Pharmaceutical compositions and methods of treating protein aggregation disorders

Abstract

The disclosure relates to compositions, pharmaceutical compositions, methods, and kits pertaining to treatment of protein aggregate disorders.

Description

DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONPHARMACEUTICAL COMPOSITIONS AND METHODS OF TREATING PROTEIN AGGREGATION DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 714,846, which was filed October 31, 2024, and is titled “Pharmaceutical Compositions and Methods of Treating Protein Aggregation Disorders” and U.S. Provisional Application No. 63 / 773,716, which was filed March 18, 2025, and is titled “Pharmaceutical Compositions and Methods of Treating Protein Aggregation Disorders,” both of which are incorporated herein by reference in their entireties.SEQUENCE LISTING

[0002] The electronic sequence listing filed herewith, titled STFD-018-PCT_SL.xml, created on October 31, 2025, and having a file size of 625,373 bytes is incorporated herein by reference in its entirety.FIELD

[0003] The disclosure relates to compositions, pharmaceutical compositions, methods, and kits pertaining to treatment of protein aggregation disorders.BACKGROUND

[0004] Transcription factors (TFs) play a central role in decoding genomic information into context-dependent gene expression patterns1’3. They do so by binding to DNA in a sequencedependent manner via DNA binding domains (DBDs) and promoting transcriptional activation or repression through effector domains that recruit cofactors. Whereas DBDs are structured, effector domains often harbor poorly structured low complexity regions (LCRs) rich in glutamine, serine, or acidic residues4’7. Across the proteome, TFs are the most enriched in LCRs, including polyglutamine (polyQ) tracts and prion-like sequences8’12. Recently, much attention has focused on LCRs in nuclear condensates linked to gene regulation13’16. However, LCRs can also drive pathological protein aggregation. For example, polyQ tracts are implicated in at least nine heritable degenerative disorders17’19. In these diseases, CAG trinucleotide repeats that encode polyQ expand past a critical length threshold - 20-40Q (SEQ ID NO: 97) depending on the protein17’19- resulting in aggregation of the mutant protein and toxicity20 21. For example, Huntington’s disease (HD) isDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION caused by polyQ expansions in the Huntingtin (HTT) protein that exceed 35 glutamine residues. Longer expansions are associated with earlier ages of onset. Surprisingly, multiple human TFs harbor polyQ tracts that equal or exceed lengths associated with pathology but are not known to form toxic aggregates (FIG.1 A), raising the question of what mechanisms prevent them from doing so.

[0005] Among wild-type human proteins, the FOXP2 transcription factor harbors the longest consecutive polyQ sequence of 40 glutamines17,22(SEQ ID NO: 98) (FIG. 1A). FOXP2 is highly evolutionarily conserved and critical for cerebellar and lung development. It has also been implicated in traits unique to humans: speech and language23,24. Heterozygous FOXP2 mutations, first reported in the ‘KE family’ from the United Kingdom, are associated with verbal dyspraxia, a severe speech and language disorder. Affected KE family members share a single amino acid substitution in FOXP2’s Forkhead domain (R553H). Mice carrying this mutation display motor and ultrasonic vocalization defects, accompanied by abnormal synaptic plasticity in striatal and cerebellar neural circuits25,26. Multiple other FOXP2 mutations have since been described in families affected by speech deficits27,28. Some produce proteins that lack the Forkhead DBD entirely. Although FOXP2 has not been implicated in glutamine expansion diseases, nuclear FOXP2 aggregates were observed in a subset of neurons in mice carrying the KE family mutation26, suggesting that the Forkhead DBD might mask a natural aggregation propensity of the protein.

[0006] FOXP2 has also been suggested to play a role in the evolution of human speech and language23,29'31. Two of the three amino acid differences between human and mouse FOXP2, T303N and N325S, arose in the human lineage following its split from the chimpanzee. Introducing these human-specific substitutions into mouse FOXP2 enhances striatal synaptic plasticity and increases vocalization32'34, leading to the proposal that these substitutions have been relevant to the evolution of human speech. Recently, dissection of the individual substitutions highlighted the predominant role of N303 in mediating the phenotypic effects in striatal synaptic plasticity35. However, molecular understanding of these variants’ functional impact is lacking. Notably, they do not fall in any annotated functional domain within FOXP2 and are far from its DNA binding domain.SUMMARYDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0007] The disclosure relates to a composition comprising a bivalent molecule comprising an aggregate targeting domain and solubility domain. In some embodiments, the solubility domain comprises at least a portion of a DNA targeting moiety or an electronegative amino acid sequence with an isoelectric point of from about 1 to about 6.5. The bivalent molecule may further comprise a linker sequence positioned between the aggregate targeting domain and the solubility domain.

[0008] In some embodiments, the bivalent molecule may comprise an aggregate targeting domain that associates with amyloid protein with a KD of from about 20 nM to about 250 nM.

[0009] In some embodiments, the solubility domain comprises an electronegative amino acid sequence comprising at least a first amino acid sequence of no less than about 7 contiguous amino acid residues. The amino acid residues are aspartic acid, glutamic acid, or an electronegative, nonnatural derivative thereof. In some embodiments, the first amino acid sequence may comprise from about 4 or more contiguous amino acid residues.

[0010] The aggregate targeting domain may comprise an antibody or antigen binding fragment thereof. The aggregate targeting domain may be an antigen binding fragment chosen from: an Fab fragment, an Fv fragment, diabody, a scFv, a minibody, a triabody, or a BiTE. In some embodiments, the aggregate targeting domain is a chemical moiety of ThioflavinT, Congo red, a derivative thereof, tautomer thereof, or pharmaceutically acceptable salt thereof, fused to the solubility domain by a linker. In some embodiments, the solubility domain comprises a nucleic acid targeting moiety chosen from: a transcription factor or functional fragment thereof, a histone protein or functional variant thereof, SSO7D or a functional variant thereof, or an HMG domain or functional variant thereof. In some embodiments, the DNA targeting moiety is SSO7D or a functional variant thereof. The DNA targeting moiety may be SOX2 or a functional variant thereof and the aggregate targeting domain. In some aspects, the DNA targeting moiety may be MAFK or a functional variant thereof. In some embodiments, the DNA targeting moiety is chosen from SSO7D, MAFK, HMG domain, or a functional fragment of any of the foregoing, and the aggregate targeting domain comprises an antibody or antibody fragment thereof.

[0011] In some embodiments, the solubility domain comprises a nucleic acid targeting moiety may comprise Hoechst, DAPI, a derivative thereof, tautomer thereof, or pharmaceutically acceptable salt thereof.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0012] In some embodiments, the solubility domain comprises one of (i) a nonspecific DNA- binding domain; (ii) a phosphomimetic patch; or (iii) a negatively charged peptide comprising no less than about 7 negatively charged amino acid residues.

[0013] In some embodiments, the solubility domain may comprise an EvoPatch or a derivative thereof. In some embodiments, the phosphomimetic patch a component of one or all domains of the human FOXP2 protein. The aggregate targeting domain may be amyloid or a derivative thereof; or Htt or a functional variant thereof.

[0014] The bivalent molecule may have aKo relative to the aggregate protein to which it is capable of binding of from about 25 nM to about 250 nM. The bivalent molecule may have a KD relative to the aggregate protein to which it is capable of binding of from about 75 nM to about 160 nM. The bivalent molecule may have a Koff relative to the aggregate protein to which it is targeted from about 1 nM to about 999 nM. In some embodiments, the bivalent molecule has a KD relative to the aggregate protein to which it is capable of binding of no more than about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 450 nM, about 400 nM, about 350 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, about 75 nM, or about 50 nM.

[0015] In some embodiments, the bivalent molecule comprises a sequence from Table 12 or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or about 99% sequence identity relative to the sequence from Table 12. The disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of the composition of any composition as described above; and a pharmaceutically acceptable carrier. In one or more embodiments, one or more methods of treating or preventing a protein aggregate disorder in a subject in need thereof may comprise administering to the subject the pharmaceutical composition described above.

[0016] The protein aggregate disorder may be chosen from: Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Fabry disease, Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FED), Prion Diseases (e.g., Creutzfeldt- Jakob Disease), Multiple System Atrophy (MSA), Lewy Body Dementia, Chronic Traumatic Encephalopathy (CTE), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Spinocerebellar Ataxia (various types), Hereditary Spastic Paraplegia (HSP), Myotonic Dystrophy, Type II Diabetes, Familial Amyloid Polyneuropathy (FAP), Systemic Amyloidosis, Gaucher Disease, Cystic Fibrosis,DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONRetinitis Pigmentosa, Alkaptonuria, Menkes Disease, Wilson’s Disease, or Hereditary ATTR Amyloidosis.

[0017] In some embodiments, the pharmaceutical composition is administered by oral administration, parenteral administration, sublingual administration, transdermal administration, rectal administration, transmucosal administration, topical administration, inhalation, buccal administration, intrapleural administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intranasal administration, intrathecal administration, and intraarticular administration, intracranial administration or combinations thereof.

[0018] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the pharmaceutical composition is administered simultaneously with, prior to, or after administration of a neuromodulatory agent.

[0019] In some embodiments, the pharmaceutical composition is administered as a dose of from about 0.1 milligrams to about 10 milligrams. In some embodiments, the disclosure relates to a method of inhibiting polymerization or assembly of an aggregate protein in a cell comprising: (i) exposing the cell to a composition of any of the compositions described herein for a time sufficient to solubilize at least a portion of the aggregate protein. In some embodiments, the time sufficient to solubilize at least a portion of the aggregate protein is from about 1 second to about 60 seconds. In some embodiments, the step of exposing is performed in vitro. In some embodiments, the step of exposing is performed in vivo.

[0020] In some embodiments, the disclosure relates to a kit comprising (i) a first container comprising the composition of any of; or one or more of: (ii) an administration element, instructions for administration of the composition into a subject, or a cell; or, if the bivalent molecule is an amino acid sequence, (i) a first container nucleic acid sequence encoding the amino acid sequence; and (ii) a cell line. In some embodiments, the administration element is a syringe.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings certain embodiments. It is understood, however,DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0022] FIGS. 1A through 1H: show that FOXP2 depends on DNA-binding for solubility: (A) PolyQ tract length distribution in the human proteome. Lengths associated with neurodegenerative disease shaded. (B) FIG. IB shows a schematic representation of FOXP2 protein highlighting distinct domains (ZF=Zinc Finger, LZ= Leucine Zipper) and mutations of interest. FIG. IB discloses SEQ ID NOS 98-99, respectively, in order of appearance. (C) Representative fluorescence micrographs of HEK293 cells with stable expression of wild-type FOXP2 and two dyspraxia-associated mutants (R553H and R328X) fused to EGFP. Contours of nuclei denoted with white dashed lines. (D) FOXP2 dimer structure bound to DNA (PDB:2A07)41. Three contact sites (Y540, R553, and R583) with DNA are highlighted in the inset. (E) Luciferase reporter assay with FOXP2-VPR fusion proteins (wild-type, Y540D, R553H, or R583D). Error bars represent standard deviation of 3 technical replicates from N=3 independent transfections. / ?<0.0001 for all pairwise comparisons (Tukey HSD post hoc test) except Y540D vs. R553H (not significant). Global ANOVAp<5* 10s. (F) Representative fluorescence micrographs of DNA-contact mutants. (G) Frequency of assembly for wild-type and mutant FOXP2. Error bars represent standard deviation from >200 cells across 3-5 fields of view. p<0.0001 for all pairwise comparisons except Y540D vs. R553H (not significant; quasi-binomial glm model pairwise tests, Benjamini-Hochberg correction, overall ANOVA p<1055). (H) Probability of assembly as a function of mean EGFP fluorescence (protein concentration proxy) for wild-type FOXP2 and variants (mean±lSE shown; fit to logistic regression with mean GFP fluorescence and FOXP2 variant as predictors). All scalebars are 10pm.

[0023] FIGS. 2A through 2E show that FOXP2 depends on DNA-binding for solubility, related to FIGS. 1 A through 1H. (2A) SConfocal micrographs of endogenous FOXP2 immunofluorescence in Cranial Neural Crest Cells. (2B) Immunoblot of wild-type or mutant FOXP2-VPR fusion proteins and a B-actin loading control in lysates from cells used for luciferase assays in FIG. IE (normalized for total protein). (2C-D) FOXP2 R553H assembly depends on polyQ and Q-rich domains. C: Representative fluorescence micrographs of full-length FOXP2 R553H, an N- terminal 328 amino acid truncation, polyQ tract deletion (40Q (SEQ ID NO: 98) +10Q (SEQ ID NO: 99)), and deletion of the entire glutamine-rich region. D: Frequency of FOXP2 assembly in these cells; error bars are standard deviations from > 1500 cells across 2-4 fields of view; £><0.0001DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION(quasibinomial model, Benjamini-Hochberg adjusted; overall ANOVA / ;« I O22), not significant for R553H 329-715aa vs. R553H del Q-rich. (2E) Schematic for analysis to measure assembly formation vs. protein level in FIG. 1H. All scalebars are 10pm. FIG. 2C discloses “50Q” as SEQ ID NO: 130.

[0024] FIGS. 3 A through 3H show that diverse DNA binding domains restore the solubility of mutant FOXP2 and other aggregation-prone TFs. (A) Representative fluorescence micrographs of FOXP2 DNA-contact mutants fused to EGFP or SSO7D-EGFP. (B) Representative fluorescence micrographs of FOXP2 R553H fused to DBDs from SOX2, MAFK, or FOXA1. (C) Heatmap of anti-EGFP ChlP-seq peaks for FOXP2 variants fused to GFP (WT, R553H, R553H-SOX2, R553H-MAFK). Unique peaks for each fusion are shown from one of two replicates. (D) Sequences enriched in unique peaks from (C) correspond to FOXP2, SOX2 and MAFK motifs. P- value, Fisher’s exact test with Bonferroni correction. (E) GO-term enrichment for top 1% of human proteins ranked by Michelitsh-Weissman (MW)45score. Terms related to nucleic acid binding are darkly shaded. (F) Ranked MW score distribution across the human proteome. (G) Representative fluorescence micrographs of SOX5 (left) or SOX5 DNA binding mutant (right), fused to EGFP(upper panels) or SSO7D-EGFP (lower panels). (H) Representative fluorescence micrographs (24h post-transfection) of EGFP fusions to MEF2D (left) or its DNA binding mutant (right). All scalebars are 10pm.

[0025] FIGS. 4A through 4L show that diverse DNA binding domains restore the solubility of mutant FOXP2 and other aggregation-prone TFs, related to FIGS. 3 Athrough 3H. (4A) Probability of assembly as a function of mean GFP fluorescence for FOXP2 Y540D and R553H mutants and SSO7D fusions to these proteins. (4B) Frequency of cells with assemblies for different FOXP2 fusion proteins in cell populations from FIG. 3B. Error bars represent standard deviation from > 800 cells across three fields. O.OOOl (quasibinomial model, Benjamini-Hochberg adjusted, overall ANOVA p« 1022) for all comparisons except R553H-MAFK vs. R553H-SOX2 which is not significant. (4C) FOXP2 forms cytosolic puncta in yeast when expressed from a centromeric plasmid. SV40-NLS fusion confers nuclear localization and solubility. (4D) FOXP2-SV40-NLS DNA-binding mutants form nuclear puncta. (4E) Fusion to SSO7D resolves these nuclear puncta. (4F) Quantification of these micrographs. FOXP2 R553H forms significantly more puncta than all other variants, p<0.0001 (quasi-binomial model, overall p<10'47, Benjamini-Hochberg correction for pairwise comparisons); other differences non-significant. (4G) GO-term enrichment top 1% ofDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION human proteins (200) by NLLR / PLAAC score47. Dark bars denote terms linked to nucleic acid binding. (4H) Ranked distribution of the NLLR score for the human proteome highlighting FOXP2, MEF2D, and SOX5 as well as the aggregation-prone proteins FUS, PRNP, and TAU. (41) Quantification of cells with assemblies of SOX5, a DNA binding deficient mutant, and SSO7D fusions to both proteins. Error bars represent standard deviation of 350-1400 cells from 4-10 fields of view. p<0.Q003 for SOX5 DNA binding mutant relative to all other variants (quasibinomial model, pairwise contrasts tests with Benjamini -Hochberg correction, overall ANOVA p<10~16); other comparisons not significant. (4J) Quantification of cells with assemblies of MEF2D and a DNA binding deficient mutant. Error bars represent standard deviation of >400 cells from 3-4 fields of view; j><0.001 (quasibinomial ANOVA). (4K and L) Immunoblot of the indicated wildtype, DBD mutant, or SSO7D rescued proteins fused to EGFP and B-actin loading controls. Equal amounts of total protein amount were loaded in each lane.

[0026] FIGS. 5A through 5D show that DNA binding solubilizes pathogenic HTT fragment. (A) Representative fluorescence micrographs of a 91Q HTT exonl expansion fused to EGFP (top) or SSO7D-EGFP (bottom). (B) Frequency-detectable assemblies (of any size) in cells harboring these fusions. Error bars represent standard deviation from >1000 cells across 4 fields of view, £><0.004 (quasibinomial ANOVA). (C) Filter trap assay of lysates from cells expressing these fusions. SDS-treated (1%) lysates were trapped on either cellulose acetate (lower panel; SDS insoluble aggregate) or nitrocellulose membranes (upper panel, total protein). HTT was visualized using anti-GFP. (D) Dependence of solubilization on both polyQ length and DNA-binding affinity. Mean frequency of assembly is shown for the indicated HTT polyQ lengths and DBD fusions. Error bars represent standard deviation from 2-4 fields of view. Assembly correlates positively with polyQ length (p< 7>I Oy) and negatively with relative affinity ( / ><<! 016, quasibinomial ANOVA). All scalebars are 10pm. FIG. 5D discloses SEQ ID NOS 129, 98, 130-132, 129, 98, 130-132, 129, 98, and 130-132, respectively, in order of appearance.

[0027] FIGS. 6A through 6C show that DNA binding solubilizes pathogenic HTT fragment, related to FIGS. 5A through 5D. (6A) Immunoblot of HTT polyQ (91Q) (SEQ ID NO: 100), or SSO7D rescued proteins fused to EGFP as well as B-actin loading controls. Equal amounts of total protein were analyzed. (6B) Filter trap assay of lysates from cells expressing FOXP2 mutants. SDS-treated (1%) lysates were trapped on either cellulose acetate (lower panel; SDS insoluble aggregate) or nitrocellulose membranes (upper panel, total protein). FOXP2 was visualized usingDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION anti-FOXP2 antibody. (6C) Representative fluorescence micrographs of stable HEK293 lines expressing HTT-polyQ of different lengths (varying from 30 to 70Q (SEQ ID NO: 101)), fused to the wild-type SOX2 DBD or SOX2 DBD mutants (K87A or W51A) with 9.3- or 17-fold lower binding affinity to DNA. Quantified in FIG. 5D

[0028] FIGS. 7A through 7F illustrate that PolyQ assemblies dissolve in mitosis. (A) Confocal immunofluorescence micrographs of interphase and mitotic cells expressing FOXP2-3xHA. (B) As in (A) for FOXP2 R553H-3xHA. (C) As in (A) FOXP2 Y540D R553H-3xHA, where mutation of two DNA-contact residues results in cytoplasmic assemblies. (D) As in (C), cells expressing FOXP2 Y540D R553H-3xHA highlighted at different stages of mitosis (white arrow). (E) Representative fluorescence micrographs of FOXP2 l-328aa and HTT-polyQ(91) EGFP fusions in asynchronous cultures and mitotic arrest (24h nocodazole). (F) Mitotic dissolution timecourse for mutant FOXP2 and HTT-polyQ assemblies. See Star Methods for details. Means (circles) and standard deviations (whiskers) fit to a 4 parameter log-logistic model (solid lines); horizontal dashed lines - lower and upper asymptotes; vertical dashed lines and open circles - 50% dissolution time for each protein variant. P-values calculated with drc R package82. All scalebars 10pm.

[0029] FIGS. 8A through 8E show that PolyQ assemblies dissolve in mitosis, related to FIGS. 7A through 7F. (8 A) Confocal immunofluorescence micrographs of cells expressing untagged FOXP2 WT and R553H or Y540D R553H mutants. Protein visualized with FOXP2 antibody; DNA with DAPI. (8B) Fluorescence micrographs of mitotic HEK293 cells expressing stable FOXP2-GFP. DNA is stained with Hoechst. (8C) Immunoblot of FOXP2 variants in asynchronous and mitotic cell cultures. Equal total protein was analyzed with anti-HA (to visualize FOXP2) or anti-B-actin, as a loading control. (8D) Representative fluorescence micrographs of HEK293 cells with stable HTT-polyQ91-EGFP expression in asynchronous culture (left) or during mitotic arrest (right, nocodazole). (8E) Experimental schematic for quantifying mitotic dissolution of polyQ assemblies in FIG. 5F. Data for R553H-EGFP shown here. FIG. 8D discloses SEQ ID NO: 133.

[0030] FIGS. 9A through 9D show that FOXP2 is hyperphosphorylated in mitosis and phosphorylation on SP / TP sites is required for its release from chromatin. (A) FOXP2 schematic highlighting phosphorylation sites enriched in mitosis including 11 canonical CDK sites (SP / TP; markers above the schematic) as well as non-SP / TP sites (light gray under the schematic) concentrated in the ‘EVO patch’ (aa292-343) and ‘Pre-FHD patch’ (aa438-465). FIG. 9A discloses SEQ ID NOS 82 and 138, respectively, in order of appearance. (B) Mitotic timecourse aDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION‘phosphodead’ FOXP2 mutant in which the 11 SP / TP sites are mutated to alanine. Representative fluorescence micrograph of a GFP fusion to this FOXP2-11 A mutant during a mitotic timecourse. DNA visualized with Hoechst. FIG. 9B discloses SEQ ID NO: 148. (C) Similar to (B), but in cells expressing a FOXP2-11D / E phosphomimetic mutant fused to EGFP FIG. 9C discloses SEQ ID NO: 139. (D) Similar to (B) and (C), but in cells expressing the FOXP2 Forkhead domain (502- 587aa) fused to SV40NLS-EGFP. All scalebars are 10pm.

[0031] FIG. 10 shows that FOXP2 is hyperphosphorylated in mitosis and modification of SP / TP sites is required for release from chromatin, related to FIGS. 9A through 9D. Mitotically-enriched phosphosites detected by mass spectrometry, grouped into SP / TP and non-SP / TP sites within the two phospho-patches. To be noted, 6 / 11 of the CDK motif SP / TP phosphosites (followed by *) are located within the two phospho-patches. Two additional phosphosites S480 and T593 -although outside of the Pre-FHD patch- were included with the Pre-FHD subsequent mutations.

[0032] FIGS. 11A through UK show concerted function of FOXP2 phosphorylation sites in mitotic solubility. (A) FOXP2 pan-phosphodead mutant does not dissolve in mitosis. Quantification, data representation, and statistical analysis as in FIG. 7F. (B) Fluorescence micrographs of HEK293 cells with stable expression of the FOXP2 pan-phosphodead mutant, harboring the indicated alanine mutations in the 30 mitotic phosphosites and R553H. DNA stained with Hoechst. FIG. 11B discloses “AAAA” as SEQ ID NO: 148. (C) Fluorescence micrographs of interphase cells stably expressing FOXP2 R553H in which EVO patch phosphorylation sites mutated to alanine (left, phosphodead) or aspartic acid (right, phosphomimic). DNA stained with Hoechst. (D) Frequency of any detectable assembly (p<0.0053) and number of puncta per cell (p<2.2x 1016, zero-inflated Poisson regression). Error bars represent standard deviation from >100 cells across three fields of view. (E) Fluorescence micrographs of interphase cells stably expressing truncated FOXP2 (l-345aa) with phosphodead and phosphomimic ‘EVO patch’ mutants. (F) Frequency of any detectable assembly from 350-600 cells across 3 fields of view (p<0.0001; quasibinomial model, Benjamini -Hochberg adjusted, overall ANOVA p<l 06 I). Error bars represent standard deviation. (G) Fluorescence micrographs of interphase cells stably expressing HTT(91Q) fused to phosphodead and phosphomimic ‘EVO patch’ mutants. (H) Frequency of any detectable assembly from >1000 cells across 7 fields of view. For all comparisons / ?<0.0001 (quasibinomial regression, pairwise contrast test, Benjamini-Hochberg correction, overall ANOVA p<l 022) Error bars represent standard deviation. (I) Assembly of FOXP2 truncation variants inDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION which the sequence preceding the ‘EVO Patch’ (l-291aa) is fused to 14 negatively charged D / E residues. Quantification as in (D), (F), (H). Error bars represent standard deviation from >2500 cells across 3 fields of view. For all differences p<0.00003 (quasibinomial ANOVA). FIG. I ll discloses SEQ ID NOS 98-99, respectively, in order of appearance. (J) Assembly of HTT polyQ (91Q (SEQ ID NO: 100)) fused to 14 D / E residues. Quantification as in (D), (F), (H), (I). Error bars represent standard deviation from >2500 cells across 4-9 fields of view The. For all differences / ?<0.0000002 (quasibinomial ANOVA). (K) Filter trap assay: SDS-treated (1%) lysates from cells expressing different HTT mutants were trapped on either cellulose acetate (lower panel; SDS insoluble aggregate) or nitrocellulose membranes (upper panel, total protein). HTT was visualized using anti-GFP. All scalebars are 10pm.

[0033] FIGS. 12A through 12H show concerted function of FOXP2 phosphorylation sites in mitotic solubility, related to FIGS. 11 A through 11K. (12A) Left panel, fluorescence micrographs of mitotic cells (white arrows) stably expressing FOXP2 R553H SP / TP 11 A, showing complete (left) or partial (right) assembly dissolution. Right panel, schematic of the 11A ‘phosphodead’ mutant in which SP / TP are mutated to alanine in the R553H background. FIG. 12A discloses “AAAA” as SEQ ID NO: 148. (12B) Representative time-lapse images of asynchronous culture stably expressing FOXP2 R553H pan-phosphodead mutant. Assemblies of this mutant do not solubilize in mitotic cells (white arrows). (12C) Fluorescence micrographs of cells with stable expression of the pan-phosphodead mutant (wild-type FOXP2 background). This mutant does not dissociate from chromatin in mitotic cells (white arrows); DNA stained with Hoechst. (12D) Representative 30 min time-lapse of cells stably expressing FOXP2 R553H with either the “EVO patch” or the “Pre-FHD patch” phosphosites mutated to alanine. Mitotic cells denoted with white arrows. (12E) Phosphodead mutations on either patch, when combined with SP / TP site 11A mutant, prevent full solubilization of FOXP2. Upper panels, schematic representations of mutant FOXP2 proteins. Lower panel, quantification, data representation, and statistical analysis as in FIG. 7F. Mitotic asymptotic assembly score does not differ significantly for all R553H mutants does not differ significantly (compParm test from drc package)82but were significantly higher than for wild-type protein (t.test, ><0.011 for 11 A R553H mutant, stronger for other mutants. . FIG. 12E discloses “AAAA” as SEQ ID NO: 148 (12F) Summary table of phosphorylation mutants in the R553H (left) or wild-type FOXP2 (right) background and their assembly status in interphase and mitosis. (12G and 12H) Immunoblot analysis of the GFP tagged-HTT polyQ91, fused to EVODOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION patch (12G), or 14D / E charged peptide (12H). Equal total protein was loaded in each lane; EGFP and B-actin levels were analyzed by immunoblot. All scalebars are 10 pm.

