) cell-penetrating peptide; antigen-binding domain that binds to α-synuclein; programmable proteasome-targeting human or mouse PEST domain. In some embodiments, the recombinant polypeptide is structured from the N-terminus to the C-terminus as follows: (start codon) leader sequence; programmable proteasome-targeting human or mouse PEST domain; antigen-binding domain that binds to α-synuclein; and cell-penetrating peptide. In other embodiments, the recombinant polypeptide is structured from the N-terminus to the C-terminus as follows: (start codon); programmable proteasome-targeting human or mouse PEST domain; antigen-binding domain that binds to α-synuclein; and cell-penetrating peptide.

[0052] In some embodiments, an intrabody useful for increasing the degradation of α-synuclein comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in any one of SEQ ID NO: 5 to SEQ ID NO: 18, SEQ ID NO: 149 to SEQ ID NO: 152, SEQ ID NO: 154 to SEQ ID NO: 158, SEQ ID NO: 161, SEQ ID NO: 163, and SEQ ID NO: 164. In some embodiments, an intrabody useful for increasing the degradation of α-synuclein consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in any one of SEQ ID NO: 5 to SEQ ID NO: 18, SEQ ID NO: 149 to SEQ ID NO: 152, SEQ ID NO: 154 to SEQ ID NO: 158, SEQ ID NO: 161, SEQ ID NO: 163, and SEQ ID NO: 164.

[0053] In some embodiments, an intrabody useful for increasing the degradation of α-tau comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 98. In some embodiments, an intrabody useful for increasing the degradation of α-tau consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in any one of SEQ ID NO: 23 to SEQ ID NO: 98, SEQ ID NO: 149 to SEQ ID NO: 152, and SEQ ID NO: 162.

[0054] In some embodiments, an intrabod is useful for increasing the degradation of hyaluronic acid and comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids as set forth in any one of SEQ ID NO: 100 to SEQ ID NO: 108, SEQ ID NO: 165, SEQ ID NO: 149 to SEQ ID NO: 152, and SEQ ID NO: 160. In some embodiments, an intrabod is useful for increasing the degradation of hyaluronic acid and consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids as set forth in any one of SEQ ID NO: 100 to SEQ ID NO: 108, SEQ ID NO: 165, SEQ ID NO: 149 to SEQ ID NO: 152, and SEQ ID NO: 160.

[0055] In some embodiments, an intrabody useful for increasing the degradation of α-synuclein, tau, or huntingtin comprises an immunoglobulin heavy chain signal peptide sequence (SS) that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in SEQ ID NO: 149. In some embodiments, an intrabody useful for increasing the degradation of α-synuclein, tau, or huntingtin comprises a cell-penetrating peptide derived from the Drosophila antennapedia homeodomain (penetratin or PEN) that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in SEQ ID NO: 150. In some embodiments, an intrabody useful for increasing the degradation of α-synuclein, tau, or huntingtin comprises a cell-penetrating peptide derived from the HIV-1 transcriptional transactivator (TA) protein (HIV-1 TAT) that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in SEQ ID NO: 151. In some embodiments, an intrabody useful for increasing the degradation of α-synuclein, tau, or huntingtin comprises a synthetic cell-penetrating peptide derived from a phage display library that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids set forth in SEQ ID NO: 152.

[0056] In some embodiments, an intrabody useful for increasing the degradation of α-synuclein, tau, or huntingtin comprises a flexible linker having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids as set forth in SEQ ID NO: 14. In some embodiments, an intrabody useful for increasing the degradation of α-synuclein comprises a flexible linker having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids as set forth in any one of SEQ ID NOs: 139-148.

[0057] In some embodiments, an epitope tag can be used to identify the expression of an intrabody. Examples of epitope tags are known in the art and can include, but are not limited to, FLAG, 6×His (SEQ ID NO: 134), HA tag, c-myc, GST, Protein A, CD, Strep-tag, maltose binding peptide (MBP), chitin binding domain (CBD), S-tag, Avitag, CBP, TAP, SF-TAP. In one embodiment, an intrabody as described herein may have an HA tag for identification of intrabody expression under experimental conditions. In some embodiments, the HA tag does not affect the function of the intrabody. In some embodiments, an intrabody useful for increasing the degradation of α-synuclein, tau, or huntingtin comprises an HA tag having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acids as set forth in SEQ ID NO: 15.

[0058] In some embodiments, an intrabod as described above comprises a single-chain antibody comprising antigen-binding fragments thereof having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the α-synuclein-specific VH domain (VH-synuclein), or the α-synuclein-specific VHH antibody (i.e., nanobody), or as set forth in SEQ ID NO: 1, or any one of SEQ ID NOs: 4-13, 16-18, 154-158, 161, 163, 164, or 166.

[0059] The antigen-binding domain of the antibody or its functional fragment can bind to unmodified or modified α-synuclein, and / or aggregated α-synuclein, with high specificity and / or high affinity. The amino acid sequence of the human α-synuclein protein (Genbank® accession number CR541653) is provided as SEQ ID NO: 4. In some embodiments, a specific number of amino acids at either the carboxy or amino terminus can be targeted by an intrabod as described herein. For example, the intrabod can target a region or portion of the α-synuclein protein, including but not limited to, for example, specific regions or groups of amino acids. In some embodiments, amino acids 53-95 of α-synuclein are targeted by an intrabod as described herein, resulting in a reduction in phosphorylation of the protein.

[0060] In some embodiments, an intrabod as described above comprises a single-chain variable fragment (scFv) that includes a tau-specific VH domain (VH-tau) or an antigen-binding fragment thereof having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acids set forth in SEQ ID NOs: 65-81. In some embodiments, the VH domain (VH-tau) may include the CDRs set forth in FIG. 16 (top). In some embodiments, an intrabod as described above comprises a single-chain variable fragment (scFv) that includes a tau-specific VL domain (VL-tau) or an antigen-binding fragment thereof having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acids set forth in SEQ ID NOs: 82-98. In some embodiments, the VL domain (VL-tau) may include the CDRs set forth in FIG. 16 (bottom). The antigen-binding domain of an intrabod, antibody or functional fragment thereof can bind phosphorylated tau, hyperphosphorylated tau, and / or aggregated tau with high specificity and / or high affinity. The amino acid sequence of the human tau protein (Genbank® accession number NP_005901) is provided as SEQ ID NO: 22.

[0061] The antigen-binding domain of the antibody or its functional fragment can bind to huntingtin and / or aggregated huntingtin with high specificity and / or high affinity. The amino acid sequence of the human huntingtin protein (Genbank® accession number NM_002111) is provided as SEQ ID NO: 99. In some embodiments, an intrabody as described above comprises an scFv comprising a huntingtin-specific VH domain (C4 VH), or an antigen-binding fragment thereof having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, an intrabody as described above comprises an scFv comprising a huntingtin-specific VL domain (C4 VL), or an antigen-binding fragment thereof having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, an intrabody as described above comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, an intrabody as described above comprises an scFv comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 165.

[0062] In some embodiments, a specific number of amino acids at either the carboxy or amino terminus can be targeted by an intrabody as described herein. For example, the intrabodies disclosed herein can target regions or portions of the huntingtin protein including, but not limited to, specific exons or introns of interest. In some embodiments, the 17 amino acids at the amino terminus of the huntingtin gene can be targeted by an intrabody as described herein. In some embodiments, the CAG trinucleotide repeat that causes Huntington's disease, as well as exon 1 containing the proline-rich region (PRR), are targeted by an intrabody as described herein.

[0063] Antigen-binding domains of intrabodies or antibodies or functional fragments thereof can include, but are not limited to, single-chain (scFv), single-chain (Fv)2 (sc(Fv)2), single-domain antibodies (dAb; VH; VL), and diabodies. ScFV antibodies and single-domain antibodies retain the binding specificity of a full-length antibody, but they can be expressed as a single gene. ScFV and single-domain VH or VL antibodies can be applied to both extracellular and intracellular (intrabodies). In some embodiments, the intrabody can be a single-chain variable fragment (scFv), variable heavy region (VH), hypervariable region, variable light region (VL), VHH antibody (i.e., nanobody), single-chain antigen-binding domain, etc. In some embodiments, the intrabody (e.g., an α-synuclein, or tau intrabody) includes a single-chain antigen-binding domain referred to herein as a nanobody.

[0064] An scFv is a single-chain polypeptide antibody obtained by linking the VH and VL of an antibody with a linker. The order of VH and VL to be linked is not particularly limited, and they can be arranged in any order. Examples of arrangements include [VH]-linker-[VL]; or [VL]-linker-[VH]. The heavy-chain variable region (VH) and light-chain variable region (VL) in the scFv can be derived from any antibody of the present disclosure (for example, an anti-α-synuclein antibody, an anti-tau antibody, or an anti-huntingtin antibody), or antigen-binding fragments thereof described herein.

[0065] An sc(Fv)2 contains two VH and two VL, which are linked by a linker to form a single chain. The sc(Fv)2 can be prepared, for example, by binding scFvs with a linker. The sc(Fv)2 can include two VH and two VL arranged in the order of VH, VL, VH, and VL starting from the N-terminus of the single-chain polypeptide ([VH]-linker-[VL]-linker-[VH]-linker-[VL]), but the order of the two VH and two VL is not limited to the above arrangement, and they can be arranged in any order. Examples of arrangements include the following. [VL]-linker-[VH]-linker-[VH]-linker-[VL] [VH]-linker-[VL]-linker-[VL]-linker-[VH] [VH]-linker-[VH]-linker-[VL]-linker-[VL] [VL]-linker-[VL]-linker-[VH]-linker-[VH] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0066] In some embodiments, when four antibody variable regions are linked, three linkers are required, and the linkers used may be the same or different. In some embodiments, the linker as described herein may be a glycine-serine linker that binds VH to VL. In some embodiments, the linker length can be optimized to allow proper folding between VH and VL in the intracellular compartment of the cell. Exemplary linkers that may be used in accordance with the present disclosure are described herein as SEQ ID NO: 14. The linker that links the VH region and the VL region of the scFv or sc(FV)2 is not particularly limited. In some embodiments, the linker is a peptide linker. Any arbitrary single-chain peptide containing about 3 to about 25 residues (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) can be used as the linker.

[0067] In other embodiments, the linker is 10 - 20, 10 - 30, 10 - 40, 10 - 50, 10 - 60, 10 - 70, 10 - 80, 10 - 90, 10 - 100, 10 - 144, or 10 - 150 amino acids in length. In certain cases, the linker contains only glycine and / or serine residues. Examples of such peptide linkers include Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO: 139); Ser Gly Gly Gly (SEQ ID NO: 140); Gly Gly Gly Gly Ser (SEQ ID NO: 141); Ser Gly Gly Gly Gly (SEQ ID NO: 142); Gly Gly Gly Gly Gly Ser (SEQ ID NO: 143); Ser Gly Gly Gly Gly Gly (SEQ ID NO: 144); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 145); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 146); (Gly Gly Gly Gly Ser)n (SEQ ID NO: 147)n (where n is one or more integers) and (Ser Gly Gly Gly Gly)n (SEQ ID NO: 148) n(wherein n is one or more integers). In some cases, the linker has multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the amino acid sequence of SEQ ID NO: 139, provided that the serine residues in each copy of the linker are replaced with another amino acid.

[0068] The amino acid sequence of VH or VL in the antigen-binding domain of an antibody or a functional fragment thereof may include modifications such as substitutions, deletions, additions, and / or insertions. For example, modifications such as substitutions, deletions, additions, and / or insertions made within the amino acid sequence of VH or VL may be present in one or more of the CDRs. In certain embodiments, the modifications include one, two, or three amino acid substitutions in one or more CDRs and / or framework regions of the VH and / or VL domains of the anti-α-synuclein antigen-binding domain of the antibody or a functional fragment thereof. Such substitutions are made to improve the binding and functional activity of the antigen-binding domain of the antibody or a functional fragment thereof (e.g., α-synuclein, tau, huntingtin) and / or to reduce immunogenicity. In certain embodiments, the substitutions are conservative amino acid substitutions. In some embodiments, one, two, or three amino acids of the CDR of the antigen-binding domain of the antibody or a functional fragment thereof (e.g., α-synuclein, tau, huntingtin) may be deleted or added as long as antigen-binding (e.g., α-synuclein, tau, huntingtin) and / or functional activity is present when VH and VL are associated. In some embodiments, the CDR may be the CDR provided in FIGS. 15A-15B and within SEQ ID NOs: 16-18.

[0069] The proteasome-targeting PEST motif is a peptide sequence containing a region rich in prolyl (P), glutamyl (E), aspartyl (D), serine (S) and threonyl (T) residues (PEST region), which is targeted for accelerating proteasomal degradation. This sequence is associated with proteins having a short intracellular half-life. Mouse ornithine decarboxylase (MODC) is one of the proteins with the shortest half-life in mammals. The constitutive degradation of MODC by the proteasome is controlled by a PEST sequence (amino acids 422-461) at its carboxy terminus.

[0070] Exemplary mouse-derived PEST motif sequences include amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence corresponding to ornithine decarboxylase (ODC) amino acids 422-461, such as those set forth in SEQ ID NO: 2 (SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV). Exemplary human-derived PEST motif sequences (hPEST) include amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence corresponding to human ornithine decarboxylase (ODC) amino acids 422-461, such as those set forth in SEQ ID NO: 3 (NPDFPPEVEEQDASTLPVSCAWESGMKRHRAACASASINV). A comparison of the mouse PEST (mPEST; SEQ ID NO: 2) and human PEST (hPEST; SEQ ID NO: 3) sequences is provided in Table 1, demonstrating 82.5% sequence homology between mouse mPEST and human hPEST.

[0071]

Table 1

[0072] The term "% identity" between two polypeptide (or polynucleotide) sequences refers to the number of identical matching positions shared by the sequences in a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position in which the same nucleotide or amino acid is presented in both the target sequence and the reference sequence. Gaps presented in the target sequence are not counted since a gap is not a nucleotide or amino acid. Similarly, gaps presented in the reference sequence are not counted since nucleotides or amino acids from the target sequence are counted and nucleotides or amino acids from the reference sequence are not counted. The percentage of sequence identity is calculated by determining the number of positions at which the identical amino acid residue or nucleic acid base appears in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software readily available for both online use and download. Appropriate software programs are available from a variety of sources for alignment of both protein and nucleotide sequences. One such program suitable for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used for comparison of nucleic acid sequences, whereas BLASTP is used for comparison of amino acid sequences.Unless otherwise noted, the BLASTP program for amino acid sequences (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) using a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix should be used to determine percent identity.

[0073] Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at ebi.ac.uk / Tools / psa. In certain embodiments, the percent identity “X” of a first amino acid sequence to a second amino acid sequence is calculated as l00×(Y / Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (when aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. When the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence is higher than the percent identity of the second sequence to the first sequence. It will be understood by those skilled in the art that the creation of sequence alignments for calculating percent sequence identity is not limited to binary sequence comparisons driven solely by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One program suitable for creating multiple sequence alignments is Clustal Omega, available from clustal.org. Another suitable program is MUSCLE, available from drive5.com / muscle. Alternatively, ClustalW2 and MUSCLE are available, for example, from the EBI.

[0074] The terms "linked" or "fused" refer to a bond via a peptide bond (e.g., genetic fusion), chemical conjugation, or other means known in the art. For example, one way a molecule or moiety can be linked is by using a peptide linker that links the molecule or moiety via a peptide bond.

[0075] The term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated with" means a covalent bond, a non-peptide bond, or a non-covalent bond. In another embodiment, the term "associated with" refers to a covalent bond, a non-peptide bond, or a non-covalent bond that is not chemically cross-linked. In another embodiment, the term "associated with" means a covalent bond excluding a peptide bond. In some embodiments, this association is indicated by a colon, i.e., (:).

[0076] Cell-penetrating peptide In some embodiments, the recombinant polypeptides described herein include a cell-penetrating peptide (CPP). Cell-penetrating peptides are described, for example, in Wolfe JM, et al. ACS Cent Sci. 2018 Apr 25;4(4):512-520. The cell-penetrating peptides of the present disclosure can be any peptide including, but not limited to, the cell-penetrating peptides described in Table 1. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 150 to 152 and SEQ ID NOs: 200 to 235.

[0077] In some embodiments, the cell-penetrating peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 150 to 152. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 150 to 152. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 150. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 151. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence set forth in SEQ ID NO: 152. In some embodiments, the cell-penetrating peptide consists of the amino acid sequence set forth in SEQ ID NO: 150. In some embodiments, the cell-penetrating peptide consists of the amino acid sequence set forth in SEQ ID NO: 151. In some embodiments, the cell-penetrating peptide consists of the amino acid sequence set forth in SEQ ID NO: 152.

[0078] In some embodiments, the cell-penetrating peptide is inherently cationic. In some embodiments, the cell-penetrating peptide is inherently amphiphilic. In some embodiments, the cell-penetrating peptide is proline-rich. In some embodiments, the cell-penetrating peptide is inherently hydrophobic. In some embodiments, when the cell-penetrating peptide is the first component of the polypeptide of the present disclosure, a methionine (M) residue at the N-terminus precedes any of the sequences in Table 1.

[0079] [Table 2] TIFF2025521084000004.tif195169

[0080] Leader sequence In some embodiments, the recombinant polypeptides described herein include a leader sequence. The leader sequence may be a signal peptide sequence or domain. The signal peptide is described, for example, in Haryadi R, et al. PLoS One. 2015 Feb 23;10(2):e0116878. The signal peptides of the present disclosure can be peptides including but not limited to the signal peptides described in Table 2. In some embodiments, the signal peptide is an Ig heavy chain signal peptide. In some embodiments, the signal peptide is a kappa light chain signal peptide.

