Protein-like polymers for tuning TAU activity

Abstract

Provided herein are protein-like polymers for tuning tau activity. In aspects, provided herein are protein-like polymers for inhibiting tau fibril propagation in a subject.

Description

LVM Ref. 102-24WO; 340492PROTEIN-LIKE POLYMERS FOR TUNING TAU ACTIVITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 743,133, filed January 8, 2025, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number AG076334 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The content of the electronic sequence listing (340492_102-24WO_US_ST 1.xml; Size: 45,201 bytes; and Date of Creation: January 7, 2026) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0004] Protein misfolding and aggregation are hallmarks of numerous neurodegenerative and systemic diseases, where aggregates spread through a prion-like mechanism, recruiting healthy proteins to adopt pathological conformations (1-6). Diseases such as Alzheimer’s(7, 8), Parkinson’s (3), amyotrophic lateral sclerosis (ALS) (9) and systemic amyloidosis(6) exhibit this pattern of progressive protein aggregation as fibrillar seeds grow, adding proteins to their propagating face. As these misfolded, aggregated proteins accumulate, they impose an increasing burden on cellular degradation pathways, such as the ubiquitin-proteasome system and autophagy, which are already compromised due to aging. The inability of these systems to effectively clear aggregates allows their unchecked growth, resulting in cellular dysfunction, toxicity, and ultimately cell death. Thus, there is a need in the art for an approach aimed at targeting the propagating face of these fibrils.SUMMARY OF THE INVENTION

[0005] Aspects disclosed herein include a polymer having a formula (FX1):LVM Ref. 102-24WO; 340492wherein: each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV); each P2independently comprises a peptide, and each instance of P2is different from each instance of P1; at least one P1independently, or in combination with other instances of P1inhibits tau fibril formation and / or tau fibril elongation; T1and T2are each independently polymer backbone terminating groups that can be the same or different; B1, B2, and B3are each independently a polymer backbone subunit; L1and L2are each independently a linking group; R1is independently a substituent; m is an integer from 2 to 1000; n is an integer from 0 to 1000; o is an integer from 0 to 1000; and each connecting line in the formula (FX1) represents a covalent linkage comprising at least one of a single bond, a double bond, one or more atoms, or any combination thereof, optionally wherein the one or more atoms comprise carbon, nitrogen, and / or oxygen atoms.

[0006] Also disclosed herein is a block copolymer having a formula (FX2):(FX2),wherein: each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV), and wherein each P1further comprises a charge modulating domain, each P2independently comprises a peptide, and each instance of P2is different from each instance of P1, and wherein each P2independently comprises a degrading agent, optionally wherein the degrading agent is or comprises an E3 ligase recruiter; each ATLVM Ref. 102-24WO; 340492independently comprises an analytical tag, optionally wherein the analytical tag is or comprises an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, or a fluorescence tag, optionally wherein the analytical tag is or comprises a fluorophore, optionally wherein the fluorophore is a cyanine dye; T2is a polymer backbone terminating group, optionally wherein T2comprises ester vinyl ether (EVE) or biotin; each of L1, L2, and L3is independently a flexible linker, optionally wherein the flexible linker comprises a flexible hydrocarbon linker, optionally wherein the flexible linker comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 2 (PGGGSK); each m is an integer from 1 to 49; n is an integer from 1 to 49; and o is an integer from 0 to 48; wherein the block copolymer has a degree of polymerization between 2 and 50, optionally between 2 and 20.

[0007] Also disclosed herein is method for inhibiting tau fibril formation and / or tau fibril elongation in a tissue, the method comprising administering to the tissue a polymer having a formula (FX1):wherein: each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV); each P2independently comprises a peptide, and each instance of P2is different from each instance of P1; T1and T2are each independently polymer backbone terminating groups that can be the same or different; B1, B2, and B3are each independently a polymer backbone subunit; L1and L2are each independently a linking group; R1is independently a substituent; m is an integer from 2 to 1000; n is an integer from 0 to 1000; o is an integer from 0 to 1000; and each connecting line in the formula (FX1) represents a covalent linkage comprising at least one of a single bond, a double bond, one or more atoms, or any combination thereof, optionally wherein the one or more atoms comprise carbon, nitrogen, and / or oxygen atoms.

[0008] Also disclosed herein is a method for treating a condition in a subject, the method comprising administering any one of the polymers disclosed herein to the subject, optionallyLVM Ref. 102-24WO; 340492wherein the condition is a 4R tauopathy, optionally wherein the 4R tauopathy is CBD, PSP, or PS19.

[0009] Also disclosed herein is a method for modeling tau fibril seeding in a human nervous system, the method comprising: fusing human iPSC-derived ventral organoids with human iPSC-derived dorsal-directed organoids to generate assembloids; treating the assembloids with fractions comprising active tau seeds; dividing the assembloids into a treatment group of assembloids and a control group of assembloids; administering a tau seeding inhibitor to the treatment group of assembloids; staining the assembloids with a conformational antibody for detecting misfolded tau; and quantifying the amount of conformational antibody in the treatment group of assembloids and the control group of assembloids with a machine learning model, thereby modeling tau fibril seeding in a human nervous system.

[0010] Without wishing to be bound by any particular theory, there may be discussion herein of beliefs or understandings of underlying principles relating to the devices and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGs. 1 A-1C: Introduction to Selected Tau Peptide Fragment for Polymerization. FIG. 1A depicts an overview of tau-originating peptide jR2R3 P301L with the PHF6 sequence underlined. To increase its flexibility in the PLP backbone a linker sequence was added and to increase its solubility and cell-penetrance two carboxy terminus lysines were added. FIG. 1B depicts that Tau fibrils can seed the fibrilization of naive tau monomers (top) or jR2R3 P301L-PLP binding can cap and inhibit fibril growth (bottom). FIG. 1C depicts a schematic of the jR2R3 P301L-PLP with the norbornene backbone in red, the flexible linker in dark orange, the jR2R3 P301L sequence in orange, and the C-terminus lysines in yellow. An optional terminating group containing a fluorophore or biotin is shown as a green star.

[0012] FIGs. 2A-2D: In Vitro Prevention of Puncta formed by jR2R3 P301L. FIG. 2A depicts jR2R3 P301 L fibrils used to seed monomeric jR2R3 P301 L peptide. In the absence of the jR2R3 P301L-PLP, the formation of long paired-helical filaments was observed. In the presence of jR2R3 P301 L-PLP, a marked absence of filaments was observed and those that were present had large globular structures decorating the end of the fibrils. 500 nm Scale Bar. FIG. 2BLVM Ref. 102-24WO; 340492depicts that increasing concentrations led to a complete observable absence of fibril formation as reported by a ThT assay. FIG. 2C depicts biotin-tagged jR2R3 P301 L-PLP, jR2R3 P301 L fibril, or jR2R3 P301L monomer were incubated with fluorescently tagged jR2R3 P301L fibril or jR2R3 P301L monomer. Pull-downs of biotin-tagged species with streptavidin beads showed that the jR2R3 P301 L-PLP interacted with both fibrils and monomers, jR2R3 P301L fibrils pulled-down little monomer or other fibrils, and jR2R3 P301 L monomers were pulled-down fibrils and to a much lesser extent other monomers. FIG. 2D depicts isothermal calorimetry measurements of the binding between the jR2R3 P301 L-PLP (titrate) and jR2R3 P301L monomer (titrant) had a tight binding of 17.8 ± 22.7 nM.

[0013] FIGs. 3A-3D: Uptake of jR2R3 P301 L-PLP Through Direct Cellular Penetration. FIG.3A depicts increasing concentrations of Cy5.5 fluorescently labeled jR2R3 P301 L-PLP (0.25 M — cyan, 0.5 pM — green, 1 pM — sand, 2.0 pM — rose) were monitored by live cell imaging every 30 minutes over 16 hours. The average intracellular fluorescence intensity normalized by total cellular area was plotted with standard error of the mean (SEM) against time (hours). A dotted line at 6 hours indicates the approximate point where the log plot plateaus, signifying a change in uptake rate. The inset shows a strong linear relationship (R2=0.95) between peptide concentration and uptake at the zero time point. FIG. 3B depicts representative live cell image at 6 hours post-PLP exposure, showing intracellular PLP accumulation and membrane-associated PLP (red). Scale bar = 10 pM. FIG. 3C depicts co-staining of PLP (red) with Lampl (green, left) and Rab7 (green, right) showing no observable colocalization between PLP and these endocytic markers. Scale bar = 10 pM. FIG. 3D depicts imaging results after cells were treated with cytochalasin D, dynasore, filipin III, or all inhibitors in combination, and PLP uptake was imaged at 24 hours. PLP uptake remained unaffected across all treatments, supporting the endocytosis-independent mechanism of cellular entry hypothesis.

[0014] FIGs. 4A-4D: Inhibition of Cellular Tau Aggregates by jR2R3 P301 L-PLP. FIG. 4A depicts a schematic representation of the assay setup. Cells stably expressing mClover3-tau187-P301L were incubated with jR2R3 P301L fibrils in the presence or absence of jR2R3 P301 L-PLP. In the absence of PLP, cells accumulated punctate tau inclusions, indicating aggregation. FIG. 4B depicts a fluorescently labeled jR2R3 P301 L-PLP (red) and fluorescently labeled jR2R3 P301L fibrils (green) were added to cells and imaged after 24 hours. Scale bar = 20 pM. FIG. 4C depicts the fraction of cells containing puncta plotted against the concentration of jR2R3 P301L-PLP (n=15 per condition). Each point represents the average of independent measurements, with error bars showing the standard error of the mean. The data were fit toLVM Ref. 102-24WO; 340492dose-response curves. After 24 hours of fibril exposure, the EC50 (the concentration of PLP at which 50% of cells exhibited a reduction in puncta) was calculated to be 104 ± 14 nM, with the percentage of cells displaying puncta approaching 0% at 4 pM PLP. At 48 hours post-fibril addition, the reduction in puncta was less pronounced, with 20% of cells still displaying puncta and an EC50 of 479 ± 100 nM for the reduction in puncta. FIG. 4D depicts representative images of cells treated with 0 pM and 2 pM jR2R3 P301 L-PLP. In the 0 pM condition, punctate inclusions are visible, while in the 2 pM PLP-treated cells, no puncta are observed. Scale bar = 10 pM.

[0015] FIGs. 5A-5D: Stability of jR2R3 P301 L-PLP. FIG. 5A depicts degradation of the PLP as monitored by PAGE at multiple time points. jR2R3 P301 L-PLP was incubated at 37°C with either human serum (top panel) or mouse striatal cell lysate (bottom panel) for 168 hours. No degradation was observed at any time point in either condition, indicating the high stability of the PLP under physiologically relevant conditions. FIG. 5B depicts the formation of tau fibrils as monitored using ThT fluorescence over seven days. In the presence of jR2R3 P301 L-PLP (purple), no ThT fluorescence was detected, suggesting complete inhibition of tau fibril formation over the entire duration. In contrast, in the absence of jR2R3 P301 L-PLP (cyan), fluorescence remained high throughout the experiment, indicating continued fibril formation. FIG. 5C depicts PLP degradation under forced conditions. jR2R3 P301 L-PLP was incubated with trypsin for 60 minutes at 37°C. T rypsin was then inhibited by TLCK, and the treated PLP was introduced to a ThT assay to monitor fibril formation. Trypsin-treated jR2R3 P301 L-PLP (purple) showed a significant increase in ThT fluorescence, indicating that degradation of the PLP compromised its ability to inhibit fibril formation. In contrast, untreated jR2R3 P301 L-PLP (cyan) continued to prevent fibril formation. FIG. 5D depicts TEM images from the ThT assays in panel C. In the absence of trypsin treatment, biotin-tagged jR2R3 P301 L-PLP was associated with tau fibrils, as indicated by streptavidin immuno-gold labeling (white arrowheads, 100 nm scale bar). However, in trypsin-treated samples, no immuno-gold staining was detected, confirming the loss of PLP binding to fibrils after forced degradation.