[0034] FIGS. 13A through 13F show that human-specific amino acid substitutions in FOXP2 promote solubility. (A) FOXP2 protein schematic highlighting evolutionary divergent amino acid substitutions including two unique to humans (N3O3 and S325) in the ‘EVO patch. FIG. 13A discloses SEQ ID NOS 98-99, respectively, in order of appearance. (B) Luciferase reporter assays of wild-type FOXP2 from different species fused to VPR, with codon usage and polyQ length adjusted to match the human protein. Transactivation capacity was tested with a reporter containing a FOXP2 promoter motif (6x TGTTTAC (SEQ ID NO: 102)). Error bars represent standard deviation from multiple independent transfections. Differences between the presence and absence of the promoter motif are significant (ANOVA p< 4^ 1043), but those between FOXP2 variants are not (ANOVA p>0.7907). (C) Distribution of assembly scores in cells expressing human, human R553H, chimpanzee, and mouse FOXP2 constructs (human codon-optimized and adjusted to the human polyQ length. Relative assembly is defined per-cell as the logarithm of the coefficient of variation measured for 300-550 cells across 3 experiments. For all comparisons / K0.0001 except chimp vs. mouse, which is not significant (pairwise U-test, Benjamini -Hochberg correction, overall Kruskal-Wallis test p«l 0 "’). (D) Probability of assembly formation as a function of mean EGFP fluorescence (protein concentration proxy) for these variants (mean±lSE shown; fit to logistic regression with mean GFP fluorescence and FOXP2 variant as predictors). For all pairwise differences, p<0.001 (post hoc Benjamini-Hochberg correction). (E) Change at residue 303 is responsible for the majority of the increased solubility of the human FOXP2. Data presentation and analysis for the indicated FOXP2 variants as in (D). Ancestralizing mutation of the N303T decreases solubility (p<0.0001, logistic regression, post hoc Benjamini-Hochberg correction) whereas S325N had no significant effect within the precision of measurement. Additionally, N3O3T does not significantly differ from the chimpanzee wild-type protein. (F) Model for how DNA binding and mitotic phosphorylation govern solubility of polyQ proteins.

[0035] FIGS. 14A through 14F show that human-specific amino acid substitutions in FOXP2 promote solubility, related to FIGS. 13Athrough 13F. (14A) Amino acid sequences of the FOXP2 EVO patch (292-343aa) in human, chimpanzee, and mouse (polymorphisms at positions 12 and 34; N303, S325; SEQ ID NOS: 82, 83, and 84 from top to bottom). (14B) FOXP2 polyQ tract (40Q (SEQ ID NO: 98) + 10Q (SEQ ID NO: 99)) in human, chimpanzee, and mouse (SEQ IDDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONNOS: 85,86, and 87 from top to bottom). Note polymorphic +1 Q in chimpanzee and -IQ in mouse. (14C) Immunoblot analysis of FOXP2 sequences, adjusted for human codon usage and polyQ length (40 (SEQ ID NO: 98)+10 (SEQ ID NO: 99)), used in the luciferase assays in FIG.11C. Equal total protein; Beta-actin as loading control. (14D) Distribution of assembly scores in cells expressing FOXP2 proteins with native polyQ lengths (as shown in 12B). Mouse vs. chimp is not significant. For all others «0.0001 (pairwise U-tests, Benjamini Hochberg correction, overall Kruskal-Wallis p«\ O’16). FIG. 14D discloses SEQ ID NOS 98-99, 98-99, 134, 99, 98, and 135, respectively, in order of appearance. (14E) Probability of assembly vs. protein concentration for FOXP2 proteins with native polyQ lengths. Mouse vs. chimp is not significant (adjusted p=0.061 ). For all others / ?«0.0001 (pairwise U-tests, Benjamini Hochberg correction). FIG. 14E discloses SEQ ID NOS 134, 99, 98 and 135, respectively, in order of appearance. (14F) Distribution of assembly scores when human-specific residues in FOXP2 are mutated to their ancestral state. Wild-type human FOXP2 has a much lower median assembly ?«0.0001 (pairwise U-tests, Benjamini-Hochberg adjusted).

[0036] FIG. 15 illustrates yeast BY4741 expressing the human aggregation-prone protein TDP43 (non-polyQ) implicated in ALS. Fusion to DNA-binding domain SSO7D removes visible aggregates formed by TDP43 when expressed in yeast.

[0037] FIG. 16 shows that FOXP2 has a plethora of isoforms.

[0038] FIG. 17 shows cloning FOXP2 from different cell stages found many of the FOXP2 isoforms.

[0039] FIG. 18 shows that three exons appear frequently in these isoforms: named 3’, 4’, and 5’.

[0040] FIG. 19 shows that EXON3’ is abundant and introduces a stop codon. FIG. 19 discloses SEQ ID NOS 140-141, respectively, in order of appearance.

[0041] FIG. 20 shows that isoform 3’ solubilize WT FOXP2 with constitutive promoter.

[0042] FIG. 21 shows that isoform 3’ solubilize WT FOXP2 with inducible promoter.

[0043] FIG. 22 shows that isoform 3’ solubilize assembly forming FOXP2 in human cells (K562).

[0044] FIG. 23 shows that isoform 3’ can be one of three possibility: Peptide, RNA, or truncated protein. FIG. 23 discloses SEQ ID NO: 142.

[0045] FIG. 24 shows that isoform 3’ does not form a truncated protein when expressed in yeast i.e. It can be one of TWO possibility: Peptide, or RNA.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0046] FIG. 25 shows that isoform 3’ does not form a truncated protein when expressed in human cells i.e. It can be one of TWO possibility: Peptide, or RNA.

[0047] FIG. 26 shows FOXP2 (Exon 3’ isoform). FIG. 26 discloses SEQ ID NO: 142.

[0048] FIG. 27 shows FOXP2 (Exon 3’ isoform).

[0049] FIG. 28 shows representations querying whether the full isoform is needed, or if the N- terminal part until exon 3’ is enough.

[0050] FIG. 29 shows observation of soluble FOXP2 with both N-terminal or full-length isoform3’.

[0051] FIG. 30 shows querying whether the different potential start codons affect the solubility induced by isoform 3’.

[0052] FIG. 31 shows that FOXP2 WT is solubilized using RNA derived from N-terminal part of isoform 3’ when expressed.

[0053] FIG. 32 shows potential RNA structure of isoform 3’ promoting FOXP2 solubility. FIG. 32 discloses SEQ ID NOS 143-144, respectively, in order of appearance.

[0054] FIG. 33 shows different RNA of mutated isoform 3 ’ maintain a general structure promoting FOXP2 solubility. FIG. 33 discloses SEQ ID NOS 145-147, respectively, in order of appearance.

[0055] FIGS. 34Aand 34B validate disease protein solubilization across different disease proteins. FIG. 34 A shows FUS P525L-GFP, IAPP S20G-GFP, Abeta42-GFP, and TDP43 Q331K-GFP expression in S. cerevisiae. FIG. 34 B shows that fusion of SSO7D DNA-binding domain to the proteins in of FIG. 34A rescue the formation of visual puncta or aggregates.

[0056] FIGS. 35Athrough 35C validate solubilization of disease protein with peptides with charge in human cells.

[0057] FIGS. 36A through 36C validate solubilization of disease protein with peptides bridging to DNA in human cells.DETAILED DESCRIPTION

[0058] Certain terminology is used in the following description for convenience only and is not limiting. The words “right,” “left,” “top,” and “bottom” designate directions in the drawings to which reference is made.

[0059] In some embodiments, the disclosure relates to a therapeutic strategy for aggregate solubilization, based on bridging aggregating protein to DNA, charged molecules, phosphorylatedDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION moieties or kinases, either by utilizing small molecules or through genetic approaches. In some embodiments, this strategy produces cures or treats aggregation-associated diseases, including, but not limited to, a number of neurodegenerative diseases.Definitions

[0060] Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0061] As used herein, the terms “a” or “an” means that “at least one” or “one or more” unless the context clearly indicates otherwise. The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in various embodiments, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0062] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of’ “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0063] As used herein, the terms “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “having” (and any form of having, such as “have” and “has”),DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION“including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0064] As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% and remain within the scope of the disclosed embodiments.

[0065] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0066] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0067] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0068] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0069] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the bivalent molecules, compositions, or methods disclosed herein. In some embodiments of the disclosed methods, the subject has been diagnosed with a need for treatment of protein aggregate disorder such as, for example, a neurodegenerative disease or a prion disease prior to the administering step. As used herein, the phrase “identified to be in needDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder. It is contemplated that the identification can, in some embodiments, be performed by a person different from the person making the diagnosis. It is also contemplated, in further embodiments, that the administration can be performed by one who subsequently performed the administration.

[0070] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various embodiments, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. The terms “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection, and infusion. The terms “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient’s system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[0071] In some embodiments, the compound is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intracardiacly, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly, intraspinally and intrastemally, by injection, or by infusion.

[0072] The term “antibody” as used herein refers to a polypeptide or group of polypeptides that are comprised of at least one binding domain that is formed from the folding of polypeptide chainsDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen. An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one “light” and one “heavy” chain. The variable regions of each light / heavy chain pair form an antibody binding site. As used herein, a “targeted binding agent” is an antibody, or antigen binding fragment thereof, that preferentially binds or associates to a target site on or within an aggregate protein, such as those on Table X. In some embodiments, the targeted binding agent is specific for only one target site. In other embodiments, the targeted binding agent is specific for more than one target site. In some embodiments, the targeted binding agent may be a monoclonal antibody and the target site may be an epitope. “Epitope” refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of an antibody. “Binding fragments” of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab’, F(ab’)2, Fv, and single-chain antibodies. An antibody other than a “bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counter-receptor when an excess of antibody reduces the quantity of receptor bound to counter-receptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay). An antibody may be oligoclonal, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody and antibodies that can be labeled in soluble or bound form as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the disclosure; exemplary fragments include Fv, Fab, Fab’, single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulfide stabilized variable region (dsFv). As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the antibodies or immunoglobulin molecules described herein. In particular, conservative amino acidDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that have related side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non- polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are an aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding function or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function.

[0073] The term “contacting” as used herein refers to bringing a disclosed compound and a cell, target receptor, or other biological entity together in such a manner that the compound can affect the activity of the target (e.g., receptor, cell, etc.), either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.

[0074] As used herein the “aggregate targeting domain” is a chemical or chemical moiety, protein, polypeptide, peptoids, lipids, carbohydrate, nucleic acid, antibody or antigen binding fragment, or any combination of the foregoing that selectively binds or associates either to specific disease- associated aggregating proteins or protein aggregates. In some embodiments, chemical or chemical moiety, protein, polypeptide, peptoids, lipids, carbohydrate, nucleic acid, antibody or antigenDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION binding fragment, or any combination of the foregoing that selectively binds or associates to a monomeric, oligomeric and / or aggregate state (specific interactors), such as poly-glutamine sequences, or, more broadly, protein aggregates (general interactor).

[0075] As used herein the “solubility domain” is a chemical or chemical moiety, protein, polypeptide, peptoids, lipids, carbohydrate, nucleic acid, antibody or antigen binding fragment, or any combination of the foregoing that selectively binds or associates to DNA or has a net negative electrical charge sufficient to solubilize an aggregate or component of an aggregate to which the solubility domain is directly or indirectly bound.

[0076] As used herein, “IC50,” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In some embodiments, an IC50 can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein.

[0077] As used herein, “EC50,” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is results in a half-maximal response (i.e., 50% of the maximum response) of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In some embodiments, an EC50 can refer to the concentration of a substance that is required to achieve 50% of the maximum response in vivo, as further defined elsewhere herein.

[0078] The bivalent molecules according to this disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc., groups as the prodrug forming moieties. For instance, the hydroxymethyl position may form mono-, di- or triphosphates and again these phosphates can form prodrugs. Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander et al., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO 2000 / 041531, p. 30). The nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the disclosure.

[0079] “Derivatives” of the bivalent molecules disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, solvates and combinations thereof. The “combinations” mentioned in this context are refer to derivatives falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio- actively labeled forms, isomers, and solvates. Examples of radio-actively labeled forms includeDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION bivalent molecules labeled with tritium, phosphorous-32, iodine-129, carbon-11, fluorine-18, and the like.

[0080] The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include sulfonate esters, including triflate, mesylate, tosylate, brosylate, and halides.

[0081] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad embodiment, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain embodiments, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0082] In defining various terms, “Al,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0083] The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine (fluoro, F), chlorine (chloro, Cl), bromine (bromo, Br), and iodine (iodo, I).

[0084] The term “aliphatic” or “aliphatic group,” as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION carbon atoms. Aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0085] The term “alkyl,” as used herein, refers to a monovalent saturated, straight or branched chain hydrocarbon radical, having unless otherwise specified, 1-6 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n propyl, isopropyl, n butyl, iso butyl, sec butyl, n-pentyl, tert-pentyl, neopentyl, sec-pentyl, 3-pentyl, sec-isopentyl, hexyl, 2-methylpentane, 3 -methylpentane, 2,2-dimethylbutane, 2,3-dimentybutane and the like. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e g., from one to four) carbon atoms. The term alkyl group can also be a Cl alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, Cl- C9 alkyl, Cl -CIO alkyl, and the like up to and including a C1-C24 alkyl.

[0086] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in one instance and a specific term such as “hydroxyalkyl” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0087] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

[0088] The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0089] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0090] The term “heteroalkyl,” as used herein refers to an alkyl group containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0091] The term “haloalkyl” includes mono, poly, and perhaloalkyl groups where the halogens are independently selected from fluorine, chlorine, bromine, and iodine.

[0092] “Alkoxy” is an alkyl group which is attached to another moiety via an oxygen linker (- O(alkyl)). Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0093] “Haloalkoxy” is a haloalkyl group which is attached to another moiety via an oxygen atom such as, e.g., but are not limited to -OCHCF2 or -OCF3.

[0094] The term “9- to 10-membered carbocyclyl” means a 9- or 10- membered monocyclic, bicyclic (e.g., a bridged or spiro bicyclic ring), polycyclic (e.g., tricyclic), or fused hydrocarbon ring system that is saturated or partially unsaturated. The term “9- to 10-membered carbocyclyl” also includes saturated or partially unsaturated hydrocarbon rings that are fused to one or more aromatic or partically saturated hydrocarbon rings (e.g., dihydroindenyl and tetrahydronaphthalenyl). Bridged bicyclic cycloalkyl groups include, without limitation, bicyclo[4.3.1]decanyl and the like. Spiro bicyclic cycloalkyl groups include, e.g., spiro[3.6]decanyl, spiro[4.5]decanyl, spiro [4.4]nonyl and the like. Fused cycloalkyl rings include, e.g., decahydronaphthalenyl, dihydroindenyl, decahydroazulenyl, octahydroazulenyl, tetrahydronaphthalenyl, and the like. It will be understood that when specified, optional substituents on a carbocyclyl (e.g., in the case of an optionally substituted cycloalkyl) may be present on any substitutable position and, include, e.g., the position at which the carbocyclyl group is attached.

[0095] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbomyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. In various aspects, the cycloalkyl group and heterocycloalkyl group can be monocyclic, bicyclic (e.g., bridged such as, for example, bicyclo[4.3.1]decanyl or spiro such as, for example, spiro[3.6]decanyl, spiro[4.5]decanyl, spiro [4.4]nonyl), polycyclic (e.g., tricyclic), or a fused hydrocarbon ring system that is saturated or partially unsaturated (e.g., decahydronaphthalenyl, dihydroindenyl, decahydroazulenyl, octahydroazulenyl, tetrahydronaphthalenyl).DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0096] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbomenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0097] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0098] The terms “heterocycle” or “heterocyclyl,” as used herein can be used interchangeably and refer to single and multi -cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle,” and “polycyclic heterocycle.” The heterocycle can be monocyclic, bicyclic (e.g., spiro or bridged), polycyclic, or a fused system that is saturated or partially saturated. Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3- oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION thiadiazole, and 1,3,4-thiadiazole, triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazine, including 1,2,4- triazine and 1,3,5-triazine, tetrazine, including 1,2, 4, 5 -tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2 heterocyclyl, C2-C3 heterocyclyl, C2-C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2- C18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodiazetyl, oxiranyl, thiiranyl, and the like. Alternatively, for example, a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

[0099] The term “bicyclic heterocycle” or “bicyclic heterocyclyl,” as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with a non-aromatic ring. Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[l,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro- 1,4-benzodioxinyl, 3,4-dihydro-2H-chromenyl, lH-pyrazolo[4,3-c]pyridin-3-yl; lH-pyrrolo[3,2- b]pyridin-3-yl; and lH-pyrazolo[3,2-b]pyridin-3-yl.

[0100] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl,DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0101] The term “9-membered fused heterocyclyl” means a 9-membered saturated or partially unsaturated fused monocyclic heterocyclic ring comprising at least one oxygen heteroatom and optionally two to four additional heteroatoms independently selected from N, O, and S. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein. A heterocyclyl ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Examples of fused saturated or partially unsaturated heterocyclic radicals compristing at least one oxygen atom include, without limitation, dihydrobenzofuranyl, dihydrofuropyridinyl, octahydrobenzofuranyl, and the like. Where specified as being optionally substituted, substituents on a heterocyclyl (e.g., in the case of an optionally substituted heterocyclyl) may be present on any substitutable position and include, e.g., the position at which the heterocyclyl group is attached.

[0102] The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized 7i electrons above and below the plane of the molecule, where the it clouds contain (4n+2) 7i electrons. A further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.

[0103] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, — NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carbon-carbon bond. For example, biaryl can be two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0104] The term “heteroaryl,” as used herein refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include,DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl. Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo[d]oxazolyl, benzo[d]thiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[l,2-b]pyridazinyl, imidazo[l,2-a]pyrazinyl, benzo[c][l,2,5]thiadiazolyl, benzo[c][l,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0105] The term “5- or 6- membered heteroaryl” refers to a 5- or 6-membered aromatic radical containing 1-4 heteroatoms selected from N, O, and S. Nonlimiting examples include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, etc. When specified, optional substituents on a heteroaryl group may be present on any substitutable position and, include, e.g., the position at which the heteroaryl is attached.

[0106] The term “aldehyde” as used herein is represented by the formula — C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C=O.

[0107] The terms “amine” or “amino” as used herein are represented by the formula — NA1A2, where Al and A2 can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is -NH2.

[0108] The term “alkylamino” as used herein is represented by the formula — NH(-alkyl) where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentyl ami no group, (tert-pentyl)amino group, hexylamino group, and the like.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0109] The term “dialkylamino” as used herein is represented by the formula — N(-alkyl)2 where alkyl is a described herein. Representative examples include, but are not limited to, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N- methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

[0110] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

[0111] The term “ester” as used herein is represented by the formula — OC(O)A1 or — C(O)OA1, where Al can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula — (AlO(O)C-A2-C(O)O)a— or — (AlO(O)C-A2-OC(O))a— , where Al and A2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.

[0112] The term “ether” as used herein is represented by the formula A1OA2, where Al and A2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by the formula — (A10-A20)a — , where Al and A2 can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0113] As described herein, bivalent molecules of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. In is also contemplated that, in certain embodiments, unlessDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION expressly indicated to the contrary, individual substituents can be further optionally substituted (z.e., further substituted or unsubstituted).

[0114] In some embodiments, a structure of a compound can be represented by a formula:which is understood to be equivalent to a formula:wherein n is typically an integer. That is, R” is understood to represent five independent substituents, R"(a), R"(b), R”(c), R"(d), R"(e). In each such case, each of the five R" can be hydrogen or a recited substituent. By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance R”(a)is halogen, then R”(b)is not necessarily halogen in that instance.

[0115] In some yet further embodiments, a structure of a compound can be represented by a formula:

[0116] wherein Ry represents, for example, 0-2 independent substituents selected from Al, A2, and A3, which is understood to be equivalent to the groups of formulae: wherein Ryrepresents 0 independent substituentswherein Ryrepresents 1 independent substituentDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONwherein Ryrepresents 2 independent substituents

[0117] Again, by “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance Ryl is Al, then Ry2 is not necessarily Al in that instance.

[0118] In some further embodiments, a structure of a compound can be represented by a formula,wherein, for example, Q comprises three substituents independently selected from hydrogen andA, which is understood to be equivalent to a formula:

[0119] Again, by “independent substituents,” it is meant that each Q substituent is independently defined as hydrogen or A, which is understood to be equivalent to the groups of formulae:DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONwherein Q comprises three substituents independently selected from H and A

[0120] In some embodiments, the disclosed bivalent molecules exists as geometric isomers. “Geometric isomer” refers to isomers that differ in the orientation of substituent atoms in relationship to a cycloalkyl ring, i.e., cis or trans isomers. When a disclosed compound is named or depicted by structure without indicating a particular cis or trans geometric isomer form, it is to be understood that the name or structure encompasses one geometric isomer free of other geometric isomers, mixtures of geometric isomers, or mixtures enriched in one geometric isomer relative to its corresponding geometric isomer. When a particular geometric isomer is depicted, i.e., cis or trans, the depicted isomer is at least about 60%, 70%, 80%, 90%, 99%, or 99.9% by weight pure relative to the other geometric isomer.

[0121] The bivalent molecules described herein may be present in the form of pharmaceutically acceptable salts. For use in medicines, the salts of the bivalent molecules described herein refer to non-toxic “pharmaceutically acceptable salts.” As noted above, the bivalent molecules of the present disclosure can be administered, inter alia, as pharmaceutically acceptable salts, esters, amides or prodrugs. The term “salts” refers to inorganic and organic salts of bivalent molecules of the present disclosure. The salts can be prepared in situ during the final isolation and purification of a compound, or by separately reacting a purified compound in its free base or acid form with a suitable organic or inorganic base or acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, palmitiate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts, and the like. The salts may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium,DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, tri ethylamine, ethylamine, and the like. See, for example, S. M. Berge, et al., “Pharmaceutical Salts,” J Pharm Sci, 66: 1-19 (1977). Examples of pharmaceutically acceptable esters of the bivalent molecules of the present disclosure include C1-C8 alkyl esters. Acceptable esters also include C5-C7 cycloalkyl esters, as well as arylalkyl esters such as benzyl. C1-C4 alkyl esters are commonly used. Esters of bivalent molecules of the present disclosure may be prepared according to methods that are well known in the art. Examples of pharmaceutically acceptable amides of the bivalent molecules of the present disclosure include amides derived from ammonia, primary Cl- C8 alkyl amines, and secondary C1-C8 dialkyl amines. In the case of secondary amines, the amine may also be in the form of a 5 or 6 membered heterocycloalkyl group containing at least one nitrogen atom. Amides derived from ammonia, C1-C3 primary alkyl amines and C1-C2 dialkyl secondary amines are commonly used. Amides of the bivalent molecules of the present disclosure may be prepared according to methods well known to those skilled in the art.

[0122] Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the bivalent molecules described herein include e.g., salts of inorganic acids (such as hydrochloric acid, hydrobromic, phosphoric, nitric, and sulfuric acids) and of organic acids (such as, acetic acid, benzenesulfonic, benzoic, methanesulfonic, and p-toluenesulfonic acids). Examples of pharmaceutically acceptable base addition salts include e.g., sodium, potassium, calcium, ammonium, organic amino, or magnesium salt.

[0123] The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodiumDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION carboxymethylcellulose, poly acrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0124] As used herein, the phrase “pharmaceutically acceptable” means those bivalent molecules, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and animals. In some embodiments, “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0125] Disease, disorder, and condition are used interchangeably herein.

[0126] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a particular organism, or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to delay their recurrence.

[0127] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. The term “preventing” refers to preventing a disease, disorder, or condition from occurring in a human or an animal that may be predisposed to the disease, disorder and / or condition, but has not yet been diagnosed as having it; and / or inhibiting the disease, disorder, or condition, i.e., arresting its development.

[0128] The term “effective amount” or “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired result (e.g., that will elicit a biological or medical response of a subject e.g., a dosage of between 0.01 - 100 mg / kg body weight / day) or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms. InDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION some embodiments, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but it is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various embodiments, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0129] As used herein, the term “salt” refers to acid or base salts of the bivalent molecules used in the methods of the present disclosure. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

[0130] The terms “subject” and “patient” may be used interchangeably, and means a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). In some embodiments, the subject is a human in need of treatment. In some embodiments, “patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a compound or pharmaceutical composition, as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONIn some embodiments, a patient or subject is human. In some embodiments, the subject is a human diagnosed with or suspected of having an aggregate protein disorder.

[0131] The term “aggregate protein disorder” is a disease or disorder caused by aggregation of a molecule that is a subunit of a larger polymerized molecule that aggregates in a cell, tissue, or organ of a subject. In some embodiments, the aggregate protein disorder is Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Fabry disease, Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), Prion Diseases (e.g., Creutzfeldt-Jakob Disease), Multiple System Atrophy (MSA), Lewy Body Dementia, Chronic Traumatic Encephalopathy (CTE), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Spinocerebellar Ataxia (various types), Hereditary Spastic Paraplegia (HSP), Myotonic Dystrophy, Type II Diabetes, Familial Amyloid Polyneuropathy (FAP), Systemic Amyloidosis, Gaucher Disease, Cystic Fibrosis, Retinitis Pigmentosa, Alkaptonuria, Menkes Disease, Wilson’s Disease, or Hereditary ATTR Amyloidosis.

[0132] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g., a aggregate protein associated disease, a symptom associated with an aggregate protein disorder) means that the disease (e.g., aggregate protein disorder) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. For example, a symptom of a disease or condition associated with a aggregation may be a symptom that results (entirely or partially) from polymerization activity of one or more monomers, dimer, or trimer compounds. As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease. For example, a disease associated with aggregation, may be treated with an agent (e.g., compound as described herein) effective for increasing the solubility of the aggregate protein or one monomer, dimer or trimer thereof.

[0133] “Control” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects.