[0081] In some embodiments, the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of the sequences described in SEQ ID NO: 149 and SEQ ID NOs: 240 to 473. In some embodiments, the signal peptide consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to any one of the sequences described in SEQ ID NO: 149 and SEQ ID NOs: 240 to 473.

[0082] In some embodiments, the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identical to the sequence described in SEQ ID NO: 149. In some embodiments, the signal peptide comprises the amino acid sequence described in SEQ ID NO: 149. In some embodiments, the cell-penetrating peptide consists of the amino acid sequence described in SEQ ID NO: 149.

[0083]

Table 3

[0084] As described herein, an "equivalent recombinant polypeptide" is a polypeptide of the present disclosure (intrabod) that does not have a cell-permeable peptide component. The equivalent recombinant peptide is targeted to the same antigen as the test polypeptide. For example, if the test polypeptide is SS-PEN-N77K-HA-hPEST that targets synuclein, the equivalent polypeptide is N77K-HA-hPEST, which also targets synuclein but does not have a penetratin (PEN) region and / or an SS-PEN region.

[0085] Methods for Producing Polypeptides The bifunctional polypeptides (or antigen-binding domains of antibodies or functional fragments thereof) described herein can be produced in bacterial cells or eukaryotic cells. To produce a polypeptide, a polynucleotide encoding the polypeptide is constructed, introduced into an expression vector, and subsequently expressed in an appropriate host cell. Standard molecular biology techniques are used to prepare a recombinant expression vector, transfect the host cell, select for transformants, culture the host cell, and recover the antibody.

[0086] If the polypeptide is to be expressed in bacterial cells (e.g., Escherichia coli), the expression vector should have properties that allow amplification of the vector in bacterial cells. Further, when Escherichia coli such as JM109, DH5α, HB101, or XL1-Blue is used as the host, the vector must have a promoter that can enable efficient expression in Escherichia coli, such as the lacZ promoter, araB promoter, or T7 promoter. Examples of such vectors include, for example, vectors of the M13 series, vectors of the pUC series, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is, in some embodiments, BL21 that expresses T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of Escherichia coli, the pelB signal sequence can be used as the signal sequence for antibody secretion. For bacterial expression, the expression vector may be introduced into bacterial cells using the calcium chloride method or the electroporation method.

[0087] In one embodiment, the polypeptide is produced in mammalian cells. Exemplary mammalian host cells for expressing the polypeptide include Chinese hamster ovary (CHO cells) (including dhfr CHO cells used with a DHFR selectable marker), human fetal kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte cell lines, such as NS0 myeloma cells and SP2 cells, and transgenic animals, such as cells derived from transgenic mammals. When the polypeptide is to be expressed in mammalian cells such as CHO, COS, 293, 293T, and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, such as the SV40 promoter, MMLV-LTR promoter, EF1α promoter, or CMV promoter. In addition to the nucleic acid sequence encoding an immunoglobulin or its domain, the recombinant expression vector may carry additional sequences such as a sequence (e.g., origin of replication) that regulates the replication of the vector in the host cell and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced. For example, usually, the selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate on the host cells into which the vector has been introduced. Examples of vectors having a selectable marker include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0088] In one case, the lentiviral system expresses a recombinant polypeptide. The system has a version of the CBA promoter called CBh, which provides long-term transgene expression in cells. The system also has a reporter protein (mCherry) derived from a separate CMV promoter. This system facilitates the monitoring of transduction efficiency.

[0089] The polypeptides described herein can be isolated from inside or outside of a host cell (e.g., a culture medium) and purified as substantially pure and homogeneous antibodies. Isolation and purification methods generally used for polypeptide purification may be used for the isolation and purification of the polypeptides, and are not limited to any specific method. The polypeptides can be isolated and purified, for example, by appropriately selecting column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, dialysis, and recrystallization and combining them. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography. Chromatography can be carried out using liquid phase chromatography such as HPLC and FPLC. Examples of columns used for affinity chromatography include protein A columns and protein G columns. Examples of columns using protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). Also disclosed are polypeptides highly purified using these purification methods. In some embodiments, the polypeptides of the present disclosure (i.e., cell-permeable intrabodies) can be purified from bacterial, insect, and mammalian cell culture systems. Also, the cell-permeable intrabodies contain a signal peptide when the polypeptides are not purified from a cell culture system.

[0090] Characterization of the antigen-binding domain of an antibody or an antigen-binding functional fragment thereof The antigen-binding properties of the polypeptides described herein (e.g., α-synuclein binding, tau binding, or huntingtin binding) can be measured by any standard method, such as the following methods: OCTET®, surface plasmon resonance (SPR), BIACORE® analysis, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and fluorescence resonance energy transfer (FRET), either singly or in combination.

[0091] The binding interaction between the protein of interest (anti-synuclein, anti-tau, or anti-huntingtin antibody-binding domain or functional fragments thereof) and the target (e.g., α-synuclein, tau, or huntingtin) can be analyzed using an OCTET® system. In this method, one of several variations of instruments from ForteBio (e.g., OCTET® QKe and QK) is used to determine protein interactions, binding specificity, and epitope mapping. The OCTET® system provides a simple way to monitor real-time binding by measuring changes in polarization as it travels through a custom chip and back to the sensor. The binding interaction between the protein of interest (anti-synuclein, anti-tau, or anti-huntingtin antibody-binding domain or functional fragments thereof) and the target (e.g., α-synuclein, tau, or huntingtin) can be analyzed using surface plasmon resonance (SPR). SPR or biomolecular interaction analysis (BIA) detects specific biological interactions in real time without labeling either of the interactants. A change in mass at the binding surface of the BIA chip (indicating a binding event) results in a change in the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The change in refractive index generates a detectable signal, which is measured as an indicator of the real-time reaction between biological molecules. Methods using SPR are known and described in the art. Information from SPR is used to determine the equilibrium dissociation constant (Kd), as well as K for the binding of a biomolecule to a target on and K offIt can provide accurate and quantitative measurement of dynamic parameters including

[0092] Also, the epitope can be directly mapped by evaluating the ability of various anti-α-synuclein antibody binding domains or functional fragments thereof to compete with each other for binding to human α-synuclein, tau, or synuclein using BIACORE chromatography techniques. When using an enzyme immunoassay, a sample containing an antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody, is added to a plate coated with an antigen. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, washed, and then an enzyme substrate such as p-nitrophenyl phosphate is added, and the absorbance is measured to evaluate the antigen-binding activity. Further general guidance for evaluating antibodies, such as Western blot and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, ed. by Harlow and Lane, Cold Spring Harbor press (1988).

[0093] Treatment method A method for treating or preventing protein aggregation caused by diseases or traumas that result in α-synuclein aggregation, such as Parkinson's disease, multiple system atrophy, spinal cord injury (SCI) or traumatic brain injury (TBI), including administration to a patient in need of treatment or prevention of gene therapy with a gene encoding an anti-α-synuclein bifunctional intrabody as described herein in a therapeutically effective amount.

[0094] In some embodiments, the methods described herein can treat or prevent protein aggregation resulting from spinal cord injury or traumatic brain injury, as described herein, in a subject or patient as described herein. Administration of a composition comprising gene therapy with a gene encoding an anti-α-synuclein intrabody, as described herein, can be in a clinical setting as described herein, or in an alternative setting considered appropriate by a clinician or practitioner.

[0095] In some embodiments, such a composition comprising gene therapy with a gene encoding an anti-α-synuclein intrabody, as described herein, may be combined with other therapies or treatments for the treatment of brain injury or spinal cord injury in a patient. Other pharmaceutical treatments may be used if considered appropriate by a clinician.

[0096] The bifunctional polypeptides described herein can be used alone or in combination with other therapies for the treatment of synucleinopathies, including, but not limited to, Parkinson's disease (PD), multiple system atrophy (MSA), Alzheimer's disease (AD), frontotemporal dementia (FTD) including frontotemporal dementia with parkinsonism on chromosome 17 (FTDP-17), Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), Lewy body disease, ganglioglioma and gangliocytoma, meningovascular angiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, and Hallervorden-Spatz disease, as well as for the treatment of traumatic injuries such as traumatic brain injury (TBI) or spinal cord injury (SCI). Such methods involve administering to a subject in need of treatment (e.g., a subject suffering from or at risk of developing a synucleinopathy) a bifunctional polypeptide comprising a first domain that includes an antigen-binding domain of an antibody or a fragment thereof that binds to an epitope of α-synuclein in a therapeutically effective amount, and a second domain that includes a programmable proteasome-targeting PEST motif. As will be understood by those skilled in the art, bifunctional polypeptides as described herein can be administered in a form that is necessary or useful for treating a subject, e.g., for treating synucleinopathies, such as by gene therapy using a gene encoding the bifunctional polypeptide.

[0097] Also provided are methods for treating or preventing protein aggregation caused by traumatic diseases that result in tau aggregation, such as Alzheimer's disease (AD), frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism on chromosome 17 (FTDP-17), Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease, ganglioglioma and gangliocytoma, meningovascular angiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, and Hallervorden-Spatz disease, but not limited thereto. Such methods involve administering to a subject in need of treatment or prevention (e.g., a subject suffering from or at risk of a tauopathy) a bifunctional intrabod y polypeptide comprising a first domain comprising an antigen-binding domain of an antibody or fragment thereof that binds to an epitope of tau, and in an amount therapeutically effective, and a second domain comprising a programmable proteasome-targeting PEST motif. As will be appreciated by those skilled in the art, the bifunctional polypeptides as described herein are administered to a subject in a form necessary or useful for the treatment of, for example, tauopathy, such as gene therapy with a gene encoding the bifunctional polypeptide.

[0098] The bifunctional polypeptides described herein can be used in the treatment, including prevention, of diseases associated with huntingtin, such as Huntington's disease. Such methods involve administering to a subject in need of treatment (e.g., a subject suffering from or at risk of Huntington's disease) a bifunctional polypeptide comprising a first domain comprising an antigen-binding domain of an antibody or fragment thereof that binds to an epitope of huntingtin, and in an amount therapeutically effective, and a second domain comprising a programmable proteasome-targeting PEST motif. As will be appreciated by those skilled in the art, the bifunctional polypeptides as described herein are administered to a subject in a form necessary or useful for the treatment of Huntington's disease, such as gene therapy with a gene encoding the bifunctional polypeptide.

[0099] Gene therapies and their uses are known in the art and, in some embodiments, may include administration of nucleic acids, such as DNA or RNA constructs, e.g., stabilized RNA constructs, or bifunctional polypeptides, in a form that enables their biological function in the cytoplasm of cells. In some embodiments, the nucleic acid may be administered in a vector, such as a gene therapy vector encoding a bifunctional polypeptide as described herein. In other embodiments, gene therapy useful for bifunctional polypeptides may be administered in a formulation or composition optimized for uptake or delivery to a particular cell type, e.g., through the use of cell-specific receptors or gene promoters. For example, in some embodiments, a gene promoter may be useful for targeting a bifunctional polypeptide as described herein to oligodendrocytes for the treatment of certain diseases. In some embodiments, the use of a particular gene promoter may enable targeted expression of a bifunctional polypeptide as described herein restricted to a particular cell type, such as neurons, astrocytes, and / or oligodendrocytes. In other embodiments, expression within a particular subpopulation of a cell type, such as dopaminergic neurons or glutamatergic neurons, may be accomplished through the use of a tyrosine hydroxylase promoter or a VGLUT1 promoter, respectively. In some embodiments, expression within a particular subpopulation of a cell type, such as excitatory neurons, may be accomplished, for example, through the use of a VGLUT1 promoter.

[0100] Also provided is a method of treating or preventing protein aggregation caused by a disease such as spinal cord injury (SCI) or traumatic brain injury (TBI), or tauopathy, comprising administration to a patient in need thereof of a gene therapy with a gene encoding an anti-tau intrabod as described herein in a therapeutically effective amount. In some embodiments, the methods described herein can treat or prevent protein aggregation resulting from spinal cord injury or traumatic brain injury, or this tauopathy, in a subject or patient as described herein. Administration of a composition comprising gene therapy with a gene encoding an anti-tau intrabody as described herein can be in a clinical setting as described herein, or in an alternative setting deemed appropriate by a clinician or practitioner. Further embodiments regarding the administration of such anti-tau intrabodies are described elsewhere herein. In some embodiments, such compositions comprising gene therapy with a gene encoding an anti-tau intrabody as described herein may be combined with other therapies or treatments for the treatment of tauopathy or TBI or spinal cord injury (SCI) in a patient.

[0101] The term "subject" refers to an animal or human, or one or more cells derived from an animal or human. Preferably, the subject is human. Additionally, the subject can include non-human primates. In some aspects, provided herein is a method of treating or preventing protein aggregation caused by a disease such as Huntington's disease or TBI or SCI, comprising administering a therapeutically effective amount of an anti-huntingtin intrabody as described herein to a patient in need of treatment or prevention.

[0102] The methods disclosed herein can treat or prevent protein aggregation resulting from spinal cord injury or traumatic brain injury, or Huntington's disease, in a subject or patient as described herein. Administration of a composition comprising an anti-huntingtin intrabody as described herein can be in a clinical setting as described herein, or in an alternative setting deemed appropriate by a clinician or practitioner. Further embodiments regarding the administration of such anti-huntingtin intrabodies are described elsewhere herein. In some embodiments, such compositions comprising an anti-huntingtin intrabody as described herein may be combined with other therapies or treatments for the treatment of Huntington's disease or related neurodegenerative or neurotraumatic conditions in a patient. Other pharmaceutical treatments may be used if considered appropriate by the clinician. Unless otherwise specified herein, the methods described herein may be performed according to the procedures exemplified herein or according to methods routinely practiced in the art. The following sections provide further guidance for performing the methods described herein.

[0103] Pharmaceutical composition As described herein, a bifunctional polypeptide as described herein can be formulated as a pharmaceutical composition, such as a gene therapy agent encoding a bifunctional polypeptide suitable for administration to a subject for treating a disorder as described herein. Typically, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. Pharmaceutical formulations are well established and known in the art. The pharmaceutical compositions described herein can exist in a variety of forms. These include, for example, liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories, among other liquid, semi-solid, and solid dosage forms. The form may depend on the intended mode of administration and therapeutic use. Typically, the compositions for the agents described herein exist in the form of injectable or infusion solutions.

[0104] In one embodiment, the gene therapy agent encoding the bifunctional polypeptide described herein is formulated using excipient materials such as sodium citrate, dibasic sodium phosphate heptahydrate, monobasic sodium phosphate, Tween-80, and a stabilizer. For example, the gene therapy can be provided in a buffer solution at an appropriate concentration and stored at 2 - 8°C. In some other embodiments, the pH of the composition is from about 5.5 to about 7.5 (such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5).

[0105] The pharmaceutical composition may also include an agent that reduces aggregation of the bifunctional polypeptide when formulated. Examples of aggregation reducers include one or more amino acids selected from methionine, arginine, lysine, aspartic acid, glycine, and glutamic acid. The pharmaceutical composition may also include a sugar (such as sucrose, trehalose, mannitol, sorbitol, or xylitol) and / or an isotonicity modifier (such as sodium chloride, mannitol, or sorbitol) and / or a surfactant (such as polysorbate-20 or polysorbate-80). Such a composition can be administered by a parenteral mode (such as intravenous, subcutaneous, intraperitoneal, or intramuscular injection). In one particular embodiment, the bifunctional polypeptide composition is administered subcutaneously. In one embodiment, the bifunctional polypeptide composition is administered intravenously. As used herein, the terms "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0106] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure that is stable at high concentrations and suitable for storage. Sterile injectable solutions can be prepared, if desired, by incorporating the required amount of the agent described herein in a suitable solvent, together with one or a combination of the components listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the agent described herein into a sterile vehicle containing a basic dispersion medium and the other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and lyophilization that yield any additional desired components from the pre-sterilized filtered solution in addition to the powder of the agent described herein. The appropriate fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and also by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition agents that delay absorption, such as monostearates and gelatin.

[0107] In certain embodiments, a composition or gene therapy agent encoding a bifunctional polypeptide as described herein may be prepared using a controlled release formulation that includes an implant, and a carrier that protects the compound, such as a microencapsulated delivery system, from rapid release. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, etc. can be used. Many of the preparation methods for such formulations are patented or generally known. In some embodiments, a composition comprising a gene therapy agent encoding a bifunctional polypeptide is formulated in sterile distilled water or phosphate buffered saline. The pH of the pharmaceutical formulation can be from about 5.5 to about 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5).

[0108] Administration of the Polypeptide A composition comprising a gene therapy agent encoding a polypeptide (e.g., a bifunctional polypeptide) as described herein can be administered to a subject, e.g., a subject in need of the composition, e.g., a human or animal subject, by a variety of methods. For many applications, the route of administration is one of intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), or intramuscular injection. Other modes of parenteral administration may also be used. Examples of such modes include intraarterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intratracheal, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injections. The route and / or mode of administration of the bifunctional polypeptide can also be adjusted for an individual case, e.g., by monitoring the subject. A composition comprising a gene therapy agent encoding a bifunctional polypeptide can be administered as a fixed dose or as a mg / kg dose. The dose can also be selected to reduce or avoid the production of antibodies against the bifunctional polypeptide. The dosing regimen is adjusted to provide a desired response, e.g., a therapeutic response or a combined therapeutic effect. Generally, the dose of the bifunctional polypeptide (and optionally, a second agent) can be used to provide the subject with a biologically available amount of the agent.