[0016] FIGs. 6A-6B: Strategies to Increase the Efficacy of jR2R3 P301L-PLP Over Extended Time Periods. FIG. 6A depicts puncta results after jR2R3 P301 L-PLP was added to cells under three different conditions: (1) a single 6-hour exposure followed by washout (cyan), (2) a continuous exposure with no washout (purple), or (3) a second dose added at 24 hours (green). Cells were imaged for puncta at 48 hours. Cells receiving two doses (green) exhibited significantly fewer puncta at 48 hours, with only 7.4% ± 2.1% of cells showing fibril formation atLVM Ref. 102-24WO; 340492a 4 pM concentration, compared to 15% for cells without washout (purple) and 19.5% ± 6.2% for cells washed out after 6 hours (cyan). FIG. 6B depicts puncta results suggesting thatjR2R3 P301L Keap1-PLP (cyan) demonstrated improved efficacy over the original jR2R3 P301L-PLP (purple) when puncta were analyzed at 48 hours. The addition of the Keapl binding peptide resulted in a more significant reduction in tau aggregates, with cells treated with the Keapl variant showing a lower percentage of cells with puncta compared to those treated with the original PLP.

[0017] FIGs. 7A-7D: jR2R3 P301L-PLP Prevents Patient- Derived Fibril Seeding. FIG. 7A depicts a comparison of tau fibril structures from various diseases to the Cryo-EM structure of jR2R3 P301L fibrils. The characteristic stem-loop-stem motif observed in 4-repeat (4R) tauopathies, such as CBD (PDB: 6VHA), PS19 (PDB: 8Q92), and PSP (PDB: 7P65), is spanned by the jR2R3 P301 L fragment (R2: green, R3: purple). This motif is absent in Alzheimer’s disease (AD) fibrils (PDB: 6VHL). FIG. 7B depicts replica exchange simulations of jR2R3 monomers which suggest that the free peptide explores various conformations that can interact with 4R fibrils. FIG. 7C depicts Dot-blot analysis using biotin-tagged jR2R3 P301L-PLP incubated with magnetic streptavidin beads bound to either PSP, PS19, CBD, or AD fibrils. The complexes were washed, eluted, and detected using a total tau antibody (Tau-5). FIG. 7D depicts the fraction of cells with puncta plotted against the concentrations of jR2R3 P301L-PLP. Fibrils from 4R tauopathies (PSP, cyan; PS19, purple; CBD, cyan) were added to cells treated with increasing concentrations of jR2R3 P301L-PLP. The fraction of cells with puncta was plotted against the concentration of jR2R3 P301 L-PLP, revealing a characteristic dose-response curve (Upper). In contrast, mixed 3-repeat (3R) / 4-repeat (4R) tau AD fibrils (purple) were added to cells treated with increasing concentrations of jR2R3 P301 L-PLP. While the fraction of cells with puncta decreased with higher doses, it did not approach baseline levels, and the decrease was unexpectedly linear (Lower). All data points represent at least 15 measurements of a minimum of 100 cells.

[0018] FIGs. 8A-8E: jR2R3 P301 L-PLP Prevents Seeding in Human Forebrain Assembloids. In each of FIGs. 8A-8E, dorsal and ventral assembloids were fused to make forebrain assembloids. Assembloids were treated with the sarkosyl insoluble fractions from 9-month-old PS19 mice brains in the presence (A, B, D) or absence (C) of 4 pM jR2R3-P301L-PLP. Organoids were stained with DAPI (Blue), the MC1 tau conformational antibody (green), and GFAP (red). FIG. 8A depicts 500 pM Scale Bar. FIG. 8B depicts 100 pM Scale Bar. FIG.8C depicts 100 pM Scale Bar. FIG. 8D depicts 15 pM Scale Bar. FIG. 8E depicts that the areaLVM Ref. 102-24WO; 340492of the signal localized to MC1 positive processes divided by the assembloid’s perimeter showed a significant difference when compared between PLP treated and untreated (independent T-Test, p = 0.017).

[0019] FIGs. 9A-9D: Characterization of peptide monomer synthesis. FIG. 9A depicts an analytical HPLC of jR2R3 P301L monomer NorAha-SEQ ID NO. 2: (PGGGSK)-SEQ ID NO: 1: (DINKHVLGGGSVQIVYKPV)-KK after purification. FIG. 9B depicts ESI-MS of jR2R3 P301L monomer, showing the following values: 1008.27 [M+3H], 756.65 [M+4H], 605.58 [M+5H], 504.71 [M+6H], FIG. 9C depicts an analytical HPLC of Keapl monomer NorAha-SEQ ID NO: 4 (LDPETGEFLRRRR) after purification. FIG. 9D depicts an ESI-MS of Keapl monomer, showing the following values: 980.49 [M+2H], 654.05 [M+3H], 490.81 [M+4H],

[0020] FIG. 10 depicts NMR kinetics of jR2R3 P301L monomer polymerization in 1M LiCI DMF.

[0021] FIGs. 11A-11C: Characterization of the Cy5.5 norbornene monomer. FIG. 11A depicts a structure of a norbornene Cy5.5 monomer. FIG. 11B depicts an ESI-MS of NorCy5.5 monomer, showing the following value: 1064.46 [M+H], FIG. 11C depicts an analytical HPLC of NorCy5.5 monomer after purification.

[0022] FIGs. 12A-12C: Synthesis and Characterization of the Biotin Terminating Agent. FIG. 12A depicts a reaction scheme of biotin terminating agent synthesis. FIG. 12B depicts an ESI-MS of biotin terminating agent, showing the following values: 779.41 [M+H], 390.30 [M+2H], FIG. 12C depicts an analytical HPLC of biotin terminating agent after purification.

[0023] FIGs. 13A-13C: Characterization of the Biotin Tagged PLP. FIG. 13A depicts a structure of jR2R3 P301L Biotin PLP. FIG. 13B depicts a molecular weight determination of end labelled polymer by SDS PAGE Gel (MW of 37,500 g / mol). FIG. 13C depicts a quantification of the degree of biotinylation of the polymer (DP of 13).

[0024] FIG. 14A-14E: SDS Page gels used for the characterization of molecular weight of the PLPs. FIG. 14A depicts jR2R3-P301L-PLP 2023 (non labeled) and jR2R3-P301L-PLP Cy5.5 labeled samples, presence of bands at about 30kD to about 50kD for both samples. FIG.14B depicts two lanes of jR2R3-P301L-PLP biotin labeled samples, presence of bands at about 30kD to about 50kD for both lanes. FIG. 14C depicts a first set of samples using Keapl (i.e., “Keap1-1” samples) jR2R3-P301L-PLP (non labeled), jR2R3-P301L-PLP-Keap1, and jR2R3-LVM Ref. 102-24WO; 340492P301L-PLP-Keap1 Cy5.5 labeled samples, presence of bands at about 25kD to about 100kD for all three samples, with the most dense bands at about 25kD to about 50kD for all three samples. FIG. 14D depicts a second set of samples using Keapl (i.e. , “Keap1-2” samples) jR2R3-P301 L-PLP (non labeled)*, jR2R3-P301L-PLP-Keap1, and jR2R3-P301 L-PLP-Keap1 Cy5.5 labeled samples, presence of bands at about 25kD to about 75kD for all samples, with the most dense bands at about 25kD to about 40kD for all samples, ‘results inconclusive. FIG.14E depicts jR2R3-P301 L-PLP 2024 samples, presence of bands at about 25kD to about 75kD.

[0025] FIGs. 15A-15D: Molecular weight determination of PLPs via SEC-MALS. LS refers to light scattering intensity trace. dRI refers to refractive index trace. These parameters are used to determine molecular weight and concentration of specific species in SEC-MALS. 1stGen PROTAC refers to a CUL5 mimic (SEQ ID NO: 14 (LWDDKQLEITKRR)). FIG. 15A depicts jR2R3-P301 L-PLP samples, reflecting MW of 52,110 g / mol and polydispersity index (PDI) of 1.026. FIG. 15B depicts jR2R3-P301 L-PLP biotin labeled samples, reflecting MW of 54,190 g / mol and PDI of 1.153. FIG. 15C depicts jR2R3-P301 L-PLP 1stGen PROTAC samples, reflecting MW of 56,340 g / mol and PDI of 1.066. FIG. 15D depicts jR2R3-P301 L-PLP PROTAC samples, reflecting MW of 44,570 g / mol and PDI of 1.626.

[0026] FIGs. 16A-16B: Dynamic light scattering (DLS) of PLPs. DLS of jR2R3-P301 L-PLP in water over the course of 1 week indicated steady aggregation. Z avg of first 24 hours is 13 nm. FIG. 16A depicts size (d.nm). FIG. 16B depicts correlation coefficient over time (ps).

[0027] FIG. 17: Circular Dichroism (CD) of PLPs. Secondary structure of PLP, Peptide and PROTAC were conserved in Tris HCI buffer.

[0028] FIGs. 18A-18C: SAXS of PLPs. SAXS of PLP (FIG. 18A), Peptide (FIG. 18B), and Fibril (FIG. 18C) in HEPES indicated aggregation.

[0029] FIGs. 19: Control dot blot. jR2R3 P301 L-PLP Cy5.5, jR2R3 P301L monomer FITC, or jR2R3 P301L fibril FITC was incubated with streptavidin magnetic beads for 30 minutes and 37°C and the supernatant, three washes and elution were imaged on a dot blot. No interaction with the beads was observed in these controls.

[0030] FIGs. 20A-20F: Residuals, VQPINK-PLP Control and jR2R3 P301L Peptide Controls. FIG. 20A depicts residuals for the 24 hour timepoint of FIG. 4C. FIG. 20B depicts Residuals for the 48 hour timepoint of FIG. 4C. FIG. 20C depicts the fraction of cells containingLVM Ref. 102-24WO; 340492puncta was plotted against the concentration of jR2R3 P301L peptide (n=15 per condition). Each point represents the average of independent measurements, with error bars showing the standard error of the mean. The data were fit to dose-response curves. FIG. 20D depicts residuals for FIG. 20C. FIG. 20E depicts the fraction of cells containing puncta was plotted against the concentration of VQPINK-PLP (n=15 per condition). Each point represents the average of independent measurements, with error bars showing the standard error of the mean. The data were fit to dose-response curves. FIG. 20F depicts residuals for FIG. 20E.

[0031] FIGs. 21A-21G: Residuals of 48 hour and patient fibril experiments FIG. 21 A depicts residuals of 48 No Washout of FIG. 6A. FIG. 21 B depicts residuals of 48 Double Treatment of FIG. 6A. FIG. 21 C depicts residuals of 48 Keapl of FIG. 6B. FIG. 21 D depicts residuals of PS19 fibrils of FIG. 7D. FIG. 21 E depicts residuals of PSP fibrils of FIG. 7D. FIG. 21 F depicts residuals of CBD fibrils of FIG. 7D. FIG. 21 G depicts residuals of AD fibrils of FIG. 7D.

[0032] FIG. 22 depicts control dot blot for disease fibrils. PSP, PS19, CBD, and AD fibrils were incubated with magnetic beads in the absence of biotin-tagged jR2R3 P301 L-PLP. The first column shows fibrils before incubation with the beads, the second column displays the supernatant containing unbound fibrils, and the last column shows the elution. The blot was stained with a total tau antibody for visualization. Across all conditions, negligible nonspecific binding of disease fibrils to the magnetic-streptavidin beads was observed.