[0134] The terms “non-natural” amino acids means any amino acid with a structure that is not naturally encoded by a cell during translation. Many of the non-naturally encoded amino acids provided herein are commercially available, eg, from Sigma-Aldrich (St. Louis, Mo., USA),DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONNovabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, Mass., USA). Those that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of skill in the art. In some embodiments, the invention relates to a method of manufacturing a polypeptide analog in which the polypeptide analog is manufactured using a synthesis technique disclosed in the following references, which are incorporated herein by reference: For organic synthesis techniques, see, eg, Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). See, also, US Patent Application Publications 2003 / 0082575 and 2003 / 0108885, which is incorporated by reference herein. In addition to unnatural (or non-natural) amino acids that contain novel side chains, unnatural amino acids that may be suitable for use in the present invention also optionally understood modified backbone structures, including but not limited to, as illustrated by the structures of Formula II and III of US Patent Application Publication 2010-0048871, wherein Z typically includes OH, NH 2 , SH, NH-R’, or SR’; X and Y, which can be the same or different, typically understood amino acids as well as hydrogen. For example, unnatural amino acids of the invention optionally include substitutions in the amino or carboxyl group as illustrated by Formulas II and III. Unnatural amino acids of this type include, but are not limited to, a -hydroxy acids, a -thioacids, a-aminothiocarboxylates, including but not limited to, with side chains corresponding to the common twenty natural amino acids or unnatural side chains. In addition, substitutions at the a-carbon optionally include, but are not limited to, L, D, or a -a- disubstituted amino acids such as D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid , and the like. Other structural alternatives include cyclic amino acids, such as proline analogs as well as 3, 4, 6, 7, 8, and 9 membered ring proline analogs, P amino acids such as substituted P- alanine.

[0135] In some embodiments, the composition or pharmaceutical compositions of the disclosure comprise a bivalent molecule, wherein the bivalent molecule comprises an amino acid sequence based upon the fragments, polypeptides, and functional derivatives disclosed herein and wherein the bivalent molecule comprises at least one or a plurality of unnatural amino acid or non-natural amino acid, wherein the unnatural amino acids based on natural amino acids, such as tyrosine, glutamine, aspartic acid, phenylalanine, and the like, and are suitable for use in the presentDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosines, orthosubstituted tyrosines, and meta substituted tyrosines, where the substituted tyrosine understood, including but not limited to, a keto group (including but not limited to, an acetyl group ), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C 6 -C 20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, an alkynyl group or the like. In addition, multiply substituted aryl rings are also contemplated.

[0136] Glutamine analogs that may be suitable for use in the present invention include, but are not limited to, a. -hydroxy derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Example phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanines, ortho- substituted phenylalanines, and metasubstituted phenylalanines, where the substitute includes, including but not limited to, a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), a benzoyl, an alkynyl group, or the like. Specific examples of unnatural amino acids that may be suitable for use in the present invention include, but are not limited to, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phenylalanine, an 0-4-allyl-L-tyrosine, a 4-propyl-L- tyrosine, a tri-O-acetyl-GlcNAcP-serine, an L-Dopa, a fluorinated phenylalanine , an isopropyl-L- phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo- phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L- phenylalanine, and a p-propargyloxy-phenylalanine, and the like. Examples of structures of a variety of unnatural amino acids that may be suitable for use in the present invention are provided in, for example, W02002 / 085923, which is incorporated by reference in it entirety.Aggregation Targeting Domain

[0137] The disclosure relates to a bivalent molecule comprising an aggregation targeting domain. In some embodiments, a nucleic acid sequence encoding an amino acid sequence that is the bivalent molecule in the form of an amino acid sequence comprising an aggregation targeting domain. In some embodiments, the aggregation targeting domain comprises a molecule, amino acid sequence or chemical moiety that binds or associates with one or more molecules identifiedDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION in Table X or functional variants thereof. Tn some embodiments, the aggregation targeting domain comprises a functional fragment of an amino acid sequence of Table X that comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity to the amino acid sequence of Table X. In some embodiments, the aggregation targeting domain comprises a functional fragment of an amino acid sequence of Table X that comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity to the amino acid sequence of Table X. In some embodiments, the aggregation targeting domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations relative to the amino acid sequence of Table X. In some embodiments, the aggregation targeting domain is a truncation mutant of one or more of the amino acid sequences of Table X. In some embodiments, the aggregation domain is a functional variant of the amino acid sequences of Table X and comprises 10, 11, 12, 23, 24, 25, 25, 26, 27, 28, 29, 30 or more contiguous or non-contiguous amino acid sequences from the amino acid sequence.Table X. Aggregation ProteinDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0138] In some embodiments, the aggregation targeting domain is a component, functional variant or monomer of the aggregation protein itself, the modification of which initiates solubilization and / or depolymerization of the aggregate protein. In some embodiments, the bivalent molecule comprises a polypeptide or functional variant of the polypeptides from Table 1.Table 1.Constructs affecting the solubility through chargeDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0139] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 1 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1 . In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:2 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2. In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:3 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:3. In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:4 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NON. In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:5 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5. In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:6 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6. In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises SEQ ID NO: or a functional variant thereof comprising atDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:7.

[0140] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises a bivalent protein comprising SEQ ID NO: 8 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:8. In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:9 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:9. In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises SEQ ID NO: 10 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 10.

[0141] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO: 1 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.

[0142] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO:2 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.

[0143] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO:3 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:3, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0144] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO:4 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.

[0145] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO:5 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.

[0146] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO:6 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.

[0147] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO:7 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:7, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.

[0148] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO: 8 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:8, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0149] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO:9 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:9, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.

[0150] In some embodiments, the composition or pharmaceutical composition of the disclosure comprises a therapeutically effective amount of a bivalent protein comprising SEQ ID NO: 10 or a functional variant thereof or a functional fragment comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 10, wherein the functional fragment consist of no more than about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids in length.Solubility Domain

[0151] The disclosure relates to a bivalent molecule that comprises a solubility domain. In some embodiments, the solubility domain comprises a DNA binding domain. In some embodiments, the DNA binding domain encodes a naturally occurring peptide or is a functional variant of a naturally occurring polypeptide and comprises a Koff, Kon, or KD to DNA that is lower than the Koff, Kon, or KD of the naturally occurring polypeptide upon which the functional variant is based.

[0152] In some embodiments, the solubility domain comprises a contiguous number of negatively or positively charged amino acid residues that are sufficient to increase the solubility needed solubilize an aggregate protein. In some embodiments, the bivalent molecule disclosed herein comprises about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acids that are any order of either glutamic acid residues and / or aspartic acid residues or non-natural derivatives thereof. If the bivalent molecule comprises positively charge amino acids, in some embodiments, the bivalent molecule comprises a solubility domain comprising an enriched segment of positively charged natural or non-natural amino acids. In some embodiments, the bivalent molecule comprises from about 2 to about 40, from about 4 to about 40, from about 6 to about 40, from about 10 to about 40, from about 15 to about 40, from about 20 to about 40 positively charged contiguous or non-DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION contiguous amino acids. In some embodiments, the solubility domain comprises one or a combination of phosphorylated sites on an amnio acid sequence within the bivalent molecule, sich that the one or more phosphorylated sites contribute to the overall net positive or negative charge affecting solubility.

[0153] In some embodiments, the solubility domains of the disclosure comprise any sequence from Tables 2, 3, or 8, Part C, or functional variants thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the sequences disclosed in Tables 2, 3, or 8, Part C. In some embodiments, the functional variants of the sequences from Tables 2, 3, or 8, Part C comprises from about 1 to about 40 amino acid mutations resulting in one or a combination of: (i) a replacement of the amino acid disclosed to a non-antrual analog of the particular amino acid; (ii) an addition of one or more natural or non-natural amino acids in the sequence; (iii) a deletion of any of the amino acids in the sequence; (iv) a truncation of a series of contiguous amino acid sequences on the amino or carboxyl end of the amino acid sequence disclosed; (v) a modification of one or more side chains of the amino acids disclosed in the aforementioned tables; or (vi) replacement of S, T, and / or Y residues with R, K, D or E residues, such that the net charge of the molecule is altered to affect solubility of the molecule.

[0154] In some embodiments, the solubility domain comprises a functional variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the sequences disclosed in Tables 2, 3, or 8, Part C, but include replacement of from about 1 to about 25 S, T, or Y residues with R, K, D, or E residues, or mutation of certain residues to include from about 1 to about 25 phosphorylation sites or phosphorylation patches. In some embodiments,

[0155] The solubility domain comprises a DNA binding region chosen from any sequence disclosed herein or a functional variant thereof.

[0156] In some embodiments, the bivalent molecule of the disclosure comprises a solubility domain comprising one of the amino acids in Table 2 or Table 3, or a functional variant thereof. In some embodiments, the bivalent molecule of the disclosure is an amino acid sequence and is encoded by a DNA construct of Table 2. In some embodiments, the bivalent molecule comprises a tag, such as a fluorescent amino acid sequence or probe. In some embodiments, the probe is a sequence complementary to the nuclei acid sequence to which the DNA binding domain binds.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0157] In some embodiments, the solubility domain comprises a contiguous number of negatively charged amino acid residues that are sufficient to increase the solubility needed solubilize an aggregate protein. In some embodiments, the bivalent molecule disclosed herein comprises about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acids that are any order of either glutamic acid residues and / or aspartic acid residues or non-natural derivatives thereof. In some embodiments, the solubility domain comprises one or a combination of the following amino acid sequences: DDDD (SEQ ID NO: 108), EEEE (SEQ ID NO: 109), DEDE (SEQ ID NO: 110), EDED (SEQ ID NO: 111).

[0158] In some embodiments, the solubility domain confers a net electronegative charge to the bivalent molecule. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.9. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.8. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.8. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.8. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.7. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.6. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.5. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.4. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.3. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.2. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.1. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 5.9. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 5.8. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 5.7. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 5.6. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 5.5. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 1 to about 5.4. In some embodiments, the bivalent moleculeDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION comprises an isoelectric point (pT) of from about 1.0 to about 5.3. Tn some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.0 to about 6.9. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.0 to about 6.0.

[0159] In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.1 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.2 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.3 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.4 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.5 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.6 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.7 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.8 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 2.9 to about 6.0. In some embodiments, the bivalent molecule comprises an isoelectric point (pl) of from about 3.0 to about 6.5.

[0160] It is generally known how to calculate pl, but an algorithm for the calculation is found at: https: / / web.expasy.org / cgi-bin / compute_pi / pi_tool.cgi, the contents of which are herein incorporated by reference in its entirety.Table 2.Constructs affecting the solubility through DNA bindingDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONTable 3. Amino Acid Sequences with Solubility DomainDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0161] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 12 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 12.

[0162] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 13 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 13.

[0163] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 14 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 14.

[0164] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 15 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 15.

[0165] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 16 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 16.

[0166] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 17 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 17.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0167] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 18 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 18.

[0168] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO: 19 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 19.

[0169] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:20 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:20.

[0170] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:21 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:21.

[0171] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:22 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:22.

[0172] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:23 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:23.

[0173] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:24 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:25.

[0174] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:26 or aDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:26.

[0175] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:27 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:27.

[0176] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:28 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:28.

[0177] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:29 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:29.

[0178] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:30 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:30.

[0179] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:31 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:31.

[0180] In some embodiments, the bivalent molecule in the composition or pharmaceutical composition disclosed herein comprises of a bivalent protein comprising SEQ ID NO:32 or a functional variant thereof comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:32.

[0181] The disclosure also relates to a bivalent molecule comprising an aggregate targeting domain and a solubility domain, wherein the aggregate targeting domain is FOXP2 or a functional variant thereof and the solubility domain is a phosphaswitch domain, or a contiguous string of D or E residues. In some embodiments, the aggregate targeting domain is the DNA binding domain of FOXP2. In some embodiments, the DNA binding domain is a FOXP2 DNA binding domainDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION with a Koff, Kon, or KD relative to DNA lower than about 20 micromolar, 19 micromolar, 18 micromolar, 17 micromolar, 16 micromolar, 15 micromolar, 14 micromolar, 13 micromolar, 12 micromolar, 11 micromolar, 10 micromolar, 5 micromolar, 4 micromolar, 3 micromolar, 2 micromolar, or 1 micromolar. In some embodiments, the DNA binding domain comprises a FOXP2 DNA binding domain, SOX2 or SS07D with a Koff, Kon, or KD relative to DNA lower than about 1000 nanomolar, 950 nanomolar, 900 nanomolar, 850 nanomolar, 800 nanomolar, 750 nanomolar, 700 nanomolar, 650 nanomolar, 600 nanomolar, 550 nanomolar, 500 nanomolar, 250 nanomolar, 200 nanomolar, 150 nanomolar, 100 nanomolar, 75 nanomolar, 50 nanomolar, 25 nanomolar.

[0182] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 20 micrmolar. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 17 micromolar.

[0183] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 15 micromolar.

[0184] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 10 micromolar.

[0185] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 5 micromolar.

[0186] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 1 micromolar. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 950 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 900 nM.

[0187] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 850 nM.

[0188] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 700 nM.

[0189] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 600 nM. In some embodiments, the bivalentDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 500 nM.

[0190] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 400 nM.

[0191] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 300 nM.

[0192] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 200 nM.

[0193] In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 100 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 25 nM to about 500 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 25 nM to about 250 nM.

[0194] In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 10 micromolar. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 5 micromolar. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 1 micromolar. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 950 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 900 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 850 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 700 nM.

[0195] In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 600 nM. In some embodiments, the bivalent molecule comprises a DNA binding domainDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 500 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 400 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 300 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA from about 1 nM to about 200 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 1 nM to about 100 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 25 nM to about 500 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain with a Koff, Kon, or KD relative to DNA from about 25 nM to about 250 nM. In some embodiments, the bivalent molecule comprises a DNA binding domain encoding an amino acid with a Koff, Kon, or KD relative to its binding affinity to DNA of no more than about 10 micromolar, 5 micromolar, 1 micromolar, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300, nM, 250 nM, 200 nM, 150 nM, 100 nM, 50 nM or about 25 nM.

[0196] Methods of calculating the Koff, Kon, or KD are generally known but can be found at the following publications, which are incorporated by reference in their respective entireties:FOXP2MMQESATETISNSSMNQNGMSTLSSQLDAGSRDGRSSGDTSSEVSTVELLHLQQQQALQAARQLLLQQQTSGLKSPKSSDKQRPLQVPVSVAMMTPQVITPQQMQQILQQQVLSPQQLQALLQQQQAVMLQQQQLQEFYKKQQEQLHLQLLQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQHPGKQAKEQQQQQQQQQQLAAQQLVFQQQLLQMQQLQQQQHLLSLQRQGLISIPPGQAALPVQSLPQAGLSPAEIQQLWKEVTGVHSMEDNGIKHGGDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONLDLTTNNSSSTTSSNTSKASPPITHHSIVNGQSSVLSARRDSSSHEETGASHTLYGHGVCK WPGCESICEDFGQFLKHLNNEHALDDRSTAQCRVQMQVVQQLEIQLSKERERLQAMMT HLHMRPSEPKPSPKPLNLVSSVTMSKNMLETSPQSLPQTPTTPTAPVTPITQGPSVITPASV PNVGAIRRRHSDKYNIPMSSEIAPNYEFYKNADVRPPFTYATLIRQAIMESSDRQLTLNEI YSWFTRTFAYFRRNAATWKNAVRHNLSLHKCFVRVENVKGAVWTVDEVEYQKRRSQKI TGSPTLVKNIPTSLGYGAALNASLQAALAESSLPLLSNPGLINNASSGLLQAVHEDLNGSL DHIDSNGNSSPGCSPQPHIHSIHVKEEPVIAEDEDCPMSLVTTANHSPELEDDREIEEEPLS EDLE (SEQ ID NO: 112)

[0197] In some embodiments, the composition comprises a biovlaent molecule, comprising FOXP2 or a functional variant thereof that is no more than about 700 amino acids in length, no more than about 600 amino acids in length, no more than about 500 amino acids in length, no more than about 400 amino acids in length, no more than about 300 amino acids in length, no more than about 200 amino acids in length, no more than about 100 amino acids in length, no more than about 50 amino acids in length. In some embodiments, the bivalent molecule further comprises one or a plurality of phosphate patches or evo patches or a string of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 non-contiguous or contiguous amino acid or derivatives thereof comprising at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 E or D residues. In some embodiments, the functional variant comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to FOXP2.SOX2MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRGQR RKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRR KTKTLMKKDKYTLPGGLL APGGNSMASGVGVGAGLGAGVNQRMD S YAHMNGW SNG SYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSM S YSQQGTPGMALGSMGSVVKSEASS SPPVVTS S SHSRAPCQAGDLRDMISMYLPGAEVP EPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHM (SEQ ID NO: 25)

[0198] In some embodiments, the composition comprises a biovlaent molecule, comprising SOX2 or a functional variant thereof that is no more than about 700 amino acids in length, no more than about 600 amino acids in length, no more than about 500 amino acids in length, no more thanDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION about 400 amino acids in length, no more than about 300 amino acids in length, no more than about 200 amino acids in length, no more than about 100 amino acids in length, no more than about 50 amino acids in length. In some embodiments, the bivalent molecule further comprises one or a plurality of phosphate patches or evo patches or a string of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 non-contiguous or contiguous amino acid or derivatives thereof comprising at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 E or D residues. In some embodiments, the functional variant comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SOX2.HttMATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPPPQLPQPPPQAQ PLLPQPQPPPPPPPPPPGPAVAEEPLHRPKKEL S ATKKDRVNHCLTICENIVAQ S VRNSPEFQ KLLGIAMELFLLC SDD AESD VRMVADECLNKVIKALMD SNLPRLQLELYKEIKKNGAPR SLRAALWRFAELAHLVRPQKCRPYLVNLLPCLTRTSKRPEESVQETLAAAVPKIMASFGN FANDNEIKVLLKAFIANLKSSSPTIRRTAAGSAVSICQHSRRTQYFYSWLLNVLLGLLVPV EDEHSTLLILGVLLTLRYLVPLLQQQVKDTSLKGSFGVTRKEMEVSPSAEQLVQVYELTL HHTQHQDHNVVTGALELLQQLFRTPPPELLQTLTAVGGIGQLTAAKEESGGRSRSGSIVE LIAGGGSSCSPVLSRKQKGKVLLGEEEALEDDSESRSDVSSSALTASVKDEISGELAASSG VSTPGSAGHDIITEQPRSQHTLQADSVDLASCDLTSSATDGDEEDILSHSSSQVSAVPSDPA MDLNDGTQASSPISDSSQTTTEGPDSAVTPSDSSEIVLDGTDNQYLGLQIGQPQDEDEEAT GILPDEASEAFRNS SMALQQ AHLLKNMSHCRQP SD S S VDKF VLRDEATEPGDQENKPCR IKGDIGQSTDDDSAPLVHCVRLLSASFLLTGGKNVLVPDRDVRVSVKALALSCVGAAVAL HPESFF SKLYK VPLDTTEYPEEQ YVSDILNYIDHGDPQ VRGATAILCGTLIC SIL SRSRFHV GDWMGTIRTLTGNTF SLADCIPLLRKTLKDE S S VTCKL ACTAVRNC VMSLC S S S YSELGL QLIIDVLTLRNSSYWLVRTELLETLAEIDFRLVSFLEAKAENLHRGAHHYTGLLKLQERVL NNVVIHLLGDEDPRVRHVAAASLIRLVPKLFYKCDQGQADPVVAVARDQSSVYLKLLMH ETQPPSHFSVSTITRIYRGYNLLPSITDVTMENNLSRVIAAVSHELITSTTRALTFGCCEALC LLSTAFPVCIWSLGWHCGVPPLSASDESRKSCTVGMATMILTLLSSAWFPLDLSAHQDAL ILAGNLLAASAPKSLRSSWASEEEANPAATKQEEVWPALGDRALVPMVEQLFSHLLKVIN ICAHVLDDVAPGPAIKAALPSLTNPPSLSPIRRKGKEKEPGEQASVPLSPKKGSEASAASR QSDTSGPVTTSKSSSLGSFYHLPSYLKLHDVLKATHANYKVTLDLQNSTEKFGGFLRSALDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDVLSQTLELATLQDIGKCVEEILGYLKSCFSREPMMATVCVQQLLKTLFGTNLASQFDGL SSNPSKSQGRAQRLGSSSVRPGLYHYCFMAPYTHFTQALADASLRNMVQAEQENDTSG WFDVLQKVSTQLKTNLTSVTKNRADKNAIHNHIRLFEPLVIKALKQYTTTTCVQLQKQV LDLLAQLVQLRVNYCLLDSDQVFIGFVLKQFEYIEVGQFRESEAIIPNIFFFLVLLSYERYH SKQIIGIPKIIQLCDGIMASGRKAVTHAIPALQPIVHDLFVLRGTNKADAGKELETQKEVV VSMLLRLIQYHQVLEMFILVLQQCHKENEDKWKRLSRQIADIILPMLAKQQMHIDSHEA LGVLNTLFEILAPSSLRPVDMLLRSMFVTPNTMASVSTVQLWISGILAILRVLISQSTEDIV LSRIQELSFSPYLISCTVINRLRDGDSTSTLEEHSEGKQIKNLPEETFSRFLLQLVGILLEDIV TKQLKVEMSEQQHTFYCQELGTLLMCLIHIFKSGMFRRITAAATRLFRSDGCGGSFYTLD SLNLRARSMITTHPALVLLWCQILLLVNHTDYRWWAEVQQTPKRHSLSSTKLLSPQMSG EEEDSDLAAKLGMCNREIVRRGALILFCDYVCQNLHDSEHLTWLIVNHIQDLISLSHEPP VQDFISAVHRNSAASGLFIQAIQSRCENLSTPTMLKKTLQCLEGIHLSQSGAVLTLYVDRL LCTPFRVL A RM VD I LAC RRVEM LL A A N LQ S SMAQLPMEELNRIQEYLQ S SGLAQRHQRL YSLLDRFRLSTMQDSLSPSPPVSSHPLDGDGHVSLETVSPDKDWYVHLVKSQCWTRSDS ALLEGAELVNRIPAEDMNAFMMNSEFNLSLLAPCLSLGMSEISGGQKSALFEAAREVTL ARVSGTVQQLPAVHHVFQPELPAEPAAYWSKLNDLFGDAALYQSLPTLARALAQYLVVV SKLPSHLHLPPEKEKDIVKFVVATLEALSWHLIHEQIPLSLDLQAGLDCCCLALQLPGLWS VVS STEFVTHAC SLIYC VHFILEAVAVQPGEQLLSPERRTNTPKAISEEEEEVDPNTQNPKY ITAACEMVAEMVESLQSVLALGHKRNSGVPAFLTPLLRNIIISLARLPLVNSYTRVPPLVW KLGWSPKPGGDFGTAFPEIPVEFLQEKEVFKEFIYRINTLGWTSRTQFEETWATLLGVLVT QPLVMEQEESPPEEDTERTQINVLAVQAITSLVLSAMTVPVAGNPAVSCLEQQPRNKPLK A LDTRFGRK L SIIRGIVEQEIQ AMVSKRENIATHHLYQ AWDP VP SL SPATTGALISHEKLLL QINPERELGSMSYKLGQVSIHSVWLGNSITPLREEEWDEEEEEEADAPAPSSPPTSPVNSR KHRAGVDIHSCSQFLLELYSRWILPSSSARRTPAILISEVVRSLLVVSDLFTERNQFELMYV TLTELRRVHPSEDEILAQYLVPATCKAAAVLGMDKAVAEPVSRLLESTLRSSHLPSRVGAL HGVLYVLECDLLDDTAKQLIPVISDYLLSNLKGIAHCVNIHSQQHVLVMCATAFYLIENY PLD VGPEF S ASIIQMCGVML SGSEESTP SIIYHC ALRGLERLLL SEQL SRLD AESLVKL S VD RVNVHSPHRAMAALGLMLTCMYTGKEKVSPGRTSDPNPAAPDSESVIVAMERVSVLFDRIRKGFPCEARVVARILPQFLDDFFPPQDIMNKVIGEFLSNQQPYPQFMATVVYKVFQTLHS TGQSSMVRDWVMLSLSNFTQRAPVAMATWSLSCFFVSASTSPWVAAILPHVISRMGKLEDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONQVDVNLFCLVATDFYRHQIEEELDRRAFQSVLEVVAAPGSPYHRLLTCLRNVHKVTTC (SEQ ID NO: 113)

[0199] In some embodiments, the composition comprises a bivlaent molecule, comprising Htt or a functional variant thereof that is no more than about 3000 amino acids in length, no more than about 2000 amino acids in length, no more than about 1000 amino acids in length, no more than about 500 amino acids in length, no more than about 300 amino acids in length, no more than about 200 amino acids in length, no more than about 100 amino acids in length, no more than about 50 amino acids in length. In some embodiments, the bivalent molecule further comprises one or a plurality of phosphate patches or evo patches or a string of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 non-contiguous or contiguous amino acid or derivatives thereof comprising at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 E or D residues. In some embodiments, the functional variant comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to Htt.TDP43MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGIL HAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQ DLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDC KLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFA DDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGG GAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPS GNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDS KSSGWGM (SEQ ID NO: 114)

[0200] In some embodiments, the composition comprises a biovlaent molecule, comprising TDP43 or a functional variant thereof that is no more than about 400 amino acids in length, no more than about 300 amino acids in length, no more than about 200 amino acids in length, no more than about 250 amino acids in length, no more than about 200 amino acids in length, no more than about 150 amino acids in length, no more than about 100 amino acids in length, no more than about 50 amino acids in length. In some embodiments, the bivalent molecule further comprises one or a plurality of phosphate patches or evo patches or a string of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 non-contiguous or contiguous amino acid or derivatives thereof comprisingDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION at least about 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 E or D residues. In some embodiments, the functional variant comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to TDP43.