[0109] A dosage unit form or "fixed dose" as used herein refers to a physically discrete unit suitable as a unit dose for the subject to be treated, each unit being calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in combination with other agents, and containing a predetermined quantity of the bifunctional polypeptide. Single or multiple dosages may be administered. Alternatively, or in addition, a composition comprising a gene therapy agent encoding a bifunctional polypeptide may be administered via continuous infusion. A composition comprising a gene therapy agent encoding a dosage of a bifunctional polypeptide can be administered in a single dose or multiple doses, e.g., at least 2 doses, 3 doses, 5 doses, 10 doses, or more, e.g., once or twice daily, or about 1 to 4 times per week, or e.g., weekly, bi-weekly (every 2 weeks), every 3 weeks, monthly, e.g., for a period of about 1 to 12 weeks, e.g., 2 to 8 weeks, e.g., about 3 to 7 weeks, and e.g., about 4, 5, or 6 weeks (course of treatment) at regular intervals sufficient to cover. Factors that can affect the dosage and timing required to effectively treat a subject include, for example, the stage or severity of the disease or disorder, the formulation, the route of delivery, previous treatments, the general health and / or age of the subject, and other diseases present. Further, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or a series of treatments.

[0110] If a subject is at risk of developing a disorder described herein, the bifunctional polypeptide can be administered, e.g., as a prophylactic measure, prior to the full onset of the disorder. The duration of such prophylactic treatment can be a single dosing of the composition or the treatment can be continued (e.g., multiple dosing). For example, a subject at risk of a disorder, or a subject having a predisposition to a disorder, can be treated with a composition as described herein for days, weeks, months, or even years to prevent the disorder from occurring or being triggered. A composition comprising a gene therapy agent encoding a bifunctional polypeptide can be administered to a patient in need thereof (e.g., a patient having or at risk of having a protein aggregation disease such as synucleinopathy, tauopathy, or Huntington's disease) alone or in combination with other therapeutic proteins (e.g., antibodies, intrabodies, polypeptides) useful for treating synucleinopathy, tauopathy, or Huntington's disease (i.e., by co - administration or sequential administration). In one embodiment, the additional therapeutic protein is included in the pharmaceutical composition described herein. Examples of therapeutic proteins that can be used to treat a subject include, but are not limited to, therapeutic proteins that target β - amyloid, α - synuclein, huntingtin, TDP - 43, and / or SOD - 1.

[0111] The composition can be administered to a patient in need thereof (e.g., a patient having or at risk of having a protein aggregation disease such as synucleinopathy, tauopathy, or Huntington's disease) in combination with other neuroprotective agents useful for treating protein aggregation diseases such as synucleinopathy or tauopathy (i.e., by co - administration or sequential administration). In one embodiment, the additional agent is included in the pharmaceutical composition described herein. Examples of neuroprotective agents include, but are not limited to, acetylcholinesterase inhibitors, glutamate receptor antagonists, kinase inhibitors, HDAC inhibitors, anti - inflammatory agents, divalproex sodium, dopamine or dopamine receptor agonists, or any combination thereof.

[0112] In some embodiments, the composition comprising a gene therapy agent encoding a bifunctional polypeptide described herein can be used in a method designed to express the bifunctional polypeptide intracellularly so as to bind to intracellular α - synuclein, tau, or huntingtin. Such a method includes delivering to the cell the bifunctional polypeptide, which can be present in any form used by those skilled in the art, e.g., as a protein, a translated RNA molecule, or a transcribed and translated DNA vector. When a polynucleotide molecule encoding a bifunctional polypeptide is used, the polynucleotide can be recombinantly engineered to be various host vector systems that can be introduced in vivo such that it is taken up by cells and induces transcription of the bifunctional polypeptide molecule. Such vectors can remain episomal or be chromosomally integrated as long as they can be expressed to produce the desired polypeptide. Such vectors can be constructed by well-known and standard recombinant DNA technology methods in the art. Vectors encoding the domain intrabodies of interest can be plasmids, viruses, or others known in the art for use in replication and expression in mammalian cells.

[0113] A variety of viral and non-viral vectors for delivery of polynucleotides encoding the bifunctional polypeptides described herein are known in the art and can be used in the production of the products and in the practice of the methods described herein. Examples of vectors include, but are not limited to, eukaryotic cell expression vectors including viral expression vectors such as those derived from the classes of retroviruses, adenoviruses, or adeno-associated viruses.

[0114] Examples of suitable viral vectors include retrovirus-based vectors (e.g., lentivirus), adenovirus, adeno-associated virus (AAV), herpes vector, and vaccinia vector. In some embodiments, the structure of the vector may be modified as necessary, for example, to optimize the expression of a recombinant polypeptide containing an expression control element (e.g., a promoter or enhancer sequence), or to achieve the desired cellular level. In some embodiments, the expression of a programmable PEST degron sequence as described herein can be accomplished using a strong promoter that results in high-speed gene transcription in cells. Various vector systems are known to those skilled in the art, and the compositions described herein can be transferred into cells, for example, using liposomes, microparticles, microcapsules, encapsulation in recombinant cells, construction of nucleic acids as part of a retrovirus, adenovirus, adeno-associated virus or other vector, DNA injection, electroporation, calcium phosphate-mediated transfection, etc.

[0115] Therapeutic devices and kits A pharmaceutical composition containing a gene therapy agent encoding a bifunctional polypeptide described herein can be administered together with a medical device. The device is designed to have features such as portability, storage at room temperature, and ease of use so that it can be used in emergencies by an immature subject or by on-site emergency responders and can be removed from medical facilities and other medical equipment. The device may include, for example, one or more housings for storing a pharmaceutical preparation containing a gene therapy agent encoding a bifunctional polypeptide, and can be set to deliver one or more unit doses of an antibody. The device can further be set to administer a second agent, e.g., a neuroprotective agent, as a single pharmaceutical composition that also contains a gene therapy agent encoding a bifunctional polypeptide, or as two separate pharmaceutical compositions.

[0116] A gene therapy agent encoding a bifunctional polypeptide can be provided in a kit. In one embodiment, the kit contains (a) a container holding a composition comprising a gene therapy agent encoding a bifunctional polypeptide as described herein, and optionally (b) informational materials. The informational materials can be explanatory, instructional, marketing, or other materials regarding the methods described herein and / or the use of the agent for therapeutic benefit. In certain embodiments, the kit also includes a second agent for treating a disorder described herein. For example, the kit includes a first container holding a composition comprising a gene therapy agent encoding a bifunctional polypeptide and a second container holding the second agent. The informational materials of the kit are not limited in their format. In one embodiment, the informational materials can include information regarding the production of the compound, the molecular weight of the compound, concentration, expiration date, batch, or production site information. In one embodiment, the informational materials relate to a method of treating a subject having or at risk of having a protein aggregation disorder such as synucleinopathy, tauopathy, or Huntington's disease described herein by administering a gene therapy agent encoding a bifunctional polypeptide in, for example, an appropriate dosage, dosage form, or mode of administration (e.g., the dosages, dosage forms, or modes of administration described herein). The information can be provided in various formats including printed text, computer-readable materials, video recordings, or audio recordings, or information providing a link or address to a substantial material, e.g., on the Internet.

[0117] In addition to the gene therapy agent encoding a bifunctional polypeptide, the composition in the kit can include other components such as a solvent or buffer, stabilizer, or preservative. The gene therapy agent encoding a bifunctional polypeptide can be provided in any substantially pure and / or sterile form, e.g., liquid, dry, or lyophilized form. If the agent is provided in a liquid solution, the liquid solution is an aqueous solution. If the agent is provided as a lyophilized product, the lyophilized powder is generally reconstituted by the addition of an appropriate solvent. A solvent, e.g., sterile water or buffer (e.g., PBS), may be provided in the kit.

[0118] The kit can include one or more containers for a composition or a composition containing an agent. In some embodiments, the kit contains separate containers, partitions or compartments for the composition and the information material. For example, the composition can be contained in a bottle, vial, or syringe, and the information material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single undivided container. For example, the composition is contained in a bottle, vial or syringe with the information material affixed in the form of a label. In some embodiments, the kit includes a plurality (e.g., a set) of individual containers, each containing one or more unit dosage forms of the agent (e.g., the dosage forms described herein). The container can include a combined unit dosage, for example, a unit containing both a gene therapy agent encoding a bifunctional polypeptide and a second agent in a desired ratio. For example, the kit includes a plurality of syringes, ampoules, foil packets, blister packs, or medical devices, for example, each containing a single combined unit dose. The containers of the kit can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-blocking. The kit may include a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device can be provided with one or both of the agents pre-loaded, or can be empty but suitable for loading.

[0119] Definitions When a range of values is provided, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of the range to one tenth of the unit of the lower limit, and any other recited or intervening value in the recited range, is to be understood as provided herein. These smaller ranges of upper and lower limits may be independently included in the smaller ranges and are provided herein subject to any specifically excluded limits in the recited range. When the recited range includes one or both of the limits, ranges excluding either or both of the included limits are also provided.

[0120] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the embodiments herein, but the preferred methods and materials are described herein. All publications mentioned herein are hereby incorporated by reference herein for the purpose of disclosing and describing the methods and / or materials related to which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the present disclosure has a right to antedate such publications by virtue of prior disclosure. Further, the provided publication dates may be different from the actual publication dates, which may need to be independently verified. Unless otherwise noted, all technical and scientific terms used herein have meanings as commonly understood by one of ordinary skill in the relevant art. Specific terms are defined particularly as follows.

[0121] As used in this specification and the appended claims, the singular forms "a", "an", and "the" are to be construed to include both the singular and the plural unless specifically stated otherwise in the context in which they are used to describe a particular embodiment (in particular, in a particular context of the appended claims). Thus, for example, "an active agent" refers to not only a single active agent but also a combination of two or more different active agents, and "a dosage form" refers to a combination of dosage forms as well as a single dosage form. In some embodiments, the term "or" is used to mean "and / or" as used in this specification including the claims, unless explicitly stated otherwise to refer only to alternatives or when the alternatives are mutually exclusive.

[0122] In some embodiments, numerical values representing amounts of ingredients, properties such as molecular weight, reaction conditions, etc. used to describe and claim certain specific embodiments described herein should be understood to be modified in some cases by the term "about". In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean value with respect to the device or method used to determine that value. In some embodiments, the numerical parameters described in the written description and the appended claims may be approximate values that can vary depending on the desired properties required to be obtained by a particular embodiment. In some embodiments, numerical parameters should be interpreted in view of the reported number of significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth broad ranges of some embodiments described herein are approximations, the numerical values set forth in the specific examples are reported as accurately as practicable. The numerical values presented in some embodiments described herein may contain certain errors that necessarily result from the standard deviation found in each of their experimental measurements. The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. In some embodiments, "about" refers to the designated value + / - 10%.

[0123] The terms "comprising," "having," and "including" are conjunctive verbs without limitation. Any one or more forms or tenses of these verbs, such as "comprise (singular form)," "comprising," "have (singular form)," "having," "include (singular form)," and "including," are also without limitation. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps, but may also encompass other unlisted steps. Similarly, any composition or device that "comprises," "has," or "includes" one or more features is not limited to possessing only those one or more features, but may also encompass other unlisted features.

[0124] As used herein, "protein" refers to a molecule consisting of amino acid residues linked by peptide bonds. In the context of the present disclosure, the protein is one involved in protein aggregation diseases, such as α-synuclein, tau, and huntingtin. As described herein, the protein is a target of an intrabody as described herein, which is degraded to a desired level due to the addition of a human programmable PEST sequence. As defined herein, the term "rate of delivery" of a protein to a proteasome refers to the rate at which the protein of interest is degraded over time in a cell in the presence of a recombinant polypeptide of the present disclosure containing a PEST domain as compared to a control (e.g., an empty vector control).

[0125] Throughout this disclosure, it will be understood that amino acids are referred to according to one-letter or three-letter codes. The one-letter and three-letter amino acid codes are provided as follows: A = Ala = alanine; C = Cys = cysteine; D = Asp = aspartic acid; E = Glu = glutamic acid; F = Phe = phenylalanine; G = Gly = glycine; H = His = histidine; I = Ile = isoleucine; K = Lys = lysine; L = Leu = leucine; M = Met = methionine; N = Asn = asparagine; P = Pro = proline; Q = Gln = glutamine; R = Arg = arginine; S = Ser = serine; T = Thr = threonine; V = Val = valine; W = Trp = tryptophan; X = Xaa = unknown [non-standard - Unk]; and Y = Tyr = tyrosine.

[0126] As used herein, "α-synuclein" refers to human α-synuclein, a protein involved in a number of neurological diseases. As described herein, α-synuclein is a target of an intrabody as described herein, which is degraded to a desired level due to the addition of a human programmable PEST sequence. In some embodiments, the term "synuclein" generally may refer to proteins of the synuclein family, such as α-synuclein, β-synuclein, or γ-synuclein. For example, an anti-synuclein antibody can bind to any member of the synuclein family, while an anti-α-synuclein antibody binds only to α-synuclein.

[0127] As used herein, the term "antibody" includes intact immunoglobulins derived from natural or recombinant sources, as well as immunoreactive portions of intact immunoglobulins (i.e., "antigen-binding domains" or "antigen-binding portions"). The antibodies described herein can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), antibody fragments (e.g., Fv, Fab, Fab', and F(ab')2), and single-chain antibodies (scFv), single-domain VH or VL antibodies, chimeric antibodies, human antibodies, and humanized antibodies. Antibody fragments (e.g., Fv, Fab, Fab’, and F(ab’)2), such as antibody fragments of an anti-α-synuclein binding antibody, can be prepared by proteolytic digestion of intact antibodies (e.g., anti-α-synuclein antibody, anti-tau antibody, or anti-huntingtin antibody). For example, antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin. Other enzymes suitable for the preparation of antibody fragments are known in the art. Papain digestion of whole antibody produces F(ab)2 or Fab fragments, pepsin digestion of whole antibody yields F(ab’)2 or Fab’, and plasmin digestion of whole antibody gives Fab fragments.

[0128] Alternatively, antibody fragments, such as antibody fragments of an anti-α-synuclein binding antibody, can be produced recombinantly. For example, a nucleic acid encoding the desired antibody fragment can be constructed, introduced into an expression vector, and expressed in a suitable host cell. For example, antibody fragments can be expressed in E. coli and secreted from E. coli, thus enabling the easy production of large amounts of these fragments. According to another approach, antibody fragments can be isolated directly from the recombinant host cell culture. As used herein, the term “epitope” refers to a specific amino acid sequence, modified amino acid sequence, or secondary or tertiary structure of a protein that is specifically recognized by an antibody. The terms “specifically recognizing,” “specifically recognize (singular),” and any grammatical variations thereof mean that an antibody or its antigen-binding molecule can specifically interact with and / or bind to at least two, at least three, or at least four amino acids of an epitope, e.g., an α-synuclein, tau, or huntingtin epitope. Such binding can be exemplified by the specificity of the “lock and key principle.” Thus, a particular motif in the amino acid sequence of the antigen-binding domain of an α-synuclein, tau, or huntingtin antibody, or its antigen-binding molecule, and an epitope bind to each other as a result of their primary, secondary, or tertiary structures, as well as as a result of secondary modifications of those structures.

[0129] As used herein, "intrabod" refers to an antibody fragment or antigen-binding domain that is active intracellularly. Intracellular antibody fragments can be, for example, single-chain variable fragments (scFv) or single-domain antibodies (also known as nanobodies; antibody fragments consisting of a single monomeric variable antibody domain). Intrabods act as neutralizing agents by direct binding to an intracellular target antigen, thereby altering protein folding, protein-protein, protein-DNA, protein-RNA interactions, and protein modifications intracellularly. In some embodiments, intrabods also include camelid nanobodies, which are small heavy-chain-only antibody fragments (VHHs) derived from naturally occurring heavy-chain-only antibodies produced in alpacas, llamas, camels, and guanacos.

[0130] As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, usually a protein, capable of inducing an immune response in a subject. For example, α-synuclein, tau, or huntingtin protein can be an antigen. The term also refers to an immunologically active protein in the sense that when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), it is capable of inducing a humoral and / or cell-type immune response against that protein. As used herein, "co-administration" refers to the simultaneous administration of one or more drugs with another drug. In other embodiments, both drugs are administered simultaneously. As described elsewhere herein, co-administration can also refer to the administration of one drug, or any particular period of administration of both drugs. For example, as described herein, a drug may be administered hours or days before the administration of another drug and still be considered co-administered. In some embodiments, co-administration can refer to the administration of one drug at any time such that both drugs are present in the patient's body simultaneously. In some embodiments, one drug may be administered before or after the other as long as both are present in the patient for a sufficient time for the patient to receive the intended clinical or pharmacological benefit.

[0131] Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). Not all residue positions within a protein will tolerate "conservative" substitutions in other contexts. For example, when an amino acid residue is essential for the function of a protein, even a conservative substitution in other contexts may disrupt its activity. For example, the specific binding of an antibody to a target epitope can be disrupted by a conservative mutation at the target epitope.

[0132] As described herein, substitution mutations that may be present in the PEST degron relative to SEQ ID NO:1 as described herein for targeting a protein to the proteasome to achieve increased degradation of the protein can be a mutation of a proline (P) residue to an alanine (A) residue, such as P426A / P427A (i.e., mutation of two consecutive P residues to two consecutive A residues), a mutation of an aspartic acid (D) residue to an A residue, such as D433A, a mutation of a serine (S) residue to an A residue, such as S445A, and / or a mutation of a lysine (K) residue to an A residue, such as K448A. In other embodiments, mutations that may be present in the PEST degron relative to SEQ ID NO:1 as described herein for targeting a protein to the proteasome to achieve reduced degradation of the protein can be a mutation of a P residue to an A residue, such as P438A, a mutation of a glutamic acid (E) residue to an A residue, such as E444A, a mutation of an S residue to an A residue, such as S440A, and / or a mutation of a threonine (T) residue to an A residue, such as T436A. In some embodiments, the mutation is one of those shown in FIGS. 15A - 15B.