[0033] FIGs. 23A-23D: TEM of Extracted Tau Fibrils. Tau fibrils of Alzheimer's (FIG. 23A), progressive supranuclear palsy (PSP) (FIG. 23B), PS19 mouse (FIG. 23C), and corticobasal degeneration (CBD) (FIG. 23D) were extracted and TEM images were taken to confirm the presence of fibrillar species. Scale bar: 200 nm.STATEMENTS REGARDING CHEMICAL COMPOUNDS AND NOMENCLATURE

[0034] The following abbreviations are used herein: RP-HPLC refers to reverse-phase high performance liquid chromatography; ESI-MS refers to electrospray ionization mass spectrometry; SEC-MALS refers to size-exclusion chromatography coupled with multiangle light scattering; PLP refers to protein-like polymer; SPPS refers to solid-phase peptide synthesis; TEM refers to transmission electron microscopy; STEM refers to scanning TEM; SE or SEM refers to scanning electron microscopy; CD refers to circular dichroism; FPLC refers to fast protein liquid chromatography; and DP refers to degree of polymerization.LVM Ref. 102-24WO; 340492

[0035] In an embodiment, a peptide, a polymer, or a composition (e.g., formulation) of the invention is isolated or purified. In an embodiment, an isolated or purified peptide, polymer, or composition (e.g., formulation) is at least partially isolated or purified as would be understood in the art. In an embodiment, the peptide, polymer, or composition (e.g., formulation) of the invention has a chemical purity of at least 95%, optionally for some applications at least 99%, optionally for some applications at least 99.9%, optionally for some applications at least 99.99%, and optionally for some applications at least 99.999% pure. The invention includes isolated and purified compositions of any of the brush block polymers described herein including the peptide brush and block copolymers and brush and brush block copolymers having one or more side chains comprising the peptide analogues, derivative, variants or fragments.

[0036] As used herein, the term “polymer” refers to a molecule composed of repeating structural units connected by covalent chemical bonds often characterized by a substantial number of repeating units (e.g., equal to or greater than 3 repeating units, optionally, in some embodiments equal to or greater than 5 repeating units, in some embodiments greater or equal to 10 repeating units) and a high molecular weight (e.g., greater than or equal to 1 kDa, in some embodiments greater than or equal to 5 kDa or greater than or equal to 50 kDa). Polymers are commonly the polymerization product of one or more monomer precursors. The term polymer includes homopolymers, or polymers consisting essentially of a single repeating monomer subunit. The term polymer also includes copolymers which are formed when two or more different types of monomers are linked in the same polymer. Copolymers may comprise two or more monomer subunits (e.g., 3 or more monomer subunits, 4 or more monomer subunits, 5 or more monomer subunits, or 6 or more monomer subunits), and include random, block, brush, brush block, alternating, segmented, grafted, tapered and other architectures. In some embodiments, copolymers of the invention comprise from 2 to 10 different monomer subunits. Useful polymers include organic polymers that may be in amorphous, semi-amorphous, crystalline or semi-crystalline states. Cross linked polymers having linked monomer chains are useful for some applications, for example linked by one or more disulfide linkages. The invention provides polymers comprising therapeutic agents, such as brush polymers having at least a portion of the repeating units comprising polymer side chains such as peptide side chains.

[0037] An “oligomer” refers to a molecule composed of repeating structural units connected by covalent chemical bonds often characterized by a number of repeating units less than that of a polymer (e.g., equal to or less than 3 repeating units) and a lower molecular weights (e.g.,LVM Ref. 102-24WO; 340492less than or equal to 1,000 Da) than polymers. Oligomers may be the polymerization product of one or more monomer precursors.

[0038] A “peptide” or “oligopeptide” herein are used interchangeably and refer to a polymer of repeating structural units connected by a peptide bond. Typically, the repeating structural units of the peptide are amino acids including naturally occurring amino acids, non-naturally occurring amino acids, analogues of amino acids or any combination of these. The number of repeating structural units of a peptide, as understood in the art, are typically less than a “protein”, and thus the peptide often has a lower molecular weight than a protein. In some embodiments, a peptide has a chain length of 3 to 150 amino acids, optionally 3 to 100 amino acids, optionally 5 to 50 amino acids, and optionally 5 to 30 amino acids.

[0039] “Block copolymers” are a type of copolymer comprising blocks or spatially segregated domains, wherein different domains comprise different polymerized monomers, for example, including at least two chemically distinguishable blocks. Block copolymers may further comprise one or more other structural domains, such as hydrophobic groups, hydrophilic groups, etc. In a block copolymer, adjacent blocks are constitutionally different, i.e., adjacent blocks comprise constitutional units derived from different species of monomer or from the same species of monomer but with a different composition or sequence distribution of constitutional units. Different blocks (or domains) of a block copolymer may reside on different ends or the interior of a polymer (e.g., [A][B]), or may be provided in a selected sequence ([A][B][A][B]). “Diblock copolymer” refers to block copolymer having two different polymer blocks. “Triblock copolymer” refers to a block copolymer having three different polymer blocks, including compositions in which two non-adjacent blocks are the same or similar. “Pentablock” copolymer refers to a copolymer having five different polymer including compositions in which two or more non-adjacent blocks are the same or similar.

[0040] “Statistical copolymers,” also generally known in the art as “random copolymers,” are copolymers in which the ordering of backbone groups is dictated by reaction kinetics. Statistical copolymers generally are antithetical to block copolymers.

[0041] “Polymer backbone group” or “polymer backbone subunit” refers to groups that are covalently linked to make up a backbone of a polymer, such as a block copolymer. Polymer backbone groups may be linked to side chain groups, such as polymer side chain groups. Some polymer backbone groups useful in the present compositions are derived fromLVM Ref. 102-24WO; 340492polymerization of a monomer selected from the group consisting of a substituted or unsubstituted norbornene, olefin, cyclic olefin, norbornene anhydride, cyclooctene, cyclopentadiene, styrene, acrylamide, and acrylate. Some polymer backbone groups useful in the present compositions are obtained from a ring opening metathesis polymerization (ROMP) reaction. Polymer backbones may terminate in a range of backbone terminating groups including hydrogen, C1-C10 alkyl, C3-C10 cycloalkyl, C5-C10 aryl, C5-C10 heteroaryl, C1-C10 acyl, C1-C10 hydroxyl, C1-C10 alkoxy, C2-C10 alkenyl, C2-C10 alkynyl, C5-C10 alkylaryl, -CO2R30, -CONR31R32, -COR33,-SOR34, -OSR35, -SO2R36,-OR37, -SR38, -NR39R40, -NR41COR42, C1-C10 alkyl halide, phosphonate, phosphonic acid, silane, siloxane, acrylamide, acrylate, or catechol; wherein each of R30-R42is independently hydrogen, C1-C10 alkyl or C5-C10 aryl.

[0042] “Polymer side chain group” (also sometimes referred to herein as “substituent,” e.g., with respect to R1) refers to a group covalently linked (directly or indirectly) to a polymer backbone group that comprises a polymer side chain, optionally imparting steric properties to the polymer. In an embodiment, for example, a polymer side chain group is characterized by a plurality of repeating units having the same, or similar, chemical composition. A polymer side chain group may be directly or indirectly linked to the polymer back bone groups. In some embodiments, polymer side chain groups provide steric bulk and / or interactions that result in an extended polymer backbone and / or a rigid polymer backbone. Some polymer side chain groups useful in the present compositions include unsubstituted or substituted peptide groups. Some polymer side chain groups useful in the present compositions comprise repeating units obtained via anionic polymerization, cationic polymerization, free radical polymerization, group transfer polymerization, or ring-opening polymerization. A polymer side chain may terminate in a wide range of polymer side chain terminating groups including hydrogen, C1-C10 alkyl, C3-C10 cycloalkyl, C5-C10 aryl, C5-C10 heteroaryl, C1-C10 acyl, C1-C10 hydroxyl, C1-C10 alkoxy, C2-C10 alkenyl, C2-C10 alkynyl, C5-C alkylaryl, -CO2R30, -CONR31R32, -COR33,-SOR34, -OSR35, -SO2R36,-OR37, -SR38, -NR39R40, -NR41COR42, C1-C10 alkyl halide, phosphonate, phosphonic acid, silane, siloxane, acrylamide, acrylate, or catechol; wherein each of R30-R42is independently hydrogen or C1-C5 alkyl.

[0043] As used herein, the term “polymer segment” (e.g., first polymer segment, second polymer segment, etc.) refers to a section (e.g., portion) of the polymer comprising a particular monomer or arrangement of monomers. A polymer segment can be a homopolymer or a copolymer. In embodiments where a polymer segment is a copolymer, the copolymer can exist in any suitable arrangement of monomers (e.g., random, block, brush, brush block, alternating,LVM Ref. 102-24WO; 340492segmented, grafted, tapered, statistical and other architectures). In some embodiments, the polymer segments are homopolymers, random copolymers, statistical copolymers, or block copolymers. Any polymer (e.g., brush polymer) described herein can have a single polymer segment or multiple polymer segments. In embodiments where the polymer has multiple polymer segments, the polymer segments can exist in any suitable arrangement (random, block, brush, brush block, alternating, segmented, grafted, tapered, statistical, and other architectures).

[0044] As used herein, the term “degree of polymerization” refers to the average number of monomer units per polymer chain. For example, for certain polymers described herein, comprising B1, B2, and / or B3backbone units, the degree of polymerization would be represented by the sum total of B1, B2, and B3backbone units. Since the degree of polymerization can vary from polymer to polymer, the degree of polymerization is generally represented by an average.

[0045] As used herein, the term “brush polymer” refers to a polymer comprising repeating units each independently comprising a polymer backbone group covalently linked to at least one polymer side chain group. A brush polymer may be characterized by brush density which refers to the percentage of the repeating units comprising polymer side chain groups. Brush polymers of certain aspects are characterized by a brush density greater than or equal to 50% (e.g., greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, or greater than or equal to 90%), optionally for some embodiments a density greater than or equal to 70%, or optionally for some embodiments a density greater than or equal to 90%. Brush polymers of certain aspects are characterized by a brush density selected from the range 50% to 100%, optionally some embodiments a density selected from the range of 75% to 100%, or optionally for some embodiments a density selected from the range of 90% to 100%. Brush polymers, such as the polymers disclosed herein (e.g., a polymer of formula (1)), can be prepared by any suitable methods including, "grafting from" methods, "grafting onto" methods, "grafting through" methods, or any combination thereof. Such suitable methods can include, for example, ring opening metathesis polymerization (ROMP) synthetic pathways and / or nonROM P synthetic pathways, such as, by way of example, reversible addition fragmentation chain transfer (RAFT) polymerization, stable free radical mediated polymerization and atom transfer radical polymerization (ATRP).LVM Ref. 102-24WO; 340492

[0046] As used herein, the term “peptide density” refers to the percentage of monomer units in the polymer chain which have a peptide covalently linked thereto, and such “peptide density” can be calculated generally for all peptides or for a specific peptide. The percentage is based on the overall sum of monomer units in the polymer chain. For example, for certain polymers described herein, the density of peptide P1(or percentage of monomer units comprising peptide P1) in a polymer having m repeat units of peptide P1, n repeat units of B2-R1, and o repeat units of peptide P2, is represented by the formula:where each variable refers to the number of monomer units of that type in the polymer chain. Polymers of certain aspects are characterized by a peptide density greater than or equal to 50% (e.g., greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, or greater than or equal to 90%), optionally for some embodiments a density greater than or equal to 70%, or optionally for some embodiments a density greater than or equal to 90%. Polymers of certain aspects are characterized by a peptide density selected from the range 50% to 100%, optionally some embodiments a density selected from the range of 75% to 100%, or optionally for some embodiments a density selected from the range of 90% to 100%. In some embodiments, the brush density is equal to the peptide density.