[0201] In some embodiments, the composition comprises a bivalent molecule, comprising an amino acid sequence of Table Y or a functional variant thereof that is no more than about 700 amino acids in length, no more than about 600 amino acids in length, no more than about 500 amino acids in length, no more than about 400 amino acids in length, no more than about 300 amino acids in length, no more than about 200 amino acids in length, no more than about 100 amino acids in length, no more than about 50 amino acids in length. In some embodiments, the bivalent molecule further comprises one or a plurality of phosphate patches or evo patches or a string of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 non-contiguous or contiguous amino acid or derivatives thereof comprising at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 E or D residues. In some embodiments, the functional variant comprises at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the respective amino acid sequence of Table Y. In some embodiments, the one or a plurality of phosphate patches or evo patches comprises the EVO patch of FOXP2 with one or more of the following mutations: T292, T293, S296, S297, S298, T299, T300, S301, S302, T304, S305, S308, S312, S315, S321, S322, S325, S330, S331, S332, T336, S339, T341, and Y343. In some embodiments, the one or a plurality of phosphate patches or evo patches comprises the EVO patch of FOXP2 with all of the following mutations: T292, T293, S296, S297, S298, T299, T300, S301, S302, T304, S305, S308, S312, S315, S321, S322, S325, S330, S331, S332, T336, S339, T341, and Y343.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0202] Further non-limiting embodiments of the DNA binding domain of the bivalent molecule include the following DNA binding proteins or functional variants thereof which may be modified to include an evo patch, string of E or D residues disclosed herein, and / or low Koff rate in respect to DNA. The embodiments are hits of a search of sequences that could include:DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONMAMLQ9NSY1 2 BMP2K Isoform 2 of BMP-2-inducible protein kinase OS=Homo sapiens GN=BMP2KQ 14686 NCOA6 Nuclear receptor coactivator 6 OS=Homo sapiens GN=NCOA6 PE=1 SV=3Q10571 MN1 Transcriptional activator MN1 OS=Homo sapiens GN=MN1 PE=1 SV=30949162 NFAT5 Isofonn A of Nuclear factor of activated T-cells 5 OS=Homo sapiens GN=NFAT5Q96L912 EP400 Isofonn 2 of El A-binding protein p400 OS=Homo sapiens GN=EP400MAMLQ96JK9 3 Mastermind-like protein 3 OS=Homo sapiens GN=MAML3 PE=1 SV=4P515312 SMCA2 Isofonn Short of Probable global transcription activator SNF2L2 OS=Homo sapiens GN=SMARCA2Q9UGI6 KCNN3 Small conductance calcium-activated potassium channel protein 3 OS=Homo sapiens GN=KCNN3 PE=2 SV=1 Q9Y6Q9Q9ULV3 2 CIZ1 Isofonn 2 of Cipl-interacting zinc finger protein OS=IIomo sapiens GN=CIZ1Q8NDV7 5 TNR6A Isofonn 5 of Trinucleotide repeat-containing gene 6A protein OS=Homo sapiens GN=TNRC6AQ96PN72 TREF1 Isofonn 2 of Transcriptional-regulating factor 1 OS=Homo sapiens GN=TRERF10147762 TCRG1 Isofonn 2 of Transcription elongation regulator 1 OS=Homo sapiens GN=TCERG1P78364 PHC1 Polyhomeotic-like protein 1 OS=Homo sapiens GN=PHC1 PE=1 SV=3P784242 PO6F2 Isofonn 2 of POU domain, class 6, transcription factor 2 OS=Homo sapiens GN=POU6F2Q86YW9DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONQ9UPQ9 TNR6B Isofonn 2 of Trinucleotide repeat-containing gene 6B protein OS=IIomo sapiens GN=TNRC6BQ3ZLR7 SP201 Transcription factor SPT20 homolog-like 1 OS=Homo sapiens GN=SUPT2OHL1 PE=2 SV=2Q96MM7 2 H6ST2 Isofonn 2 of Heparan-sulfate 6-O-sulfotransferase 2 OS=Homo sapiens GN=HS6ST2Q6Y7W6Q8NFD5 2 ARI1B Isoform 2 of AT-rich interactive domain-containing protein IB OS=Homo sapiens GN=ARID1BP42694 HELZ Probable helicase with zinc finger domain OS=Homo sapiens GN=HELZ PE=1 SV=2CLOC015516 K Circadian locomoter output cycles protein kaput OS=Homo sapiens GN=CLOCK PE=1 SV=1Q6PJG2 EMSA1 ELM2 and SANT domain-containing protein 1 OS=Homo sapiens GN=ELMSAN1 PE=1 SV=2Q2TAL8 QRIC1 Glutamine-rich protein 1 OS=Homo sapiens GN=QRICH1 PE=1 SV=1Q9UGU0Q9UQL6 2 HDAC5 Isofonn 2 of Histone deacetylase 5 OS=Homo sapiens GN=HDAC5Q7Z2Y52 NRK Isofonn 2 of Nik-related protein kinase OS=Homo sapiens GN=NRKQ92804 RBP56 TATA-binding protein-associated factor 2N OS=Homo sapiens GN=TAF15 PE=1 SV=1Q155322 SSXT Isofonn 2 of Protein SSXT OS=Homo sapiens GN=SS18DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONP314832 TIAl Isofonn Short of Nucleolysin TIA-1 isoform p40 OS=IIomo sapiens GN=TIA1Q148142 MEF2D Isofonn MEF2DA’B of Myocyte-specific enhancer factor 2D OS=Homo sapiens GN=MEF2DQ4VCS5 AMOT Angiomotin OS=Homo sapiens GN=AMOT PE=1 SV=1P20265 PO3F2 POU domain, class 3, transcription factor 2 OS=Homo sapiens GN=POU3F2 PE=1 SV=4Q139503 RUNX2 Isofonn 3 of Runt-related transcription factor 2 OS=Homo sapiens GN=RUNX2Q86XK2 4 FBX11 Isofonn 4 of F-box only protein 11 OS=Homo sapiens GN=FBXO11Q9H1B7 I2BPL Interferon regulator - factor 2-binding protein-like OS=Homo sapiens GN=IRF2BPL PE=1 SV=1Q9UPA5 BSN Protein bassoon OS=Homo sapiens GN=BSN PE=1 SV=4MAMLNANGQ8N7R0 2 Putative homeobox protein NANOG2 OS=Homo sapiens GN=NANOGP1 PE=5 SV=1Q8IWX8 CHERP Calcium homeostasis endoplasmic reticulum protein OS=Homo sapiens GN=CHERP PE=1 SV=3P35869 AHR Aryl hydrocarbon receptor OS=Homo sapiens GN=AHR PE=1 SV=2Q8N4C82 MINK1 Isofonn 1 of Misshapen-like kinase 1 OS=Homo sapiens GN=MINK1Q96JA42 M4A14 Isofonn 2 of Membrane-spanning 4-domains subfamily A member 14 OS=Homo sapiens GN=MS4A14Q9UKV0 0 HDAC9 Isoform 10 of Histone deacetylase 9 OS=Homo sapiens GN=HDAC9Q3L8U12 CHD9 Isofonn 2 of Chromodomain-helicase-DNA-binding protein 9 OS=Homo sapiens GN=CHD9Q9P0K82 FOXJ2 Isofonn FOXJ2.S of Forkhead box protein .12 OS=Homo sapiens GN=FOXJ2 Q9Y2K5 2 R3HD2 Isofonn 2 of R3H domain-containing protein 2 OS=Homo sapiens GN=R3HDM2P42858 HD Huntingtin OS=Homo sapiens GN=HTT PE=1 SV=2Q9UBL0 3 ARP21 Isofonn 3 of cAMP-regulated phosphoprotein 21 OS=Homo sapiens GN=ARPP21Q150322 R3HD1 Isofonn 2 of R3H domain-containing protein 1 OS=Homo sapiens GN=R3HDM1Q9H2F5 EPCI Enhancer of polycomb homolog 1 OS=Homo sapiens GN=EPC1 PE=1 SV=1Q9NQV6 PRD10 Isofonn 1 of PR domain zinc finger protein 10 OS=Homo sapiens GN=PRDM10Q9ULU4 0 PKCB1 Isofonn 10 of Protein kinase C-binding protein 1 OS-Homo sapiens GN-ZMYND8075909 CCNK Isofonn 3 of Cyclin-K OS=Homo sapiens GN=CCNKP526552 TF2AA Isofonn 37 kDa of Transcription initiation factor IIA subunit 1 OS=Homo sapiens GN=GTF2A1Q9Y6V0 2 PCLO Isofonn 2 of Protein piccolo OS=Homo sapiens GN=PCLOQ9UN79 SOX13 Transcription factor SOX-13 OS=Homo sapiens GN=SOX13 PE=1 SV=3AMOLQ8IY632 1 Isofonn 2 of Angiomotin-like protein 1 OS=Homo sapiens GN=AMOTL1Isofonn 2 of Membrane-associated guanylate kinase, W W and PDZ domain-containing protein 1 OS=Homo sapiensQ96QZ72 MAGI1 GN=MAGHP357112 SOX5 Isofonn 2 of Transcription factor SOX-5 OS=Homo sapiens GN=SOX5Q8IV76 PASD1 Circadian clock protein PASD1 OS=Homo sapiens GN=PASD1 PE=1 SV=1P54259 ATN1 Atrophin-1 OS=Homo sapiens GN=ATN1 PE=1 SV=3Q6N021 TET2 Methylcytosine dioxygenase TET2 OS=Homo sapiens GN=TET2 PE=1 SV=3Q6KC792 NIPBL Isofonn 2 of Nipped-B-like protein OS=Homo sapiens GN=NIPBLQ020782 MEF2A Isofonn MEFA of Myocyte-specific enhancer factor 2A OS=Homo sapiens GN=MEF2AQ68CP93 ARID2 Isofonn 2 of AT-rich interactive domain-containing protein 2 OS=Homo sapiens GN=ARID2Q144442 CAPRI Isofonn 2 of Caprin-1 OS=IIomo sapiens GN=CAPRIN1DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONQ9Y2K2 2 SIK3 Isoform 2 of Serine / threonine-protein kinase SIK3 OS=Homo sapiens GN=SIK3MAMDQ134953 1 Isofonn 2 of Mastermind-like domain-containing protein 1 OS=Homo sapiens GN=MAMLD1Q01826 SATB1 DNA-binding protein SATB1 OS=Homo sapiens GN=SATB1 PE=1 SV=1 Q9UPN9 2 TRI33 Isoform Beta of E3 ubiquitin-protein ligase TRIM33 OS=Homo sapiens GN=TRIM33P55197 AF10 Protein AF-10 OS=Homo sapiens GN=MLLT10 PE=1 SV=2Q96BD52 PF21A Isofonn 2 of PHD finger protein 21A OS=Homo sapiens GN=PHF21AQ127722 SRBP2 Isofonn 2 of Sterol regulatory element-binding protein 2 OS=Homo sapiens GN=SREBF2Q96PP8 GBP5 Guanylate-binding protein 5 OS=Homo sapiens GN=GBP5 PE=1 SV=1GOGAQ083792 2 Isofonn 2 of Golgin subfamily A member 2 OS=Homo sapiens GN=GOLGA2000461 GOLI4 Golgi integral membrane protein 4 OS=Homo sapiens GN=GOLIM4 PE=1 SV=1P07476 INVO Involucrin OS=Homo sapiens GN=IVL PE=1 SV=2Q9BUJ22 HNRL1 Isofonn 2 of Heterogeneous nuclear ribonucleoprotein LT-like protein 1 OS=Homo sapiens GN=HNRNPUL1 Q9Y3Y4 2 PYGO1 Isofonn 2 of Pygopus homolog 1 OS=Homo sapiens GN=PYGO10433902 HNRPR Isofonn 2 of Heterogeneous nuclear ribonucleoprotein R OS=Homo sapiens GN=HNRNPRWWTRQ9GZV5 1 WW domain-containing transcription regulator protein 1 <)S Homo sapiens GN_WWTR1 PE_1 SV_1060885 BRD4 Bromodomain-containing protein 4 OS=Homo sapiens GN=BRD4 PE=1 SV=2Q9ULH7 4 MKL2 Isofonn 4 of MKL / inyocardin-like protein 2 OS=Homo sapiens GN=MKL2Q8IX12 CCAR1 Cell division cycle and apoptosis regulator protein 1 OS=Homo sapiens GN=CCAR1 PE=1 SV=2Q9H2G4 TSYL2 Testis-specific Y-encoded-like protein 2 OS=Homo sapiens GN=TSPYL2 PE=1 SV=1NANOQ9H9S0 G Homeobox protein NANOG OS=Homo sapiens GN=NANOG PE=1 SV=2Q9NPJ4 PNRC2 Proline-rich nuclear receptor coactivator 2 OS=Homo sapiens GN=PNRC2 PE=1 SV=1 Q8NEM7 2 SP20H Isofonn 2 of Transcription factor SPT20 homolog OS=Homo sapiens GN=SUPT20HQ7Z5892 EMSY Isofonn 2 of BRCA2-interacting transcriptional repressor EMSY OS=Homo sapiens GN=EMSYQ9HCK8 2 CHD8 Isofonn 2 of Chromodomain-helicase-DNA-binding protein 8 OS=Homo sapiens GN=CHD8P19484 TFEB Transcription factor EB OS=Homo sapiens GN=TFEB PE=1 SV=3P0C7V6 SP202 Putative transcription factor SPT20 homolog-like 2 OS=Homo sapiens GN=SUPT20HL2 PE=5 SV=10949672 WDR47 Isofonn 2 of WD repeat-containing protein 47 OS=Homo sapiens GN=WDR47P357122 SOX6 Isofonn 2 of Transcription factor SOX-6 OS=Homo sapiens GN=SOX6Q96KQ4 ASPP1 Apoptosis-stimulating of p53 protein 1 OS=Homo sapiens GN=PPP1R13B PE=1 SV=3Q9P2E92 RRBP1 Isofonn 1 of Ribosome-binding protein 1 OS=Homo sapiens GN=RRBP1NUMAQ149802 1 Isofonn 2 of Nuclear mitotic apparatus protein 1 OS-Homo sapiens GN-NUMA1Q15075 EEA1 Early endosome antigen 1 OS=Homo sapiens GN=EEA1 PE=1 SV=2P356372 FUS Isofonn Short of RN A-binding protein FUS OS=Homo sapiens (iX ITSQ131484 TADBP Isofonn 2 of TAR DNA-binding protein 43 OS=Homo sapiens GN=TARDBPQ9P2D12 CHD7 Isofonn 2 of Chromodomain-helicase-DNA-binding protein 7 OS=Homo sapiens GN=CHD7Q8IZD22 KMT2E Isofonn 2 of Histone-lysine N-methyltransferase 2E OS=Homo sapiens GN=KMT2EQ997002 ATX2 Isofonn 2 of Ataxin-2 OS=Homo sapiens GN=ATXN2Q928962 GSLG1 Isofonn 2 of Golgi apparatus protein 1 OS=Homo sapiens GN=GLG1043765 SGTA Small glutamine-rich tetratricopeptide repeat-containing protein alpha OS=Homo sapiens GN=SGTA PE=1 SV=1DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0203] An aggregating disease protein, or the aggregating domains thereof, may form a part of a bivalent molecule herein. An aggregating protein comprising a poly rich region or polyQ sequence may form a part of a bivalent molecule herein. In some embodiments, the protein is selected from the Table below.

[0204] In some embodiments, the protein is fused to a solubilizing moiety or solubilization domain as described herein. The solubilizing moiety (or solubility domain, used interchangeably), in some embodiments, can be a DNA targeting moiety, a transcription factor, a peptide comprising phosphorylation sites or phosphorylation patches, an EVO patch, or the like. In some embodiments, the solubilization domain comprises an EVO patch of FOXP2 or a functional variant thereof having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% sequence identity to the EVO patch pf FOXP2. In some embodiments, the solubilization domain comprises the EVO patch of FOXP2 with one or more of the following mutations: T292, T293, S296, S297, S298, T299, T300, S301, S302, T304, S3O5, S308, S312,DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONS315, S321, S322, S325, S330, S331, S332, T336, S339, T341, and Y343. In some embodiments, the solubilization domain comprises the EVO patch of FOXP2 with all of the following mutations: T292, T293, S296, S297, S298, T299, T300, S301, S302, T304, S3O5, S308, S312, S315, S321, S322, S325, S330, S331, S332, T336, S339, T341, and Y343. Further non-limiting embodiments of the bivalent molecule include the following DNA binding proteins or functional variants thereof which may be modified to include an evo patch, string of E or D residues disclosed herein, or low Koff rate in respect to DNA. The embodiments are hits of a search of sequences that could includeProtein DiseaseFUS ALSFUS P525L ALSFUS G156E ALSIAPP human Wt mature peptide Diabetes typellIAPP Rat no agg .mature peptide Diabetes typellIAPP human S20G more agg. mature peptide Diabetes typellAbeta42 human ADAbeta42 human E22G arctic mutant more agg ADAbeta42 human L17P artificial mutation slower agg ADTDP43 ALSTDP43 Q331KMutant ALSTAU WT ADTAU T212E, R406W, T23 IE, S262E ADTAU double T212E, R406W ADSNCAA53T PDSNCAA30P PDSNCAWt PD

[0205] In some embodiments, a DNA binding domain herein is selected from DNA binding domains of the proteins of Table 12 or functional variants thereof. In some embodiments, aggregation domains are selected from aggregation domains of the proteins of Table 12 orDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION functional variants thereof comprising at least about 75% sequence identity to the proteins of Table 12.Linkers

[0206] In some embodiments, the bivalent molecule comprises a linker that bridges the aggregate targeting domain and the solubility domain. In some embodiments, the linker comprises a linker of Table 4 or a functional variant thereof comprising at least about 75% sequence identity to the proteins of Table 4.Table 4. LinkersDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONTable 5* Minimum construct that decreased aggregation had 4 negative charges ( Construct 9)Table 6DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0207] In some embodiments, a characteristic feature of solubilizing tags is the DNA binding strength, which can be expressed as kD, kOn, or kOFF.

[0208] FOXP2 DNA binding domain kD (just the binding domain, not full protein) was measured in Thulo, M. et al. “The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function” (2021) Biosci. Rep. 41(1): BSR20202128. Thulo et al. reported the FOXP2 DNA bind domain kD to be from 1745.7 ± 15.9 nM to 1904.0 ± 25.1 nM.

[0209] SOX2 kD was measured in Yang, W.C. and Swartz, J.R. “A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA” (2011) Anal. Biochem. 415(2): 168-74. Yang and Swarts reported the SOX2 kD to be 54.2±9nM.

[0210] SSO7D kD was measure in Kalichuk, V et al. “The archaeal ‘7 kDa DNA-binding’ proteins: extended characterization of an old gifted family” (2016) Sci. Rep. 6: 37274. Kalichuk et al found the SSO7D kD to be 17 pM. While Baumann H. et al. “Solution structure and DNA- binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus” (1994) Nat. Struct. Biol. 1(11): 808-19, reported the SSO7D kD to be <10 pM.

[0211] In some embodiments, methods for measuring KD include those in Table 7.Table 7DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0212] In some embodiments, the disclosure relates to a composition comprising (A) a polyQ binding peptide or a generally aggregating protein binding peptide; (B) a linker; and (C) a DNA- binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules; or a combination of two or more of the foregoing. In this case, (C) represents a solubility domain. The (A) a polyQ binding peptide or a generally aggregating protein binding peptide may be selected from any polyQ binding peptide or a generally aggregating protein binding peptide disclosed herein. The linker may be selected from any linker herein. The (C) DNA-binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules may be selected from any DNA- binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules herein.

[0213] Non-limiting examples of the (A) a polyQ binding peptide or a generally aggregating protein binding peptide are listed in Table 8 and referred to as Part A. Likewise, non-limiting examples of (B) a linker; and (C) a DNA-binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules are listed in in Table 8 and referred to as Parts B and C, respectively.

[0214] In some embodiments, the two or more of (A) a polyQ binding peptide or a generally aggregating protein binding peptide; (B) a linker; and (C) a DNA-binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules together comprise an amino acid sequence. In some embodiments, the amino acid sequence comprises two or more of (A) a polyQ binding peptide or a generally aggregating protein binding peptide; (B) a linker; and (C) a DNA-binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules contiguousDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION with one another. In some embodiments, the amino acid sequence comprises two or more of (A) a polyQ binding peptide or a generally aggregating protein binding peptide; (B) a linker; and (C) a DNA-binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules non-contiguous with one another. The designation of Parts A, B, and C does not limit the arrangement of (A) a polyQ binding peptide or a generally aggregating protein binding peptide; (B) a linker; and (C) a DNA-binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules within the amino acid sequence to A then B and then C. Any order of A, B, and C is contemplated. In some embodiments, the arrangement is A then B and then C. In some embodiments, the arrangement is C then B then C.

[0215] In some embodiments, the bivalent molecule comprises A- B - C domains comprising any order of contiguous natural or non-natural amino acids. In some embodiments, the bivalent polypeptide comprises from about 20 to about 100 amino acids, and wherein domain C comprises any functional variant of any amino acid sequence in Tables 2, 3, or 8, Part C and from about 1 to about 40 amino acid mutations resulting in one or a combination of: (i) a replacement of the amino acid disclosed to a non-natural analog of the particular amino acid; (ii) an addition of one or more natural or non-natural amino acids in the sequence; (iii) a deletion of any of the amino acids in the sequence; (iv) a truncation of a series of contiguous amino acid sequences on the amino or carboxyl end of the amino acid sequence disclosed; (v) a modification of one or more side chains of the amino acids disclosed in the aforementioned tables; or (vi) replacement of S, T, and / or Y residues with R, K, D or E residues, such that the net charge of the molecule is altered to increase solubility of the molecule relative to the original sequence upon which it is based. In some embodiments, the bivalent molecule comprises an aggregation targeting domain A, and a solubility domain C, optionally including a linker B, wherein the domain C comprises a functional variant comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the sequences disclosed in Tables 2, 3, or 8, Part C, but includes replacement of from about 1 to about 25 S, T, or Y residues with R, K, D, or E residues, or mutation of certain residues to include from about 1 to about 25 phosphorylation sites or phosphorylation patches. In some embodiments,

[0216] The solubility domain comprises a DNA binding region chosen from any sequence disclosed herein or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 91%,DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the DNA-binding amino acid sequences disclosed herein.Table 8DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0217] Table 9 lists exemplary bivalent molecules. In some embodiments, the disclosure relates to a bivalent molecule of Table 9 or a functional variant thereof. In some embodiments, the disclosure relates to a composition comprising one or more of the bivalent molecules of Table 9 or a functional variant thereof.Table 9DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0213] The disclosure relates to methods of making one or more amino acid sequences of the bivalent molecule. In some embodiment, the method of making comprises a method of manufacturing the bivalent molecule by recombinant production of the amino acid sequence through expression of a nucleic acid molecule comprising a nucleic acid sequence encoding one or more domains of the bivalent molecule; or by solid state synthesis of the one or more amino acid sequences of the bivalent molecule. In some embodiments, the invention relates to methods of manufacturing any one of the aforementioned compositions, pharmaceutical compositions, or a pharmaceutical salt derived therefrom comprising catalyzing a reaction between at least a first and a second amino acid.

[0214] The disclosure relates to the manufacturing of a synthetic bivalent molecule that is a polypeptide which is an amino acid sequence that corresponds to the sequence of theDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION polypeptides disclosed herein or fragment thereof Tn some embodiments, the synthetic polypeptides comprises a repeated pattern of aaa but does not comprise any other repeated pattern of any alpha or beta amino acids. In some embodiments, the invention relates to an animal cell comprising any of the polypeptides disclosed herein. In some embodiments, animal cells can be contacted with the synthetic polypeptide to induce the biochemical pathway or biological activity ordinarily induced by the naturally occurring polypeptide upon which the analog is based.

[0215] The compositions of the disclosure may be prepared by the synthetic chemical procedures described herein, as well as other procedures similar to those which may be used for making amino acid peptides. Such procedures include both solution and solid phase procedures, e.g., using either Boc and Fmoc methodologies. The compounds of the invention may be synthesized using solid phase synthesis techniques. Fmoc-N- Protected-amino acids can be used to synthesize polypeptides by conventional manual solid-phase synthesis procedures under standard conditions on any number of solid supports, including ortho-chloro-trityl chloride resin. Esterification of Fmoc- -amino acids with the ortho-chloro-trity can be performed according to the method of Barios et. al., Tetrahedron Lett., 1989, 30, 3943. The resin (150 mg, 1.05 mmol Cl) is swelled in 2 ml CH2Ch for 10 min. A solution of the Fmoc-protected -amino acid in CH2Ch and iPr2EtN are then added successively and the suspension is mixed under argon for 4 h. Subsequently, the resin is filtered and washed with CH2Ch / MeOH / iPr2EtN (17:2: 1, 3x3 min), CH2Ch (3x3 min), DMF (2x3 min), CH2Ch (3x3 min), and MeOH (2x3 min). The substitution of the resin is determined on a 3 mg sample by measuring the absorbance of the dibenzofulvene adduct at 300 nm. The Fmoc group is removed using 20% piperidine in DMF (4 ml, 2x20 min) under Ar bubbling. The resin is then filtered and washed with DMF (6x3 min). For each coupling step, a solution of the -amino acid (3 equiv ), BOP (3 equiv.) and HOBT (3 equiv.) in DMF (2 ml) and iPr2EtN (9 eq) are added successively to the resin and the suspension is mixed for 1 h under Ar. Monitoring of the coupling reaction is performed with 2,4,6-trinitrobenzene-sulfonic acid (TNBS) (W.S. Hancock and J.E. Battersby, Anal. Biochem. (1976), 71, 260). In the case of a positive TNBS test (indicating incomplete coupling), the suspension is allowed to react for a further 1 h. The resin is then filtered and washed with DMF (3x3DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION min) prior to the following Fmoc deprotection step. After the removal of the last Fmoc protecting group, the resin is washed with DMF (6x3 min), CH2Ch (3x3 min), Et20 (3x3 min) and dried under vacuum for 3 h. Finally the peptides are cleaved from the resin using 2% TFA in CH2Ch (2 ml, 5x15 min) under Ar. The solvent is removed and the oily residues are triturated in ether to give the crude a- / -polypeptides. The compounds are further purified by HPLC.

[0216] The compositions of the disclosure may be prepared by the synthetic chemical procedures described herein, as well as other procedures similar to those which may be used for making -amino acid peptides. Such procedures include both solution and solid phase procedures, e.g., using either Boe or Fmoc methodologies. The compounds of the invention may be synthesized using solid phase synthesis techniques. Fmoc-N- Protected -amino acids can be used to synthesize poly-a / -peptides by conventional manual solid-phase synthesis procedures under standard conditions on any number of solid supports, including ortho- chloro-trityl chloride resin, Wang resin (NovaBiochem 0.75mmol substitution) and Rink amid resin (NovaBiochem .55mmol substitution). Resin is typically swelled in 100% DMF for 30minutes then deprotected using 20% piperidine in DMF for 2 minutes at 80° (3x). Fmoc-protected amino acids (natural or non-natural) can then be coupled to the resin using a cocktail of AA:HATU:DIEA:Resin (3:2.5:4:1, LiCL 0.8M final concentration) in DMF for 2 minutes at 70° (3x). The resin is then washed (3x) with DMF, DCM (dichloromethane) (3x) and again with DMF (3x) between deprotection and coupling steps. Monitoring of the coupling reaction is performed with 2,4,6-trinitrobenzene-sulfonic acid (TNBS) (W.S. Hancock and J.E. Battersby, Anal. Biochem. (1976), 71, 260). In the case of a positive TNBS test (indicating incomplete coupling), the suspension is allowed to react for another three times. This process is repeated until the desired product has been achieved. After the removal of the last Fmoc protecting group, the resin is washed with DMF (3x), CH2Ch (3x) and DMF again (3x). The remaining free-amine group is then acetylated using a cocktail of DIEA:Ac2O (1: 1) for 5 minutes at room temperature. Full-length peptides were then cleaved from solid support using TFA:TIS:H2O (95:2.5:2.5) for 150 minutes, precipitated in cold ethyl ether and lyophilized. The polymer was reconstituted in a 1 :1 solution of A:B (A: H20, 0.1% TFA) (B: 90: 10:0.1DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION acetonitrile / H2O / TFA). The compositions described herein may be prepared by successive amide bond- forming procedures in which amide bonds are formed between the -amino group of a first-amino acid residue or a precursor thereof and the a- carboxyl group of a second-amino acid residue or alpha-amino acid residue or a precursor thereof. The amide bond-forming step may be repeated as many times, and with specific a-amino acid residues and / or -amino acid residues and / or precursors thereof, as required to give the desired a / -polypeptide. Also analogs comprising two, three, or more amino acid residues (a- or -)may be joined together to yield larger analogs comprising any combination of a-, or -amino acids. Cyclic compounds may be prepared by forming peptide bonds between the N-terminal and C- terminal ends of a previously synthesized linear polypeptide or through the disulfide crosslinking of sidechains of non-adjacent residues. In some embodiments, amino acids are produced enantioselectively from corresponding alpha-amino acids. For instance, by Arndt- Eisert homologation of N-protected a-amino acids. Homologation may be followed by coupling of the reactive diazoketone intermediate of the Wolff rearrangement with a - amino acid residue.