[0133] In some embodiments, conservative amino acid substitutions, e.g., substituting one acidic or basic amino acid for another, can often be made without affecting the biological activity of the recombinant polypeptides as described herein. Minor variations in the sequences of this nature can be made in any of the peptides disclosed herein, provided that these changes do not substantially (e.g., by 15% or more) alter the desired activity of the protein.

[0134] As used herein, "degron" refers to a part of a protein that is important in the regulation of proteolysis rate. Known degrons include short amino acid sequences, structural motifs, and exposed amino acids (e.g., lysine or arginine) located anywhere in a protein. In some embodiments, some proteins contain multiple degrons. As used herein, PEST degron refers to a sequence useful for targeting a specific protein to the proteasome for degradation. As described herein, useful PEST degrons may be of mouse or human origin and may have the consensus sequence described herein as SEQ ID NO: 1.

[0135] As used herein, "diabody" refers to a non-covalent dimer of single-chain Fv (scFv) fragments consisting of heavy-chain variable (VH) and light-chain variable (VL) regions linked by a small peptide linker. In some embodiments, the diabody is two intrabodies linked or joined using a linker, e.g., Vh--linker--Vh. In other embodiments, the diabody is a single-chain (Fv)2 in which two scFv fragments are covalently bonded to each other.

[0136] The pharmaceutical compositions described herein that contain a gene therapy agent encoding a bifunctional polypeptide may contain a "therapeutically effective amount" of a bifunctional polypeptide as described herein. The terms "therapeutically effective amount," "pharmacologically effective dosage," "pharmacologically effective amount," or simply "effective amount" may be used interchangeably and refer to the amount of an agent effective to produce the intended pharmacological, therapeutic, or prophylactic result, e.g., the amount necessary to achieve a desired level of a protein such as α-synuclein, tau, or huntingtin. A pharmacologically effective amount results in improvement of one or more symptoms of a disorder, or prevents progression of a disorder, or causes regression of a disorder, or prevents a disorder. Such effective amount can be determined based on the effect of the administered agent, or the combined effect of the agents if more than one agent is used. The therapeutically effective amount of an agent also varies according to factors such as the stage, condition, age, sex, and weight of the individual's disease, and the ability of the compound to elicit a desired response in the individual, e.g., improvement of at least one disorder parameter or improvement of at least one symptom of a disorder. The therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. Generally, this amount is sufficient to clearly target the protein target to the proteasome for degradation. When administered to a subject, generally a dosage is used that achieves the target tissue concentration that has been shown to achieve a desired level of proteolysis (i.e., a desired level of the protein being targeted / sent to the proteasome), e.g., in neurons. In several examples, an "effective amount" is one that treats (including prevents) one or more symptoms and / or the underlying cause of any protein aggregation disorder or disease. In one example, an effective amount is a therapeutically effective amount. In one example, an effective amount is an amount that prevents the onset of one or more signs or symptoms of a particular disease or condition.

[0137] As used herein, "epitope" refers to an antigenic determinant. An epitope is a specific chemical group or peptide sequence on a molecule that is antigenic such that it elicits a specific immune response. For example, an epitope is a region of an antigen to which B cells and / or T cells respond. Epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of a protein. As used herein, "exogenous sequence" refers to a nucleic acid sequence that is derived from outside of a host cell. The exogenous sequence may be a DNA sequence, an RNA sequence, or a combination thereof. As would be understood by one of skill in the art, any type of nucleic acid available in the art can be used. Such nucleic acid sequences can be obtained from a species different from the species of the cell into which it is delivered, such as a mouse, or from the same species. In some embodiments, the exogenous nucleic acid sequence can encode a PEST degron sequence for targeting a desired protein to the proteasome for degradation as described herein, suitable for administration to a subject or patient. Such recombinant polypeptides can be administered to a subject or patient to treat or prevent protein aggregation diseases or protein accumulation in specific cells, tissues, organs, etc.

[0138] As used herein, "gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene transfer and can include targeting, binding, uptake, transport, localization, replicon integration, and expression. In some embodiments, gene delivery can refer to the introduction of the encoded product of a gene, i.e., a polypeptide or protein, such as a bifunctional polypeptide described herein. As used herein, "gene transfer" refers to the introduction of an exogenous polynucleotide into a cell that can include targeting, binding, uptake, transport, localization, and replicon integration, but does not imply subsequent expression of the gene, as opposed to subsequent expression of the gene. As used herein, "gene expression" or "expression" refers to the processes of gene transcription, translation, and post-translational modification.

[0139] "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the composition in which it is contained. When the term "pharmaceutically acceptable" is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient meets the required criteria of toxicological and manufacturing testing, or that the carrier or excipient is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration. "Pharmacologically active" (or simply "active") as seen in "pharmacologically active" (or simply "active") derivatives or analogs refers to derivatives or analogs that have the same type of pharmacological activity as the parent compound and to an approximately equal degree. The term "pharmaceutically acceptable salts" includes, for example, acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be obtained from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0140] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible. The compositions can include pharmaceutically acceptable salts, for example, acid addition salts or base addition salts.

[0141] As used herein, "programmable" as in "programmable PEST" or "programmable PEST degron" or "programmable proteasome targeting PEST motif" refers to a PEST degron that can be modified or altered in such a way as to introduce certain mutations (i.e., amino acid substitutions described elsewhere herein) that can increase or decrease the degradation of a protein (such as α-synuclein, tau, huntingtin, etc.) that is the target of an antigen-binding domain fused to the PEST degron, as compared to an unmodified or unaltered version of the same PEST degron. In some embodiments, the PEST motif can have various mutations that increase the level of degradation of a protein (such as α-synuclein, tau, or huntingtin) in cells from a baseline level, for example, from a low level reduction from the baseline (e.g., 5%) to a high level increase from the baseline (e.g., 100%). This increased degradation can be confirmed by comparison to a control such as an empty vector control.

[0142] As used herein, "increase in degradation" refers to an increase or enhancement in the targeting of a protein for degradation to the proteasome or for transport or delivery to the proteasome, by the addition of a PEST sequence to the protein as described herein. Similarly, "decrease in degradation" or "reduction in degradation" refers to a reduction or decrease in the targeting of a protein for degradation to the proteasome or for transport or delivery to the proteasome, by the addition of a PEST sequence to the protein as described herein. Mutations in the PEST consensus sequence that can be useful in achieving an increase or decrease in the degradation of a protein, such as α-synuclein, tau, or huntingtin, are described herein. The degradation of a protein, such as huntingtin, is described herein.

[0143] When comparing levels of proteolysis as described herein, an increase or decrease in the proteolysis of a target protein can be compared to an empty vector, wild-type hPEST, hPEST scramble, or an antigen control (B8-hPEST). As used herein, "scrambled control PEST" or "Scr" refers to a randomized polypeptide having the same number of amino acids as the programmable PEST, but which does not target the protein to the proteasome. This experimental proteolysis control allows for quantification of how efficiently a particular PEST degron degrades a target protein, such as alpha-synuclein, tau, huntingtin. For example, the PEST degrons described herein can increase protein proteolysis by a certain percentage compared to the scrambled control PEST, or the PEST degrons described herein can decrease protein proteolysis by a certain percentage compared to the scrambled control PEST. In some embodiments, a particular PEST degron can be compared to an empty vector control, referred to herein as "EV-CON" or "EV". The empty vector control, as used herein, refers to an experimental control for comparing or quantifying the level of proteolysis in which the vector used for transfection of cells with a construct encoding a PEST degron, or an intrabody fused to a PEST degron, lacks the sequence encoding the PEST degron or intrabody. In some embodiments, a particular PEST degron may be compared to an unmodified or unchanged version of the same PEST degron (i.e., the wild-type PEST sequence).

[0144] As used herein, "reducing" refers to decreasing or lessening, such as reducing cytotoxicity after spinal cord injury (SCI), or reducing the amount or concentration of a protein as described herein, such as α-synuclein, tau, or huntingtin. In some embodiments, administration of a bifunctional polypeptide as described herein can result in "reduction" or alleviation of protein aggregation or related symptoms in a patient as compared to a patient not administered such bifunctional polypeptide. "Reducing" can also refer to reduction of disease symptoms, alone or co-administered with another drug, as a result of a treatment as described herein.

[0145] As used herein, "subject" or "individual" or "patient" refers to any patient for whom therapy is desirable, and generally refers to the recipient of therapy. "Subject" or "patient" refers to any animal classified as a mammal, such as a human and non-human mammals other than humans. Examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Unless otherwise noted, the terms "patient" or "subject" are used interchangeably herein. In some embodiments, a subject suitable for therapeutic application can be a primate, such as a human and non-human primates. As used herein, "tau" refers to human tau, a protein involved in a number of neurological diseases. As described herein, tau is an intrabodily target as described herein, which is degraded to a desired level due to the addition of a human programmable PEST sequence.

[0146] As used herein, administration of a polynucleotide or vector to a host cell or subject refers to introduction into the cell or subject via any routine method. This includes "transduction", "transfection", "transformation", or "transducing", as are well known in the art. All of these terms refer to standard processes for introduction of an exogenous polynucleotide into a host cell, resulting in expression of the polynucleotide in the cell, such as a transgene, and including the use of plasmids and / or recombinant viruses for introduction of the exogenous polynucleotide into the host cell. Transduction, transfection, or transformation of a polynucleotide in a cell can be determined by methods well known to those skilled in the art, including, but not limited to, measurement of protein expression (including steady state levels) by ELISA, flow cytometry, and Western blot, assays such as Northern blot, Southern blot, reporter function (Luc) assay, and / or DNA and RNA measurement by gel shift mobility assay. Methods for introduction of exogenous polynucleotides include well-known techniques such as bacterial and / or viral infection or transfection, lipofection, transformation, and electroporation, as well as other non-viral gene delivery techniques such as introduction of stabilized RNA molecules. The introduced polynucleotide can be maintained stably or transiently in the host cell.

[0147] As used herein, "transcription regulatory sequence" or "TRS" generally includes at least one transcription promoter and may also include one or more enhancers and / or terminators of transcription. "Operably linked" refers to the arrangement of two or more components where the components are in a relationship that allows them to function cooperatively. By way of example, a transcription regulatory sequence or promoter is operably linked to a coding sequence when the TRS or promoter facilitates transcription of the coding sequence. A TRS that is operably linked is generally cis-linked to the coding sequence, but is not necessarily directly adjacent to it.

[0148] As used herein, the terms "treating" and "treatment" or "alleviating" refer to reducing or lessening the severity and / or frequency of a symptom, eliminating the symptom and / or underlying cause, and improving or repairing an injury. In certain embodiments, the terms "treating" and "treatment" as used herein refer to preventing the occurrence of a symptom. In other embodiments, the terms "treating" and "treatment" as used herein refer to preventing the underlying cause of a symptom associated with a disease or condition such as spinal cord injury (SCI). The phrase "administering to a patient" refers to the process of introducing a composition or dosage form to a patient via an introduction means recognized in the art. "Treating" or "alleviating" also encompasses administering a compound or agent to a subject to prevent or delay the onset of symptoms, complications, or biochemical markers of a disease (e.g., SCI), alleviate the symptoms, or stop or inhibit further development of a disease, condition, or disorder. Subjects in need of treatment include subjects already suffering from a disease or condition, as well as humans at risk of developing a disease or condition. Treatment can be prophylactic (to prevent or delay the onset of a disease or condition or to prevent its clinical or asymptomatic manifestations) or therapeutic suppression, or alleviation of symptoms following the manifestations of a disease or condition.

[0149] A "vector" is a nucleic acid, with or without a carrier that can be introduced into a cell. A vector capable of inducing the expression of a gene encoding one or more polypeptides is referred to as an "expression vector". Examples of suitable vectors include, for example, viral vectors, plasmid vectors, liposomes, and other gene delivery vehicles.

[0150] All of the methods described in this specification can be implemented in any suitable order, unless otherwise specified herein or unless the context clearly dictates otherwise in other circumstances. All examples provided with respect to a particular embodiment herein, or the use of exemplary language (e.g., "such as"), are merely intended to clarify the embodiment more clearly and are not intended to limit the scope of the claimed embodiments in other circumstances. No language in the specification should be construed as indicating any non-claimed element essential to the implementation of the embodiments.

[0151] The grouping of alternative elements or embodiments disclosed herein should not be construed as a limitation. Each member of a group can be referred to individually or in any combination with members of the group or other elements found herein and can be claimed. One or more members of a group can be included in or deleted from the group for reasons of convenience or patentability. Although the embodiments have been described in detail, it is clear that modifications, variations, and equivalent embodiments are possible without departing from the scope defined in the appended claims. Furthermore, it should be understood that all examples described herein are provided as non-limiting examples.

[0152]

Table 4

Example

[0153] Examples of the embodiments described in this specification are provided in the following examples. The following examples are presented for illustrative purposes only and to assist those skilled in the art. The examples are not intended to limit the scope of the present embodiment in any way.

[0154] (Example 1) - Identification and research overview of an anti-α-synuclein intrabody that most efficiently targets synuclein to the proteasome for degradation. As described herein, a bifunctional proteasome-targeting intrabody has been developed that prevents protein misfolding and targets their bound cargo to the proteasome for degradation through fusion to the PEST degron (mPEST) from mouse ornithine decarboxylase. As described herein, the level of α-synuclein reduction can be controlled with the human PEST degron. This has been demonstrated to work in multiple systems. Without being bound by any theory, targeted degradation of α-synuclein protein using the cell's normal protein removal process reduces the amount of α-synuclein available for folding and thus may reduce the physiological effects of synucleinopathies. Targeted degradation of synuclein protein using the cell's normal protein removal process reduces the amount of misfolded synuclein available and reduces cytotoxicity due to synuclein-related neurodegenerative diseases or after SCI.

[0155] (Example 2) Humanization of a bifunctional anti-synuclein-PEST intrabody. The PEST degron was optimized for human use. To achieve this goal, the murine PEST degron (mPEST) was replaced with the human PEST (hPEST) degron. A comparison of murine and human PEST degrons derived from the ornithine decarboxylase (ODC) gene is shown in Table 3. The hPEST degrons were transferred into a GFP transcriptional reporter and their half-lives could be reduced to a level similar to that of the GFP-mPEST reporter. Fusion of the hPEST degrons to the anti-synuclein intrabodies induced synuclein to the proteasome for degradation with the same efficiency as the mPEST degrons. Therefore, the hPEST degron was cloned from human ornithine decarboxylase into the anti-α-synuclein intrabody VH14. VH14-hPEST reduced synuclein-GFP fluorescence to a level similar to that of VH14-mPEST in murine ST14A neural progenitor cells (Figure 2). α-Synuclein-GFP was co-transfected with either VH14-mPEST or VH14-hPEST. 72 hours after transfection, live cell imaging of the cells was performed.

[0156] Western blot analysis confirmed the results of live cell imaging such that VH14-hPEST reduced the steady-state protein level of human α-synuclein-GFP to a similar extent as VH14-mPEST. As shown in Figure 2, α-synuclein-GFP monomers in VH14-mPEST- and VH14-hPEST-transfected cells were reduced compared to the empty vector control. VH14-mPEST and VH14-hPEST also reduced the presence of high molecular weight species of α-synuclein-GFP observed in the empty vector control samples. Actin was probed as a loading control. The graph shows a densitometric analysis of the Western blot signals. Each bar represents the pan-synuclein / actin loading control expressed as a percentage of the empty vector control.

[0157] Patient-derived induced pluripotent stem cells (iPSCs) from patients with Parkinson's disease were established with an increased copy number variation in the SNCA gene (SNCA triplication (RUCDR; ND50040) encoding α-synuclein). Patients with this mutation develop autosomal dominant Parkinson's disease. Subsequently, optimization was performed with respect to a human iPSC three-dimensional (3D) brain organoid protocol developed by Sergiu Pasca and known in the art. Cultures using this protocol yield brain organoids that are spheres approximately 1-3 mm in diameter of human neurons consisting mainly of neurons but also including macroglia. This protocol was selected for its high reproducibility and its ability to produce all major cerebral cortical cell types, including CTIP2-positive neurons present in cortical layer V, and send their axons to deep brain structures such as the thalamus (corticothalamic neurons), striatum (corticostriatal neurons), pons (corticopontine neurons), tectum (corticotectal neurons), and spinal cord (corticospinal motor neurons, CSMN). Overexpression of endogenous synuclein was verified in 60-day-old cortical neurons of 3D organoids derived from human iPSCs with SNCA gene triplication (3X SNCA) compared to wild-type (WT) healthy controls by Western blotting (Figure 3). As shown in the figure, 3X-SNCA and WT iPSCs were differentiated into 3D forebrain organoids. After 60 days in vitro (DIV), the organoids were harvested for Western blotting. 20 μg of total protein (left 4 lanes) or 10 μg (lanes 5 and 6) was separated by gel electrophoresis and transferred to a PVDF membrane. Endogenous synuclein was detected using the MJFR1 anti-synuclein antibody (1:1000; Abcam #ab138501). GAPDH was used as a loading control (1:10,000; Abcam, #ab181602).