[0047] In an aspect, the polymer side chain groups (e.g., also termed substituents herein) can have any suitable spacing on the polymer backbone. Typically, the space between adjacent polymer side chain groups is from 3 angstroms to 30 angstroms, and optionally 5 to 20 angstroms and optionally 5 to 10 angstroms. By way of illustration, in certain embodiments having a brush density of 100%, the polymer side chain groups typically are spaced 6 ± 5 angstroms apart on the polymer backbone. In some embodiments the brush polymer has a high a brush density (e.g., greater than 70%), wherein the polymer side chain groups are spaced 5 to 20 angstroms apart on the polymer backbone.

[0048] As used herein, the term "sequence homology" or "sequence identity" means the proportion of amino acid matches between two amino acid sequences of interest in two different peptides considering the ordering of the amino acids. Matches occur when amino acids are in the same order in one peptide compared to the other peptide. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the fraction of matches over theLVM Ref. 102-24WO; 340492length of sequence that is compared to some other sequence, considering the amino acid order. Gaps (in either of the two sequences) are permitted to maximize matching; for example, wherein gap lengths of 5 amino acids or less, optionally 3 amino acids or less, are usually used. In other words, a sequence having 75% or greater sequence identity to an amino acid sequence with 9 amino acids can indicate that the 9 amino acid sequence can have one or two point mutations (i.e., amino acid change), one or two amino acid deletions, one or two amino acid additions, one point mutation and one amino acid deletion, or one point mutation and one amino acid addition. Even with two such amino acids being different, 7 out of 9 amino acids still match in the correct order, such that there is greater than 75% sequence identity. For clarity, the analysis of whether there sequence homology between two amino acid sequences of interest is conducted with respect to a particular portion of one peptide or protein (i.e., a first amino acid sequence of interest) relative to a particular portion of another peptide or protein (i.e., a second amino acid sequence of interest), and is not conducted relative to all amino acids present in a peptide or protein (i.e., the analysis does not include amino acids outside of the particular amino acid sequence of interest).

[0049] As used herein, the term “amino acid composition similarity” or “amino acid similarity” means the proportion of amino acid matches between two amino acid sequences of interest in two different peptides regardless of the ordering of the amino acids. Matches occur when amino acids are present in both amino acid sequences regardless of order. When amino acid composition similarity is expressed as a percentage, e.g., 50%, the percentage denotes the fraction of matches over the length of sequence that is compared to some other sequence, regardless of amino acid order. Gaps (in either of the two sequences) are permitted to maximize matching; for example, wherein gap lengths of 5 amino acids or less, optionally 3 amino acids or less, are usually used. By way of example, if two amino acid sequences each containing ten amino acids have three amino acids in common, in any order, then there is 30% amino acid composition similarity between the sequences. For clarity, the analysis of whether there is amino acid composition similarity between two amino acid sequences of interest is conducted with respect to a particular portion of one peptide or protein (i.e., a first amino acid sequence of interest) relative to a particular portion of another peptide or protein (i.e., a second amino acid sequence of interest), and is not conducted relative to all amino acids present in a peptide or protein (i.e., the analysis does not include amino acids outside of the particular amino acid sequence of interest).LVM Ref. 102-24WO; 340492

[0050] The term “fragment” refers to a portion, but not all of, a composition or material, such as a peptide composition or material. In an embodiment, a fragment of a peptide refers to 50% or more of the sequence of amino acids, optionally 70% or more of the sequence of amino acids and optionally 90% or more of the sequence of amino acids.

[0051] “Polymer blend” refers to a mixture comprising at least one polymer, such as a brush polymer, e.g., brush block copolymer, and at least one additional component, and optionally more than one additional component. In some embodiments, for example, a polymer blend of the invention comprises a first brush copolymer and one or more addition brush polymers having a composition different than the first brush copolymer. In some embodiments, for example, a polymer blend of the invention further comprises one or more additional brush block copolymers, homopolymers, copolymers, block copolymers, brush block copolymers, oligomers, solvent, small molecules (e.g., molecular weight less than 500 Da, optionally less than 100 Da), or any combination of these. Polymer blends useful for some applications comprise a first brush polymer, and one or more additional components comprising polymers, block copolymers, brush polymers, linear block copolymers, random copolymers, homopolymers, or any combinations of these. Polymer blends of the invention include mixture of two, three, four, five and more polymer components.

[0052] As used herein, the term “compound” can be used to refer to any of the peptides or polymers described herein. Alternatively, or additionally, the term compound can refer to any of the synthetic precursors, reagents, additives, excipients, etc. used in preparation of or formulation with the peptides or polymers described herein.

[0053] As used herein, the term “group” may refer to a functional group of a chemical compound. Groups of the present compounds refer to an atom or a collection of atoms that are a part of the compound. Groups of the present invention may be attached to other atoms of the compound via one or more covalent bonds. Groups may also be characterized with respect to their valence state. The present invention includes groups characterized as monovalent, divalent, trivalent, etc. valence states.

[0054] As used herein, the term “substituted” generally refers to a compound wherein a hydrogen is replaced by another functional group, unless otherwise contradicted by context.

[0055] Unless otherwise specified, the term “average molecular weight” or “molecular weight” refers to number average molecular weight. Number average molecular weight is theLVM Ref. 102-24WO; 340492defined as the total weight of a sample volume divided by the number of molecules within the sample. As is customary and well known in the art, peak average molecular weight and weight average molecular weight may also be used to characterize the molecular weight of the distribution of polymers within a sample.

[0056] As used herein, the term “K18” refers to a recombinant Tau protein fragment having the four domains responsible for microtubule binding and amyloid fibril formation. The 129-residue-long chain is considered the core peptide of Tau, or the active Tau monomer.

[0057] As used herein, “mimic,” “mimicking,” “mimetic,” and grammatically equivalent variations in reference to a compound, oligomer, and / or polymer mimicking a given species (e.g., “proteomimetic”), such as one or more oligo- or poly-peptides (e.g., proteins), means that the compound, oligomer, and / or polymer has a portion with a similar and / or corresponding amino acid sequence to a portion of the given species. In some aspects, the similar and / or corresponding portion typically relates to there being a certain level of sequence homology and / or amino acid composition similarity between the given species and the one or more oligo-or poly-peptides (e.g., proteins). In aspects, a mimetic refers to a material capable of imitating key structures and / or functions of a peptide or protein. Mimetics may be synthetically produced and modified to comprise specific properties depending on desired outcome, including variable size, greater stability, greater affinity, protease-resistance and improved solubility. In aspects, mimetic refers to a protein-like polymer (PLP) designed to engage proteins and the quality control machinery within cells. Callmann CE et al., Poly(peptide): Synthesis, Structure, and Function of Peptide-Polymer Amphiphiles and Protein-like Polymers. Acc Chem Res 2020;53:400-13; Gianneschi NC et al., Biomolecular Densely Grafted Brush Polymers:Oligonucleotides, Oligosaccharides and Oligopeptides. Angew Chemie Int Ed 2020; Blum AP, Kammeyer JK, Gianneschi NC, each of which is incorporated by reference herein in its entirety, and more specifically to facilitate the understanding of PLPs, to the extent not inconsistent with the description herein.

[0058] In aspects of the invention, a mimetic may be modified to comprise a residue-specific modification, a peptide backbone modification, an N-terminal modification, a C-terminal modification, or any combination thereof. In examples, the modification may improve peptide stability, alter peptide structure, incorporate imaging and / or detection agents, improve solubility, enhance non-specific enzyme resistance, reduce steric hindrance, increase cellular penetration, improve binding affinities to targets, enhance safety, or any combination thereof. For example, aLVM Ref. 102-24WO; 340492modification may include one or more of: biotin labeling, contrast agent labeling such as Gd-DOTA labeling, fluorescent dye labeling such as cyanine labeling, fluorescein and 7-methoxycoumarin acetic acid labeling, dansyl and / or 2, 4-dinitrophenyl labeling, EDANS labeling, coumarin labeling, and / or rhodamine labeling, one or more point mutations, introduction of one or more spacers, isotopic labeling, introduction of one or more chelating agents, acetylation, amidation, methylation, palmitylation, hydroxylation, glycosylation, sulfation and sulfonation, esterification, phosphorylation, peptide stapling, lipidation, cyclization, or any combination thereof. In aspects, the peptide sequences described herein (e.g., P1and P2) are modified to incorporate arginine or lysine residues at the N- or at the C- terminus. In aspects, the arginine or lysine residues are added at the C-terminus of the peptide. In aspects, one or two arginine or lysine residues are added at the C-terminus of the peptide sequence. In aspects, two lysine residues are added at the C-terminus of the peptide. In aspects, two arginine residues are added at the C-terminus of the peptide.

[0059] As used herein, the phrase “charge modulating domain” refers to one or more amino acids added to the peptide sequences described herein to modulate the charge of the peptide. For example, the charge modulating domain can be a TAT sequence (e.g., SEQ ID NO: 16 (YGRKKRRQRRR), an Arg8 (e.g., SEQ ID NO: 17 (RRRRRRRR)) a glycine-serine domain (e.g., SEQ ID NO: 13 (GSGSGS), SEQ ID NO: 18 (GSGSG)) a cationic residue domain, or a combination thereof, or optionally a glycine-serine domain, a cationic residue domain, or a combination thereof. In certain embodiments, the charge modulating domain has from 2 to 7 amino acid residues. The 2 to 7 amino acids can be added in a single block containing from 2 to 7 amino acid residues or more than one block containing from 1 to 6 amino acid residues. In some embodiments, the charge modulating domain is a cationic residue domain having from 2 to 7 amino acid residues selected from lysine, arginine, histidine, or a combination thereof. In some embodiments, the charge modulating domain comprises an aspartic acid residue.Generally, the charge modulating domain modulates the charge of the peptide to have a net positive charge. Without wishing to be bound by any particular theory, it is believed that the net positive charge increases the cellular uptake of the peptide or polymer comprising the peptide. The overall charge of the peptide or copolymer comprising the peptide can be determined by any suitable means. For example, the overall charge can be determined by (i) structural analysis of the functional residues on the peptide sequence and their respective pKa, (ii) physical characterization by measuring the zeta potential, and / or (iii) by virtue of the material moving towards a negative pole in an electrophoresis polymer gel. In certain embodiments, theLVM Ref. 102-24WO; 340492overall charge of the peptide or copolymer comprising the peptide is determined by measuring the zeta potential.

[0060] As used herein, a “degrader agent” or “degron” refers to a class of agents capable of directly or indirectly facilitating the regulation of protein degradation. For example, the regulation may comprise promoting degradation, inhibiting degradation, increasing the rate of degradation, and / or decreasing the rate of degradation of a protein. In embodiments, such as wherein the degrader agent is incorporated in a PLP, the degrader agent facilitates specific degradation of a targeted protein. In some aspects of the invention, the degrader agent facilitates ubiquitin recruitment. Without subscribing to a particular theory, it is believed that in aspects wherein the degrader agent facilitates ubiquitin recruitment, the degrader agent participates in the polyubiquitination process to target proteins, or fragments thereof, for degradation by a proteasome. In these aspects, the degrader agent may be referred to herein as a “proteasome recruiter.” In embodiments, the degrader agent comprises a proteasome-targeting chimera (“PROTAC”). In aspects, the degrader agent comprises any suitable number of amino acid units so long as the peptide comprises a sequence having 75% or greater (e.g., 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100%) sequence identity of SEQ ID 6: (ALAPYIP) or SEQ ID: 7 (ALAPYIPR). In aspects, the degrader agent comprises any suitable number of amino acid units so long as the peptide comprises a sequence having 75% or greater (e.g., 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100%) sequence identity of SEQ ID NO: 4 (LDPETGEFLRRRR) or SEQ ID 5:(LDPETGEYL).