[0217] In some embodiments, the method of manufacturing comprises assembling two or more of (A) a polyQ binding peptide or a generally aggregating protein binding peptide; (B) a linker; and (C) a DNA-binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules as described above. In some embodiments, the method comprises expressing a nucleic acid encoding an amino acid sequence comprising two or more of (A) a polyQ binding peptide or a generally aggregating protein binding peptide; (B) a linker; and (C) a DNA- binding peptide, a charged peptide, a peptide that can be phosphorylated (to be charged), or a peptide that recognizes charged molecules as described above.

[0218] In some embodiments, the disclosure relates to a composition comprising one or more bridging molecule that bridges an aggregation protein to DNA. In some embodiments, the one or more bridging molecule comprises ThioflavinT-Hoechst. In some embodiments, the one or more bridging molecule comprises Methelene blue- Hoechst.

[0219] In some embodiments, the disclosure relates to a nucleic acid sequenceDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION encoding any one or more amino acid sequence herein. In some embodiments, the disclosure relates to a nucleic acid sequence encoding one or more of SEQ ID NOS: 1 through 87 or a variant comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to one of SEQ ID NOS: 1 through 87. In some embodiments, the disclosure relates to a nucleic acid molecule comprising one or more nucleic acid sequence herein. In some embodiments, the disclosure relates to an amino acid sequence comprising one of SEQ ID NOS: 1 through 87 or a variant comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to one of SEQ ID NOS: 1 through 87. In some embodiments, the disclosure relates to an amino acid sequence comprising one or more of SEQ ID NOS: 1 through 87 or a variant comprising at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to one of SEQ ID NOS: 1 through 87.Pharmaceutical Compositions

[0220] One of skill in the art will recognize that the appropriate dosage of the compositions and pharmaceutical compositions may vary depending on the individual being treated and the purpose. For example, the age, body weight, and medical history of the individual patient may affect the therapeutic efficacy of the therapy. Further, a lower dosage of the composition may be needed to produce a transient cessation of symptoms, while a larger dose may be needed to produce a complete cessation of symptoms associated with the disease, disorder, or indication. A competent physician can consider these factors and adjust the dosing regimen to ensure the dose is achieving the desired therapeutic outcome without undue experimentation. It is also noted that the clinician and / or treating physician will know how and when to interrupt, adjust, and / or terminate therapy in conjunction with individual patient response. Dosages may also depend on the strength of the particular bivalent molecule chosen for the pharmaceutical composition.

[0221] The dose of the composition or pharmaceutical compositions may vary. The dose of the composition may be once per day. In some embodiments, multiple doses may be administered to the subject per day. In some embodiments, the total dosage is administered in at least two application periods. In some embodiments, the period can be an hour, a day, a month, a year, aDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION week, or a two-week period. In an additional embodiment of the invention, the total dosage is administered in two or more separate application periods, or separate doses.

[0222] In some embodiments, subjects can be administered the composition in which the composition is provided in a daily dose range of about 0.0001 mg / kg to about 5000 mg / kg of the weight of the subject. The dose administered to the subject can also be measured in terms of total amount of bivalent molecule administered per day. In some embodiments, a subject is administered from about 0.001 to about 3000 milligrams of bivalent molecule per day. In some embodiments, a subject is administered up to about 2000 milligrams of bivalent molecule per day. In some embodiments, a subject is administered up to about 1800 milligrams of bivalent molecule per day. In some embodiments, a subject is administered up to about 1600 milligrams of bivalent molecule per day. In some embodiments, a subject is administered up to about 1400 milligrams of bivalent molecule per day. In some embodiments, a subject is administered up to about 1200 milligrams of bivalent molecule per day. In some embodiments, a subject is administered up to about 1000 milligrams of bivalent molecule per day. In some embodiments, a subject is administered up to about 800 milligrams of bivalent molecule per day. In some embodiments, a subject is administered from about 0.001 milligrams to about 700 milligrams of bivalent molecule per dose. In some embodiments, a subject is administered up to about 700 milligrams of bivalent molecule per dose. In some embodiments, a subject is administered up to about 600 milligrams of bivalent molecule per dose. In some embodiments, a subject is administered up to about 500 milligrams of bivalent molecule per dose. In some embodiments, a subject is administered up to about 400 milligrams of bivalent molecule per dose. In some embodiments, a subject is administered up to about 300 milligrams of Bivalent molecule per dose. In some embodiments, a subject is administered up to about 200 milligrams of bivalent molecule per dose. In some embodiments, a subject is administered up to about 100 milligrams of bivalent molecule per dose. In some embodiments, a subject is administered up to about 50 milligrams of bivalent molecule per dose.

[0223] In some embodiments, subjects can be administered the composition in which the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dose range of about 0.0001 mg / kg to about 5000 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 450 mg / kg of the weight of the subject. In some embodiments, the composition comprising a BivalentDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 400 mg / kg of the weight of the subject. In some embodiments, the composition including a ibvalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 350 mg / kg of the weight of the subject. In some embodiments, the composition comprising a bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 300 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 250 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 200 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 150 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 100 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 50 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 25 mg / kg of the weight of the subject.

[0224] In some embodiments, the composition comprising a Bivalent molecule or

[0225] pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 10 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 5 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 1 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 0.1 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in a daily dosage of up about 0.01 mg / kg of the weight of the subject. In some embodiments, the composition comprising a Bivalent molecule or pharmaceutically acceptable salt thereof is administered in aDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION daily dosage of up about 0.001 mg / kg of the weight of the subject. The dose administered to the subject can also be measured in terms of total amount of Bivalent molecule administered per day.Methods

[0226] The bivalent molecules and pharmaceutical compositions of the disclosure are useful in treating or controlling protein aggregate disorders. To treat or control the disorder, the bivalent molecules and pharmaceutical compositions comprising the bivalent molecules are administered to a subject in need thereof, such as a vertebrate, e.g., a mammal, a fish, a bird, a reptile, or an amphibian. The subject can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The subject is preferably a mammal, such as a human. Prior to administering the bivalent molecules or compositions, the subject can be diagnosed with a need for treatment of the protein aggregate disorder. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of the bivalent molecules or a nucleic acid sequence encoding the bivalent molecule.

[0227] The bivalent molecules or compositions comprising the same, or nucleic acid sequence encoding the foregoing can be administered to the subject according to any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. A preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. A preparation can also be administered prophylactically; that is, administered for prevention of a disease or condition.

[0228] The therapeutically effective amount or dosage of the compound can vary within wide limits. Such a dosage is adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition beingDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg or more, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, as a continuous infusion. Single dose compositions can contain such amounts or submultiples thereof of the compound or composition to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0229] The disclosure relates to a method of solubilizing an aggregate protein comprising exposing one or a plurality of bivalent molecules disclosed herein with one or a plurality of aggregate proteins and allowing a sufficient time for the bivalent molecule to associate with the one or plurality of bivalent molecules disclosed herein and to alter the net charge and solubility of the aggregate protein.

[0230] The bivalent molecules disclosed herein are useful for treating or controlling disorders associated with aggregation protein formation polymerization. Thus, provided is a method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a disclosed bivalent molecule to the subject.

[0231] Accordingly, in some embodiments, the present disclosure provides methods of treating or preventing a protein aggregation disorder in a subject comprising administering to the subject one or more bivalent molecules disclosed herein, or a pharmaceutically acceptable salt thereof, of any one of the bivalent molecules described herein or a pharmaceutical composition comprising one or more of the bivalent molecules described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the result of the step of administering comprises ameliorating symptoms in the subject by stimulating depolymerization of an existing polymer or aggregate protein and / or solubilizing an aggregate or polymer by altering the net charge of the target protein complex to be more electronegative or carry more negative charge. In the case of preventing a protein aggregation disorder, in some embodiments, the step of administering prevents the presentation of clinical symptoms by altering the net charge of the target protein complex to be more electronegative or carry more negative charge thereby preventing the formation or polymerization of an aggregate protein.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0232] In some embodiments, the present disclosure provides methods of treating or preventing Huntington’s disease, ALS, Parkinson’s disease, Alzheimer’s disease in a subject comprising administering to the subject one or more bivalent molecules, or a pharmaceutically acceptable salt thereof, of any one of the bivalent molecules described herein or a pharmaceutical composition comprising one or more of the bivalent molecules described herein, or pharmaceutically acceptable salt thereof, wherein the aggregation targeting domain is capable of associating with an aggregation protein associated with Huntington’s disease, ALS, Parkinson’s disease, and Alzheimer’s disease, and wherein the solubility domain comprises a nucleic acid binding domain or an amino acid sequence comprising an isoelectric point of from about 1 to about 6.5. In some embodiments, the treating of the mitochondrial disease comprises ameliorating symptoms by stimulating the solubilization of the aggregate protein associated with Huntington’s disease, ALS, Parkinson’s disease, and Alzheimer’s disease.

[0233] In some embodiments, the present disclosure provides methods of treating or preventing a prion disease in a subject in need there of comprising administering to the subject one or more bivalent molecules, or a pharmaceutically acceptable salt thereof, of any one of the bivalent molecules described herein or a pharmaceutical composition comprising one or more of the bivalent molecules described herein, or pharmaceutically acceptable salt thereof. In some embodiments, the treating of prion disease comprises ameliorating symptoms by stimulating depolymerization and / or solubilization of a plurality of prions.

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[0235] The references cited throughout this application are incorporated herein by reference in their entireties for all purposes apparent herein and in the references themselves. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0236] It is understood, therefore, that the disclosure is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope as defined by the appended claims; the above description; and / or shown in the attached drawings.EXAMPLES

[0237] Polyglutamine expansion is associated with pathogenic protein aggregation in neurodegenerative disorders. However, long polyglutamine tracts are also found in many transcription factors (TFs), such as FOXP2, a TF implicated in human speech. Here, we explore how FOXP2 and other glutamine-rich TFs avoid unscheduled assembly and focus on FOXP2 as a model for how native glutamine-rich TFs avoid forming pathological assemblies. Throughout interphase, DNA binding, irrespective of sequence specificity, has a solubilizing effect. During mitosis, multiple phosphorylation events promote FOXP2’s eviction from chromatin and supplant the solubilizing function of DNA. Further, human-specific amino-acid substitutions linked to the evolution of speech map to a mitotic phospho-patch, the ‘EVO patch’, and reduce the propensity of the human FOXP2 to assemble. Fusing the pathogenic form of Huntingtin to either a DNA binding domain, a phospho-mimetic variant of this ‘EVO patch’ or a negatively charged peptide is sufficient to diminish assembly formation, suggesting that hijacking mechanisms governing solubility of glutamine-rich TFs may offer new strategies for treatment of polyQ expansion diseases.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0238] We identified DNA binding and mitotic phosphorylation as mechanisms that maintain the solubility of aggregation-prone TFs and show that these mechanisms can be leveraged to eliminate aggregates implicated in polyQ expansion diseases. We further demonstrate that human-specific amino acid substitutions reduce FOXP2’s propensity to aggregate. Remarkably, enhanced solubility of human FOXP2 compared to that of the chimpanzee protein can be primarily attributed to the T303N substitution, suggesting a functional link between enhanced FOXP2 proteostasis and physiological effects of T303N on synaptic plasticity35, with potential implications for the evolution of human speech.Results

[0239] FOXP2 harbors the longest native polyQ tract in the human proteome

[0240] We searched the human proteome for proteins that normally harbor extended stretches of consecutive glutamine residues (Q >10) (SEQ ID NO: 126), recovering 67 such proteins (FIG. 1A). The longest was 40 consecutive glutamine residues (SEQ ID NO: 98) in the FOXP2 transcription factor36(FIG.1A). FOXP2 contains a Forkhead DNA binding domain (FHD) and a large glutamine-rich domain harboring the aforementioned 40Q stretch (SEQ ID NO: 98) followed by a 10Q patch (SEQ ID NO: 99) (FIG. IB). Although this polyQ length exceeds thresholds that spark pathological aggregation in HTT and other proteins, wild-type FOXP2 fused to EGFP showed diffuse nuclear localization pattern when stably expressed in HEK293 cells (FIG. IB). We observed similar diffuse nuclear staining of endogenous FOXP2 in neural crest cells, an embryonic cell population that expresses this protein (FIG. 2A).

[0241] By contrast, two FOXP2 variants associated with verbal dyspraxia - R553H23,24and R328X28- encode proteins that aggregate26,37'39, suggesting that these mutations unmask FOXP2’s capacity to assemble. The R553H mutation alters a conserved residue within the DBD, whereas the R328X truncation removes the DBD and additional domains, including two nuclear localization signals [amino acids 476-480 (RRRHS (SEQ ID NO: 127))40and 582-587 (KRRSQK (SEQ ID NO: 128))37,40]. Both dyspraxia-associated mutants retain the glutamine-rich domain (FIG. IB and28). We tagged both of them with EGFP and stably expressed the fusion proteins in HEK293 cells. Unlike wild-type FOXP2, FOXP2(R553H)-EGFP fusion formed multiple small nuclear puncta (FIG. 1C, quantified in FIG. 1G). Deleting : (i) the polyQ tract, or (ii) the glutamine- rich region including this tract, or (iii) the first 328 amino acids of FOXP2, restored the diffuse nuclear pattern of the R553H mutant, either partially or fully, demonstrating that assembly dependsDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION on the glutamine-rich domain (FIG. 2C and 2D). In agreement, FOXP2(R328X)-EGFP, which contains the glutamine-rich region but lacks a DBD and NLS, typically assembled into a large cytosolic punctum, with additional small cytosolic puncta observed in some cells (FIGS. 1C, 1G). We hereafter refer to these puncta as ‘assemblies,’ with no assumptions regarding their physical state or properties. Conversely, we refer to the proteins showing diffuse localization pattern as ‘soluble.’ Together, our observations suggest that wild-type FOXP2 has features that prevent assembly formation driven by its glutamine-rich domain.

[0242] DNA binding protects FOXP2 from assembly

[0243] The dyspraxia-associated KE family mutation, R553H, impairs FOXP2’s ability to bind DNA3739. Since this mutation also causes FOXP2 to form nuclear puncta, we hypothesized that DNA binding might prevent assembly. Examining the structure of a FOXP2 FHD dimer-DNA complex41, we noted that R553 and two other residues (Y540 and R583) directly contact DNA (FIG. ID). Mutating Y540 or R583 to aspartic acid did not affect protein stability (FIG. 2B), but attenuated luciferase reporter activity driven by a FOXP2 consensus motif in transactivation assays (FIG. IE). To increase dynamic range, we fused FOXP2 to a strong activation domain, VPR, using luciferase activity as an indirect measure of sequence-dependent recruitment to the reporter DNA (rather than FOXP2’s native transactivation capacity). As expected, wild-type FOXP2-VPR strongly activated reporter transcription, dependent on the presence of the FOXP2 consensus motif. Conversely, each DNA contact site mutant (R553H, Y540D, or R583D) impaired this activity (FIG. IE), consistent with reduced DNA binding.

[0244] EGFP fusions to Y540D and R583D also formed nuclear assemblies (FIG. IF), supporting a connection between DNA binding and FOXP2 solubility. The R583D mutant, which retained some activity in our luciferase reporter assays (FIG. IE), showed fewer assemblies (FIG. IF). To further explore this relationship we took advantage of the variability in protein levels inherent to transient overexpression, quantifying the relationship between the probability of assembly formation by FOXP2-EGFP fusions and the mean EGFP signal per cell as a proxy for protein concentration (FIG. 2E). Consistent with our observations from stable cell lines, all DNA contact mutants showed a higher propensity for assembly formation compared to wild-type FOXP2, with Y540D being the most assembly-prone and R583D the least (FIG. 1H). Together, these results suggest that DNA binding is necessary to prevent FOXP2 assembly.

[0245] Diverse DNA binding domains restore solubility to mutant FOXP2 assembliesDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0246] To test whether DNA binding per se was responsible for solubility, we fused the mutant proteins to SSO7D, a strong non-specific DBD from the hypertherm ophilic archaeon Sulfolobus solfataricus42 44. Addition of this domain decreased the assembly propensity of the R553H and Y540D mutants, restoring their diffuse nuclear localization pattern (FIGS. 3A, 4A). Next, we explored FOXP2 solubility in a distantly related model organism, Saccharomyces cerevisiae, where human FOXP2 was mainly cytoplasmic, suggesting its endogenous nuclear localization sequences (NLS) were not recognized. Separated from chromatin in this context, FOXP2-GFP formed visible puncta (FIG. 4C). Introducing a strong SV40-NLS sequence promoted diffuse nuclear signal. In contrast, R553H and Y540D mutants formed nuclear assemblies even when fused to SV40-NLS (FIG. 4D). Just as in human cell culture, adding SSO7D DBD to these mutants resulted in diffuse nuclear localization (FIG.4E, 4F). However, SSO7D fusion did not restore solubility without the SV40-NLS, establishing that it is not simply a generic solubility tag (FIG. 4C).

[0247] To investigate whether DNA binding, mediated by unrelated, sequence-specific human TFs, could also restore the solubility to FOXP2 R553H, we generated fusions of FOXP2 R553H mutant to three human TFs. Two of these factors, SOX2 and MAFK (FIG. 3B, quantified in FIG. 4B), restored solubility, whereas another, FOXA1, did not, indicating that not all DBDs can prevent assembly formation (FIG. 3B, FIG. 4B). We next asked whether solubility conferred by SOX2 or MAFK is associated with recruitment to chromatin. We used ChlP-seq to analyze genomic occupancy of the wild-type FOXP2, FOXP2 R553H, and FOXP2 R553H fused to SOX2 or MAFK. As expected, R553H mutation was associated with loss of genomic binding at many sites. In contrast, SOX2 and MAFK fusions restored chromatin occupancy. Although a subset of sites occupied by these fusions overlapped with those bound by wild-type FOXP2, others were distinct and consistent with the DNA binding specificity of these TFs (FIG. 3C). Indeed, ChlP-seq peaks unique to wild-type FOXP2 were enriched for the FOXP2 motif, whereas those unique to R553H- SOX2 or R553H-MAFK were enriched for SOX2 or MAFK motifs, respectively (FIG. 3D).

[0248] Altogether, these results demonstrate that restoring FOXP2’s interaction with chromatin, irrespective of specific DNA sequence recognition, is sufficient to counteract assembly formation.

[0249] DNA binding promotes solubility of aggregation-prone transcription factors

[0250] Based on these observations, we hypothesized that DNA binding may help TFs tolerate aggregation-prone sequences. Across the proteome, we calculated two metrics linked to glutamineDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION enrichment and aggregation propensity (Table 11): the Michelitsh-Weissman (MW) score45, which counts the number of Q / N residues in an 80 amino acid sliding window, and the PLAAC score4647, which predicts prion-like self-templating capacity from multiple sequence features. Analyzing the functional annotations of proteins within the top 1% of all MW and PLAAC scores, we found that proteins involved in “DNA-binding,” “transcription regulation,” or related terms were the most enriched (FIG. 3E and FIG. 4G).

[0251] We selected two top-scoring TFs, MEF2D and SOX5, for further analysis (FIG. 3F and FIG. 4H). Prior structural studies of both protein families allowed us to identify and mutate residues in MEF2D and SOX5 that contact DNA48,49. At comparable expression levels (FIG. 4K and 4L), EGFP fusions of these mutant proteins formed assemblies, whereas fusions with the wildtype TFs did not (FIG. 3G, 3H, 41 and 4J). Similarly to our observations for FOXP2, addition of the SSO7D DBD restored solubility to mutant SOX5 (FIG. 3G and 41). Collectively, these data show that DNA binding facilitates the solubility of many glutamine-rich or otherwise aggregation- prone TFs.

[0252] DNA binding solubilizes pathogenic HTT fragment

[0253] We next explored whether SSO7D DBD could facilitate the solubilization of a pathogenic polyQ expansion. We stably expressed Huntington’s disease-associated 9 IQ HTT fused to EGFP alone or to EGFP and SSO7D; both fusions were expressed at comparable levels (FIG. 6A). As expected, HTT-polyQ-EGFP formed large cytoplasmic puncta consistent with aggregation. In contrast, HTT-polyQ-EGFP-SSO7D showed a far more diffuse pattern, and the few remaining puncta were smaller (FIG. 5A and 5B).

[0254] HTTpolyQ forms amyloid aggregates, defined by strong resistance to SDS50-52. We performed a filter trap assay53to assess whether DBD fusion can render these aggregates SDS soluble. As a readout, we compared signal on (i) a nitrocellulose membrane that detects all proteins regardless of assembly status; and (ii) a cellulose acetate filter that traps large aggregates. Whereas HTT-polyQ in these cell lysates was retained on the cellulose acetate membrane after incubation with 1% SDS, HTTpolyQ-SSO7D was almost entirely soluble (FIG. 5C). Notably, nuclear and cytosolic FOXP2 mutant proteins were not resistant to SDS despite their robust assembly capacity (FIG. 6B). Thus, DBD fusion can broadly promote solubility, whether of SDS-sensitive protein assemblies or of SDS-resistant amyloids.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0255] We hypothesized that this mechanism would depend on both DNA binding affinity and polyQ length. To test this, we constructed a series of hybrid proteins containing HTT-polyQ of varying lengths (30Q (SEQ ID NO: 129), 40Q (SEQ ID NO: 98), 50Q (SEQ ID NO: 130), 60Q (SEQ ID NO: 131), and 70Q (SEQ ID NO: 132)) and SOX2 DBDs of differing DNA binding affinities (i.e., wild-type, K87A with 9.3-fold lower affinity, and W51A with 17-fold lower affinity34). Sequences of these hybrid proteins are provided in the below Table 10. Wild-type SOX2 DBD rescued the solubility of HTT expansions, irrespective of polyQ length (FIG. 5D and 6C, top). By contrast, the intermediate affinity mutant rescued only partially, and poorly for longer polyQ sequences (FIG. 5D and 6C, middle). The low-affinity mutant had no impact on HTT aggregation (FIG. 5D and 6C, bottom). Together, our results indicate that, even in an ectopic context, DNA binding can facilitate the solubility of polyQ proteins, in a manner that depends on the DBD binding affinity and the properties of the aggregating protein.

[0256] Solubility depends on affinity to DNA.Table 10DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0257] FOXP2 assemblies and polyQ aggregates are dissipated in mitosis

[0258] Although DNA binding solves the TF solubility problem in the interphase nucleus, it raises the question of how aggregation-prone TFs remain soluble during mitosis, when most are evicted from DNA35. To investigate, we returned to FOXP2 and examined its association with chromatinDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION and solubility during this cell cycle stage. Immunofluorescence showed wild-type FOXP2 was indeed excluded from mitotic chromosomes, but did not form assemblies in the cytoplasm (FIG. 7A). Live imaging supported this conclusion (FIG. 8B), though exclusion was less prominent due to different imaging modalities (confocal in FIG. 7A vs. widefield fluorescence in FIG. 8B). Surprisingly, even the R553H ‘KE family’ mutant showed a diffuse pattern during mitosis (FIG. 7B and FIG. 8E), suggesting a different solubilization mechanism during this cell cycle stage.

[0259] We first considered whether the distinct chemical environment of the cytoplasm might promote solubility. Serendipitously, we found that mutating both DNA contact residues, R553H and Y540D, resulted in cytosolic puncta during interphase (FIG. 7C). These puncta disappeared upon entering prophase and remained soluble through mitosis (FIG. 7C, 7D), establishing that prior nuclear localization is not required for assembly dissipation. Protein levels of wild-type and mutant FOXP2 remained stable, suggesting active solubilization (FIG. 8C). Untagged protein showed similar mitotic dissipation (FIG. 8A). Interestingly, the apraxia-associated FOXP2 R328X truncation mutant and pathogenic HTT expansions (Q91 (SEQ ID NO: 100) and Q103 (SEQ ID NO: 133)) were also solubilized during mitosis (FIG. 7E, FIG.8D).

[0260] We next followed the disassembly kinetics of R553H and the polyQ domains of FOXP2 and HTT fused to EGFP, inducing mitotic arrest with nocodazole in an asynchronous culture and tracking individual cells (FIG. 8E). We observed that all three proteins began to dissipate upon mitotic entry. The FOXP2 R553H mutant disassembled rapidly and completely (FIG. 7F). Assemblies formed by FOXP2 R328X and aggregates formed by HTT dissolved more gradually and sometimes partially, with occasional small foci remaining even during mitosis (FIG. 7F). Yet, remarkably, most assemblies, including HTT aggregates, were solubilized during mitosis.

[0261] FOXP2 is hyperphosphorylated during mitosis

[0262] Because we and others have observed that mitotic phosphorylation can regulate protein assembly36'60, we hypothesized that this modification might contribute to FOXP2 solubilization. To explore this possibility, we purified HA-tagged FOXP2 from asynchronous and mitotically arrested cell extracts and analyzed its phosphorylation by mass spectrometry obtaining nearly full peptide coverage, and identifying a significant number of phosphopeptides in the mitotic samples. To further investigate, we repeated the experiment using data-independent acquisition (DIA-MS) to obtain label-free quantification. Most phosphorylation sites were detected in both experiments and enriched between 2.4- and 6.1-fold in the mitotic samples (Table 11).DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONTable 11DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0263] Overall, we detected 30 phosphosites on FOXP2 enriched in mitotic cells (FIG. 10 and Table 11). Eleven were SP / TP motifs typical of mitotic CDK-mediated phosphorylation, distributed throughout the protein. The remaining sites were concentrated within two S / T-rich patches (FIG. 9A). The first (aa292-343) surrounds the two human-specific amino acid residues in FOXP2 (N303 and S325), which we term the ‘EVO patch’. The second (aa438-465) is a low complexity region upstream of the Forkhead domain, hereafter referred to as the ‘pre-FHD patch’ (FIG. 9A). We also identified the following mutations in the EVO patch T292, T293, S296, S297, S298, T299, T300, S301, S302, T304, S305, S308, S312, S315, S321, S322, S325, S330, S331, S332, T336, S339, T341, and Y343. A bivalent molecule comprising these mutations dissolved FOXP2.