[0158] To evaluate the targeted degradation of α-synuclein in human neurons, WT and 3X-SNCA 3D cortical organoids were transduced with an inducible lentivirus expressing either anti-α-synuclein VH14 with a human PEST degron (VH14-hPEST) or VH14-hPEST with a scrambled PEST degron control (VH14-hPEST-Scr). Figure 4A shows the design of the Tet-On inducible anti-synuclein lentiviral construct. Briefly, the anti-synuclein mPEST and hPEST intrabodies were subcloned into pTet-O-Ngn2-puro (Addgene plasmid #52047). The Ngn2 insert was replaced with VH14-mPEST, VH14-hPEST, VH14-mPEST-scrambled control, VH14-hPEST-scrambled control, VHH-B8-mPEST, and VHH-B8-hPEST. The 5’ cloning site was EcoRI and the 3’ cloning site was XbaI. Thirty days after treatment, VH14-hPEST significantly reduced endogenous α-synuclein levels compared to the empty virus control and VH14-hPEST-Scr control (Figure 4B). Briefly, VH14-hPEST reduced the levels of endogenous human α-synuclein in 3X SNCA forebrain organoids. Immunofluorescence shows a reduction in α-synuclein (MJFR1; Abcam, Green) levels in lentivirus-transduced (n = 3) organoids carrying VH14-hPEST compared to organoids treated with an empty vector (EV) and VH14 fused to a scrambled (Scr) PEST. Densitometric quantification of the α-synuclein signal in the organoids confirms a statistically significant reduction of the target protein in both VH14-treated cohorts. (All ****p < 0.0001). These experiments represent a significant improvement over previous in - body drug screening models that rely on immortalized tumor - derived cell lines, which may contain genetic and metabolic abnormalities due to their mode of derivation. Furthermore, experiments with such cell lines are typically limited to a short time frame (2 - 4 days) due to their continuous growth. Cultures derived from human iPSCs overcome these limitations by more faithfully simulating human neurons and disease phenotypes.

[0159] (Example 3) - Development of control in - body for in - vivo studies. To control the effects of both protein overexpression and proteasomal degradation of the PEST - tagged in - body, a control single - domain in - body was generated against an antigen that is not expressed in any of the test systems. VHH - B8 is a well - characterized camelid nanobody that binds to botulinum neurotoxin and exhibits excellent intracellular solubility in the test system.

[0160] As expected, the bifunctional intrabody (B8-hPEST) targeting botulinum toxin (B8) with hPEST did not alter the removal of the steady-state protein level of squid DY-synuclein~GFP (DY-syn~GFP) (Figure 5). Briefly, ST14A neuron cells were co-transfected with either DY-syn~GFP and B8-hPEST or an empty vector control. 48 hours after transfection, live cell imaging of the cells was performed, followed by harvesting for Western blotting. Live cell imaging of DY-syn~GFP (scale bar 200 μm). Representative Western blotting was as described above. To determine the target association of the intrabodies with the proteasome for endogenous human α-synuclein and the hPEST degron, N77D-mPEST, N77D-hPEST, N77D-mPEST-scrambled control, and N77D-hPEST-scrambled control intrabodies were cloned into a tetracycline-inducible lentiviral vector with puromycin resistance to enable stable gene selection (Table 3).

Table 5

[0161] (Example 4) - Identification of candidate intrabodies targeting synuclein in humans and model organisms. The axolotl expresses three synuclein isoforms (DY-synuclein~GFP, FD-synuclein~GFP, and syn3~GFP) that have significant homology to human synuclein. The synuclein isoform most abundantly expressed in RS neurons is DY-synuclein, which is γ-synuclein and is approximately 70% identical and approximately 90% similar to the first 90 amino acids of human α-synuclein. Human α-synuclein and axolotl synuclein are highly conserved in their N-terminal domains (see the figure of the human α-synuclein protein in FIG. 6), and published studies have confirmed that they have similar biochemical properties (e.g., lipid membrane binding) and functional effects in neurons (e.g., synaptic vesicle transport). To identify optimal therapeutic targets for reducing human α-synuclein toxicity after SCI, a series of bifunctional anti-α-synuclein intrabodies were generated that were directed to each of the major regions of α-synuclein and fused to a proteasome targeting motif (FIG. 6). The positions of the proposed intrabody binding sites of VH14, VHH-4C, VHH-4C-N77D, and DB1 to the non-amyloid component (NAC) hydrophobic domain of α-synuclein, which tends to aggregate, have been shown to be important for misfolding.

[0162] The starry eel DY-synuclein~GFP, FD-synuclein~GFP, and syn3-synuclein~GFP were cloned into the mammalian pcDNA3.1 expression plasmid. After sequence verification of these plasmids, their expression in the ST14A neuronal cell line was verified (Figure 7). The figure shows the expression of starry eel synuclein. DY-syn~GFP, FD-syn~GFP, and Syn3~GFP were separately transfected into ST14A neurons. Forty-eight hours after transfection, live cell imaging of the cells was performed. In a subset of cells, DY-synuclein forms spots that show α-synuclein aggregation (insert in Figure 7). Since DY-synuclein is the major variant of starry eel synuclein, it was the initial focus in preliminary studies. Established intrabodies against human α-synuclein d5PEST, VH14PEST, NAC32PEST, syn2PEST, and syn87PEST were screened against starry eel DY-synuclein, but no significant turnover of starry eel DY-synuclein~GFP was observed in the culture system. Antibodies against antigenic proteins from different species sharing 75% sequence homology are generally predicted to cross-react. The lack of DY-synuclein degradation is likely due to the difference in sequence homology between human α-synuclein and starry eel DY-synuclein, as they only share 67% sequence homology with each other.

[0163] (Example 5) - Development of novel anti-synuclein nanobodies. To identify an intrabody that can cross-react with human α-synuclein and DY-synuclein of squid, VHH nanobodies were produced by the Hybribody service. Camelid single-domain nanobodies were screened against DY-synuclein. Camelids produce a unique type of immunoglobulin lacking a light chain and thus are called heavy-chain antibodies (HCAbs). Camelid HCAb shows a binding affinity similar to that of conventional antibodies for many antigens. However, unlike conventional antibodies, HCAb uses a single variable heavy chain (VHH) to bind to an epitope, eliminating the need for a hinge structure characteristic of single-chain antibody Fv fragments composed of both variable heavy and light chains. Camelid VHH nanobodies were selected because they have a broad antigen-binding repertoire and possess properties highly suitable for therapeutic research applications, such as their small size, high solubility, thermal stability, refolding ability, good tissue penetration in vivo, and ability to bind to unique epitopes. By this approach, an antibody named DB1 was identified (SEQ ID NO: 5): MAEVQLQASGGGFVQPGGSLRLSCAASGFTSWEDTMGWFRQAPGKEREFVSAISFDANDLSDTSVYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAVASFEILLYGESLHIYWGQGTQVTVSS

[0164] (Example 6) -Intrabody screening. Two additional anti-synuclein-VHH single-domain intrabodies, VHH-4C and VHH-4C-N77D (referred to herein as N77D), were screened. These intrabodies were derived from a phagemid synuclein alpaca VHH immune library (Addgene #1000000071) immunized as used above, but were isolated via identification of functional ligand binding by Tat-based recognition of the associated protein. N77D was developed by computational affinity maturation and differs from its parent by one amino acid (N77D). N77D showed enhanced nanomolar affinity for α-synuclein compared to the micromolar affinity of VHH-4C, through an increase in the association rate verified by surface plasmon resonance (SPR) experiments. N77D-mPEST reduced the steady-state levels of leech DY-synuclein~GFP (Figure 8A) and human α-synuclein~GFP (Figure 8B) by approximately 40%.

[0165] In this synuclein overexpression system, VHH-4C-PEST increased the soluble monomer level of DY-synuclein compared to the empty vector control (Figure 8A). VHH-4C-PEST also increased the level of α-synuclein compared to the empty vector control (Figure 8B). Without wishing to be bound by any theory, the enhanced levels may be due to a decrease in the ability to recruit synuclein to the proteasome. Based on cell-free experiments and without wishing to be bound by any theory, VHH-4C can bind to DY-synuclein but does not have sufficient affinity to re-induce DY-synuclein to the proteasome. This binding reaction may potentially reduce the normal turnover of the protein. However, no obvious signs of cell death that would be toxic were observed, and no signs of DY-synuclein~GFP puncta reflecting aggregated synuclein were observed as shown in Figure 7. The enhanced affinity of N77D for synuclein may be involved in an increase in intracellular functional activity compared to VHH-4C.

[0166] After verifying that N77D-PEST significantly increases the degradation of DY-synuclein~GFP (Figure 8A) and α-synuclein~GFP (Figure 8B), the PEST degron from human ornithine decarboxylase (hPEST) was cloned into VHH-N77D, and it was determined that N77D-hPEST could re-induce the degradation of lamprey DY-synuclein~GFP to the proteasome for degradation in ST14A rat medium spiny neuron progenitor cells (Figure 9).

[0167] The specificity of N77D-hPEST versus off-target binding was examined. β-synuclein and γ-synuclein were cloned into the GFP-pcDNA3.1(-) plasmid to generate β-synuclein~GFP and γ-synuclein~GFP fusion proteins. Next, ST14A cells were co-transfected with either β-synuclein~GFP or γ-synuclein~GFP and an empty vector control, N77D-hPEST, N77D-hPEST-Scr control, or B8-hPEST control. N77D-hPEST did not significantly change the degradation of either β-synuclein~GFP (Figure 10A) or γ-synuclein~GFP (Figure 10B).

[0168] (Example 7) -Controlled degradation of intracellular proteins using human PEST. This method uses human ODC and controls the level of degradation by creating mutations at specific locations (Figures 15A - 15B). The inventors have shown that various levels of degradation of human α - synuclein can be achieved by changing the PEST sequence at the designated site. C441A inactivates the PEST sequence and causes a significant increase in the observed α - synuclein compared to the empty vector control. A significant increase in degradation relative to the empty vector control was observed when using VH14 - hPEST modified S445A and D433A (Figure 11). A significant increase in degradation compared to S445A and D433A was observed when using VH14 - hPEST modified P426A and P427A (compound mutations) (Figure 11). This indicates that various levels of degradation are achievable with various PEST modifications.

[0169] As shown in Figures 15A - 15B, mutations that may be present in the PEST degron relative to SEQ ID NO: 1 for targeting a protein to the proteasome to achieve an increase in protein degradation can be a mutation of one or more proline (P) residues to alanine (A) residues, such as P426A / P427A (i.e., mutation of two consecutive P residues to two consecutive A residues), a mutation of one or more aspartic acid (D) residues to a residue, such as D433A, a mutation of one or more serine (S) residues to A residues, such as S445A, and / or a mutation of one or more lysine (K) residues to A residues, such as K448A, etc. In some embodiments, additional mutations may be made at one or more amino acid residues of the human ODC PEST degron to enhance the degradation of a protein, such as α - synuclein. In other embodiments, mutations that may be present in the PEST degron relative to SEQ ID NO: 1 as described herein for targeting a protein to the proteasome to achieve a reduction in protein degradation can be a mutation of a P residue to an A residue, such as P438A, a mutation of a glutamic acid (E) residue to an A residue, such as E444A, a mutation of an S residue to an A residue, such as S440A, and / or a mutation of a threonine (T) residue to an A residue, such as T436A.

[0170] (Example 8) - Human ornithine decarboxylase (ODC) PEST (hPEST) degron variant. A panel of intrabodies was developed that target α-synuclein to the proteasome for degradation via a human proline (P), glutamate (E), aspartate (D), serine (S), and threonine (T) (PEST) degron fusion. Figures 15A - 15B show the PEST degron variants identified in this study. Certain mutations (highlighted in gray) within the hPEST degron alter the target degradation of the intrabodies and their binding antigens. The PEST degron is shaded gray in the upper panel (ODC amino acids 423 - 450).

[0171] (Example 9) - Efficacy tests regarding mutations that increase or decrease the degradation of α-synuclein protein. Efficacy tests of mutations to increase the degradation of α-synuclein are performed using any method such as protein binding, translocation, etc. For example, mutations including but not limited to P426A / P427A, D433A, S445A, and / or K448A are introduced into the human PEST degron sequence (SEQ ID NO: 1) as described herein. For visualization, it is possible to visualize the translocation of the protein to the proteasome using a GFP marker or other suitable screenable marker. Appropriate controls are used for comparison and to determine the success of the mutations in increasing the transport of the protein to the proteasome. The mutation from cysteine (C) to alanine (A) at residue 441 (C441A or C20A) can be used as a control because this mutation has no therapeutic effect.

[0172] Furthermore, using the same method, a subset of mutations using glycine (G) instead of alanine (A) are created and tested.

[0173] (Example 10) Verification of the target association of intrabodies against α-synuclein and hPEST degron to the proteasome in iPSC-derived cortical neurons. As described herein, PEST degrons are used in iPSC-derived cortical and midbrain organoid systems as shown in Figure 13. Patient-derived induced pluripotent stem cells (iPSCs) (SNCA triplication (RUCDR; ND50040)) from patients with Parkinson's disease having an increased copy number variation in the SNCA gene encoding α-synuclein and iPSCs from healthy donors are used as controls. Patients with this mutation develop autosomal dominant Parkinson's disease. The assay system used for this optimization screening is the lentiviral transduction of wild-type iPSC-derived cortical forebrain and midbrain organoids with a bifunctional anti-α-synuclein-hPEST intrabody. Candidate intrabodies described herein are subcloned into a tetracycline-inducible pTetO-puromycin resistance lentiviral vector and subsequently transduced into cortical or midbrain organoids on day 30. Endogenous bifunctional α-synuclein degradation is verified by immunofluorescence staining and by quantitative Western blotting using the anti-synuclein monoclonal antibody MJFR1 (1:1,000). HA-tagged intrabodies are probed with monoclonal anti-HA (1:5,000, Covance). Samples are normalized against either actin or GAPDH housekeeping proteins with monoclonal (anti-actin; 1:1,000, Sigma or anti-GAPDH; 1:10,000 Abcam) antibodies. Densitometry is quantified with Image J software. Samples with n = 3 per treatment group are analyzed.

[0174] (Example 11) -Controlled degradation of intracellular synuclein protein using a bifunctional anti-synculein intrabody with a human PEST degron in rat ST14A neural progenitor cells. To identify hPEST degron variants that alter tau degradation to the desired level, ST14A neuronal cells were transfected with α-synuclein~GFP and either an empty vector control (EV CON), VH14-hPEST, VH14-hPEST degron variants P426A / P427A, D433A, C441A, S445A, an inactive scrambled PEST degron control (SCR), or an antigen control (B8-hPEST). 72 hours after transfection, samples were collected for: (A) live cell image analysis; (B) Western blot; (C) relative protein expression was determined by the ratio of α-synuclein to an internal standard control (GAPDH). Samples were then normalized to the EV control. Human PEST degron variants resulted in altered protein degradation levels compared to the empty vector control (EV CON) (Figure 12). The combined mutation variant P426A / P427A resulted in altered synuclein expression compared to VH14-hPEST and significant (p<0.001) protein degradation levels compared to the empty vector control (EV CON). VH14-hPEST and VH14-hPEST variant D433A significantly reduced synuclein compared to EV CON (P<0.01).

[0175] Example 12 -Controlled degradation of intracellular proteins using anti-synuclein intrabody antibodies bearing human PEST degron in iPSC-derived midbrain neurons. This method uses human ODC and controls the level of degradation by creating mutations at specific locations (Figures 15A - 15B). The inventors have shown that various levels of degradation of human α - synuclein can be achieved by changing the PEST sequence at the designated site. The PEST degron as described herein was used in the iPSC - derived midbrain organoid system as shown in Figure 13. Patient - derived induced pluripotent stem cells (iPSCs) from patients with Parkinson's disease having an increased copy number variation in the SNCA gene encoding α - synuclein (SNCA triplication (RUCDR; ND50040)) and iPSCs from healthy donors were used as controls. Patients with this mutation develop autosomal - dominant Parkinson's disease. The assay system used for this optimization screening was lentiviral transduction of wild - type iPSC - derived midbrain organoids with a bifunctional anti - α - synuclein - hPEST intrabody. The candidate intrabodies described herein were subcloned into a tetracycline - inducible pTetO - puromycin - resistant lentiviral vector and subsequently transduced into midbrain organoids on day 30. Endogenous bifunctional α - synuclein - mediated degradation was verified by immunofluorescence staining on day 60. Samples with n = 3 per treatment group were analyzed. A significant increase in degradation was observed for VH14 - hPEST compared to the empty vector control and VH14 - PEST - SCR (inactive PEST degron control). The VH14 - hPEST variants S445A, D433A, and P426A and P427A (compound mutation) showed an increase in degradation compared to VH14 - PEST (Figure 13). This indicates that various levels of degradation are achievable when using various PEST modifications after long - term post - expression in disease - relevant cell types.

[0176] For Example 11 and Example 12, rat precursor cells (ST14A) showing transfection-neuron characteristics were used for transfection. ST14A cells were cultured using a standard protocol. The cells were cultured in 12-well plates for transfection. Co-transfection was performed using 0.75 μg of prk5-GFP-α-synuclein per well and 2.25 μg of anti-α-synuclein-hPEST or anti-α-synuclein-hPEST variant 2 expressed in the pcDNA3.1(-) expression intraboddy expression vector per well. A PEI DNA transfection reagent was used to transiently transfect the cells. 72 hours after transfection, imaging of all cultures was performed, and then the cultures were harvested for Western blot analysis.