[0061] In some embodiments, the degrader agent is a degrader peptide or component or fragment thereof. In embodiments, the degrader agent may be a degrader peptide having a chain length of 3 to 150 amino acids, optionally of 3 to 100 amino acids, optionally 5 to 50 amino acids, optionally 5 to 20 amino acids, and optionally 4 to 10 amino acids. In some embodiments, the degrader agent may comprise a small molecule degrader. In examples, the small molecule degrader comprises a low molecular weight organic compound having a molecular weight of less than or equal to 2 kDa, optionally less than or equal to 1.5 kDa, or optionally less than or equal to 1 kDa. In some embodiments, the degrader agent is characterized by molecular weight between 100 Da and 2000 Da. In some embodiments, the degrader agent is characterized by molecular weight between 250 Da and 1500 Da.LVM Ref. 102-24WO; 340492

[0062] As used herein, “HYDRAC” refers to Heterofunctional polYmeric DegRading Chimeras (HYDRACs). In aspects, HYDRACS are a subclass of PLPs which contain heterologous side chains with distinct functionalities, wherein one domain binds to a protein of interest and a second targets it for degradation.

[0063] As used herein, the terms “alkylene” and “alkylene group” are used synonymously and refer to a divalent group derived from an alkyl group as defined herein. The invention includes compounds having one or more alkylene groups. Alkylene groups in some compounds function as linking and / or spacer groups. Compounds of the invention may have substituted and / or unsubstituted C1-C20 alkylene, C1-C10 alkylene and C1-C5 alkylene groups, for example, as one or more linking groups (e.g., L1, L2, L3).

[0064] As used herein, the terms “cycloalkylene” and “cycloalkylene group” are used synonymously and refer to a divalent group derived from a cycloalkyl group as defined herein. The invention includes compounds having one or more cycloalkylene groups. Cycloalkyl groups in some compounds function as linking and / or spacer groups. Compounds of the invention may have substituted and / or unsubstituted C3-C20 cycloalkylene, C3-C10 cycloalkylene and C3-C5 cycloalkylene groups, for example, as one or more linking groups (e.g., L1, L2, L3).

[0065] As used herein, the terms “arylene” and “arylene group” are used synonymously and refer to a divalent group derived from an aryl group as defined herein. The invention includes compounds having one or more arylene groups. In some embodiments, an arylene is a divalent group derived from an aryl group by removal of hydrogen atoms from two intra-ring carbon atoms of an aromatic ring of the aryl group. Arylene groups in some compounds function as linking and / or spacer groups. Arylene groups in some compounds function as chromophore, fluorophore, aromatic antenna, dye and / or imaging groups. Compounds of the invention include substituted and / or unsubstituted C3-C30 arylene, C3-C20 arylene, C3-C10 arylene and C1-C5 arylene groups, for example, as one or more linking groups (e.g., L1, L2, L3).

[0066] As used herein, the terms “heteroarylene” and “heteroarylene group” are used synonymously and refer to a divalent group derived from a heteroaryl group as defined herein. The invention includes compounds having one or more heteroarylene groups. In some embodiments, a heteroarylene is a divalent group derived from a heteroaryl group by removal of hydrogen atoms from two intra-ring carbon atoms or intra-ring nitrogen atoms of a heteroaromatic or aromatic ring of the heteroaryl group. Heteroarylene groups in someLVM Ref. 102-24WO; 340492compounds function as linking and / or spacer groups. Heteroarylene groups in some compounds function as chromophore, aromatic antenna, fluorophore, dye and / or imaging groups. Compounds of the invention include substituted and / or unsubstituted C3-C30 heteroarylene, C3-C20 heteroarylene, C1-C10 heteroarylene and C3-C5 heteroarylene groups, for example, as one or more linking groups (e.g., L1, L2, L3).

[0067] As used herein, the terms “alkenylene” and “alkenylene group” are used synonymously and refer to a divalent group derived from an alkenyl group as defined herein. The invention includes compounds having one or more alkenylene groups. Alkenylene groups in some compounds function as linking and / or spacer groups. Compounds of the invention include substituted and / or unsubstituted C2-C20 alkenylene, C2-C10 alkenylene and C2-C5 alkenylene groups, for example, as one or more linking groups (e.g., L1, L2, L3).

[0068] As used herein, the terms “cycloalkenylene” and “cycloalkenylene group” are used synonymously and refer to a divalent group derived from a cycloalkenyl group as defined herein. The invention includes compounds having one or more cycloalkenylene groups.Cycloalkenylene groups in some compounds function as linking and / or spacer groups.Compounds of the invention include substituted and / or unsubstituted C3-C20 cycloalkenylene, C3-C10 cycloalkenylene and C3-C5 cycloalkenylene groups, for example, as one or more linking groups (e.g., L1, L2, L3).

[0069] As used herein, the terms “alkynylene” and “alkynylene group” are used synonymously and refer to a divalent group derived from an alkynyl group as defined herein. The invention includes compounds having one or more alkynylene groups. Alkynylene groups in some compounds function as linking and / or spacer groups. Compounds of the invention include substituted and / or unsubstituted C2-C20 alkynylene, C2-C10 alkynylene and C2-C5 alkynylene groups, for example, as one or more linking groups (e.g., L1, L2, L3).

[0070] As used herein, the term “halo” refers to a halogen group such as a fluoro (-F), chloro (-CI), bromo (-Br), iodo (-I) or astato (-At).

[0071] The term "heterocyclic" refers to ring structures containing at least one other kind of atom, in addition to carbon, in the ring. Examples of such heteroatoms include nitrogen, oxygen and sulfur. Heterocyclic rings include heterocyclic alicyclic rings and heterocyclic aromatic rings. Examples of heterocyclic rings include, but are not limited to, pyrrolidinyl, piperidyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, quinolyl, isoquinolyl,LVM Ref. 102-24WO; 340492pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, benzoxadiazolyl, benzothiadiazolyl, triazolyl and tetrazolyl groups. Atoms of heterocyclic rings can be bonded to a wide range of other atoms and functional groups, for example, provided as substituents.

[0072] The term “carbocyclic” refers to ring structures containing only carbon atoms in the ring. Carbon atoms of carbocyclic rings can be bonded to a wide range of other atoms and functional groups, for example, provided as substituents.

[0073] The term “alicyclic ring” refers to a ring, or plurality of fused rings, that is not an aromatic ring. Alicyclic rings include both carbocyclic and heterocyclic rings.

[0074] The term “aromatic ring” refers to a ring, or a plurality of fused rings, that includes at least one aromatic ring group. The term aromatic ring includes aromatic rings comprising carbon, hydrogen and heteroatoms. Aromatic ring includes carbocyclic and heterocyclic aromatic rings. Aromatic rings are components of aryl groups.

[0075] The term “fused ring” or “fused ring structure” refers to a plurality of alicyclic and / or aromatic rings provided in a fused ring configuration, such as fused rings that share at least two intra ring carbon atoms and / or heteroatoms.

[0076] As used herein, the term "alkoxyalkyl" refers to a substituent of the formula alkyl-O-alkyl.

[0077] As used herein, the term "polyhydroxyalkyl" refers to a substituent having from 2 to 12 carbon atoms and from 2 to 5 hydroxyl groups, such as the 2,3-dihydroxypropyl, 2,3,4-trihydroxybutyl or 2,3,4, 5-tetrahydroxypentyl residue.

[0078] As used herein, the term "polyalkoxyalkyl" refers to a substituent of the formula alkyl-(alkoxy)n-alkoxy wherein n is an integer from 1 to 10, preferably 1 to 4, and more preferably for some embodiments 1 to 3.

[0079] Amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, asparagine, glutamine, glycine, serine, threonine, serine, threonine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid. As used herein, reference to “a side chain residue of a natural a-amino acid” specifically includesLVM Ref. 102-24WO; 340492the side chains of the above-referenced amino acids. Peptides are comprised of two or more amino acids connected via peptide bonds.

[0080] Alkyl groups include straight-chain, branched and cyclic alkyl groups. Alkyl groups include those having from 1 to 30 carbon atoms. Alkyl groups include small alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length alkyl groups having from 4-10 carbon atoms. Alkyl groups include long alkyl groups having more than 10 carbon atoms, particularly those having 10-30 carbon atoms. The term cycloalkyl specifically refers to an alky group having a ring structure such as ring structure comprising 3-30 carbon atoms, optionally 3-20 carbon atoms and optionally 2 - 10 carbon atoms, including an alkyl group having one or more rings. Cycloalkyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring(s) and particularly those having a 3-, 4-, 5-, 6-, or 7-member ring(s). The carbon rings in cycloalkyl groups can also carry alkyl groups. Cycloalkyl groups can include bicyclic and tricycloalkyl groups. Alkyl groups are optionally substituted. Substituted alkyl groups include among others those which are substituted with aryl groups, which in turn can be optionally substituted. Specific alkyl groups include methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all of which are optionally substituted. Substituted alkyl groups include fully halogenated or semihalogenated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and / or iodine atoms. Substituted alkyl groups include fully fluorinated or semifluorinated alkyl groups, such as alkyl groups having one or more hydrogens replaced with one or more fluorine atoms. An alkoxy group is an alkyl group that has been modified by linkage to oxygen and can be represented by the formula R-0 and can also be referred to as an alkyl ether group.Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy and heptoxy. Alkoxy groups include substituted alkoxy groups wherein the alky portion of the groups is substituted as provided herein in connection with the description of alkyl groups. As used herein MeO- refers to CH3O-. Compositions of some embodiments of the invention comprise alkyl groups as terminating groups, such as polymer backbone terminating groups and / or polymer side chain terminating groups.

[0081] Alkenyl groups include straight-chain, branched and cyclic alkenyl groups. Alkenyl groups include those having 1 , 2 or more double bonds and those in which two or more of the double bonds are conjugated double bonds. Alkenyl groups include those having from 2 to 20 carbon atoms. Alkenyl groups include small alkenyl groups having 2 to 3 carbon atoms.LVM Ref. 102-24WO; 340492Alkenyl groups include medium length alkenyl groups having from 4-10 carbon atoms. Alkenyl groups include long alkenyl groups having more than 10 carbon atoms, particularly those having 10-20 carbon atoms. Cycloalkenyl groups include those in which a double bond is in the ring or in an alkenyl group attached to a ring. The term cycloalkenyl specifically refers to an alkenyl group having a ring structure, including an alkenyl group having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring(s) and particularly those having a 3-, 4-, 5-, 6- or 7-member ring(s). The carbon rings in cycloalkenyl groups can also carry alkyl groups. Cycloalkenyl groups can include bicyclic and tricyclic alkenyl groups. Alkenyl groups are optionally substituted.Substituted alkenyl groups include among others those which are substituted with alkyl or aryl groups, which groups in turn can be optionally substituted. Specific alkenyl groups include ethenyl, prop-1-enyl, prop-2-enyl, cycloprop- 1-enyl, but-1-enyl, but-2-enyl, cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl, pent-2-enyl, branched pentenyl, cyclopent- 1-enyl, hex-1-enyl, branched hexenyl, cyclohexenyl, all of which are optionally substituted. Substituted alkenyl groups include fully halogenated or semihalogenated alkenyl groups, such as alkenyl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and / or iodine atoms. Substituted alkenyl groups include fully fluorinated or semifluorinated alkenyl groups, such as alkenyl groups having one or more hydrogen atoms replaced with one or more fluorine atoms. Compositions of some embodiments of the invention comprise alkenyl groups as terminating groups, such as polymer backbone terminating groups and / or polymer side chain terminating groups.