[0264] SP / TP phosphosites mediate the release of FOXP2 from chromatin

[0265] We first explored the function of the broadly distributed SP / TP sites, generating FOXP2 mutants in which all 11 sites were mutated to alanine (11 A, ‘phosphodead’) or aspartic / glutamic acid (11D / E, ‘phosphomimetic’). Like wild-type FOXP2 (FIG. 1C), both mutants showed diffuse nuclear localization in asynchronous culture (FIG. 9B and 9C). However, in mitotic cells, the SP / TP phosphodead mutant tracked mitotic chromosomes (FIG. 9B), whereas the phosphomimetic mutant was evicted, similarly to the wild-type protein (FIG. 9C). FOXP2 FHD also remained associated with mitotic chromatin (FIG. 9D). Our data suggest that the release of FOXP2 from mitotic chromosomes is not merely a passive by-product of chromatin condensation. Instead, it requires active phosphorylation coincident with mitosis.

[0266] FOXP2 hyperphosphorylation regulates mitotic solubility

[0267] To understand whether SP / TP phosphorylation contributes to the mitotic solubility of FOXP2, we introduced the 11 A phosphodead mutant in the R553H background. Because R553H impairs DNA binding, this combined mutant allowed us to separate the roles of SP / TP phosphorylation in chromatin eviction and mitotic solubility. FOXP2-11A-R553H-GFP formed nuclear puncta, which were only partially solubilized during mitosis (FIG. 12A, quantified in FIG. 11 A). To investigate further, we generated a pan-phosphodead FOXP2 R553H mutant, where all 11 SP / TP sites and remaining phosphorylation sites in the “EVO patch” and “Pre-FHD patch” were mutated to alanines. This ‘pan-phosphodead’ variant, containing 30 phosphosite mutations, failed to solubilize in mitosis (FIG. 11A and 11B, FIG. 12B). Importantly, introducing the same 30 mutations in wild-type FOXP2 did not affect the protein’s diffuse nuclear staining duringDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION interphase, demonstrating that the extensive mutations do not cause protein aggregation themselves (FIG. 12C). As expected, during mitosis the pan-phosphodead mutant in the wild-type background remained associated with chromosomes (FIG. 12C).

[0268] To assess how the individual phosphorylation patches contribute to mitotic solubility, we generated FOXP2 R553H variants in which either the EVO patch phosphosites or Pre-FHD phosphosites were mutated to alanines (absent SP / TP mutations). These mutants were solubilized during mitosis (FIG. 12D). Moreover, we observed partial solubility when alanine mutations of one or the other patch were combined with the 11A R553H (FIG. 12E; effects of all mutants are summarized in FIG. 12F). Thus, concerted modification in all three phosphorylation sites - SP / TP, the EVO patch and the Pre-FHD patch - contribute to FOXP2’s mitotic solubility.

[0269] Amyloids decrease with increasing charge.

[0270] EVO patch phosphorylation promotes solubility of polyQ proteins

[0271] Next, we examined whether modification of the EVO might influence FOXP2 solubility outside of mitosis. In interphase cells, we investigated the influence of EVO patch ‘phosphomimetic’ or ‘phosphodead’ mutants on FOXP2 R553H assembly. Remarkably, most nuclear puncta were solubilized when FOXP2 R553H was combined with the EVO patch phosphomimetic, but not phosphodead mutations (FIG. 11C-11D). We also tested whether the EVO patch is alone sufficient to control the solubility of cytosolic polyQ aggregates, examining the solubility ofFOXP2 truncations (1-345) containing the N-terminal glutamine-rich domain and a wild-type, phosphomimetic, or phosphodead EVO patch. In parallel, we examined HTT-polyQ- EGFP fused to these same EVO patch variants. The phosphomimetic EVO patch suppressed cytoplasmic assembly of these polyQ proteins, whereas the phosphodead mutants enhanced it (FIG. 11E-11H).

[0272] Our data suggest that the EVO patch phosphomimetic could be harnessed to solubilize pathological polyQ aggregates. We hypothesized this effect might be mediated by the negative charge from phosphorylation. To test this, we replaced the EVO-patch in the prior constructs with a short stretch of 14 negatively charged amino acids (D / E). Indeed, the 14D / E peptide suppressed assembly formation by both truncated FOXP2 and HTT polyQ (FIG. HI and 11 J). Additionally, fusions to the EVO patch phosphomimetic or 14D / E peptide significantly reduced SDS-resistant HTT-polyQ amyloids (FIG. UK, 12G, 12H). This suggests a potential strategy to dissolve pathological assemblies in post-mitotic cells by recruiting polyQ to negatively charged entities.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0273] Human-specific EVO patch substitutions protect FOXP2 from assembly

[0274] FOXP2 is extraordinarily well conserved: Human and mouse proteins differ by only three amino acids. Two of these substitutions fall within the EVO patch and are not present in great apes (FIG. 13A, 14A). These changes have been hypothesized to be linked to the acquisition of speech and language in humans, but their molecular function remains unknown32'34. Our discovery that the EVO patch modulates FOXP2 solubility led us to explore whether these human-specific substitutions impact assembly.

[0275] Some genome annotations also indicate minor differences of + / - 1 Q in polyQ length of FOXP2 (FIG. 14B). To ensure clear results, we synthesized chimpanzee and mouse FOXP2 cDNAs with equal polyQ length (40Q (SEQ ID NO: 98) + 10Q (SEQ ID NO: 99)) and using human codons. For simplicity, we refer to these constructs, which have similar expression levels in HEK293 cells (FIG. 14C) and comparable transactivation capacity in FOXP2 motif-driven luciferase reporter assays (FIG. 13B), as human, chimp, and mouse FOXP2.

[0276] Next, we assessed the assembly capacity of FOXP2 from these three species. We did so by quantifying two metrics in transient overexpression assays (described in FIG. 1H and 2E): (i) the per-cell coefficient of variation in EGFP signal as a measure of assembly and (ii) the per-cell mean EGFP fluorescence as a proxy for protein concentration. We then modeled the relationship between mean GFP fluorescence and assembly probability for each protein using a logistic regression. FOXP2 R553H, a positive control, had a higher propensity to assemble than wild-type human FOXP2 (FIG. 13C and D). Interestingly, mouse and chimp FOXP2 also formed more assemblies (FIG. 13C) at lower concentrations (FIG. 13D) compared to human FOXP2. To examine if these differences might be influenced by subtle variation in polyQ length, we also compared polyQ variants annotated in some chimp (41Q (SEQ ID NO: 134) + 10Q (SEQ ID NO: 99)) and mouse (40Q (SEQ ID NO: 98) +9Q (SEQ ID NO: 135)) proteomes to the wild-type human FOXP2 (40Q (SEQ ID NO: 98) + 10Q (SEQ ID NO: 99)). Again, we observed that the human protein was substantially less assembly-prone (Fig 14D and 14E).

[0277] Our results show that the two amino acid substitutions in FOXP2 that occurred uniquely in the human lineage are associated with increased protein solubility. Introducing these variants into mice enhances striatal synaptic plasticity and vocalization32'34; a recent study demonstrated that T303N substitution is the primary driver of physiological effects in the striatum35. Intrigued, we investigated the effects of the individual amino acid substitutions on assembly, using wild-typeDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION human and chimp FOXP2 as a reference. Remarkably, the N303 to T mutation showed a much stronger effect than S325 to N, as quantified by the probability of assembly formation with increasing concentration (FIG. 13E and 14F). Together, our results identify a molecular function for human-specific substitutions in FOXP2, and show a concordance between the effects of individual amino acid substitutions on assembly formation and striatal synaptic plasticity.

[0278] Testing other disease proteins validates the approach across aggregating disease proteins.

[0279] Experiments were conducted with disease alleles of different aggregation-linked diseases. Expression in S. cerevisiae leads to aggregation, but fusing these proteins to SSO7D increase localization to nucleus and no observable aggregates.

[0280] FIG. 34A illustrates FUS P525L-GFP, IAPP S20G-GFP, Abeta42-GFP, and TDP43 Q331K-GFP expression in S. cerevisiae. Aggregates form in cytosol or nucleus dependent on the localization of each protein. Abeta42-GFP has been described in literature not to have GFP expression or form visual aggregates when expressed as the Aggregating Abeta42 inhibit the folding of GFP and its visualization, soluble version of Abeta42-GFP can show GFP expression on the contrary. FIG. 34B illustrates fusion of SSO7D DNA-binding domain to the proteins in of FIG. 34A rescue the formation of visual puncta or aggregates. For Abeta42, the GFP signal is recovered, indicating conversion to the soluble version of the protein.

[0281] Validation that peptides with charge inhibit aggregation in human cells.

[0282] Charged Peptide binding to HTT 91Q decreases the aggregation of HTT in inductiondependent manner. FIG. 35A shows cells comprising constitutive HTT 91Q-EGFP, FIG. 35B shows cells comprising constitutive HTT 91Q-EGFP plus a construct allowing inducible expression of a charged peptide where the cells were induced. FIG. 35B shows cells comprising constitutive HTT 91Q-EGFP plus a construct allowing inducible expression of a charged peptide where the cells were induced but then induction was ceased for six days. Cells expressing HTT 91Q-EGFP show aggregates (FIG. 35 A). Upon induction of charged peptide with affinity to polyQ (14DE-20Q-14DE-3xHA) the number of visual aggregates decreases (FIG. 35B). 14DE is a stretch containing 14 mixed negatively charged amino acids D (Aspartic acid) or E (Glutamic acid), 20Q is a stretch of 20 glutamine amino acid (SEQ ID NO: 136), 3xHA is 3 copies of HA tag used for biochemical assays. However, stopping the induction of the peptide leads to formation of more aggregates (FIG. 35C)DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0283] Validation that peptides bridging to DNA inhibit aggregation in Human cells.

[0284] Peptides bridging HTT 9 IQ to DNA decreases the aggregation of HTT in inductiondependent manner. FIG. 36A shows cells comprising constitutive HTT 91Q-EGFP, FIG. 35B shows cells comprising constitutive HTT 91Q-EGFP plus a construct allowing inducible expression of a peptide (bridging to DNA) where the cells were induced. FIG. 35B shows cells comprising constitutive HTT 91Q-EGFP plus a construct allowing inducible expression of a peptide (bridging to DNA) where the cells were induced but then induction was ceased for six days. Cells expressing HTT 91Q-EGFP shows aggregates (FIG. 36A). Upon induction of peptide- DNA bridge (20Q-SSO7D-3xHA), the number of visual aggregates decreases (FIG. 36B). 20Q is a stretch of 20 glutamine amino acids (SEQ ID NO: 136), SSO7D is a DNA-binding domain, 3xHA is 3 copies of the HA tag for biochemical assays. Stopping the induction of the peptide led to formation of more aggregates (FIG. 36C).Discussion

[0285] In this study, we demonstrate that TFs rich in polyglutamine tracts are dependent on DNA binding for solubility. When DNA binding capacity is perturbed, these TFs form assemblies, similar to the polyQ expansion proteins linked to neurodegenerative diseases. The solubilizing effect of DNA may explain how TFs can tolerate much longer polyQ stretches than other proteins, and have indeed coopted glutamine-rich domains as mediators of protein-protein interactions and transactivation4'7. Previous in vitro studies suggested that some nucleic acids might have chaperone activity61'64. Furthermore, the 23 S rRNA is critical for the ribosome’s capacity to catalyze protein folding65. Our work now highlights an unappreciated role of DNA in preventing the formation of nuclear assemblies. Given that ligand binding typically increases a protein’s conformational stability, we considered the possibility that sequence-dependent DNA binding to the cognate motif might stabilize aggregation-prone TFs. However, our results demonstrate that DBDs with diverse sequence specificities can solubilize assemblies formed by aggregation-prone TFs and even dissipate aggregates associated with polyQ expansion (FIG. 13F). Thus, other mechanisms underlying DNA’s solubilizing function are likely at play, such as electrostatic interactions and / or physical dispersion of the protein through DNAbinding, which may also reduce the unbound protein concentration below the critical threshold for assembly.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0286] Our results argue that rather than providing a ‘silver bullet’, solubilization is dependent on concentration, DNA binding affinity, and properties of the aggregating protein. An interesting case in point is TATA-binding protein (TBP), an abundant DNA-binding protein associated with spinocerebellar ataxia driven by polyQ expansion66. Interestingly, the wild-type sequence of this key transcriptional regulator harbors the second longest polyQ stretch in the proteome (38Q (SEQ ID NO: 149)), which is nonetheless compatible with solubility and function. Interestingly, TBP’s expansion threshold for aggregation is among the highest for polyQ-associated diseases, 47-63 glutamines66(SEQ ID NO: 137). This suggests that DNA binding may indeed help TBP tolerate a long polyQ tract, but past a certain polyQ length threshold cannot prevent aggregation of this abundant protein.

[0287] Our observations may seem at odds with a large body of emerging literature implicating nucleic acids in the formation of nuclear condensates. For example, coactivator proteins such as the Mediator complex can form condensates at super-enhancer DNA in a manner that depends upon their intrinsically disordered regions (IDRs) and can be regulated by R.N AI 3J 6'7'69. Similarly, nucleic acids can scaffold certain protein assemblies70'76. Nevertheless, IDRs and other LCRs found in TFs and their cofactors likely differ vastly in their capacity to drive assembly, regulation, and the physical and biochemical properties of the assemblies they do form (e.g., liquid vs solid, self-templating or not). Distinct transcriptional regulators may utilize interactions with DNA to promote or prevent the formation of nuclear assemblies. Due to the high risk of pathology associated with long polyQ tracts, regulation that counteracts assemblies may be particularly relevant for glutamine-rich TFs.

[0288] Utilizing DNA binding to ensure solubility presents a unique problem during mitosis when most transcriptional machinery is stripped from DNA75. We document an efficient solution to this problem for FOXP2, whereby mitotic phosphorylation both coordinates the protein’s release from the chromatin and promotes its solubility when away from the DNA (FIG. 13F). SP / TP phosphosites distributed across the protein are required for chromatin eviction, indicating that release of FOXP2 FHD from mitotic chromosomes is an actively regulated process rather than a byproduct of chromatin condensation. These SP / TP phosphorylation sites also act with two concentrated phospho-patches to regulate the mitotic solubility of FOXP2. Given the sheer number of modifications, their broad distribution across the protein, and concerted effects on assembly, it is tempting to speculate that the mitotic solubility of FOXP2 is mediated by the negative charge.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONHowever, we cannot exclude the possibility that mitotic phosphorylation may also regulate the association of FOXP2 with specific solubilizing factors that remain to be identified.

[0289] Interestingly, even without phospho-patches, HTT polyQ puncta dissolve in mitosis, albeit incompletely and with slower kinetics than full-length FOXP2. This suggests that, in addition to regulation by mitotic phosphorylation, protein aggregates formed by polyQ may generally become more soluble in mitosis. The mechanisms involved remain enigmatic and might involve cell-cycle- regulated chaperone activity77,78. Moreover, our observation is in line with a previous report that the thermal stability of many proteins increases during mitosis79. It may also provide a partial explanation for the long-standing observation that toxicity in polyQ expansion diseases is limited to post-mitotic cells, even when the causative protein is broadly expressed.

[0290] PolyQ expansion diseases are terminal and currently uncurable. Our study reveals that mechanisms safeguarding native glutamine-rich transcription factors from assembly could inform novel treatments for polyQ expansion. We show that ectopic addition of either a DBD, a phosphomimetic EVO patch, or a short negatively charged peptide greatly diminishes the formation of amyloid aggregates formed by the pathogenic form of HTT. Importantly, these strategies also promote HTT solubility outside of mitosis, and thus might be applied in post-mitotic cells.

[0291] Interestingly, human-specific substitutions within the EVO patch significantly reduce FOXP2’s capacity to self-assemble. These substitutions enhance vocalization and modulate striatal synaptic plasticity when introduced to mice, with a predominant role attributed to N3O3 in mediating these phenotypes35. These observations have led to the proposal that human-specific substitutions in FOXP2 may have been directly involved in the evolution of human speech, although whether they have undergone a recent positive selection in humans remains controversial, as does the question of whether these coding changes or rather, cis-regulatory divergence at the F0XP2 locus, may have played a more important role in this process32,80. We have now uncovered a molecular function for the human-specific substitutions in promoting the solubility of FOXP2. Moreover, concordant effects of the N303 residue on striatal synaptic plasticity and enhanced solubility suggest a functional connection between the two.

[0292] Our results may reconcile some controversies in the field and point toward a synergistic role of coding and non-coding F0XP2 changes in the evolution of human speech. The amino acid substitutions leading to enhanced solubility of human FOXP2 may have allowed speech-relevant neurons to accommodate elevated levels of FOXP2 without toxicity associated with proteinDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION aggregation. This increased tolerance in turn may have enabled cis-regulatory changes leading to increased FOXP2 expression to be selected in the human lineage. Such cis-regulatory changes often occur over short evolutionary timeframes and in a highly cell type-selective manner. Consistent with such possibility, human-specific upregulation of F0XP2 transcripts was noted in two neuronal subtypes of human brains, as compared to those of chimps and macaques, suggesting cis-regulatory evolution facilitating increased F0XP2 transcription in these subtypes81. Finally, the increased levels of F0XP2 in specific human neuronal subtypes may have led to the augmented regulation of its speech-relevant downstream targets. Regardless of the specific underlying mechanism, our study, for the first time, links enhanced protein solubility to the human-specific amino acid substitution postulated to play a key role in the evolution of a unique human trait.Methods

[0293] Experimental model and subject details

[0294] Cell culture

[0295] Female Hek293 cells (ATCC, CRL-1573, RRID:CVCL_0045) were cultured in DMEM high glucose (Cytiva SH30243.FS) supplemented with 10%FBS (Sigma-Aldrich, F4135), lx MEM non-essential amino acid (Stem Cell Technologies, 07600) IxGlutamax (Gibco, 35050061) , lx Anti / Anti(Life Technologies, 15240062)) and passaged using trypsin.

[0296] Female H9 hESCs (WiCell, WA09, RRID:CVCL_9773) were cultured in mTeSRl medium (Stem Cell Technologies, 85850) on Matrigel Matrix (Coming, 356231) and passaged using ReLeSR (Stem Cell Technologies, 05872). CNCC differentiation was performed as described83-85

[0297] Yeast cultures BY4741 strain was grown in synthetic SD media (0.17% yeast nitrogen base, 2% glucose, 0.5% NH4-sulfate and amino acids as described86.

[0298] Mitotic cultures

[0299] Mitotic arrested cultures were induced by adding IpM Nocodazole (sigma M1404) for 18- 24h. mitotic cells arrested with nocodazole round-up and can be visually assessed by DIC microscope (for Immuno-precipitation or western blot) or by condensation of chromatin with Hoechst (live cell imaging) or D API (immunofluorescence staining).

[0300] Stable cell populationsDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION

[0301] In one well of 6-wells plate (Fisher Scientific, 08-772-1B) growing HEK293, 2.5 pg of vector were transfected using lipofectamine 2000 (Invitrogen, 11668019) (750ng of piggybac vector , 1 pg of super piggybac transposase(System Biosciences, PB210PA-1), and 750ng of pGEMT carrier DNA) at a ratio of 2.8 ul lipofectamine per ug of DNA according to manufacturer protocol, 48hours after transfection, puromycin (2 pg / ml) was added to start selection. In 3-7 days post-selection a resistant population was obtained and frozen for further experiments. All images in this study are from stable populations of cells unless specified otherwise.

[0302] HEK293 cells were used for all figures with exception to 2A (H9 hESCs) and 4 C-F (Saccharomyces cerevisiae BY 4741).

[0303] For HEK293 transfections, all constructs used were cloned in a piggybac vectors used to integrate the constructs in the Hek293 genome to obtain stable populations as described.

[0304] Transient transfections (without super piggybac transposase) were used in the following experiments:

[0305] For FIG. IE and 13B: For luciferase assay, samples were collected 24h post transfection.

[0306] For FIG.3H: as in stable population but cells were imaged 24-48h post-transfection.

[0307] For FIG. 4K, 4L, 6A: as in stable population but cells were lysed for WB 48h posttransfection.

[0308] For FIG. 1H and FIG. 13: In one well of 6-wells plate growing HEK293, 2.5 pg of piggybac vector at a ratio of 2.8 ul lipofectamine per ug of DNA were transfected using lipofectamine 2000 (Invitrogen, 11668019) according to manufacturer protocol.

[0309] For yeast transformations, centromeric plasmids from pRS 413 series expression FOXP2 constructs under Tefl promoter were used for yeast transformation and cells were grown under amino acid selection.

[0310] Analysis of Q length

[0311] Using Homo sapiens proteome from uniport (UP000005640), we searched for each amino acid the list of proteins that contains the length of >=10 consecutive stretches of the respective amino acid. For glutamine, the distribution of these stretches is presented in Figure 1A.