[0177] 72 hours after Western blotting - transfection, imaging of ST14A cells for GFP expression was performed. After imaging, samples were collected from 6-well plates by trypsin treatment. The cell samples were washed with 1×PBS and then added to RIPA buffer and cell lysis was performed using 1× protease inhibitor cocktail (50 mM Tris pH7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 2% SDS). Subsequently, the samples were sonicated for 10 minutes. A DC protein assay was performed on the samples and protein concentration data was generated. From the protein assay, the sample concentration was normalized to 1 ng / mL in 2× denaturing sample buffer (125 mM Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, pH 6.8) and heated to ensure protein denaturation. 10 μg of each lysate sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4-20% Criterion precast gel (Bio-Rad #456-1095). The proteins were blotted onto a PVDF membrane (Millipore) at 24-27 V for 30 minutes using a Trans-Blot semi-dry (SD) electroblotter (Bio-Rad). The PVDF membrane was probed for total α-synuclein (MJFRI or synI; 1:1,000) and GAPDH (as a loading control, Abcam; 1:5,000).

[0178] (Example 13) - Bifunctional VH14-PEST enhances viability in iPSC-derived midbrain cultures with 3X SNCA mutations. To test the safety and efficacy of the anti-synuclein bifunctional intrabodies, patient-derived iPSCs with 3X SNCA gene triplication and iPSCs from healthy controls (WT) were differentiated into 3D midbrain organoids, a brain region affected in Parkinson's disease. On day 30, the organoids were transduced with either an empty vector control (EV), VH14-hPEST, VH14-hPEST degron variants P426A / P427A, D433A, S445A, or a scrambled PEST degron control (SCR). On day 60, cell death was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling staining (DeadEnd Fluorometric TUNEL System; Promega #G3250), also known as the TUNEL assay. As expected, mutant 3X SNCA midbrain organoids showed an increase in TUNEL reactivity compared to WT midbrain organoids (Figure 14). Minimal TUNEL reactivity was seen in WT or 3X SNCA midbrain organoids treated with VH14-hPEST and VH14-hPEST degron variants P426A / P427A, D433A, S445A (Figure 14). VH14-hPEST, which showed the lowest level of synuclein reduction in immortalized cells (Figures 11 and 12) and midbrain organoids (Figure 13), was as effective as the most potent synuclein-reducing VH14-hPEST degron variant P426A / P427A in preserving cell viability after long-term treatment. Thus, a minimal level of synuclein reduction to provide a therapeutic effect can be achieved using this technology.

[0179] (Example 14) - Identification and study summary of an anti-tau intrabody that most efficiently targets tau to the proteasome for degradation. Tau is a protein involved in many neurodegenerative diseases such as tauopathies including Alzheimer's disease (AD) and frontotemporal dementia (FTD), as well as traumatic brain injury (TBI) and spinal cord injury (SCI). Tauopathy occurs when tau protein accumulates into aggregates, resulting in neurological symptoms as a consequence of neuronal and glial cell dysfunction and death. Therefore, targeted degradation of abnormal tau protein is an important therapeutic target. Intrabodies can be designed and selected to bind to various protein conformations and epitopes on their targets. Furthermore, they can be further engineered to relocate the target protein to various cellular compartments such as the nucleus, endoplasmic reticulum, and proteasome (Figure 1).

[0180] As described herein, a bifunctional proteasome-targeting intrabody was developed that prevents protein misfolding and targets their bound cargo for degradation to the proteasome via fusion to the PEST degron from human ornithine decarboxylase (hPEST). As described herein, the level of tau reduction can be controlled with the human PEST degron. This has been demonstrated herein to function in multiple systems. Without being bound by any theory, targeted degradation of tau protein using the cell's normal protein removal process may reduce the amount of tau available for folding and thus reduce the physiological impact of tauopathy.

[0181] (Example 15) - Humanization of the bifunctional anti-tau PEST intrabody. The PEST degron can be optimized for human use by replacing the mouse PEST degron (mPEST) with the human PEST (hPEST) degron. A comparison of the mouse and human PEST degrons from the ornithine decarboxylase (ODC) gene is shown in FIG. 17. The hPEST degrons can be transferred into a GFP transcriptional reporter and their half-lives can be reduced to levels similar to those of the GFP-mPEST reporter. Fusion of the hPEST degrons to anti-tau intrabodies can induce tau to the proteasome for degradation as efficiently as the mPEST degrons. Thus, the hPEST degrons are cloned from human ornithine decarboxylase onto the anti-tau intrabodies.

[0182] Previous experiments using immortalized tumor-derived cell lines have generated valuable information, but they present major limitations due to their derivation. Patient-derived induced pluripotent stem cells (iPSCs) can overcome these limitations by more faithfully simulating the phenotypes of human diseases observed in the CNS. iPSC lines derived from patients with tauopathy are established with mutations in the gene encoding tau. Next, optimization is performed using a human iPSC three-dimensional (3D) brain organoid protocol developed by Sergiu Pasca and known in the art. Cultures using this protocol yield brain organoids that are spheres approximately 1-3 mm in diameter of human neurons consisting mainly of neurons but also including macroglia. This protocol was selected for its high reproducibility and its ability to produce all major neocortical cell types, including CTIP2-positive neurons present in cortical layer V, and send their axons to deep brain structures such as the thalamus (corticothalamic neurons), striatum (corticostriatal neurons), pons (corticopontine neurons), tectum (corticotectal neurons), and spinal cord (corticospinal motor neurons, CSMN). Expression of endogenous tau with mutations as described herein is verified in three-dimensional (3D) 60-day-old cortical neurons with mutations compared to wild-type (WT) healthy controls by Western blotting.

[0183] Figure 28 shows a schematic diagram of the MAPT (tau) gene along with mutations in specific exons and introns. These mutations include, but are not limited to, the following mutations: Exon 1: R5H and R5L; Exon 2: G55R; Exon 3: V75A and A91V; Exon 4: Q124E; Exon 7: A152T, D177V, and A178T; Exon 9: G201S, R221Q, A239T, K257T, I260V, T263P, L266V, G272V, and G273R; Intron 9: I9-10 G>T, I9+33 G>A, and I9-15 T>C; Exon 10: N279K, ΔK280, L284L, L284R, S285R, N286N, V287I, N296D, N296H, N296N, ΔN296, K298E, P301L, P301S, P301T, G303V, G304S, S305I, S305N, and S305S; Intron 10: I10+3 G>A, I10+4 A>C, I10+11 T<C, I10+12 C>T, I10+13 A>G, I10+14 C>T, I10+16 C>T, I10+19 C>G, I10+25 C>T, and I10+29 G>A; Exon 11: L315L, L315R, K317M, K317N, S320F, and P332S; Exon 12: G335S, G335V, Q336R, V337M, E342V, D348G, Q351R, S352L, S356T, V363A, V363I, P364S, G366R, and K369I; Exon 13: G389R, R406W, N410H, D418N, Q424K, and T427M.

[0184] iPSCs have been prepared to date for a number of the mutations enumerated above, including A152T, N279K, P301L, S305N, IVS10+16, G335S, G335V, V337M, G389R, and R406W (Karch PMID:31631020).

[0185] (Example 16) - Intracellular protein controlled degradation using human PEST variants. As described herein, the level of tau reduction can be controlled with the human PEST degron by changing the PEST sequence at the designated site. As shown in Figure 18, the hPEST degron significantly reduced tau more than the mPEST degron in immortalized mouse ST14A neural progenitor cells (92% reduction vs 84% reduction). Briefly, ST14A cells were transfected with either GFP-tau and empty vector control (EV CON), V-mPEST, or V-hPEST. 72 hours after transfection, samples were collected for the following: (A) live cell imaging. (B) Western blot. (C) Relative protein expression was determined by the ratio of total tau to internal standard control (GAPDH). Samples were then normalized to the EV control.

[0186] To optimize the level of intracellular tau protein reduction, the inventors generated a series of anti-tau-hPEST intrabodies, V-hPEST, N-hPEST, F-hPEST, and A-hPEST selected against tau amino acids 151-441. Subsequently, the inventors generated hPEST degron variants P426A / P427A, E428A-E430A-E431A, D433A, P438A, S435A, S440A, E444A, K448A, S445A, C441A and hPEST-scrambled anti-tau-hPEST intrabodies (V, N, and F). Furthermore, the inventors generated hPEST variants P426A / P427A, D433A, S445A, C441A, and hPEST-scrambled for A-hPEST. Next, the intrabodies were subcloned into the pcDNA3.1(-) expression vector. Figure 19 shows the controlled degradation of tau by varying the hPEST construct sequences of the V-hPEST and N-hPEST intrabodies. ST14A cells were co-transfected with either V-hPEST or N-hPEST, or their respective hPEST degron variants P426A / P427A, D433A, S445A, and C441A. As control constructs, an empty vector (EV) and an inactive human PEST degron generated by the inventors by mutating C441, a residue previously identified as important for proteasome recognition in the mouse PEST degron, to A, which has been shown to inactivate the human degron, are mentioned. Next, the V-hPEST and N-hPEST degron variants D433A, S445A, and the composite variant P426A / P427A were compared. 72 hours after co-transfection with GFP-tau, the composite V-hPEST-P426A / P427A and N-hPEST-P426A / P427A variants reduced tau by approximately 25% compared to EV-CON. The V-hPEST-D433A, N-hPEST-D433A, N-hPEST-S445A and N-hPEST-S445A-hPEST variants resulted in an approximately 40% reduction of total tau compared to EV-CON. This indicates that various levels of degradation are achievable when using various PEST modifications across multiple intrabodies.

[0187] Mutations that may be present in the PEST degron for SEQ ID NO: 1 as described herein for targeting a protein to the proteasome to achieve a reduction in protein degradation are mutations of one or more proline (P) residues to alanine (A) residues, such as P426A / P427A (i.e., mutations of two consecutive P residues to two consecutive A residues), mutations of one or more aspartic acid (D) residues to A residues, such as D433A, mutations of one or more serine (S) residues to A residues, such as S445A, and the like.

[0188] In other embodiments, mutations that may be present in the PEST degron for SEQ ID NO: 1 as described herein for targeting a protein to the proteasome to achieve a reduction in protein degradation are mutations of P residues to A residues, such as P438A, mutations of glutamic acid (E) residues to A residues, such as E444A, mutations of S residues to A residues, such as S440A, and / or mutations of threonine (T) residues to A residues, such as T436A.

[0189] Rat progenitor cells (ST14A) showing transfection-neuron characteristics were used for transfection. ST14A cells were cultured using standard protocols. Cells were cultured in 6-well plates for transfection. Co-transfection was performed using prk5-GFP-tau per well and anti-tau-hPEST or anti-tau-hPEST variants expressed in a pcDNA3.1(-) expression intrabod expression vector per well. The cells were transiently transfected using a PEI DNA transfection reagent. 72 hours after transfection, imaging of all cultures was performed, and then the cultures were harvested for Western blot analysis.

[0190] 72 hours after Western blotting - transfection, imaging of ST14A cells for GFP expression was performed. After imaging, samples were collected from 6-well plates by trypsin treatment. The cell samples were washed with 1×PBS and then added to RIPA buffer and cell lysis was performed using 1× protease inhibitor cocktail (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 2% SDS). Subsequently, the samples were sonicated for 10 minutes. A DC protein assay was performed on the samples and protein concentration data was generated. From the protein assay, the sample concentration was normalized to 1 ng / mL in 2× denaturing sample buffer (125 mM Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, pH 6.8) and heated to ensure protein denaturation. 10 μg of each lysate sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4-20% Criterion precast gel (Bio-Rad #456-1095). The proteins were blotted onto a PVDF membrane (Millipore) at 24-27 V for 30 minutes using a Trans-Blot semi-dry (SD) electroblotter (Bio-Rad). The PVDF membrane was probed for total tau (DA9; 1:1,000) and GAPDH (as a loading control, Abcam; 1:5,000). Subsequently, the readout of tau cleavage from immunofluorescence staining was verified by Western blotting using a pan-specific anti-tau monoclonal antibody (1:1,000), and the HA-tagged intrabodies were probed with monoclonal anti-HA (1:5,000, Covance). The samples were normalized to either actin or GAPDH housekeeping proteins with monoclonal (anti-actin; 1:1000, Sigma or anti-GAPDH; 1:10,000 Abcam) antibodies. Densitometry was quantified with Image J software.

[0191] (Example 17) - Human ornithine decarboxylase (ODC) PEST (hPEST) degron variants. We developed a panel of intrabodies that target tau to the proteasome for degradation via human proline (P), glutamic acid (E), aspartic acid (D), serine (S) and threonine (T) (PEST) degron fusions. Figures 29A-B show the PEST degron variants identified in this study. The PEST degron is shaded at the top (ODC amino acids 423-450). Key single (A) and combined (B) mutations within the hPEST degron (highlighted in yellow) are predicted to alter the targeted degradation of the intrabody and its bound antigen. This is demonstrated by those highlighted in green in Figures 29A-B.

[0192] (Example 18) -Efficacy studies of mutations that increase or decrease the degradation of tau protein. Testing the effectiveness of mutations as described herein to increase tau degradation is performed using any method for protein binding, translocation, etc. For example, mutations including but not limited to P426A / P427A, D433A, S445A, etc. are introduced into the human PEST degron sequence (SEQ ID NO: 1) as described herein. For visualization, a GFP marker or other suitable screenable marker can be used to visualize the translocation of the protein to the proteasome. Appropriate controls are used for comparison and to determine the success of the mutations in increasing the trafficking of the protein to the proteasome. A cysteine ​​(C) to A mutation at residue 441 (C441A) can be used as a control since this mutation has no therapeutic effect. Additionally, using the same method, a subset of mutations using glycine (G) instead of alanine (A) is generated and tested.

[0193] (Example 19) -Validation of targeted association of bifunctional anti-tau-PEST intrabodies to endogenous human tau in iPSC-derived cortical neurons. Organoid differentiation - Human induced pluripotent stem cells (iPSCs) derived from FTD patients are used to validate bifunctional anti-tau - hPEST and hPEST variants. A human iPSC three-dimensional (3D) brain organoid protocol developed by Sergiu Pasca and known in the art. Cultures using this protocol yield brain organoids, which are spheres approximately 1 mm in diameter of human neurons consisting mainly of neurons but also including macroglia (Figure 37). This protocol was selected because of its high reproducibility and its ability to produce all major neocortical cell types, including CTIP2-positive neurons present in cortical layer V, and send their axons to deep brain structures such as the thalamus (corticothalamic neurons), striatum (corticostriatal neurons), pons (corticopontine neurons), tectum (corticotectal neurons), and spinal cord (corticospinal motor neurons, CSMN).

[0194] To evaluate the target engagement of a bifunctional anti-tau-PEST intrabody to endogenous human tau produced by iPSCs, 3D cortical organoids from healthy donors (WT) were transduced with an inducible lentivirus expressing anti-tau together with mouse PEST degron (tau-mPEST) intrabodies V-mPEST, N-mPEST, and F-mPEST. Twenty-one days after treatment, tau levels were compared to empty virus controls and untreated controls by Western blotting (see methods below). As shown in Figure 37, V-PEST, N-PEST, and F-PEST significantly (p<0.05) reduced endogenous tau protein levels compared to untreated controls (CON) and empty vector (EV) controls. Furthermore, this result demonstrates that the bifunctional anti-tau-PEST intrabodies function in disease-relevant cells. These experiments represent a significant improvement over previous intrabody drug screening models that rely on immortalized tumor-derived cell lines that may contain genetic and metabolic abnormalities due to their mode of derivation. Furthermore, experiments in such lines tend to be limited to a shorter time frame (2–4 days) due to their continuous growth. By more accurately simulating the phenotypes of human diseases, human iPSC-derived cultures overcome these limitations and serve as a platform for long-term testing of therapeutic agents.

[0195] Twenty-one days after Western blotting-lentiviral transduction, the organoid samples were washed with 1×PBS and then added to RIPA buffer and lysed using 1× protease inhibitor cocktail (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 2% SDS). The samples were sonicated for 10 minutes. A DC protein assay was performed on the samples to generate protein concentration data. From the protein assay, the sample concentration was normalized to 1 ng / mL in 2× denaturing sample buffer (125 mM Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, pH 6.8) and heated to ensure protein denaturation. 10 μg of each lysate sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4-20% Criterion precast gel (Bio-Rad #456-1095). The proteins were blotted onto a PVDF membrane (Millipore) at 24-27 V for 30 minutes using a Trans-Blot semi-dry (SD) electroblotter (Bio-Rad). The PVDF membrane was probed for total tau (DA9; 1:1,000) and GAPDH (as a loading control, Abcam; 1:5,000). The readout of tau degradation from immunofluorescent staining was verified by Western blotting using the anti-tau monoclonal antibody DA9 (1:1,000) that recognizes all tau isoforms, and the HA-tagged intrabodies were probed with monoclonal anti-HA (1:5,000, Covance). The samples were normalized to the GAPDH housekeeping protein with a monoclonal (anti-GAPDH; 1:10,000 Abcam) antibody. Densitometry was quantified with Image J software as previously described.

[0196] (Example 20) -Regulated degradation of tau using the bifunctional anti-tau intrabody V with a human PEST degron in ST14A neural progenitor cells. To identify hPEST degron variants that change tau degradation to the desired level, ST14A neuronal cells were transfected with GFP-tau-(0N4R) and either an empty vector control (EV CON), V-hPEST, V-hPEST degron variants E428A / E430A / E431A, D433A, S435A, P438A, S440A, C441A, E444A, S445A, or an inactive scrambled PEST degron control (SCR). 72 hours after transfection, samples were collected for: (A) live cell imaging. (B) Western blot. (C) Relative protein expression was determined by the ratio of total tau to an internal standard control (GAPDH). Samples were then normalized to the EV control. Human PEST degron variants resulted in changes in proteolysis levels compared to the empty vector control (EV CON) (Figure 20). V-PEST variants E428A / E430A / E431A, D433A, S435A, P438A, S440A, C441A, and E444A reduced GFP-tau by 75-100% compared to the control. V-PEST and V-PEST variant S445A reduced GFP-tau to 50-75% of the control.