[0082] Aryl groups include groups having one or more 5-, 6- or 7- member aromatic rings, including heterocyclic aromatic rings. The term heteroaryl specifically refers to aryl groups having at least one 5-, 6- or 7- member heterocyclic aromatic rings. Aryl groups can contain one or more fused aromatic rings, including one or more fused heteroaromatic rings, and / or a combination of one or more aromatic rings and one or more nonaromatic rings that may be fused or linked via covalent bonds. Heterocyclic aromatic rings can include one or more N, O, or S atoms in the ring. Heterocyclic aromatic rings can include those with one, two or three N atoms, those with one or two O atoms, and those with one or two S atoms, or combinations of one or two or three N, O or S atoms. Aryl groups are optionally substituted. Substituted aryl groups include among others those which are substituted with alkyl or alkenyl groups, which groups in turn can be optionally substituted. Specific aryl groups include phenyl, biphenyl groups, pyrrolidinyl, imidazolidinyl, tetra hydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl,LVM Ref. 102-24WO; 340492pyridinyl, benzoxadiazolyl, benzothiadiazolyl, and naphthyl groups, all of which are optionally substituted. Substituted aryl groups include fully halogenated or semihalogenated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and / or iodine atoms. Substituted aryl groups include fully fluorinated or semifluorinated aryl groups, such as aryl groups having one or more hydrogens replaced with one or more fluorine atoms. Aryl groups include, but are not limited to, aromatic group-containing or heterocylic aromatic group-containing groups corresponding to any one of the following: benzene, naphthalene, naphthoquinone, diphenylmethane, fluorene, anthracene, anthraquinone, phenanthrene, tetracene, tetracenedione, pyridine, quinoline, isoquinoline, indoles, isoindole, pyrrole, imidazole, oxazole, thiazole, pyrazole, pyrazine, pyrimidine, purine, benzimidazole, furans, benzofuran, dibenzofuran, carbazole, acridine, acridone, phenanthridine, thiophene, benzothiophene, dibenzothiophene, xanthene, xanthone, flavone, coumarin, azulene or anthracycline. As used herein, a group corresponding to the groups listed above expressly includes an aromatic or heterocyclic aromatic group, including monovalent, divalent and polyvalent groups, of the aromatic and heterocyclic aromatic groups listed herein are provided in a covalently bonded configuration in the compounds of the invention at any suitable point of attachment. In embodiments, aryl groups contain between 5 and 30 carbon atoms. In embodiments, aryl groups contain one aromatic or heteroaromatic six-membered ring and one or more additional five- or six-membered aromatic or heteroaromatic ring. In embodiments, aryl groups contain between five and eighteen carbon atoms in the rings. Aryl groups optionally have one or more aromatic rings or heterocyclic aromatic rings having one or more electron donating groups, electron withdrawing groups and / or targeting ligands provided as substituents. Compositions of some embodiments of the invention comprise aryl groups as terminating groups, such as polymer backbone terminating groups and / or polymer side chain terminating groups.

[0083] Arylalkyl groups are alkyl groups substituted with one or more aryl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. Specific alkylaryl groups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups. Alkylaryl groups are alternatively described as aryl groups substituted with one or more alkyl groups wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted. Specific alkylaryl groups are alkyl-substituted phenyl groups such as methylphenyl. Substituted arylalkyl groups include fully halogenated or semihalogenated arylalkyl groups, such as arylalkyl groups having one or more alkyl and / or aryl groups havingLVM Ref. 102-24WO; 340492one or more hydrogens replaced with one or more fluorine atoms, chlorine atoms, bromine atoms and / or iodine atoms. Compositions of some embodiments of the invention comprise arylalkyl groups as terminating groups, such as polymer backbone terminating groups and / or polymer side chain terminating groups.

[0084] As to any of the groups described herein which contain one or more substituents, it is understood that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. Optional substitution of alkyl groups includes substitution with one or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or aryl groups are optionally substituted. Optional substitution of alkenyl groups includes substitution with one or more alkyl groups, aryl groups, or both, wherein the alkyl groups or aryl groups are optionally substituted. Optional substitution of aryl groups includes substitution of the aryl ring with one or more alkyl groups, alkenyl groups, or both, wherein the alkyl groups or alkenyl groups are optionally substituted.

[0085] Optional substituents for any alkyl, alkenyl and aryl group includes substitution with one or more of the following substituents, among others: halogen, including fluorine, chlorine, bromine or iodine; pseudohalides, including -ON;

[0086] -COOR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted;

[0087] -COR where R is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted;

[0088] -CON(R)2where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted; and where R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;

[0089] -OCON(R)2 where each R, independently of each other R, is a hydrogen or an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group all of which groups are optionally substituted; and where R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;LVM Ref. 102-24WO; 340492

[0090] -N(R)2where each R, independently of each other R, is a hydrogen, or an alkyl group, or an acyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, phenyl or acetyl group, all of which are optionally substituted; and where R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;

[0091] -SR, where R is hydrogen or an alkyl group or an aryl group and more specifically where R is hydrogen, methyl, ethyl, propyl, butyl, or a phenyl group, which are optionally substituted;

[0092] -SO2R, or -SOR where R is an alkyl group or an aryl group and more specifically where R is a methyl, ethyl, propyl, butyl, or phenyl group, all of which are optionally substituted;

[0093] -OCOOR where R is an alkyl group or an aryl group;

[0094] -SO2N(R)2where each R, independently of each other R, is a hydrogen, or an alkyl group, or an aryl group all of which are optionally substituted and wherein R and R can form a ring which can contain one or more double bonds and can contain one or more additional carbon atoms;

[0095] -OR where R is H, an alkyl group, an aryl group, or an acyl group all of which are optionally substituted. In a particular example R can be an acyl yielding -OCOR” where R” is a hydrogen or an alkyl group or an aryl group and more specifically where R” is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted.

[0096] Specific substituted alkyl groups include haloalkyl groups, particularly trihalomethyl groups and specifically trifluoromethyl groups. Specific substituted aryl groups include mono-, di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalene groups; 3- or 4-halo-substituted phenyl groups, 3- or4-alkyl-substituted phenyl groups, 3- or 4-alkoxy-substituted phenyl groups, 3- or 4-RCO-substituted phenyl, 5- or6-halo-substituted naphthalene groups. More specifically, substituted aryl groups include acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups, particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups, particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups, particularly 4-methylphenyl groups; and methoxyphenyl groups, particularly 4-methoxyphenyl groups.LVM Ref. 102-24WO; 340492

[0097] As to any of the above groups which contain one or more substituents, it is understood that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible.

[0098] The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for the present invention. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5, which is combined with buffer prior to use.

[0099] Thus, the compounds, oligomers, or polymers disclosed herein may exist as salts, such as with pharmaceutically acceptable acids. Examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof includingLVM Ref. 102-24WO; 340492racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid. These salts may be prepared by methods known to those skilled in the art.

[0100] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0101] In addition to salt forms, the present invention provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0102] Certain compounds, oligomers, or polymers disclosed herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds disclosed herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the disclosed compounds, oligomers, or polymers.

[0103] As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present invention. 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.

[0104] Certain compounds of the present invention possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as ( / ?)- or (S)- or, as d- or I- for amino acids, and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those which are known in art to be too unstable to synthesize and / or isolate. The present invention is meant to include compounds in racemic and optically pureLVM Ref. 102-24WO; 340492forms. Optically active (R)- and (S)-, or d- or I -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

[0105] As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms. Isomers include structural isomers and stereoisomers such as enantiomers.

[0106] The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[0107] It will be apparent to one skilled in the art that certain compounds of this invention may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the invention.

[0108] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

[0109] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by13C- or14C-enriched carbon are within the scope of this invention.

[0110] As is customary and well known in the art, hydrogen atoms in formulas (e.g., FX1, FX2) are not always explicitly shown, for example, hydrogen atoms bonded to the carbon atoms of aromatic, heteroaromatic, and alicyclic rings are not always explicitly shown in formula (FX1). The structures provided herein, for example in the context of the description of formulas (e.g., FX1, FX2) and schematics and structures in the drawings, are intended to convey to one of reasonable skill in the art the chemical composition of compounds of the methods and compositions of the invention, and as will be understood by one of skill in the art, the structuresLVM Ref. 102-24WO; 340492provided do not indicate the specific positions and / or orientations of atoms and the corresponding bond angles between atoms of these compounds.

[0111] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125l), or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0112] The symboldenotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0113] The terms “treating” or “treatment” refers to any indicia of success in the treatment or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to a subject, such as a patient in need of treatment; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a subject's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and / or a psychiatric evaluation.

[0114] An “effective amount” is an amount sufficient to accomplish a stated purpose (e.g., achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce transcriptional activity, increase transcriptional activity, reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occurLVM Ref. 102-24WO; 340492only after administration of a series of doses. Thus, a prophylactical ly effective amount may be administered in one or more administrations. An “activity decreasing amount,” as used herein, refers to an amount of antagonist (inhibitor) required to decrease the activity of an enzyme or protein (e.g., transcription factor) relative to the absence of the antagonist. An “activity increasing amount,” as used herein, refers to an amount of agonist (activator) required to increase the activity of an enzyme or protein (e.g., transcription factor) relative to the absence of the agonist. A “function disrupting amount,” as used herein, refers to the amount of antagonist (inhibitor) required to disrupt the function of an enzyme or protein (e.g., transcription factor) relative to the absence of the antagonist. A “function increasing amount,” as used herein, refers to the amount of agonist (activator) required to increase the function of an enzyme or protein (e.g., transcription factor) relative to the absence of the agonist. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0115] As used herein, the term “inhibition,” “inhibit,” “inhibiting,” and grammatically equivalent variations in reference to a compound, oligomer, and / or polymer inhibiting aggregation means disrupting, preventing, or otherwise negatively affecting (e.g., decreasing) the ability of a given species, such as one or more oligo- or poly-peptides (e.g., proteins), to bind together (e.g., noncovalently) or otherwise associate relative to the level of association of such species in the absence of the compound, oligomer, and / or polymer. As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor (e.g., antagonist) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In some embodiments inhibition refers to reduction of a disease or symptoms of disease. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein.

[0116] As defined herein, the term “activation”, “activate”, “activating” and the like in reference to a protein-activator (e.g., agonist) interaction means positively affecting (e.g., increasing) the activity or function of the protein.LVM Ref. 102-24WO; 340492

[0117] The term “modulator” refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule.

[0118] “Patient”, “subject”, 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 nonmammalian animals. In some embodiments, a patient is human. In some embodiments, a patient is a mammal. In some embodiments, a patient is a mouse. In some embodiments, a patient is an experimental animal. In some embodiments, a patient is a rat. In some embodiments, a patient is a test animal.

[0119] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCI, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0120] The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0121] As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular,LVM Ref. 102-24WO; 340492intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). In embodiments, administration includes direct administration to a tumor. Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g., anti-cancer agent or chemotherapeutic). The compound of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm.Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In another embodiment, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellularLVM Ref. 102-24WO; 340492membrane or are endocytosed, i.e. , by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. QA& Q- , 1989).

[0122] As used herein, the term “conjugated” when referring to two moieties means the two moieties are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent. In embodiments, the two moieties are covalently bonded to each other (e.g., directly or through a covalently bonded intermediary). In embodiments, the two moieties are non-covalently bonded (e.g., through ionic bond(s), van der waal's bond(s) / interactions, hydrogen bond(s), polar bond(s), or combinations or mixtures thereof).