[0312] PLAAC and MW score plots

[0313] A pre-computed list of human proteome PLAAC scores87using PLAAC software47was used to plot the ranking of Michelitsh-Weissman score MW score45which represents the maximum number of N + Q in a window of at most 80 AA. Or NLLR score (PLAAC score) which representsDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION the normalized LLR score (max sum of PLAAC log-likelihood ratios (base 4) in window of size c)47(Table 12) Table 12 illustrates a selected set of this data; whole data set not shown.Table 12 is organized with a UniProt number, MW Score and NLLRscore (the scores used in the FIG. 3F), Each UniPrto number represent a single protein separates by a semicolon, the sequence for each protein identified by UniProt is incorporated by reference in its entirety.Table 12Q8IZL2 68 0.945;Q930742 67 0.945;O146863 67 0.875;O154092 63 0.855;Q96RN52 56 0.695;O154052 54 0.703 ;P0CG23 53 0.378;P202262 51 0.77;Q6ZW49 47 0.496;Q9NSY12 45 0.598;Q14686 43 0.647;Q10571 41 0.583;O949162 41 0.58;Q96L912 41 0.53;Q96JK9 40 0.57;P515312 40 0.565;Q9UGI6 40 0.553;Q9Y6Q92 40 0.537;Q2M2I82 40 0.446;Q9ULV32 40 0.338;Q8NDV75 38 0.671;Q96PN72 38 0.411;O147762 38 0.382;P78364 38 0.329;P784242 37 0.407;Q86YW94 37 0.362;Q159112 37 0.337;Q8TF682 36 0.496;Q96EK4 36 0.495;P54253 35 0.535;Q927932 35 0.501;G951042 35 0.467;P102753 35 0.345;Q02446 35 0.31;Q9H3346 35 0.175;Q9UPQ9 34 0.164;Q3ZLR7 34 0.129;Q96MM72 33 0.475;Q6Y7W63 33 0.43;Q5JV73 33 0.397;Q7Z739 33 0.376;Q68DE3 33 0.322;Q128302 33 0.174;Q09472 32 0.488;Q15596 32 0.44;Q9P267 32 0.357;Q86UP32 32 0.29;Q8WV24 32 0.272;P148592 32 0.182;Q8IVH22 32 0.082;Q927342 31 0.525;O144972 31 0.49;Q8NFD52 31 0.44;P42694 31 0.412;O15516 31 0.299;Q6PJG2 31 0.295;Q2TAL8 31 0.17;Q9UGU02 30 0.43;Q157882 30 0.412;P080472 30 0.275;Q9H3P7 30 0.268;P235112 30 0.229;Q139522 30 0.218;Q3V6T22 30 0.171;O751772 29 0.612;Q92794 29 0.525;Q7Z5J42 29 0.315;Q9HCJ02 29 0.285;Q5KU26 29 0.25;Q157143 29 0.232;Q024472 29 0.189;Q9UQL62 29 -0.036;Q7Z2Y52 29 -0.104;Q92804 28 0.574;Q155322 28 0.546;P314832 28 0.37;Q148142 28 0.339;Q4VCS5 28 0.309;P20265 28 0.292;Q139503 28 0.283;Q86XK24 28 O.278;Q9H1B7 28 0.25;Q9UPA5 28 0.213;Q92585 28 0.175;Q14119 28 0.164;Q8N7R0 28 0.145;Q8IWX8 28 0.096;P35869 28 0.074;Q8N4C82 28 -0.006;Q96JA4228 - 0.076;Q9UKV00 28 -0.121;Q3L8U12 27 0.442;Q9P0K82 27 0.432;Q9Y2K52 27 0.371;P42858 270.348;Q9UBL03 270.339;Q150322270.326;Q9H2F5 270.312;Q9NQV6270.297;Q9ULU40 27 0.247;O75909 27 0.229;P526552 27 0.151;Q9Y6V02 27 0.118;Q9UN79 27 0.075;Q8IY632 27 0.044;Q96QZ72 27 0.011 ;P35711227 -0.041;Q8IV76 27 -0.107;P54259 26 0.369;Q6N021 26 0.336;Q6KC792 26 0.277;Q020782 26 0.271;Q68CP93 26 0.242;Q144442 26 0.227;Q9Y2K22DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION26 0.171;Q134953 26 0.168;Q01826 26 0.165;Q9UPN92 26 0.123;P55197 26 0.091;Q96BD52 26 0.074;Q127722260.073;Q96PP8 26 -0.132;Q083792 26 -0.158;000461 26 -0.195;P0747626 -0.366;Q9BUJ22 25 0.484;Q9Y3Y42 25 0.408;043390225 0.294;Q9GZV5 25 0.251;O60885 25 0.185;Q9ULH74 25 0.18;Q8IX12 25 0.178;Q9H2G4 25 0.174;Q9H9S0 25 0.166;Q9NPJ4 25 0.132;Q8NEM72 25 0.119;Q7Z5892 25 0.116;Q9HCK82 25 0.11;P19484 25 0.092;P0C7V6 25 0.083;O94967225 0.046;P35712225 -0.016;Q96KQ425 -0.032;Q9P2E9225 -0.051;Q14980225 -0.105;Q15075 25 -0.298;P35637224 0.605;Q131484 24 0.452;Q9P2D12 24 0.409;Q8IZD22 24 0.36;Q997002 24 0.339;Q928962 24 0.331;O43765 24 0.313;P561793 24 0.305;Q9BWU12 24 0.298;P461002 24 0.296;Q995902 24 0.294;P02810 24 0.275;P50616 24 0.25;Q6AI39 24 0.241;Q020862 24 0.235;P46531 24 0.222;Q925402 24 0.22;Q5SZQ82 24 0.211;Q8NEZ42 24 0.19;Q9UMX03 24 0.184;P63128 24 0.182;Q6NSW7 24 0.166;Q9Y6R0 24 0.165;Q8TF012 24 0.162;Q8IXK04 24 0.135;Q6PIJ62 24 0.133;O151642 24 O.13;B1AL88 24 0.118;P54098 24 0.098;Q52LR7 24 0.092;Q9NZ56 24 0.058;075064 24 0.048;P411802 24 0.04;0607632 24 0.003;P0CG43 24 -0.061;P55198 24 -0.063;Q8IZD42 24 -0.1;Q9Y2I62 24 -0. 11;Q9UPN32 24 - 0.112;P0CJ92 24 -0.146;Q9Y2642 24 -0.149;Q8IZU3 24 -0.209;Q9BV732 24 -0.233;Q07283 24 -0.4;Q146712 23 0.408;Q86VE33 23 0.307;Q86UU02 23 0.289;P469373 23 0.288;Q6XPR3 23 0.272;Q6P3W7 23 0.262;Q8TB722 23 0.246;Q146772 23 0.216;P87889 23 0.19;Q9Y5A92 23 0.186;Q96EV2 23 0.185;P62683 23 0.182;P62684 23 0.182;P63126 23 0.182;Q7LDI9 23 0.182;P63145 23 0.163;Q99218 23 0.15;P63130 23 0.142;P50553 23 0.139;Q151542 23 0.124;Q15233 23 0.115;Q8IZQ82 23 0.099;Q8NDX52 23 0.091 ;Q71SY52 23 0.085;Q9UPN62 23 0.083;Q9NR972 23 0.075;O75179223 0.061;O94842223 0.04;Q6NUN723 0.036;A4UGR94 23 0.025;Q151492 23 0.012;0146074 23 -0.002;P570712 23 -0.038;A6NE01 23 - 0.046;Q8N9N52 23 -0.047;O951712 23 -0.058;C9JC47 23 -0.066;P547253 23 -0.072;A6NMD2 23 -0.146;Q9H4E7 23 -O.168;Q96PI1 23 -0.24;Q5JU672 23 -0.248;Q5T655 23 -0.352;O149792 22 0.569;Q8WYB52 22 0.542;Q9ULJ62 22 0.376;O94913 22 0.241;Q9H4A34 22 0.24;O14994 22 0.24;Q5TAX3 220.234;Q9H3D4022 0.198;Q96KN3222 0.178;Q96JM23 22 0.166;Q2KHR3 22 0.166;Q9UHD9220.158;Q9YNA8220.157;O1555022 0.129;Q8IYU4220.128;O94986222 0.121;Q9Y5202 22 O.113;Q3C1VO 22 0.099;Q9NX702 22 0.094;P483802 22 0.093;Q9NRR5 22 0.08;Q9NVW2 22 0.07;Q9HDB9 22 O.O68;A1E959 22 0.035;Q8TF662 22 0.034;P10153 22 0.033;Q99743 22 0.032;P547272 22 0.025;P436993 22 0.021;P090865 22 0.007;Q15007 22 0.003;Q9H0Kl 22 -0.005;Q9P0J7 22 -0.006;Q9Y6183 22 -0.014;014965 22 -0.036;Q6P995 22 -DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONO.O42;Q8N1NO2 22 -0.067;Q05469 22 -0.074;P0CG42 22 -0.092;Q141882 22 -0.117;Q5VVM6 22 -0.126;O750523 22 -0.141;H3BSY2 22 -0.146;Q147892 22 -0.154;Q15643 22 -0.16;O151233 22 -0.16;P407632 22 -0.163;Q8NBJ42 22 -0.166;Q8TC92 22 -0.174;Q083782 22 - 0.185;Q6ZUS5222 -0.19;O7511622 -0.21;Q8NHQ1222 -0.216;Q9BXX3 22 -0.222;A6NN73 22 -0.233;Q0D2H9 22 -0.233;H7BZ55 22 -0.234;Q969H0 22 -0.251;Q8N4C60 22 -0.265;O760643 22 -0.275;Q9H7C4 22 -0.298;Q9BZW7 22 -0.309;Q15431 22 -0.331;Q928782 22 - 0.334;Q96NL62 22 -0.334;Q141032 21 0.544;Q008392 21 0.433;Q8WU792 21 0.345;O149642 21 0.278;Q9NZW4 21 0.257;P232462 21 0.247;O95639 21 0.227;O602812 21 0.221;Q9BYW2 21 0.208;Q9Y4E8 21 0.204;060741 21 O.2;Q9NRM1 21 0.199;Q9Y6N76 21 0.196;P48436 21 0.185;Q9NS71 21 0.181;Q010852 21 0.177;Q992172 21 0.17;Q9BYJ92 21 0.156;Q928022 21 0.152;Q9BSE4 21 0.143;Q9BXT5 21 0.127;Q136252 21 0.118;094900 21 0.117;0605062 21 0.109;O953192 21 0.109;Q928792 21 0.084;Q930732 21 0.083;P15822 21 0.073;015063 21 0.071;Q8IVL10 21 0.07;Q08999 21 0.068;Q9BYH82 21 0.065;0603333 21 0.058;Q68DL7 21 0.048;P542522 21 0.047;Q96JP2 21 0.043;Q5T749 21 0.039;Q7Z3K34 21 0.036;Q8WU58 21 0.034;Q09MP3 21 0.033;Q9UBG3 21 0.032;P477102 21 0.023;O15372 21 0.019;P207942 21 - 0.006;P56715 21 -0.011;Q145152 21 -0.031;043167 21 -0.032;Q86Y82 21 -0.033;Q15459 21 - 0.035;Q9UJ55 21 -0.035;0150162 21 -0.038;Q9BYF12 21 -0.038;P565242 21 -0.046;P042792 21 -0.051;Q86WS421 -0.053;P24043 21 -0.056;Q5VU43321 -0.064;Q9P21921 -0.073;Q995502 21 -0.081;Q7Z7A12 21 -0.088;Q92786 21 -0.092;Q8NB253 21 -0.133;Q9P0K72 21 - 0.133;P49454 21 -0.139;O75420 21 -0.14;Q9BRK4 21 -0.143;P21854 21 -0.145;Q6UWE02 21 - 0.147;P422292 21 -0.148;A6NC78 21 -0.154;D6RF30 21 -0.154;Q6A163 21 -0.163;Q2VIQ3 21 -0.163;Q49A883 21 -0.165;Q8N573 21 -0.18;Q9UPS82 21 -0.195;Q8TC20221 -0.208;Q6UXK5 21 -0.212;O94804 21 -0.216;Q146672 21 -0.218;Q9NXG02 21 -0.22;Q8NBR6 21 - 0.231;Q8NEH6 21 -0.236;Q9BXG8 21 -0.241;P0CG33 21 -0.252;Q8TD312 21 -0.252;A6NDK9 21 -0.253;Q9Y2282 21 -0.254;A6NDN3 21 -0.282;Q5TB802 21 -0.285;Q9NYA3 21 - 0.29;Q8IWJ2 21 -0.304;Q92805 21 -0.307;Q9NVI72 21 -0.31;Q96MC2 21 -0.327;Q9H2G22 21 -0.329;P49901 21 -0.354;Q129067 20 0.512;Q9HCD5 20 0.311;P404263 20 0.278;Q9C0J8 20 0.263;Q7Z3532 20 0.246;Q9NYJ82 20 0.234;Q8N5C82 20 0.229;P3324020 O.2O6;Q6P1W52 20 0.188;P497503 20 0.186;Q9UPY32 20 0.169;P35908 20 0.169;P04280 20 0.164;Q7Z794 20 O.162;Q9HCM1 20 0.154;Q9BTL3 20 0.148;Q14157 20 0.148;Q7Z4172 20 0.147;Q7Z5Q18 20 0.147;P22670 20 0.146;Q96I24 20 0.125;O435242 20 0.124;Q92922 20 0.118;P356802 20DOCKET NO. 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STFD-018-PCT INTERNATIONAL APPLICATION17 -0.255;P355792 17 -0.258;Q162222 17 -O.258;Q9H4K1 17 -O.262;Q156422 17 - 0.263;Q6P3R8 17 -0.265;Q9UKX3 17 -0.265;P32455 17 -0.265;Q9UN81 17 -0.268;Q128462 17 -0.268;O604472 17 -0.268;Q8N6G52 17 -0.269;Q5BJE12 17 -0.269;Q96JB22 17 -0.27;Q9H0J9 17 -0.27;Q9Y234 17 -0.271;Q8IY18 17 -0.273;Q96T512 17 -0.276;Q6P9F02 17 -0.278;Q02818 17 -0.279;O956132 17 -0.28;Q08043 17 -0.28;Q081172 17 -0.28;P78380 17 -O.283;Q49AM1 17 -0.287;A8MW95 17 -0.288;Q8NEP33 17 -0.293 ;Q9BZD4 17 -0.294;P073322 17 -0.295;Q8IYEl 17 -0.296;Q58EX72 17 -0.299;Q86T292 17 -0.301;Q8TCGl 17 -0.301;Q9BY27 17 - 0.305;Q9BYX4 17 -0.305;Q8TD162 17 -0.31;Q8TDY22 17 -0.311;Q8IVM02 17 -0.311;P52732 17 -0.312;Q8WXW32 17 -0.314;Q3SXY82 17 -0.319;Q8WYA03 17 -O.32;Q5F1R6 17 - 0.326;Q86VW0 17 -0.328;Q96FL92 17 -0.329;Q2M243 17 -0.329;O751452 17 -0.333;O76013 17 -0.341 ;P0C221 17 -0.344;Q9BSW22 17 -0.348;Q9P031 17 -0.348;Q6P3W6 17 -0.351;Q15323 17 -0.354;Q9UJC32 17 -0.357;D6REC4 17 -O.363;Q96BH1 17 -0.364;Q14129 17 -0.372;076011 17 -0.389;Q9NQ482 17 -0.393;Q8N4Y2 17 -0.419;Q4G0S7 17 -0.426;Q8WWB32 17 - 0.48;Q86YZ3 16 0.31;Q9NX072 16 0.309;0002672 16 0.27;Q96PK6 16 0.243 ;P48431 16 0.241;Q9UH732 16 0.23;P24928 16 0.221;Q12965 16 0.217;Q9UMZ28 16 0.205;P404243 16 0.202;Q9BZC13 16 0.191;P525943 16 0.185;Q16650 16 0.182;Q9NXJ02 16 0.176;Q9UBF9 16 0.164;Q9UBV8 16 0.162;Q9Y6Y8 16 0.152;Q9NXR8 16 0.144;P026712 16 0.144;Q96P474 16 0.143;O15162 16 0.142;Q9H2E62 16 0.14;Q9NUK02 16 0.131;Q71RC22 16 0.12;0005552 16 0.12;Q4KMG02 16 0.119;Q5DX212 16 0.107;P07498 16 0.097;Q995043 16 0.097;Q132272 16 0.091;P355562 16 0.091;O435813 16 0.09;Q99856 16 0.089;O434322 16 0.089;P10071 16 0.083;Q93008 16 0.083;P263672 16 0.083;A8TX702 16 0.078;Q9UPU92 16 0.077;Q96T58 16 0.077;Q14774 16 0.074;Q96G252 16 0.074;P08579 16 0.073;Q9NR562 16 0.073;A6NCI8 16 0.069;Q007222 16 0.067;Q8TAQ22 16 0.066;Q15078 16 0.061;P40199 16 0.061;P10070 16 0.059;060318 16 0.055;Q96JE72 16 0.054;Q9Y490 16 0.048;Q96S82 16 0.047;O152262 16 0.047;Q133303 16 0.047;Q140042 16 0.043;0147704 16 0.042;Q9Y385 16 0.041;000507 16 0.039;P136880 16 0.038;Q9HCJ32 16 0.035;Q6ZU65 16 0.034;Q6UWI4 16 0.033;060494 16 0.033;P09630 16 0.032;Q15842 16 0.026;0954906 16 0.023;P469342 16 0.022;Q96BU12 16 0.02;Q5JSZ53 16 0.019;Q9C0A62 16 0.018;O152652 16 0.017;Q9UNA12 16 0.017;P102429 16 0.014;Q5TC822 16 0.012;Q02413 16 0.012;Q12864 16 0.011;Q8IZD9 16 0.009;A6NMK8 16 0.007;Q014842 16 0.006;P518262 16 0.004;P28300 16 0.002;Q9NRJ4 16 0.001;0602292 16 - 0.001;Q8NHV9 16 -0.008;Q130992 16 -0.008;Q9UJF22 16 -0.011;Q9BX69 16 -0.013;Q9Y572DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION16 -0.014;Q96AY4 16 -0.016;Q8NFC6 16 -0.017;Q8IYH52 16 -0.017;Q5T4S72 16 - 0.018;Q8N5683 16 -0.02;Q134722 16 -0.02;Q8WWI14 16 -0.02;P313273 16 -0.02;Q9C0402 16 -0.021;P00533 16 -0.022;P159272 16 -0.023;Q9Y467 16 -0.025;P0DMB2 16 -0.026;A0AVK6 16 -0.026;Q7L7X33 16 -0.026;Q7RTS92 16 -0.028;Q53FD02 16 -0.028;Q9UKZ42 16 -0.029;Q6ZNGl 16 -0.029;P02533 16 -0.03;P073332 16 -0.031;Q86UAl 16 -0.032;A0AUZ92 16 -0.032;Q9BTC0 16 -0.032;Q9UPW52 16 -0.032;Q7Z5N43 16 -0.032;Q8WZ732 16 -0.034;Q7Z3G6 16 -0.034;Q9BZV3 16 -0.034;O146462 16 -O.O35;Q5T1H1 16 -0.035;Q5UIP02 16 -0.036;Q8TC27 16 -0.036;Q9Y5F8 16 -0.038;Q9Y5G2 16 -0.038;Q9Y5G5 16 -0.038;Q9Y5H0 16 -0.038;060563 16 -0.039;Q6ZRS22 16 -0.039;O951552 16 -0.041 ;Q9NZM42 16 - 0.041;Q7Z6Z72 16 -0.042;Q9NXL62 16 -0.042;Q5VUJ62 16 -0.042;P518142 16 -0.043;P227942 16 -0.043;Q6ZU672 16 -0.046;Q6IE37 16 -0.047;O757172 16 -0.049;Q9BRl l 16 -0.05;Q8IZF22 16 -0.052;Q9HAR24 16 -0.052;P265992 16 -0.052;Q5T3J3 16 -0.053;P550552 16 - 0.053;Q9UL49 16 -0.053;Q9Y2H5 16 -0.053;Q96IP42 16 -0.053;Q96QP12 16 -0.054;O43186 16 -0.055;Q9UJV32 16 -0.056;Q2LD372 16 -0.057;Q53TQ32 16 -0.057;Q8N7W22 16 - 0.057;Q9UPY62 16 -0.06;B5MCYl 16 -0.061;Q154682 16 -0.061;0001412 16 -0.062;P14410 16 -0.062;Q6UX392 16 -0.063;Q7RTS7 16 -0.063;Q99795 16 -0.064;P60508 16 -0.064;0004822 16 -0.064;Q9Y2J42 16 -0.065;Q2NKX8 16 -0.065;Q7LBE32 16 -0.065;Q9H114 16 -0.065;O95758 16 -0.067;0750772 16 -0.067;Q6IE36 16 -0.068;O94929 16 -0.068;Q9Y6X6 16 -0.07;P08922 16 -0.071;Q8NAP3 16 -0.072;Q16820 16 -0.072;Q9H8M52 16 -0.073;Q9Y2225 16 -0.074;Q96MT72 16 -0.075;Q8NB12 16 -0.076;P198272 16 -0.076;Q5JU852 16 -0.077;A6NJ88 16 -0.077;B4DYI2 16 -0.077;Q9BY443 16 -0.078;Q9UIW2 16 -0.079;Q9P2K33 16 -0.079;Q9BZY9 16 -0.079;Q5JWR5 16 -0.081;Q153292 16 -0.082;Q9NQ603 16 -0.083;Q8NHY22 16 -0.083;Q96PZ0 16 -0.084;Q9NQW5 16 -0.084;Q502W64 16 -0.084;Q9UJA32 16 -0.085;Q9HBW9 16 -0.085;P526304 16 -0.086;Q96DT5 16 -0.086;A3KN832 16 -0.086;Q5T5U3 16 -0.086;Q9ULB5 16 -0.086;Q6ZSGl 16 -0.087;Q9NNZ3 16 -0.087;Q9C0H92 16 -0.087;075051 16 -0.087;Q9H347 16 -0.087;Q9ULL82 16 -0.088;Q96PZ2 16 -0.089;Q04721 16 -O.O89;B1AJZ93 16 -0.089;Q86SQ42 16 -0.09;Q133222 16 -0.09;Q9UJ78 16 -0.091;Q9GZX5 16 -0.091;Q13443 16 -0.092;Q928702 16 -0.092;Q13698 16 -0.093;0604773 16 -0.093;Q6SJ932 16 -0.094;P15088 16 -0.094;Q96NI6 16 -0.095;Q86XM0 16 -0.095;Q8IVW4 16 -0.096;Q86W10 16 -O.O97;Q8N1L42 16 -0.097;Q9C0D22 16 -0.098;Q8N7Z5 16 - 0.099;Q9NYQ62 16 -0.099;P158122 16 -0.099;Q6ZSB92 16 -0.099;P161571 16 -0.1;Q9Y4A8 16DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION-0.1 ;Q9Y5P32 16 -0.101;Q9HDB52 16 -0.101;Q9Y4C03 16 -0.101 ;P822792 16 -0.102;Q6RI452 16 -0.102;P51888 16 -0.102;Q9Y2502 16 -0.103;043303 16 -0.104;Q9H8K7 16 -0.105;P220032 16 -0.105;Q8WVD3 16 -0.106;015083 16 -0.106;P190222 16 -0.107;Q8IZ07 16 - 0.108;Q3MJ402 16 -0.109;Q3LI83 16 -0.109;Q9NX05 16 -0.11;A6NCI4 16 -0.11;Q9Y4G8 16 - 0.11;Q029282 16 -0.112;Q141232 16 -0.112;Q7Z2Q7 16 -0.113;Q8WWF3 16 -0.114;P542772 16 -0.114;Q6UB982 16 -0.116;Q8NI352 16 -0.116;Q68CQ4 16 -0.117;P35900 16 -O.117;Q9ULL1 16 -0.118;P293502 16 -0.118;Q9UQ164 16 -0.118;Q8NA66 16 -0.119;P17661 16 -0.119;A2VCL2 16 -0.119;P61647 16 -0.119;Q96M85 16 -0.12;Q8IWZ5 16 -0.122;Q14CW9 16 -0.122;Q6PIU22 16 -0.122;Q9NQV7 16 -0.123;Q2M3C72 16 -0.123;Q9NZI8 16 -0.124;P460872 16 - 0.124;Q8TB69 16 -0.125;Q5VZ892 16 -0.125;P08670 16 -0.125;Q6DHV5 16 -0.125;Q8NAT2 16 -0.126;Q9Y2F5 16 -0.126;P16885 16 -0.126;Q9UN76 16 -O.126;A6NM11 16 -0.127;Q53H473 16 -0.129;Q86TB3 16 -0.13;Q5T6X52 16 -0.133;Q52LW3 16 -0.134;O956703 16 - 0.134;Q9C0H62 16 -0.134;Q8N5D04 16 -0.136;Q9Y4E54 16 -0.136;Q9H0784 16 - 0.136;Q96GX12 16 -0.137;Q166102 16 -0.137;O60235 16 -0.137;A8MT65 16 -0.137;060309 16 -0.137;Q96A04 16 -0.138;Q9Y6Q23 16 -0.138;0002062 16 -0.139;P00451 16 -0.14;0153209 16 -0.14;Q5M7752 16 -0.142;P35247 16 -0.142;Q9P2Y5 16 -0.142;Q9UIE0 16 -0.142;Q9Y2832 16 -0.143 ;P010422 16 -0.143;Q96H552 16 -0.144;P54132 16 -0.145;P08172 16 -0.146;Q8TE773 16 -0.147;Q5FVE42 16 -0.147;O602422 16 -0.147;Q7Z4012 16 -0.148;A6NDX5 16 - O.148;Q9HCG1 16 -0.148;A8MVM7 16 -O.148;Q6P1J63 16 -0.148;Q9P2D72 16 -0.148;P22415 16 -0.149;O602252 16 -0.149;P59052 16 -0.15;Q8TAM23 16 -0.15;Q04695 16 -0.15;Q9UKU9 16 -0.151;Q9UBW52 16 -0.151;Q5TC84 16 -0.151;Q8TE73 16 -0.151;000425 16 - 0.152;Q9C0B7 16 -0.153;Q9UFE4 16 -0.153;O751705 16 -0.153;P24592 16 -0.153;Q5HYA8 16 -0.154;P981983 16 -0.154;Q135762 16 -0.154;Q9Y2I92 16 -0.155;P15036 16 -0.155;P01031 16 -0.156;O953472 16 -0.156;Q8TDM62 16 -0.156;P13521 16 -0.156;Q9Y4I52 16 -0.156;Q927502 16 -0.157;Q9H195 16 -0.157;Q9UMR2 16 -0.157;A4D0V72 16 -0.158;Q9Y6X32 16 - 0.158;Q9UHH9 16 -0.158;Q5VYJ5 16 -0.159;P412192 16 -0.159;Q6R2W3 16 -0.16;Q9Y5X52 16 -0.16;Q9NQX0 16 -0.16;000161 16 -0.16;Q8NE093 16 -0.16;Q05BQ5 16 -0.162;P541082 16 -0.162;Q7Z4S65 16 -0.163;Q5SNV92 16 -0.163;Q9H9H4 16 -0.163;Q96QE3 16 -0.164;Q93091 16 -0.164;Q9UNK9 16 -0.164;Q9UKU6 16 -0.164;Q7Z2F62 16 -0.164;Q5XKR94 16 - 0.165;A6NMS7 16 -0.167;Q006102 16 -0.167;P542892 16 -0.167;Q92858 16 -0.167;Q9NSC52 16 -0.169;Q9UHX13 16 -0.169;Q9HBX8 16 -0.169;P63132 16 -0.17;Q6ZN28 16 -0.17;Q86XR82DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION16 -0.17;Q9H3W5 16 -0.17;A6NLI5 16 -0.171 ;Q96PX8 16 -O.171;B1 APH42 16 -0.173;Q070022 16 -0.173;Q127742 16 -0.173;P16109 16 -0.174;Q9BQP9 16 -0.174;Q8IZP7 16 - 0.175;Q5W0A02 16 -0.175;Q047272 16 -0.175;Q8TDZ22 16 -0. 176;Q9NSI62 16 - 0.176;Q6PEZ83 16 -0.176;Q92508 16 -0.176;Q5TID72 16 -0.177;O434242 16 -0.177;O94892 16 -O.178;Q1X8D72 16 -0.178;Q49A262 16 -0.179;Q8TE23 16 -0.179;014980 16 -0.179;Q9H0R12 16 -0.179;Q86XU0 16 -0.18;Q5T7B84 16 -0.18;Q8IYY4 16 -0.181;O75899 16 -0.183;Q5THK12 16 -0.184;Q8WYP52 16 -0.185;Q8IXTl 16 -0.186;Q2TAC22 16 -0.187;O60346 16 - 0.187;Q8IYB42 16 -0.187;Q63HN84 16 -0.187;Q96PD4 16 -0.187;Q96PC5 16 -0.187;P10266 16 -0.188;P63133 16 -0.188;P63136 16 -0.188;Q9BXR3 16 -0.188;Q9UQG0 16 -0.188;Q9WJR5 16 -0.188;P19622 16 -0.188;P981712 16 -O.188;Q96AQ1 16 -0.189;Q9NUQ82 16 -0.19;Q8IWZ62 16 -0.191;Q6PJP8 16 -0.191 ;Q3LIE5 16 -0.191;Q8IUZ02 16 -0.191;O439332 16 -0.192;O753812 16 -0.192;Q96LY2 16 -0.192;Q9UGV22 16 -0.192;Q140082 16 -0.193;Q8IW19 16 - 0.193;O751642 16 -0.194;Q9GZR12 16 -0.194;P552592 16 -0.194;Q9BXL6 16 -0.195;Q085542 16 -0.195;O94826 16 -0.195;Q9HCH02 16 -0.196;P54802 16 -0.197;076031 16 -0. 198;Q9BY123 16 -0.198;A6NI47 16 -0.198;Q6S5H5 16 -0.198;O75044 16 -0.198;P21506 16 -0.198;Q9UIA9 16 -0.198;Q6S545 16 -0.2;Q8TDR0 16 -0.201;Q7Z6K4 16 -0.201;Q17RD72 16 -0.201;042043 16 - 0.202;Q9NZN52 16 -0.202;A6NEMl 16 -0.202;Q2TAC62 16 -0.202;Q925522 16 -0.202;Q15058 16 -O.2O3;Q8N5P1 16 -0.204;Q8N3R92 16 -0.205;P355802 16 -0.205;Q6ZWH52 16 - 0.205;Q128882 16 -0.205;Q131832 16 -0.206;Q9UGL12 16 -0.206;P785042 16 - 0.206;Q8NEU82 16 -0.206;Q9ULS5 16 -0.207;0150202 16 -0.207;Q6U8412 16 -0.208;Q5R3F8 16 -0.208;Q9NQR7 16 -0.208;A6NI86 16 -0.208;Q9NWD9 16 -0.209;Q9NZC42 16 - 0.209;Q9H4I0 16 -0.21;P0CB33 16 -0.211;Q86VH42 16 -0.212;P239452 16 -0.212;Q6XZB02 16 -0.212;000629 16 -0.212;P68543 16 -0.213;C9JE40 16 -0.213;Q8TAB5 16 -0.214;075071 16 - 0.214;Q7Z6B72 16 -0.214;H0YKK7 16 -0.214;Q6PJQ5 16 -O.215;Q9NZU1 16 -0.215;A4FU692 16 -0.215;Q9BXT8 16 -0.215;O602822 16 -0.216;Q133422 16 -0.216;Q5TH69 16 - 0.216;Q9NPA52 16 -0.217;Q9NTW74 16 -0.217;Q92628 16 -0.217;Q49AM3 16 -0.217;P109112 16 -0.217;P112772 16 -0.218;Q8TCU5 16 -0.219;Q9BT92 16 -0.219;Q6ZN16 16 -0.22;Q86T902 16 -0.22;Q96K49 16 -0.22;0609023 16 -0.22;Q9NNX60 16 -0.22;000505 16 -0.22;Q8N8V2 16 -0.221,Continuation of Table 12 with sequences listed>sp|Q8IZL2|MAML2_HUMAN Mastermind-like protein 2 OS=Homo sapiens OX=9606 GN=MAML2 PE=1 SV=2 (SEQ ID NO: 273)DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONMGDTAPPQAPAGGLGGASGAGLLGGGSVTPRVHSAIVERLRARIAVCRQHHLSCEGRYERGRAESSDRERESTLQLLSLVQHGQGARKAGKHTKATATAATTTAPPPPPAAPPAASQAAATAAPPPPPDYHHHHQQHLLNSSNNGGSGGINGEQQPPASTPGDQRNSALIALQGSLKRKQWNLSPANSKRPNGFVDNSFLDIKRIRVGENLSAGQGGLQINNGQSQIMSGTLPMSQAPLRKTNTLPSHTHSPGNGLFNMGLKEVKKEPGETLSCSKHMDGQMTQENIFPNRYGDDPGEQLMDPELQELFNELTNISVPPMSDLELENMINATIKQDDPFNIDLGQQSQRSTPRPSLPMEKIVIKSEYSPGLTQGPSGSPQLRPPSAGPAFSMANSALSTSSPIPSVPQSQAQPQTGSGASRALPSWQEVSHAQQLKQIAANRQQHARMQQHQQQHQPTNWSALPSSAGPSPGPFGQEKIPSPSFGQQTFSPQSSPMPGVAGGSGQSKVMANYMYKAGPSAQGGHLDVLMQQKPQDLSRSFINNPHPAMEPRQGNTKPLFHFNSDQANQQMPSVLPSQNKPSLLHYTQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQSSISAQQQQQQQSSISAQQQQQQQQQQQQQQQQQQQQQQQQQQQPSSQPAQSLPSQPLLRSPLPLQQKLLLQQMQNQPIAGMGYQVSQQQRQDQHSWGQNTGPSPSPNPCSNPNTGSGYMNSQQSLLNQQLMGKKQTLQRQIMEQKQQLLLQQQMLADAEKIAPQDQINRHLSRPPPDYKDQRRNVGNMQPTAQYSGGSSTISLNSNQALANPVSTHTILTPNSSLLSTSHGTRMPSLSTAVQNMGMYGNLPCNQPNTYSVTSGMNQLTQQRNPKQLLANQNNPMMPRPPTLGPSNNNNVATFGAGSVGNSQQLRPNLTHSMASMPPQRTSNVMITSNTTAPNWASQEGTSKQQEALTSAGVRFPTGTPAAYTPNQSLQQAVGSQQFSQRAVAPPNQLTPAVQMRPMNQMSQTLNGQTMGPLRGLNLRPNQLSTQILPNLNQSGTGLNQSRTGINQPPSLTPSNFPSPNQSSRAFQGTDHSSDLAFDFLSQQNDNMGPALNSDADFIDSLLKTEPGNDDWMKDINLDEILGNNS>sp|Q93074|MED12_HUMAN Mediator of RNA polymerase II transcription subunit 12 OS=Homo sapiens OX=9606 GN=MED12 PE=1 SV=4 (SEQ ID NO: 274)MAAFGILSYEHRPLKRPRLGPPDVYPQDPKQKEDELTALNVKQGFNNQPAVSGDEHGSAKNVSFNPAKISSNFSSIIAEKLRCNTLPDTGRRKPQVNQKDNFWLVTARSQSAINTWFTDLAGTKPLTQLAKKVPIFSKKEEVFGYLAKYTVPVMRAAWLIKMTCAYYAAISETKVKKRHVDPFMEWTQIITKYLWEQLQKMAEYYRPGPAGSGGCGSTIGPLPHDVEVAIRQWDYTEKLAMFMFQDGMLDRHEFLTWVLECFEKIRPGEDELLKLLLPLLLRYSGEFVQSAYLSRRLAYFCTRRLALQLDGVSSHSSHVISAQSTSTLPTTPAPQPPTSSTPSTPFSDLLMCPQHRPLVFGLSCILQTILLCCPSALVWHYSLTDSRIKTGSPLDHLPIAPSNLPMPEGNSAFTQQVRAKLREIEQQIKERGQAVEVRWSFDKCQEATAGFTIGRVLHTLEVLDSHSFERSDFSNSLDSLCNRIFGLGPSKDGHEISSDDDAWSLLCEWAVSCKRSGRHRAMWAKLLEKRQAEIEAERDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONCGESEAADEKGSIASGSLSAPSAPIFQDVLLQFLDTQAPMLTDPRSESERVEFFNLVLLFCELIRHDVFSHNMYTCTLISRGDLAFGAPGPRPPSPFDDPADDPEHKEAEGSSSSKLEDPGLSESMDIDPSSSVLFEDMEKPDFSLFSPTMPCEGKGSPSPEKPDVEKEVKPPPKEKIEGTLGVLYDQPRHVQYATHFPIPQEESCSHECNQRLWLFGVGKQRDDARHAIKKITKDILKVLNRKGTAETDQLAPIVPLNPGDLTFLGGEDGQKRRRNRPEAFPTAEDIFAKFQHLSHYDQHQVTAQVSRNVLEQITSFALGMSYHLPLVQHVQFIFDLMEYSLSISGLIDFAIQLLNELSWEAELLLKSSDLVGSYTTSLCLCIVAVLRHYHACLILNQDQMAQVFEGLCGWKHGMNRSDGSSAERCILAYLYDLYTSCSHLKNKFGELFSDFCSKVKNTIYCNVEPSESNMRWAPEFMIDTLENPAAHTFTYTGLGKSLSENPANRYSFVCNALMHVCVGHHDPDRVNDIAILCAELTGYCKSLSAEWLGVLKALCCSSNNGTCGFNDLLCNVDVSDLSFHDSLATFVAILIARQCLLLEDLIRCAAIPSLLNAACSEQDSEPGARLTCRILLHLFKTPQLNPCQSDGNKPTVGIRSSCDRHLLAASQNRIVDGAVFAVLKAVFVLGDAELKGSGFTVTGGTEELPEEEGGGGSGGRRQGGRNISVETASLDVYAKYVLRSICQQEWVGERCLKSLCEDSNDLQDPVLSSAQAQRLMQLICYPHRLLDNEDGENPQRQRIKRILQNLDQWTMRQSSLELQLMIKQTPNNEMNSLLENIAKATIEVFQQSAETGSSSGSTASNMPSSSKTKPVLSSLERSGVWLVAPLIAKLPTSVQGHVLKAAGEELEKGQHLGSSSRKERDRQKQKSMSLLSQQPFLSLVLTCLKGQDEQREGLLTSLYSQVHQIVNNWRDDQYLDDCKPKQLMHEALKLRLNLVGGMFDTVQRSTQQTTEWAMLLLEIIISGTVDMQSNNELFTTVLDMLSVLINGTLAADMSSISQGSMEENKRAYMNLAKKLQKELGERQSDSLEKVRQLLPLPKQTRDVITCEPQGSLIDTKGNKIAGFDSIFKKEGLQVSTKQKISPWDLFEGLKPSAPLSWGWFGTVRVDRRVARGEEQQRLLLYHTHLRPRPRAYYLEPLPLPPEDEEPPAPTLLEPEKKAPEPPKTDKPGAAPPSTEERKKKSTKGKKRSQPATKTEDYGMGPGRSGPYGVTVPPDLLHHPNPGSITHLNYRQGSTGLYTQNQPLPAGGPRVDPYRPVRLPMQKLPTRPTYPGVLPTTMTGVMGLEPSSYKTSVYRQQQPAVPQGQRLRQQLQQSQGMLGQSSVHQMTPSSSYGLQTSQGYTPYVSHVGLQQHTGPAGTMVPPSYSSQPYQSTHPSTNPTLVDPTRHLQQRPSGYVHQQAPTYGHGLTSTQRFSHQTLQQTPMISTMTPMSAQGVQAGVRSTAILPEQQQQQQQQQQQQQQQQQQQQQQQQQQYHIRQQQQQQILRQQQQQQQQQQQQQQQQQQQQQQQQQQHQQQQQQQAAPPQPQPQSQPQFQRQGLQQTQQQQQTAALVRQLQQQLSNTQPQPSTNIFGRY>sp|O14686|KMT2D_HUMAN Histone-lysine N-methyltransferase 2D OS=Homo sapiens OX=9606 GN=KMT2DPE=1 SV=2 (SEQ ID NO: 275)MDSQKLAGEDKDSEPAADGPAASEDPSATESDLPNPHVGEVSVLSSGSPRLQETPQDCSGDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONGPVRRCALCNCGEPSLHGQRELRRFELPFDWPRCPWSPGGSPGPNEAVLPSEDLSQIGFPEGLTPAHLGEPGGSCWAHHWCAAWSAGVWGQEGPELCGVDKAIFSGISQRCSHCTRLGASIPCRSPGCPRLYHFPCATASGSFLSMKTLQLLCPEHSEGAAYLEEARCAVCEGPGELCDLFFCTSCGHHYHGACLDTALTARKRAGWQCPECKVCQACRKPGNDSKMLVCETCDKGYHTFCLKPPMEELPAHSWKCKACRVCRACGAGSAELNPNSEWFENYSLCHRCHKAQGGQTIRSVAEQHTPVCSRFSPPEPGDTPTDEPDALYVACQGQPKGGHVTSMQPKEPGPLQCEAKPLGKAGVQLEPQLEAPLNEEMPLLPPPEESPLSPPPEESPTSPPPEASRLSPPPEELPASPLPEALHLSRPLEESPLSPPPEESPLSPPPESSPFSPLEESPLSPPEESPPSPALETPLSPPPEASPLSPPFEESPLSPPPEELPTSPPPEASRLSPPPEESPMSPPPEESPMSPPPEASRLFPPFEESPLSPPPEESPLSPPPEASRLSPPPEDSPMSPPPEESPMSPPPEVSRLSPLPWSRLSPPPEESPLSPPPEESPTSPPPEASRLSPPPEDSPTSPPPEDSPASPPPEDSLMSLPLEESPLLPLPEEPQLCPRSEGPHLSPRPEEPHLSPRPEEPHLSPQAEEPHLSPQPEEPCLCAVPEEPHLSPQAEGPHLSPQPEELHLSPQTEEPHLSPVPEEPCLSPQPEESHLSPQSEEPCLSPRPEESHLSPELEKPPLSPRPEKPPEEPGQCPAPEELPLFPPPGEPSLSPLLGEPALSEPGEPPLSPLPEELPLSPSGEPSLSPQLMPPDPLPPPLSPIITAAAPPALSPLGELEYPFGAKGDSDPESPLAAPILETPISPPPEANCTDPEPVPPMILPPSPGSPVGPASPILMEPLPPQCSPLLQHSLVPQNSPPSQCSPPALPLSVPSPLSPIGKWGVSDEAELHEMETEKVSEPECPALEPSATSPLPSPMGDLSCPAPSPAPALDDFSGLGEDTAPLDGIDAPGSQPEPGQTPGSLASELKGSPVLLDPEELAPVTPMEVYPECKQTAGQGSPCEEQEEPRAPVAPTPPTLIKSDIVNEISNLSQGDASASFPGSEPLLGSPDPEGGGSLSMELGVSTDVSPARDEGSLRLCTDSLPETDDSLLCDAGTAISGGKAEGEKGRRRSSPARSRIKQGRSSSFPGRRRPRGGAHGGRGRGRARLKSTASSIETLVVADTDSSPSKEEEEEDDDTMQNTWLFSNTDKFVLMQDMCWCGSFGRGAEGHLLACSQCSQCYHPYCVNSKITKVMLLKGWRCVECIVCEVCGQASDPSRLLLCDDCDISYHTYCLDPPLLTVPKGGWKCKWCVSCMQCGAASPGFHCEWQNSYTHCGPCASLVTCPICHAPYVEEDLLIQCRHCERWMHAGCESLFTEDDVEQAADEGFDCVSCQPYVVKPVAPVAPPELVPMKVKEPEPQYFRFEGVWLTETGMALLRNLTMSPLHKRRQRRGRLGLPGEAGLEGSEPSDALGPDDKKDGDLDTDELLKGEGGVEHMECEIKLEGPVSPDVEPGKEETEESKKRKRKPYRPGIGGFMVRQRKSHTRTKKGPAAQAEVLSGDGQPDEVIPADLPAEGAVEQSLAEGDEKKKQQRRGRKKSKLEDMFPAYLQEAFFGKELLDLSRKALFAVGVGRPSFGLGTPKAKGDGGSERKELPTSQKGDDGPDIADEESRGLEGKADTPGPEDGGVKASPVPSDPDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONEKPGTPGEGMLSSDLDRISTEELPKMESKDLQQLFKDVLGSEREQHLGCGTPGLEGSRTPLQRPFLQGGLPLGNLPSSSPMDSYPGLCQSPFLDSRERGGFFSPEPGEPDSPWTGSGGTTPSTPTTPTTEGEGDGLSYNQRSLQRWEKDEELGQLSTISPVLYANINFPNLKQDYPDWSSRCKQIMKLWRKVPAADKAPYLQKAKDNRAAHRINKVQKQAESQINKQTKVGDIARKTDRPALHLRIPPQPGALGSPPPAAAPTIFIGSPTTPAGLSTSADGFLKPPAGSVPGPDSPGELFLKLPPQVPAQVPSQDPFGLAPAYPLEPRFPTAPPTYPPYPSPTGAPAQPPMLGASSRPGAGQPGEFHTTPPGTPRHQPSTPDPFLKPRCPSLDNLAVPESPGVGGGKASEPLLSPPPFGESRKALEVKKEELGASSPSYGPPNLGFVDSPSSGTHLGGLELKTPDVFKAPLTPRASQVEPQSPGLGLRPQEPPPAQALAPSPPSHPDIFRPGSYTDPYAQPPLTPRPQPPPPESCCALPPRSLPSDPFSRVPASPQSQSSSQSPLTPRPLSAEAFCPSPVTPRFQSPDPYSRPPSRPQSRDPFAPLHKPPRPQPPEVAFKAGSLAHTSLGAGGFPAALPAGPAGELHAKVPSGQPPNFVRSPGTGAFVGTPSPMRFTFPQAVGEPSLKPPVPQPGLPPPHGINSHFGPGPTLGKPQSTNYTVATGNFHPSGSPLGPSSGSTGESYGLSPLRPPSVLPPPAPDGSLPYLSHGASQRSGITSPVEKREDPGTGMGSSLATAELPGTQDPGMSGLSQTELEKQRQRQRLRELLIRQQIQRNTLRQEKETAAAAAGAVGPPGSWGAEPSSPAFEQLSRGQTPFAGTQDKSSLVGLPPSKLSGPILGPGSFPSDDRLSRPPPPATPSSMDVNSRQLVGGSQAFYQRAPYPGSLPLQQQQQQLWQQQQATAATSMRFAMSARFPSTPGPELGRQALGSPLAGISTRLPGPGEPVPGPAGPAQFIELRHNVQKGLGPGGTPFPGQGPPQRPRFYPVSEDPHRLAPEGLRGLAVSGLPPQKPSAPPAPELNNSLHPTPHTKGPTLPTGLELVNRPPSSTELGRPNPLALEAGKLPCEDPELDDDFDAHKALEDDEELAHLGLGVDVAKGDDELGTLENLETNDPHLDDLLNGDEFDLLAYTDPELDTGDKKDIFNEHLRLVESANEKAEREALLRGVEPGPLGPEERPPPAADASEPRLASVLPEVKPKVEEGGRHPSPCQFTIATPKVEPAPAANSLGLGLKPGQSMMGSRDTRMGTGPFSSSGHTAEKASFGATGGPPAHLLTPSPLSGPGGSSLLEKFELESGALTLPGGPAASGDELDKMESSLVASELPLLIEDLLEHEKKELQKKQQLSAQLQPAQQQQQQQQQHSLLSAPGPAQAMSLPHEGSSPSLAGSQQQLSLGLAGARQPGLPQPLMPTQPPAHALQQRLAPSMAMVSNQGHMLSGQHGGQAGLVPQQSSQPVLSQKPMGTMPPSMCMKPQQLAMQQQLANSFFPDTDLDKFAAEDIIDPIAKAKMVALKGIKKVMAQGSIGVAPGMNRQQVSLLAQRLSGGPSSDLQNHVAAGSGQERSAGDPSQPRPNPPTFAQGVINEADQRQYEEWLFHTQQLLQMQLKVLEEQIGVHRKSRKALCAKQRTAKKAGREFPEADAEKLKLVTEQQSKIQKQLDQVRKQQKEHTNLMAEYRNKQQQQQQQQQQQQQQHSAVLALSPSQSPRLLTKLPGQLLPGHGLQPPQGPPGGQAGGLRLTPGDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONGMALPGQPGGPFLNTALAQQQQQQHSGGAGSLAGPSGGFFPGNLALRSLGPDSRLLQERQLQLQQQRMQLAQKLQQQQQQQQQQQHLLGQVAIQQQQQQGPGVQTNQALGPKPQGLMPPSSHQGLLVQQLSPQPPQGPQGMLGPAQVAVLQQQHPGALGPQGPHRQVLMTQSRVLSSPQLAQQGQGLMGHRLVTAQQQQQQQQHQQQGSMAGLSHLQQSLMSHSGQPKLSAQPMGSLQQLQQQQQLQQQQQLQQQQQQQLQQQQQLQQQQLQQQQQQQQLQQQQQQQLQQQQQQLQQQQQQQQQQFQQQQQQQQMGLLNQSRTLLSPQQQQQQQVALGPGMPAKPLQHFSSPGALGPTLLLTGKEQNTVDPAVSSEATEGPSTHQGGPLAIGTTPESMATEPGEVKPSLSGDSQLLLVQPQPQPQPSSLQLQPPLRLPGQQQQQVSLLHTAGGGSHGQLGSGSSSEASSVPHLLAQPSVSLGDQPGSMTQNLLGPQQPMLERPMQNNTGPQPPKPGPVLQSGQGLPGVGIMPTVGQLRAQLQGVLAKNPQLRHLSPQQQQQLQALLMQRQLQQSQAVRQTPPYQEPGTQTSPLQGLLGCQPQLGGFPGPQTGPLQELGAGPRPQGPPRLPAPPGALSTGPVLGPVHPTPPPSSPQEPKRPSQLPSPSSQLPTEAQLPPTHPGTPKPQGPTLEPPPGRVSPAAAQLADTLFSKGLGPWDPPDNLAETQKPEQSSLVPGHLDQVNGQWPEASQLSIKQEPREEPCALGAQSVKREANGEPIGAPGTSNHLLLAGPRSEAGHLLLQKLLRAKNVQLSTGRGSEGLRAEINGHIDSKLAGLEQKLQGTPSNKEDAAARKPLTPKPKRVQKASDRLVSSRKKLRKEDGVRASEALLKQLKQELSLLPLTEPAITANFSLFAPFGSGCPVNGQSQLRGAFGSGALPTGPDYYSQLLTKNNLSNPPTPPSSLPPTPPPSVQQKMVNGVTPSE...