[0197] (Example 21) -Controlled degradation of tau using bifunctional anti-tau intrabodies N with human PEST degrons in ST14A neural progenitor cells. To identify hPEST degron variants that change tau degradation to a desired level, ST14A neuronal cells were transfected with GFP-tau-(0N4R) and either an empty vector control (EV CON), N-hPEST, N-hPEST degron variants E428A / E430A / E431A, D433A, S435A, P438A, S440A, C441A, E444A, S445A, or an inactive scrambled PEST degron control (SCR). 72 hours after transfection, samples were collected for: (A) live cell imaging. (B) Western blot. (C) Relative protein expression was determined by the ratio of total tau to an internal standard control (GAPDH). Samples were then normalized to the EV control. Human PEST degron variants resulted in changes in proteolysis levels compared to the empty vector control (EV CON) (Figure 21). N-PEST reduced GFP-tau by 50 - 75% compared to the control, and N-PEST variants P426A / P427A, P438A, E444A, and K448A / R449A / H450A reduced GFP-tau by 25 - 50% compared to the control. N-PEST variants E428A / E430A / E431A, S435A, S440A, and S445A reduced GFP-tau by 0 - 25% compared to the control. B8-PEST, N-PEST-SCR, and N-PEST variants D433A and C441A increased the level of GFP-tau compared to EV CON.

[0198] (Example 22) -Controlled degradation of tau using bifunctional anti-tau intrabodies F with human PEST degrons in ST14A neural progenitor cells. To identify hPEST degron variants that change tau degradation to the desired level, ST14A neuron cells were transfected with either GFP-tau-(0N4R) and empty vector control (EV CON), F-hPEST, F-hPEST degron variants P246A / P427A, E428A / E430A / E431A, D433A, S435A, P438A, S440A, C441A, E444A, S445A, K448A / R449A / H450A, or an inactive scrambled PEST degron control (SCR). 72 hours after transfection, samples were collected for: (A) live cell imaging. Scale bar = 50 μm. (B) Western blot. (C) Relative protein expression was determined by the ratio of total tau to an internal standard control (GAPDH). Samples were then normalized to the EV control. Human PEST degron variants resulted in changes in proteolysis levels compared to the empty vector control (EV CON) (Figure 22). F-PEST and F-PEST variants E428A / E430A / E431A, D433A, P438A, E444A, S445A, and K448A / R449A / H450A reduced GFP-tau by 50 - 75% compared to the control. The F-PEST variant P426A / P427A reduced GFP-tau by 25 - 50% compared to the control. F-PEST-SCR expressed similar levels of GFP-tau compared to EV CON.

[0199] (Example 23) - Establishment of a rigorous cell death assay using a series of neuron cells derived from donor-derived human iPSC lines carrying the disease-causing MAPT V337M mutation and their corresponding gene-corrected control V337V. This cell death assay is useful for both modeling FTD and screening for therapeutic molecules for tauopathy. V337M and control V337V iPSCs were differentiated into neural progenitor cells (NPCs) that exhibit signatures characteristic of forebrain identity on day 20 (Figure 23). Using a protocol developed by the Temple lab, these NPCs were differentiated into highly enriched cortical neurons of all major cortical cell subtypes by 45 days. Next, the survival of V337M mutants relative to control neurons was performed. To date, studies have shown that A152T MAPT mutant neurons are more sensitive to stressors such as rotenone and exhibit increased cell death (Silva, Cheng et al. (2016) Stem Cell Reports 7(3): 325-340). Notably, this vulnerability to stressors only emerged after the cultures had been approximately 100 days old. Therefore, the inventors compared the levels of cell death in 110-day-old V337M cortical neurons to V337V control cortical neurons with and without rotenone treatment. Cell death was quantified using the ethidium homodimer assay (Thermo Fisher), in which dying cells are indicated by the uptake of nuclear dye into damaged DNA. A significant increase in cell death was seen in 110-day-old V337M mutant neurons compared to isogenic V337V controls (Figure 23). The presence of dead cells that have lost membrane integrity can be detected by measuring markers that leak from the cytoplasm, such as lactate dehydrogenase (LDH), into the culture medium (Riss, Niles et al. (2004) Assay Guidance Manual. S. Markossian, G. S. Sittampalam, A. Grossman et al. Bethesda (MD)). Cell death was significantly increased in V337M mutant cultures by the LDH-Glo™ cytotoxicity assay (Promega) kit (Figure 23).Ehrlich et al. recently showed that patient-derived iPSC lines with the MAPT V337M mutation generate neurons with enhanced tau fragmentation and phosphorylation, decreased neurite outgrowth, and enhanced sensitivity to oxidative stress (Ehrlich, Hallmann et al. (2015) Stem Cell Reports 5(1): 83-96). Surprisingly, as shown, the inventors did not observe a significant increase in cell death when cells were stressed with rotenone, an environmental toxin that inhibits mitochondrial electron transport chain (ETC) complex I. The main difference between the studies was that the inventors generated forebrain neurons as compared to the midbrain neurons produced by Ehrlich. This is important because a subset of patients with the V337M mutation have severe frontal lobe atrophy with high densities of NFTs, pretangles, and neuropil threads (Spina, Schonhaut et al. 2017), while the substantia nigra (located in the midbrain region) in the same patients showed mild atrophy and NFT pathology (Spina, Schonhaut et al. (2017) Neurology 88(8): 758-766). Combining these results with clinical data suggests selective vulnerability in forebrain neurons to the V337M mutation alone without further stressors.

[0200] (Example 24) - The proteasome is impaired in iPSC-derived cortical neurons with the MAPT V337M mutation as compared to the isogenic V337V control. To monitor proteasome function, mutant (V337M) and isogenic control (V337V) cortical cultures were transfected with ubiquitin G76V GFP (Ub G76V(GFP) reporter was transfected. This reporter is widely used for monitoring the role of ubiquitin / proteasome-dependent proteolysis in various disorders and for testing the effectiveness of compounds on the ubiquitin / proteasome system. V337V and V337M cortical cultures were transfected on day 90. Live cell imaging was performed on day 110 of culture (Figure 23), which is the time point when cell death increases in MAPT V377M mutant cultures compared to the isogenic V337V control, to measure the expression level of the Ub G76V GFP reporter. In healthy V337V control cultures, the Ub G76V GFP (arrow) reporter is rapidly degraded, suggesting that the function of the ubiquitin proteasome system is impaired in MAPT V337M mutant cortical neurons (Figure 24), and accumulation of the UB G76V GFP reporter is observed, potentially contributing to the cell death observed in Figure 23.

[0201] (Example 25) -The bifunctional anti-tau-PEST intrabodies alleviate proteasome impairment in induced pluripotent stem cell (iPSC)-derived cortical neurons with the MAPT V337M mutation compared to isogenic V337V controls. To determine whether the anti-tau-PEST intrabodies can counteract the proteasome impairment caused by the mutant V337M tau, mutant (V337M) and isogenic control (V337V) cortical cultures were transfected with the ubiquitin G76V GFP (Ub G76V GFP) reporter and either an empty vector control (EV), V-hPEST, N-hPEST, or F-hPEST. Twenty days after treatment, live cell imaging was performed to measure the fluorescence expression level of the Ub G76V GFP reporter. In V337M mutant cultures treated with the anti-tau-hPEST intrabodies, the Ub G76VThe GFP reporter is rapidly degraded (Figure 25). This demonstrates that a bifunctional anti-tau-PEST intrabody that undergoes ubiquitin-independent proteolysis can counteract the proteasome impairment caused by V337M tau toxicity.

[0202] (Example 26) - Anti-tau-hPEST intrabodies, V-hPEST, and N-hPEST reduced cell death in human iPSC-derived cortical neurons carrying the MAPT V337M mutation. After showing that V337M mutant cortical cultures exhibit increased cell death (Figure 23) and proteasome impairment (Figure 24), which can be counteracted by anti-tau-PEST intrabodies (Figure 25), cell death was evaluated after treatment with the bifunctional anti-tau-PEST intrabodies. V337M cortical cultures were transduced at day 90 with either an empty vector control (EV-CON), V-hPEST, N-hPEST, or the B8-hPEST control intrabody against the unrelated antigen botulinum toxin. At day 110, ethidium homodimer (EtHD) assays were used to detect dead and / or dying cells. As expected, cell death levels were reduced to control levels by the V-hPEST and N-hPEST intrabodies (Figure 26). Cell death was significantly reduced by V-hPEST ( * , p = 0.0271), while N-hPEST was comparable in significance to the V337M cultures compared to the isogenic V337V control (p = 0.0517).

[0203] (Example 27) - Using programmable target antigen proteolysis (P-TAP) technology, the lowest effective level of tau cleavage to achieve neuroprotection using anti-tau-PEST intrabodies can be determined in disease-related human iPSC-derived cortical neurons. To determine the level of tau reduction necessary to achieve neuroprotection in human cells, mutant MAPT V337M cortical cultures were transduced with either the potent tau reducer (50 - 75%; Figure 21) N-hPEST or the low tau reducer (0 - 25%; Figure 21) N-hPEST degron variant S445A and compared to either an empty vector control or N-hPEST with a scrambled inactive PEST degron. In this experiment, after transducing the cultures on day 60, cell death was evaluated on day 90 using the ethidium homodimer (EtHD) assay as described in (Figures 23 and 26). Consistent with Figure 26, cell death levels were significantly ( * , p < 0.05) reduced by the potent tau reducer (50 - 75%) N-hPEST compared to EV CON (Figure 27). The low tau reducer (0 - 25%) N-hPEST degron variant S445A significantly ( * , p < 0.05) reduced cell death in V337M cortical cultures compared to EV CON (Figure 27). This novel finding demonstrates that long-term expression of the following, with only approximately 25% tau reduction, is sufficient to achieve neuroprotection.

[0204] (Example 28) - Identification of a bifunctional anti-huntingtin - human - PEST variant that controls huntingtin degradation. Huntingtin is the protein that causes Huntington's disease. An expansion of the CAG repeat in exon 1 of the HTT gene results in a protein with an abnormal polyglutamine (polyQ) extension at the N-terminus. This polyQ extension adopts a number of conformations including α-helices, random coils, and extended loops. Huntington's disease develops when the mutant huntingtin protein aggregates, leading to neurological symptoms as a result of neuronal cell death. Therefore, the targeted degradation of abnormal huntingtin protein is an important therapeutic target. Intrabodies can be designed and selected to bind to various protein conformations and epitopes on their targets. Furthermore, intrabodies can be further engineered to relocate the target protein to various cellular compartments such as the nucleus, endoplasmic reticulum, and proteasome (Figure 1). As described herein, the inventors have developed bifunctional proteasome-targeting intrabodies that prevent protein misfolding and at the same time target their bound cargo to the proteasome for degradation via fusion to the PEST degron (hPEST) derived from human ornithine decarboxylase. The overall hypothesis of this study is that targeted degradation of the huntingtin protein using the cell's normal protein removal process reduces the amount of huntingtin available for folding and thus reduces the physiological effects of Huntington's disease or other diseases associated with mutations and / or aggregation of huntingtin.

[0205] (Example 29) -Controlled degradation of intracellular proteins using human PEST. As described herein, the level of huntingtin reduction can be controlled with the human PEST degron. This has been demonstrated herein to function in multiple systems. Without being bound by any theory, targeted degradation of the huntingtin protein using the cell's normal protein removal process reduces the amount of huntingtin available for folding and thus may reduce the physiological effects of Huntington's disease. For example, it is shown that modification of degrons enables control of the level of huntingtin degradation in cells in culture (e.g., ST14A rat neural progenitor cells). The assumptions made were that (1) antigen-intrabody association was given, and (2) degradation of the antigen-intrabody-degron complex was proportional to that of unbound intrabody-degron. Huntingtin protein is targeted herein, which is a naturally occurring protein, and the intrabody has a PEST degron derived from human ODC.

[0206] As shown in FIGS. 35A-35B, mutations that may be present in the PEST degron relative to SEQ ID NO: 1 as described herein for targeting a protein to the proteasome to achieve a change in protein degradation may be a mutation of a proline (P) residue to an alanine (A) residue, e.g., P426A / P427A (i.e., a compound mutation of two consecutive P residues to two consecutive A residues), a single mutation of an aspartic acid (D) residue to an A residue, e.g., D433A, a single mutation of a serine (S) residue to an A residue, e.g., S445A, and / or a mutation of a lysine (K) residue to an A residue, e.g., K448A.

[0207] Testing of the effectiveness of mutations as described herein to increase huntingtin degradation is performed using any method for protein binding, translocation, etc. For example, mutations including but not limited to P426A / P427A, D433A, S445A, and / or K448A are introduced into the human PEST degron sequence (SEQ ID NO: 1) as described herein. For visualization, it is possible to visualize the translocation of the protein to the proteasome using a GFP marker or other suitable screenable marker. Appropriate controls are used for comparison and to determine the success of the mutation in increasing the transport of the protein to the proteasome. A mutation from cysteine (C) to A at residue 441 (C441A) can be used as a control since this mutation has no therapeutic effect.

[0208] Cloning - Anti - huntingtin - PEST and their respective PEST variants (SEQ ID NO: 100 - SEQ ID NO: 105) are sub - cloned into pAAV - MCS. To identify the expression of the intrabodies, a hemagglutinin (HA) epitope tag (amino acid sequence YPYDVPDYA) is fused to the C - terminus of the intrabodies. To target the intrabodies and their cargo to the proteasome, a canonical PEST motif corresponding to amino acids 422 - 461 from human ODC (GenBank accession number AH002917.2) is added to the C - terminus of the HA tag. The scFv intrabodies are arranged as 5’ - VH - (G4S)3 - V L -HA - PEST - 3’. The intrabodies are sub - cloned into pAAV - MCS using standard cloning techniques according to the following cloning strategy: XbaI - intrabody - NotI - HA - PEST degron - HindIII. All expression plasmids are verified by Sanger DNA sequencing (Genewiz, NJ) and prepared according to the manufacturer's protocol using the Nucleobind Xtra Midi Endotoxin free (Takara #740420.5) preparation kit.

[0209] Transfection - Rat progenitor cells (ST14A) that exhibit neuronal characteristics are used for transfection. ST14A cells are cultured using standard protocols. The cells are plated in 6 - well plates for transfection. Co - transfection is performed using 0.75 μg of mHTTex1 - 72 - eGFP - pcDNA3.1 per well and 2.25 μg of anti - huntingtin - hPEST or anti - huntingtin - hPEST variant expressed from the pAAV expression vector per well. The PEI DNA transfection reagent is utilized to transiently transfect the cells. 72 hours after transfection, imaging of all cultures is performed, and then the cultures are harvested for Western blot analysis.

[0210] 72 hours after Western blotting - transfection, image ST14A cells for GFP expression. After imaging, collect the samples from the 6-well plate by trypsin treatment. Wash the cell samples with 1×PBS and then add them to RIPA buffer and perform cell lysis using 1× protease inhibitor cocktail (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 2% SDS). Sonicate the samples for 10 minutes. Perform a DC protein assay on the samples and generate protein concentration data. From the protein assay, normalize the sample concentration to 1 ng / mL in 2× denaturing sample buffer (125 mM Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, pH 6.8) and heat to ensure protein denaturation. Separate 10 μg of each lysate sample by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4-20% Criterion precast gel (Bio-Rad #456-1095). Transfer the proteins to a PVDF membrane (Millipore) at 24-27 V for 30 minutes using a Trans-Blot semi-dry (SD) electroblotter (Bio-Rad). Probe the PVDF membrane for mutant huntingtin (EM48; 1:1,000) and GAPDH (as a loading control, Abcam; 1:5,000). Quantify by densitometry using Image J software.

[0211] (Example 30) - Humanization of the bifunctional anti-HTT-PEST intrabody. The PEST degron was optimized for human use. To achieve this goal, the mouse PEST degron (mPEST) was replaced with the human PEST (hPEST) degron. A comparison of mouse and human PEST degrons derived from the ornithine decarboxylase (ODC) gene is shown in Table 3. The fusion of the hPEST degron to the anti-HTT intrabodies induces the mutant HTT exon 1 protein fragment to the proteasome for degradation as efficiently as the mPEST degron. Thus, the hPEST degron was cloned from human ornithine decarboxylase into the anti-HTT C4 scFv intrabodies and the VL12.3 single domain intrabodies. mHTTex1-72Q-eGFP was co-transfected with either an empty vector control (CON), C4-PEST, VL12.3-PEST, or C4 and VL12.3 with an inactive scrambled PEST degron that does not promote proteolysis (C4-PEST-SCR), or (VL12.3-PEST-SCR). 72 hours after transfection, the cells were imaged and harvested for Western blot. As shown in Figure 31, live cell imaging revealed that mHTTex1-72Q-GFP readily formed aggregates (foci) in EV CON cells. In C4-hPEST and VL12.3-hPEST treated cells, the fluorescent signal for mHTTex1-72Q-GFP was almost undetectable. C4-PEST and VL12.3-PEST prevent the aggregation of mHTTex1-72Q-GFP compared to the control. Western blot analysis confirmed the results of live cell imaging. Soluble and insoluble (high molecular weight species) mHTTex1-72Q-GFP detected with the monoclonal antibody EM48 (Millipore, Cat #MAB5374) was reduced in C4-PEST and VL12.3-PEST treated cells compared to the empty vector control and the C4-PEST-SCR and VL12.3-PEST-SCR controls (Figure 31).