[0123] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In some aspects, about means within a standard deviation using measurements generally acceptable in the art. In some aspects, about means a range extending to + / - 10% of the specified value. In embodiments, about means the specified value.

[0124] Various polymers disclosed herein are characterized, in part, by the relative amounts of distinct functional side chains present in the polymer. In aspects, the relative amounts of distinct functional side chains is represented as an average ratio defined herein as “peptide ratio.” Since the degree of polymerization can vary from polymer to polymer, the composition of monomers (and functional side chains of said monomers) can also vary. Therefore, the peptide ratio should be understood as an average. It will be appreciated by one having skill in the art that polymerization methods and subsequent analysis methods are subject to random, experimental error, and the peptide ratios should therefore be read to encompass reasonable variations from the stated value. Specifically, in some aspects, peptide ratios associated with functional side chains of polymers include variations of ±20% of the stated ratio. In keeping with this aspect, a P1:P2ratio of 2:1 includes variations of P1:P2ratios of 1.6:1 to 2.4:1 (e.g., 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1) and 2:0.8 to 2:1.2 (e.g., 2:0.8, 2:0.9, 2:1, 2:1.1, 2:1.2). In some aspects, peptide ratios associated with functional side chains of polymers include variations of ±10% of the stated ratio. In some aspects, peptide ratios associated with functional side chains ofLVM Ref. 102-24WO; 340492polymers include variations of ±5% of the stated ratio. In some aspects, peptide ratios associated with functional side chains of polymers include variations of ±1% of the stated ratio.

[0125] As used herein, the term “acceleration,” “accelerate,” “accelerating,” and grammatically equivalent variations in reference to a compound, oligomer, and / or polymer accelerating aggregation means promoting, facilitating, or otherwise positively affecting (e.g., increasing or speeding up) the ability of a given species, such as one or more oligo- or polypeptides (e.g., proteins), to bind together (e.g., noncovalently) or otherwise associate relative to the level of association of such species in the absence of the compound, oligomer, and / or polymer. Acceleration of aggregation is relevant, for example, in the context of speeding up aggregation past a toxic species, such as nanofibirils, towards higher order aggregates that are less toxic, so as to reduce overall toxicity of the system.

[0126] As used herein, the term “bind,” “binding,” and grammatically equivalent variations in reference to a compound, oligomer, and / or polymer binding to a given species, such as one or more oligo- or poly-peptides (e.g., proteins), means that the compound, oligomer, and / or polymer makes multiple noncovalent bonds to the given species. In some aspects, the multiple noncovalent bonds are possible because the compound, oligomer, and / or polymer has a portion with a similar and / or corresponding amino acid sequence to a portion of the given species.

[0127] As used herein, the phrase “at least a portion of each instance of P1independently comprises at least” a specified percentage of amino acid composition similarity and / or sequence homology, and similar phrasing, refers to a discrete segment of P1having the indicated amino acid composition similarity and / or sequence homology. For clarity, the specified percentage is not determined by references to single amino acids taken from unrelated segments of P1.

[0128] As used herein, the phrase “at least a portion of each instance of P1independently is or comprises at least one of” specified amino acid sequences, and similar phrasing, means that each P1can be any of the specified amino acid sequences or any combination of the specified amino acid sequences.

[0129] As used herein, the term “tau-binding peptide” refers to a peptide or a peptide fragment that specifically binds to a tau protein, a tau polypeptide, and / or a tau aggregate (including, for example, tau monomers, tau oligomers, tau protofibrils, and tau fibrils).LVM Ref. 102-24WO; 340492

[0130] As used herein, “triggering an organism’s ubiquitination cellular machinery,” or similar phrasing, means activating an organism’s molecular components, such as enzymes, to cause ubiquitination of an oligo- or poly-peptide (e.g., protein) of interest, such as tau.

[0131] As used herein, an “aggregative region” of an oligo- or poly-peptide (e.g., protein) is the portion thereof that is prone to aggregate with the same or similar oligo- or poly-peptides to form, for example, aggregated species up to and including amyloids (e.g., of tau proteins).

[0132] As used herein, “metaphilic” means a compound, oligomer, or polymer that is transiently amphiphilic, such as byway of a hydrophobic backbone with hydrophilic side chains. In some aspect, the metaphilicity leads to globular but fluxional structures, which may be useful for penetrating cell walls.DETAILED DESCRIPTION OF THE INVENTION

[0133] In the following description, numerous specific details of the devices, device components and methods of the present invention are set forth in order to provide a thorough explanation of the precise nature of the invention. It will be apparent, however, to those of skill in the art that the invention can be practiced without these specific details.

[0134] The misfolding, aggregation, and spread of tau protein fibrils underlie tauopathies, a diverse class of neurodegenerative diseases for which effective treatments remain elusive. Among these diseases are corticobasal dementia (CBD) and progressive supranuclear palsy (PSP), canonical examples of 4-repeat (4R) tauopathies characterized by tau isoforms with four microtube-binding repeat domains. In aspects, this 4R tau isoform-specific mechanism is targeted, focusing on the misfolded tau’s distinctive stem-loop-stem structural motif formed by the alternatively spliced exon encoding this fourth repeat domain. It was hypothesized that a synthetic peptide based on this stem-loop-stem sequence could induce aggregation and spread in an isoform specifical manner. A protein-like polymer (PLP) was developed in which multiple copies of this synthetic peptide were observed to form a comb-like structure capable of preventing tau aggregation by binding and capping fibril ends in vitro, in human brain organoids, and in cellular models with an EC50 of 105 ± 14 nM. In aspects, the PLP demonstrates robust activity against fibrils derived from CBD and PSP patient brains and a PS19 mouse tauopathy model. In aspects, the PLP is optimized for cellular uptake, water solubility, flexibility, or any combination thereof. Preferably, the PLP is optimized for cellular uptake, water solubility, and flexibility. In aspects, this PLP-based approach provides a highly specific and potentially safeLVM Ref. 102-24WO; 340492therapeutic avenue for tauopathies. Previous tau-targeted treatments have primarily focused on broad tau clearance, aggregation inhibition, or microtubule stabilization, often lacking isoform specificity and precision. In contrast, approaches described herein target the 4R tau isoform’s unique structural motif, offering a more tailored therapeutic intervention for diseases like CBD and PSP. Supported by PLP’s blood-brain barrier penetrance and safety profile in prior studies, the findings discussed herein offer a promising translational path toward clinical applications in tauopathy treatment.

[0135] The polymers discussed herein comprise one or more tau-binding peptides. In some aspects, the tau-binding peptide is capable of associating with tau via one or more non-covalent interactions (e.g., electrostatic interactions, hydrogen bonding, hydrophobic interactions, TT-TT interactions) and may thereby localize to tau species in solution or in an aggregated stated. In some aspects, “binds” and “binding” when used in the context of a tau-binding peptide, refer to formation of a detectable complex between the tau-binding peptide and tau (or a tau aggregate) under physiological or assay conditions, as measured by any suitable methodology. In some aspects, the tau-binding peptide preferentially binds tau relative to non-tau proteins and / or relative to other amyloidogenic proteins (e.g., A , a-synuclein), for example as evidenced by a lower dissociation constant for tau than for a non-tau protein under the same conditions, or by a higher fraction bound to tau at a given peptide concentration. In some aspects, the tau-binding peptide binds one or more tau isoforms and / or tau fragments that participate in aggregation. In some aspects, the tau-binding peptide comprises, consists essentially of, or consists of amino acids and may be a linear peptide, cyclic peptide, constrained peptide, or stabled peptide. In some aspects, the tau-binding peptide comprises L-amino acids, D-amino acids, or a combination thereof, and may include non-natural amino acids, amino acid analogs, peptidomimetics, or backbone modifications (e.g., N-methylation) provided the peptide retains tau-binding activity. In some aspects, the tau-binding peptide comprises from about 3 to about 150 amino acids (e.g., about 4 to about 150 amino acids, about 4 to about 100 amino acids, about 4 to about 75 amino acids, about 4 to about 50 amino acids, about 5 to about 150 amino acids, about 5 to about 100 amino acids, about 5 to about 75 amino acids, about 5 to about 50 amino acids, or about 5 to about 25 amino acids). In some aspects, the tau-binding peptide comprises terminal modifications (e.g., N-terminal acetylation, C-terminal amidation), and / or may be conjugated to a linker, carrier, polymer, or other moiety, provided the tau-binding peptide retains tau-binding activity. In some aspects, the tau-binding peptide is a peptide side chain of a molecular construct, for example, a side chain of the PLPs discussed herein.LVM Ref. 102-24WO; 340492

[0136] In some aspects, the tau-binding peptide refers to a peptide that binds tau fibrils and / or tau protofibrils. In some aspects, the tau-binding peptide binds an end region of a tau fibril and is capable of inhibiting, reducing, or delaying tau fibril elongation (e.g., by end-capping) relative to a control lacking the peptide.

[0137] In some aspects, the tau-binding peptide is or comprises a peptide that is capable of capping and inhibiting tau fibril growth. For example, in some aspects, the tau-binding peptide reduces a tau fibril elongation rate and / or reduces formation of higher-order tau aggregates in an in vitro aggregation assay, relative to an otherwise identical condition lacking the tau-binding peptide. In some aspects, such reduction is statistically significant and / or is at least about 10%, 25%, 50%, 75%, or 90% relative to control, as measured by an assay such as ThT fluorescence kinetics, turbidity, electron microscopy / AFM-based fibril length distributions, seeded growth assays, or other suitable aggregation metrics.

[0138] In some aspects, the tau-binding peptide comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV). In some aspects, the tau-binding peptide consists of SEQ ID NO: 1.

[0139] In some aspects, the tau-binding peptide is or comprises a variant of SEQ ID NO: 1 that retains tau-binding activity and / or the ability to cap and inhibit tau fibril growth. In some aspects, the variant has at least about 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 1 over the full length of SEQ ID NO: 1. In some aspects, the variant comprises one or more conservative amino acid substitutions relative to SEQ ID NO: 1. In some aspects, the tau-binding peptide comprises SEQ ID NO: 1 with one or more terminal modifications (e.g., N-acetylation and / or C-amidation), one or more amino acid substitutions, deletions, or additions that do not materially reduce tau-binding and / or capping activity, or a truncated fragment of SEQ ID NO: 1 that retains tau-binding activity. In some aspects, the tau-binding peptide is or comprises SEQ ID NO: 1 , a functional fragment thereof, or a functional variant thereof, wherein “functional” means capable of binding tau and inhibiting tau fibril growth in an assay.