Claims

1. DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATIONCLAIMS1. A composition comprising a bivalent molecule, or pharmaceutically acceptable salt thereof, comprising an aggregate targeting domain and solubility domain, wherein the solubility domain comprises at least a portion of a DNA targeting moiety or an electronegative amino acid sequence with an isoelectric point of from about 2 to about 6.5.

2. The composition of claim 1, wherein the bivalent molecule further comprises a linker sequence positioned between the aggregate targeting domain and the solubility domain.

3. The composition of claims 1 or 2, wherein the bivalent molecule comprises an aggregate targeting domain that associates with amyloid protein with a KD of from about 20 nM to about 250 nM.

4. The composition of any of claims 1 through 3, wherein the solubility domain comprises an electronegative amino acid sequence comprising at least a first amino acid sequence of no less than about 7 contiguous amino acid residues, wherein the amino acid residues are aspartic acid, glutamic acid, or electronegative, non-natural derivative thereof.

5. The composition of claim 4, wherein the first amino acid sequence comprises from about 7 to about 80 contiguous amino acid residues.

6. The composition of any of claims 1 through 5, wherein the aggregate targeting domain comprises an antibody or antigen binding fragment thereof.

7. The composition of claim 6, wherein the aggregate targeting domain is an antigen binding fragment chosen from: an Fab fragment, an Fv fragment, diabody, a scFv, a minibody, a triabody, or a BiTE.

8. The composition of any of claims 1 through 7, wherein the aggregate targeting domain is chemical moiety of ThioflavinT, Congo red, a derivative thereof, tautomer thereof, or pharmaceutically acceptable salt thereof, fused to the solubility domain by a linker.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION9. The composition of any of claims 1 through 8, wherein the solubility domain comprises a nucleic acid targeting moiety chosen from: a transcription factor or functional fragment thereof, a histone protein or functional fragment thereof, SSO7D or a functional variant thereof, or an HMG domain or functional fragment thereof.

10. The composition of claim 9, wherein the DNA targeting moiety is SSO7D or a functional variant thereof and the aggregate targeting domain comprises FOXP2 or a functional variant thereof.

11. The composition of any of claims 1 through 8, wherein the solubility domain comprises a nucleic acid targeting moiety comprises Hoechst, DAPI, a derivative thereof, tautomer thereof, or pharmaceutically acceptable salt thereof.

12. The composition of any of claims 1 through 11, wherein the solubility domain comprises one of: (i) a nonspecific DNA-binding domain; (ii) a phosphomimetic patch; or (iii) a negatively charged peptide comprising no less than about 7 negatively charged amino acid residues.

13. The composition of claim 12, wherein the solubility domain comprises EvoPatch or a derivative thereof.

14. The composition of claim 12, wherein the phosphomimetic patch is from the human FOXP2 protein.

15. The composition of any of claims 1 through 14, wherein the aggregate targeting domain is amyloid or a derivative thereof; or Htt or a functional variant thereof.

16. The composition of any of claims 1 through 15, wherein the bivalent molecule has a KD relative to the aggregate protein to which it is capable of binding of from about 25 nM to about 250 nM.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION17. The composition of any of claims 1 through 15, wherein the bivalent molecule has a KD relative to the aggregate protein to which it is capable of binding of from about 75 nM to about 160 nM.

18. The composition of any of claims 1 through 17, wherein the bivalent molecule has a Kotr relative to the aggregate protein to which it is targeted from about 1 nM to about 999 nM.

19. The composition of any of claims 1 through 18, wherein the bivalent molecule comprises a sequence from Table 12 or a functional variant thereof.

20. A pharmaceutical composition comprising a therapeutically effective amount of the composition of any of claims 1 through 19; and a pharmaceutically acceptable carrier.

21. A method of treating or preventing a protein aggregate disorder in a subj ect in need thereof comprising:(i) administering to the subject the pharmaceutical composition of claim 20.

22. The method of claim 21, wherein the protein aggregate disorder is chosen from: Alzheimer's disease, Huntington’s disease, Parkinson’s disease, Fabry disease, Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), Prion Diseases (e g., Creutzfeldt-Jakob Disease), Multiple System Atrophy (MSA), Lewy Body Dementia, Chronic Traumatic Encephalopathy (CTE), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Spinocerebellar Ataxia (various types), Hereditary Spastic Paraplegia (HSP), Myotonic Dystrophy, Type II Diabetes, Familial Amyloid Polyneuropathy (FAP), Systemic Amyloidosis, Gaucher Disease, Cystic Fibrosis, Retinitis Pigmentosa, Alkaptonuria, Menkes Disease, Wilson’s Disease, or Hereditary ATTR Amyloidosis.

23. The method of either of claims 21 or 22, wherein the pharmaceutical composition is administered by oral adminstration, parenteral administration, sublingual administration, transdermal administration, rectal administration, transmucosal administration, topical administration, inhalation, buccal administration, intrapleural administration, intravenousDOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intranasal administration, intrathecal administration, and intraarticular administration, intracranial adminstration or combinations thereof.

24. The method of any of claims 21 through 23, wherein the subject is a mammal.

25. The method of any of claims 21 through 24, wherein the subject is a human.

26. The method of any of claims 21 through 25, wherein the pharmaceutical composition is administered simultaneously with, prior to or after administration of a neuromodulatory agent.

27. The method of any of claims 21 through 26, wherein the pharmaceutical composition is administered as a dose of from about 0.1 milligrams to about 10 milligrams.

28. The method of any of claims 26, wherein the neuromodulatory agent is chosen from.

29. A method of inhibiting polymerization or assembly of an aggregate protein in a cell comprising: (i) exposing the cell to a composition of any of claims 1 through 19 for a time sufficient to solubilize at least a portion of the aggregate protein.

30. The method of claim 29, wherein the time sufficient to solubilize at least a portion of the aggregate protein is from about 1 second to about 60 seconds.

31. The method of claim 29 or 30, wherein the step of exposing is performed in vitro.

32. The method of claim 29 or 30, wherein the step of exposing is performed in vivo.

33. A kit comprising:(i) a first container comprising the composition of any of claims 1 through 19; and one or more of:(ii) an administration element, instructions for administration of the composition into a subject, or a cell; or, if the bivalent molecule is an amino acid sequence:DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION(i) a first container comprising a nucleic acid sequence encoding the amino acid sequence; and(ii) a cell line, optionally in a second container.

34. The kit of claim 33, wherein the administration element is a syringe.

35. A method of solubilizing an aggregate protein in a cell comprising: (i) exposing the cell to a composition of any of claims 1 through 19 for a time sufficient to solubilize at least a portion of the aggregate protein.

36. The method of claim 35, wherein the time sufficient to solubilize at least a portion of the aggregate protein is from about 1 second to about 60 seconds.

37. The method of claim 35 or 36, wherein the step of exposing is performed in vitro.

38. The method of claim 35 or 36, wherein the step of exposing is performed in vivo.

39. A composition comprising a nucleic acid sequence encoding a bivalent molecule, or a pharmaceutically acceptable salt thereof, comprising an aggregate targeting domain and solubility domain, wherein the solubility domain comprises at least a portion of a DNA targeting moiety or an electronegative amino acid sequence with an isoelectric point of from about 2 to about 6.5.

40. A composition comprising a bivalent molecule comprising an aggregate targeting domain and solubility domain, wherein the solubility domain comprises at least a portion of a DNA targeting moiety or an electric positive amino acid sequence at a neutral pH from about 7 to about 7.4, or a pharmaceutically acceptable salt thereof; or a nucleic acid sequence encoding a bivalent molecule comprising an aggregate targeting domain and solubility domain, wherein the solubility domain comprises at least a portion of a DNA targeting moiety or an electric positive amino acid sequence at a neutral pH from about 7 to about 7.4, or a pharmaceutically acceptable salt thereof.DOCKET NO. STFD-018-PCT INTERNATIONAL APPLICATION41. A method of treating a protein aggregation disorder in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the compositions of claims 39 or 40 and a pharmaceutically acceptable carrier.

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