[0212] (Example 31) Mutant HTT exon 1 protein fragments with either -46Q or 72Q repeats impair the function of the ubiquitin proteasome system. To determine whether the ubiquitin-proteasome is impaired by toxic mutant HTT protein fragments, ST14A cells were co-transfected with ubiquitin G76V GFP (ubiquitin G76V GFP) reporter and either an empty vector control (EV CON), mHTTex1-25Q-RFP (46Q-RFP), mHTTex1-46Q-RFP (46Q-RFP), or mHTTex1-72Q-RFP (72Q-RFP). 72 hours after transfection, live cell imaging of the cells was performed for mHTT aggregation and UB G76V GFP accumulation (Figure 32). In healthy cells, ubiquitin G76V GFP is rapidly degraded by the proteasome, and the ubiquitin G76V GFP reporter accumulates if the proteasome is impaired. As expected, in healthy EV CON cells, ubiquitin G76V GFP is rapidly degraded by the proteasome. In cells transfected with non-pathogenic polyglutamine repeat length 25Q-RFP, the reported UB G76V GFP was similarly efficiently degraded. However, in cells treated with pathogenic polyglutamine repeat lengths 46Q-RFP and 72Q-RFP, the UB G76V GFP reporter accumulated and was also incorporated into mHTT aggregates. In summary, this suggests that the ubiquitin proteasome system is impaired by mHTT aggregation.

[0213] (Example 32) - The bifunctional intrabody C4-PEST counteracts proteasome impairment caused by mHTT exon 1 protein fragments. To determine whether inhibition of mHTTex1-72Q-RFP aggregation and its removal by the proteasome are due to ubiquitin-independent proteolysis via C4-hPEST, ST14A cells were transfected with ubiquitinG76V GFP (ubiquitin G76V GFP) reporter, mHTTex1-72Q-RFP (72Q-RFP), and either an empty vector control (EV CON), C4 with a human PEST degron (C4-PEST), or C4 with an inactivated scrambled human PEST degron (C4-PEST-SCR) were co-transfected. 72 hours after transfection, live cell imaging of the cells was performed for mHTT aggregation and UB G76V GFP accumulation. As shown in Figure 33, the proteasome was impaired in function by Q72-RFP aggregation. Also, as shown in Figure 32, UB G76V GFP reporter accumulated in these cells and was incorporated into 72Q-RFP aggregates, but in cells treated with C4-PEST-SCR that only inhibited 72Q-RFP aggregation, UB G76V GFP reporter was rapidly degraded by the proteasome. Furthermore, in cells Q72-RFP treated with C4-PEST that inhibited Q72-RFP aggregation and induced Q72-RFP to the proteasome via ubiquitin-independent proteolysis, both Q72-RFP and UB G76V GFP reporter were effectively removed by the cells (Figure 33).

[0214] (Example 33) -Controlled degradation of toxic intracellular mHTT fragments using a bifunctional anti-HTT intrabody C4 with a human PEST degron in rat ST14A neural progenitor cells. To identify hPEST degron variants that alter mHTT degradation to the desired level, ST14A neuronal cells were transfected with human mHTTex1-72Q-GFP and either empty vector control (EV CON), C4, C4-PEST, and C4-PEST variants (T436A, P438A, S440A, E444A), compound mutant variants (E428A / E430A / E431A), or inactive scrambled PEST degron control (SCR). 72 hours after transfection, cells were live imaged and harvested for Western blotting. As shown in Figure 34, live cell imaging revealed that mHTTex1-72Q-GFP readily formed aggregates (puncta) in EV CON cells. Consistent with Figure 31, C4 and C4-PEST-SCR prevented mHTTex1-72Q-GFP aggregation. In C4-PEST and C4-PEST variant T436A treated cells, the presence of soluble mHTT was almost undetectable. Cells treated with C4-PEST variants E428A / E430 / E431A, E444A, and S440A showed increased levels of soluble mHTTex1-72Q-GFP compared to C4-PEST. Western blotting was used to confirm the live cell imaging results (Figure 31). For Western blotting: mHTTex1-72Q-GFP was detected using monoclonal antibody EM48 (Millipore, Cat #MAB5374). Intrabodies were detected by probing for their HA tag with anti-HA tag, and GAPDH was probed as a loading control. Relative protein expression was determined by the ratio of soluble mHTTex1-72Q-GFP (EM48) to the internal standard control (GAPDH). Samples were then normalized to EV-CON. The human PEST degron variants T436A, S440A, E444A, and the combined mutation variant E428 / 430 / 431A result in altered levels of protein degradation compared to EV CON. C4-PEST and the C4-PEST variant T436A reduced mHTT by 75-100% compared to controls.The C4-PEST variants E428A / E430 / E431A and E444A reduced mHTT to 50 - 75% of the control. The C4-PEST variant S440A reduced mHTT to 25 - 0% of the control, and C4, C4-PEST-SCR, and the C4-PEST variant P438A all increased the percentage of mHTT compared to the control. These results indicate that various levels of mHTT degradation are achievable with various PEST modifications.

[0215] (Example 34) - Degradation of intracellular synuclein protein using a cell-permeable bifunctional anti-α-synuclein VHH-hPEST intrabody in rat ST14A neural progenitor cells. Using rat ST14A progenitor cells that exhibit neural characteristics, a conditioned medium containing a cell-permeable intrabody or a control was generated. Separate sets of ST14A cells were transfected with α-Syn~eGFP to determine whether a conditioned medium containing the cell-permeable anti-α-synuclein bifunctional intrabody (SS-PEN-N77D-PEST) could enter recipient cells and degrade intracellular α-Syn~eGFP compared to the control. For transfection, cells were seeded and cultured in 6-well plates according to a standard protocol. Day 0, generation of conditioned medium containing the cell-permeable intrabody: Transient transfection of ST14A cells with either the cell-permeable intrabody SS-PEN-N77D-PEST (+), the PEN-N77D-PEST (-) control, or the empty vector control was performed using the PEI DNA transfection reagent with 3.0 μg of DNA per expression vector per well. Day 1. Generation of recipient ST14A cells expressing α-Syn~eGFP. ST14A cells were transfected with 3.0 μg of α-Syn~eGFP expressed in pcDNA3.1. Four hours after transfection, the cells were treated with conditioned medium from each intrabody donor group. In the SS-PEN-N77D-PEST (+)-treated cells, the cell-permeable intrabody was secreted into the conditioned medium, whereas in the PEN-N77D-PEST (-) control group, the intrabody remained inside the cells. Next, the Syn~eGFP cells were treated with the conditioned medium for an additional 3 days. On day 4, live imaging of the cells was performed (Figure 39B), followed by processing for Western blotting (Figure 39C). The graph shows the relative densitometric quantification of the synuclein Western blot band intensity (Figure 39D). The expression of the α-Syn~eGFP protein was reduced by approximately 20% in the SS-PEN-N77D-PEST (+)-treated cells compared to the EV control.

[0216] Western blotting - 72 hours after treatment with the conditioned medium, ST14A cells were imaged for Syn~eGFP expression. After imaging, samples were collected from the 6-well plates by trypsin treatment. The cell samples were washed with 1×PBS and then added to RIPA buffer and lysed using 1× protease inhibitor cocktail (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 2% SDS). Subsequently, the samples were sonicated for 10 minutes. A DC protein assay was performed on the samples to generate protein concentration data. From the protein assay, the sample concentration was normalized to 1 ng / mL in 2× denaturing sample buffer (125 mM Tris, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, pH 6.8) and heated to ensure protein denaturation. Lysate samples (10 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–20% Criterion precast gel (Bio-Rad #456-1095). Proteins were blotted onto a PVDF membrane (Millipore) at 24–27 V for 30 minutes using a Trans-Blot semi-dry (SD) electroblotter (Bio-Rad). The PVDF membrane was probed for total α-synuclein (MJFRI or syn1; 1:1,000) and GAPDH (as a loading control, Abcam; 1:5,000). Synuclein densitometry was quantified using Image J software.

[0217] (Example 35) - The cell-permeable bifunctional anti-α-synuclein VHH-hPEST intrabodies reduce endogenous α-synuclein in iPSC-derived neuron cultures with 3X SNCA mutations. Patient-derived induced pluripotent stem cells (iPSCs) (SNCA triplication (RUCDR; ND50040)) from a Parkinson's disease patient with an increased copy number variation in the SNCA gene encoding α-synuclein and iPSCs from healthy donors were used as controls. Patients with this mutation develop autosomal dominant Parkinson's disease. The assay system used for this optimization screening was lentiviral transduction of wild-type iPSC-derived cortical forebrain cultures with the cell-permeable bifunctional anti-α-synuclein VHH-hPEST intrabody. The cell-permeable intrabodies and controls described herein were cloned into a puromycin-resistant lentiviral vector and subsequently transduced into 60-day-old cortical cultures. Endogenous bifunctional α-synuclein-mediated degradation was verified by quantitative Western blotting using the anti-synuclein monoclonal antibody MJFR1 (1:1,000; Abcam). HA-tagged intrabodies were probed with monoclonal anti-HA (1:5,000, Covance). Samples were normalized to the GAPDH housekeeping protein with a monoclonal (1:10,000; Abcam) antibody. Densitometry was quantified with Image J software.

[0218] To generate conditioned media containing cell-permeable intrabodies, 2-month-old induced pluripotent stem cell (iPSC)-derived cortical neurons from healthy controls were transduced with lentivirus expressing either SS-PEN-N77D-PEST, SS-PEN-DB1-PEST, or the antigen control SS-PEN-B8-PEST (botulinum neurotoxin light chain A). 72 hours after transduction, recipient 3X SNCA triplication cortical cultures were treated with conditioned media for an additional 7 days and subsequently processed for Western blotting (Figure 40B). Endogenous synuclein levels were reduced by approximately 40% and approximately 30% respectively in 3X SNCA recipient cells treated with conditioned media containing the cell-permeable intrabodies SS-PEN-N77D-hP and SS-PEN-DB1-hP compared to the antigen control (SS-PEN-B8-hP) (Figure 40C).

[0219] (Example 36) - Cell - permeable bifunctional anti - α - synuclein VHH - hPEST intrabodies having various cell - penetrating peptides (CPPs) reduce endogenous α - synuclein. The delivery ability of three CPP sequences (penetratin, TAT, and 435b) by the bifunctional N77D-hPEST (N77D-hP) anti-synuclein intrabodies was examined in human cortical neurons derived from iPSCs with 3X SNCA gene triplication. To generate conditioned media with cell-permeable intrabodies, 2-month-old iPSC-derived cortical neurons from healthy controls were transduced with lentivirus carrying either SS-PEN-N77D-hPEST, SS-TAT-N77D-hPEST, or the SS-N77D-hPEST control lacking the CPP domain. 72 hours after transduction, the recipient 3X SNCA triplication cortical cultures were treated with conditioned media from each donor for an additional 7 days and then processed for Western blotting (Figure 41A). Endogenous α-synuclein (Syn) and HA-tagged intrabody expression were evaluated by Western blotting. 10 μg of total protein was separated by gel electrophoresis and transferred to a nitrocellulose membrane. Endogenous α-synuclein was detected using the MJFR1 anti-α-synuclein antibody (1:1,000; Abcam #ab138501). HA-HRP was used to detect the HA-tagged intrabodies (1:1,000; Thermo-Fisher #26183-HRP). GAPDH was used as a loading control (1:10,000; Abcam, #ab181602). SS-PEN-N77D-hP, SS-TAT-N77D-hP, and SS-435b-N77D-hP reduced endogenous α-synuclein by approximately 40%, 20%, and 40% compared to the SS-N77K-hP control (Figures 41B - 41C). The HA-tagged cell-permeable intrabodies SS-PEN-N77D-hP, SS-TAT-N77D-hP, and SS-435b-N77D-hP were present in the 3X SNCA recipient lysates compared to the SS-N77D-hP control-treated samples (Figure 41B).

[0220] (Example 37) - The cell-permeable bifunctional anti-mutant HTT scFv C4-hPEST intrabody reduces the aggregation of the toxic mHTTex1-72Q-eGFP fragment. The cell-permeable anti-HTT bifunctional intrabody was designed as shown in Figure 38. An immunoglobulin heavy chain signal peptide sequence (SS; MEFGLSWLFLVAILKGVQG; SEQ ID NO: 149) was added to the N-terminus to direct the intrabody complex into the secretory pathway. To facilitate translocation of the secreted bifunctional intrabody across the cell membrane, the cell-permeable peptide PEN (RQIKIWFQNRRMKWKK; SEQ ID NO: 150) was added to the N-terminus of scFv C4-hPEST. To determine whether the cell-permeable bifunctional anti-mutant HTT scFv C4-hPEST intrabody could reduce the aggregation of the toxic mHTTex1-72Q-eGFP fragment, ST14A intrabody donor cells were transfected with either SS-PEN-C4-PEST or the C4-PEST control. In donor-treated cells, the cell-permeable intrabody is secreted into the conditioned medium, whereas in the C4-PEST control group, the intrabody remains inside the cells. On day 1, the intrabody acceptor ST14A cells were transfected with mHTTex1-72Q-eGFP. Four hours after transfection, the cells were treated with conditioned medium from each intrabody donor group. Next, the recipient cells were treated with conditioned medium for an additional 3 days. Four hours after each medium change, live imaging of the recipient cells was performed to evaluate mHTTex1-72Q-eGFP aggregation. Live cell imaging 52 hours after treatment revealed a reduction in mHTTex1-72Q-eGFP aggregation in cells treated with conditioned medium containing SS-PEN-C4-hPEST compared to conditioned medium from the C4-hPEST control (Figure 42B). Arrows indicate the presence of diffuse mHTTex1-72QGFP in SS-PEN-C4-hPEST-treated cells. SS-PEN-C4-hPEST-treated cells showed a significant (P<0.01) reduction in mHTTex1-72QGFP aggregation after 76 hours (Figures 42C–42D).

[0221] (Example 38) - The cell-permeable bifunctional anti-tau intrabody (PEN-N-hPEST) significantly (p<0.01) reduces endogenous human tau in differentiated neurons. To demonstrate that protein-based cell-permeable bifunctional intrabodies can reduce their intracellular targets, cell-permeable intrabodies and control antibodies (Figure 43C) were purified from Rosetta-gami™ 2(DE3) Escherichia coli (Millipore Cat# 71351) transformed with the pET-6X-His plasmid encoding the antibody of interest. Purification was performed using a spin column loaded with Ni-NTA resin to pull down His-tagged proteins. The purified proteins were dialyzed against PBS. To evaluate non-viral protein-based delivery of intrabodies, human neuron cell cultures were generated from SH-SY5Y neuroblastoma cells, which produced 3R and 4R tau isoforms 17 days after differentiation (Shipley et al., 2016). Cells were seeded at 8.4×10 per well in Neurobasal medium supplemented with B-27, Anti-Anti, 2 mM GlutaMax, 50 ng / mL BDNF, 20 mM KCl, 2 mM db-c-AMP, and 10 μM retinoic acid. 4Cells were seeded at a density of individual cells in 96-well plates coated with Matrigel. The inventors serially diluted purified antibodies with or without the cell-penetrating peptide (penetratin, PEN) sequences (PEN-N-HA-hPEST and N-HA-hPEST, respectively) in SH-SY5Y medium to produce concentrations in the range of 5 μM to 8 nM and a final volume of 100 μL per concentration (Figure 43B). The medium was aspirated from the cells and replaced with 100 μL of the antibody serial dilutions. The treated cells were returned to an incubator at 37 °C and 5% CO2 for 2 days and then fixed in 4% paraformaldehyde for 10 minutes at room temperature. To assess endogenous tau protein levels, the fixed cells were immunostained. The cells were permeabilized with 0.1% Triton-X-100 and blocked with 3% bovine serum albumin, 10% normal goat serum, and 0.1% Triton-X-100 in PBS. The cells were stained with a HA-tagged purified antibody using a mouse anti-HA IgG1 monoclonal antibody (Invitrogen #26183) and a rabbit anti-human tau polyclonal antibody (Dako #A0024) overnight at 4 °C, and then incubated with AlexaFluor™ 488-conjugated goat anti-mouse (Invitrogen #A32723) and Cy3-conjugated goat anti-rabbit (Jackson #111-545-144) secondary antibodies for 1 hour at room temperature. After secondary antibody incubation, cell nuclei were counterstained with DAPI (ThermoFisher #D1306) for 5 minutes at room temperature. Cells were imaged at a magnification of 320× using the following exposure conditions for each channel: phase contrast, 280 milliseconds; 488, 1100 milliseconds; CY3, 3000 milliseconds; DAPI, 358 milliseconds. Figure 43C shows representative staining 48 hours after treatment with 5 μM of PEN-N-hPEST, N-hPEST (non-cell permeant control), or vehicle control (PBS). (Scale bar 20 μm). The cell permeant anti-tau intrabodies (PEN-N-hPEST) significantly ( * p < 0.05, ** p < 0.01) reduced endogenous tau protein in human SHSY-5Y differentiated neurons compared to the non-cell permeant control (N-hPEST) and vehicle control, respectively (Figure 43D).

[0222] Other embodiments The present invention has been described in conjunction with its detailed description, but the foregoing description is for the purpose of illustrating the scope of the invention as defined by the appended claims and is not intended to be limiting. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

[Claim 1] From the N-terminus to the C-terminus, I. (a) Optional signal peptide domains; (b) Cell-permeable peptides; (c) Antigen-binding domain that binds to α-synuclein; and (d) Programmable proteasome-targeting human or mouse PEST domains; or II. (a) Optional signal peptide domains; (b) Programmable proteasome-targeting human or mouse PEST domains; (c) Antigen-binding domain that binds to α-synuclein; and (d) Cell-permeable peptides Recombinant polypeptides containing [specific components].