[0140] In aspects, the polymers discussed herein comprise one or more linking groups (also referred to herein as a linker or a linker group) between a polymer backbone subunit (e.g., a norbornyl polymer backbone subunit) and a peptide (e.g., a tau-binding peptide). The length of a linking group may be selected for certain features. For example, a longer link group may beLVM Ref. 102-24WO; 340492used in order to separate the peptide from the polymer backbone, thereby making the peptide more flexible and sterically accessible. In aspects, the linkers are cleavable under appropriate biological conditions or by exogenous sources. Appropriate pH- or UV-sensitive linkers may be included in the polymers. In aspects, polymer-drug conjugates improve drug solubility, circulation time (through the properties of the polymer carrier), and drug targeting (via the use of appropriate linkers that can respond to changes in physiological conditions such as temperature, pH, and the presence of enzymes). In aspects, the linking group is a peptidic moiety. In aspects, the linking group is a non-peptidic moiety. In aspects, the linking group is a flexible linker. In aspects, suitable flexible linkers include, but are not limited to, peptide sequences that are unstructured and hydrophilic, peptide sequences that are unstructured and hydrophobic, GS type linkers (e.g., SEQ ID NO: 13 (GSGSGS)), simple hydrocarbon linkers, PEG linkers, and / or neutral noncanonical amino acid sequences. In aspects, the linking group is oligoethylene glycol (OEG), polyethylene glycol) 5000 (PEG 5000), or polyethylene glycol) 2000 (PEG 2000). In aspects, suitable linking groups include, but are not limited to, SEQ ID NO: 2 (PGGGSK), a degron moiety, (e.g., SEQ ID NO: 9 (RRRG)), SEQ ID NO: 13 (GSGSGS), SEQ ID NO: 18 (GSGSG), SEQ ID NO: 19 (RGSGSG), SEQ ID NO: 20 (RRGSGSG), SEQ ID NO: 21 (GSGSGR), SEQ ID NO: 22 (GSGSGRR), SEQ ID NO: 23 (GSGSGK), and SEQ ID NO: 24 (GSGSGKK). In aspects, the polymer comprises at least one linker comprising a sequence having 75% or greater sequence identity, optionally 85% or greater, 95% or greater, or 99% or greater sequence identity of SEQ ID NO: 2 (PGGGSK) or SEQ ID NO: 13 (GSGSGS). In aspects, the polymer comprises at least one linker comprising a sequence having 100% sequence identity of SEQ ID NO: 2. In aspects, the polymer comprises at least one linker comprising a sequence having 100% sequence identity of SEQ ID NO: 13.

[0141] In aspects, the polymers discussed herein comprise one or more analytical tags. In aspects, the analytical tag is or comprises an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, a fluorescence tag, or any combination thereof. In aspects, the analytical tag is or comprises a fluorophore, optionally wherein the fluorophore is or comprises a cyanine dye. In aspects, suitable analytical tags include, but are not limited to: an AviTag, a peptide allowing biotinylation by the enzyme BirA and so the protein can be isolated by streptavidin (e.g., SEQ ID NO: 25 (GLNDIFEAQKIEWHE)); a Calmodulin-tag, a peptide bound by the protein calmodulin (e.g., SEQ ID NO: 26 (KRRWKKNFIAVSAANRFKKISSSGAL)); a polyglutamate tag, a peptide binding efficiently to anion-exchange resin, such as Mono-Q (SEQ ID NO: 27 (EEEEEE)); an E-tag, a peptide recognized by an antibody (e.g., SEQ ID NO: 28LVM Ref. 102-24WO; 340492(GAPVPYPDPLEPR)); a FLAG-tag, a peptide recognized by an antibody (e.g., SEQ ID NO: 29 (DYKDDDDK); an HA-tag, a peptide recognized by an antibody (e.g., SEQ ID NO: 30 (YPYDVPDYA)); a His-tag, 5-10 histidines bound by a nickel or cobalt chelate (e.g., SEQ ID NO: 31 (HHHHHH)); a Myc-tag, a short peptide recognized by an antibody (e.g., SEQ ID NO: 32 (EQKLISEEDL)); an S-tag (e.g., SEQ ID NO: 33 (KETAAAKFERQHMDS)); an SBP-tag, a peptide which binds to streptavidin (e.g., SEQ ID NO: 34 (MDEKTTGWRGGHWEGLAGELEQLRARLEHHPQGQREP)); a Softag 1, for mammalian expression (e.g., SEQ ID NO: 35 (SLAELLNAGLGGS)); a Softag 3, for prokaryotic expression (e.g., SEQ ID NO: 36 (TQDPSRVG)); a Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (e.g., a Strep-tag II: (SEQ ID NO: 37 (WSHPQFEK))); a TC tag, a tetracysteine tag that is recognized by FIAsH and ReAsH biarsenical compounds (e.g., SEQ ID NO: 38 (CCPGCC)); a V5 tag, a peptide recognized by an antibody (e.g., SEQ ID NO: 39 (GKPIPNPLLGLDST)); a VSV-tag, a peptide recognized by an antibody (e.g., SEQ ID NO: 40 (YTDIEMNRLGK)); or an Xpress tag (e.g., SEQ ID NO: 41 (DLYDDDDK). In aspects, the analytical tag is or comprises a covalent peptide tag. In aspects, suitable covalent peptide tags include, but are not limited to: an Isopeptag, a peptide which binds covalently to pilin-C protein (e.g., SEQ ID NO: 42 (TDKDMTITFTNKKDAE)); a SpyTag, a peptide which binds covalently to SpyCatcher protein (e.g., SEQ ID NO: 43 (AHIVMVDAYKPTK)). In aspects, the analytical tag is or comprises a protein tag. In aspects, suitable protein tags include, but are not limited to: BCCP (Biotin Carboxyl Carrier Protein), a protein domain biotinylated by BirA enabling recognition by streptavidin; Glutathione-S-transferase-tag, a protein which binds to immobilized glutathione; Green fluorescent protein-tag, a protein which is spontaneously fluorescent and can be bound by nanobodies; Halo-tag, a mutated hydrolase that covalently attaches to the HaloLink™ Resin (Promega); Maltose binding protein-tag, a protein which binds to amylose agarose; Nus-tag; and Thioredoxin-tag; Fc-tag, derived from immunoglobulin Fc domain, which allow dimerization and solubilization.

[0142] Aspects of the Invention

[0143] Various aspects are contemplated herein, several of which are set forth in the paragraphs below. It is explicitly contemplated that any aspect or portion thereof can be combined to form an aspect. Furthermore, although the aspects below are subdivided into aspects A, B, C, D, and so forth, it is explicitly contemplated that aspects in each of subdivisions A, B, C, D, etc. can be combined in any manner. Moreover, the term “any preceding aspect” means any aspect that appears prior to the aspect that contains such phrase (in other words,LVM Ref. 102-24WO; 340492the sentence “Aspect B13: The method of any one of aspects B1-B12, or any preceding aspect, ...” means that any aspect prior to aspect B13 is referenced, including aspects B1-B12 and all of the “A” aspects, including any subdivision thereof (e.g., A1, A2, A3, A1a, A1b, A1c, etc.). For example, it is contemplated that, optionally, any method, polymer or composition of any of the below aspects may be useful with or combined with any other aspect provided below. Further, for example, it is contemplated that any embodiment described elsewhere herein, including above this paragraph, may optionally be combined with any of the below listed aspects. In some instances in the aspects below, or elsewhere herein, two open ended ranges are disclosed to be combinable into a range. For example, “at least X” is disclosed to be combinable with “less than Y” to form a range, in which X and Y are numeric values. For the purposes of forming ranges herein, it is explicitly contemplated that “at least X” combined with “less than Y” forms a range of X-Y inclusive of value X and value Y, even through “less than Y” in isolation does not include Y. Unless otherwise stated, ranges recited herein are explicitly contemplated to include the modifier “about” (as defined herein) for each value. For example, a range of “between 1 and 5” is intended to include a range of “between about 1 and about 5”.

[0144] Aspect A1. A polymer having a formula (FX1):wherein:each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV);each P2independently comprises a peptide, and each instance of P2is different from each instance of P1;at least one (e.g., at least one, at least two, at least three, at least four, at least five) P1independently, or in combination with other instances of P1, inhibits tau fibril formation and / or tau fibril elongation;T1and T2are each independently polymer backbone terminating groups that can be the same or different;LVM Ref. 102-24WO; 340492B1, B2, and B3are each independently a polymer backbone subunit;L1and L2are each independently a linking group;R1is independently a substituent;m is an integer from 2 to 1000 (e.g., from 2 to 1000, 2 to 500, 2 to 250, 2 to 100, 2 to 50, 2 to 30, 5 to 1000, 5 to 500, 5 to 250, 5 to 100, 5 to 50, or 5 to 30);n is an integer from 0 to 1000 (e.g., from 0 to 1000, 0 to 500, 0 to 250, 0 to 100, 0 to 50, 0 to 30, 2 to 1000, 2 to 500, 2 to 250, 2 to 100, 2 to 50, 2 to 30, 5 to 1000, 5 to 500, 5 to 250, 5 to 100, 5 to 50, or 5 to 30);o is an integer from 0 to 1000 (e.g., from 0 to 1000, 0 to 500, 0 to 250, 0 to 100, 0 to 50, 0 to 30, 2 to 1000, 2 to 500, 2 to 250, 2 to 100, 2 to 50, 2 to 30, 5 to 998, 5 to 500, 5 to 250, 5 to 100, 5 to 50, or 5 to 30); andeach connecting line in the formula (FX1) represents a covalent linkage comprising at least one of a single bond, a double bond, one or more atoms, or any combination thereof, optionally wherein the one or more atoms comprise carbon, nitrogen, and / or oxygen atoms.

[0145] Aspect A1 a. The polymer of aspect A1, wherein the polymer has a degree of polymerization of between 5 and 50, for example between 5 and 50, 5 and 30, 5 and 25, 5 and 20, 5 and 15, 7 and 30, 7 and 25, 7 and 20, or 7 and 15.

[0146] Aspect A1b. The polymer of aspect A1a, wherein the polymer is a block copolymer and wherein:m is an integer from 2 to 29, for example from 2 to 29, 2 to 25, 2 to 20, 2 to 15, 2 to 10, or 2 to 8;n is an integer from 0 to 27, for example from 0 to 27, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, 1 to 27, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5; ando is an integer from 1 to 28, for example from 1 to 28, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 8.

[0147] Aspect A1c. The polymer of aspect A1a or A1b, wherein the polymer is a block copolymer having a degree of polymerization between 7 and 25, for example between 7 and 25, 7 and 20, 7 and 18, 7 and 16, 7 and 15, 10 and 20, 10 and 18, 10 and 16, or 10 and 15, and wherein:m is an integer from 5 to 24, for example from 5 to 24, 5 to 20, 5 to 15, 5 to 10, or 5 to 8;LVM Ref. 102-24WO; 340492n is an integer from 0 to 27, for example from 0 to 27, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, 1 to 27, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5; ando is an integer from 1 to 28, for example from 1 to 28, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 8.

[0148] Aspect Aid. The polymer of any one of aspects A1a-A1c, wherein the polymer is a block copolymer having a degree of polymerization between 10 and 20, for example between 10 and 20, 10 and 18, 10 and 16, or 10 and 15, and wherein:m is an integer from 5 to 15, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; n is an integer from 0 to 5, for example 0, 1, 2, 3, 4, or 5, preferably wherein n is 0 or 1; ando is an integer from 5 to 10, for example 5, 6, 7, 8, 9, or 10.

[0149] Aspect A2. The polymer of aspect A1 , wherein at least one, optionally all instances of, P1further comprises a charge modulating domain.

[0150] Aspect A3. The polymer of aspect A1 , or any preceding aspect, wherein the charge modulating domain has from 2 to 30 (e.g., from 2 to 30, 2 to 20, 2 to 10, or 2 to 7) amino acid residues that can be the same or different, optionally wherein the amino acid residues are lysines, glycines, serines, or arginines, optionally wherein the amino acid residues are lysines or arginines, optionally wherein the amino acid residues are lysines.

[0151] Aspect A3a. The polymer of aspect A1, or any preceding aspect, wherein the charge modulating domain has from 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues that can be the same or different, optionally wherein the amino acid residues are lysines, glycines, serines, or arginines, optionally wherein the amino acid residues are lysines or arginines, optionally wherein the amino acid residues are lysines.

[0152] Aspect A3b. The polymer of aspect A3, or any preceding aspect, wherein the charge modulating domain has 2 lysine residues or 2 arginine residues or the charge modulating domain has 1 lysine residue and 1 arginine residue.

[0153] Aspect A4. The polymer of aspect A2 or A3, or any preceding aspect, wherein the charge modulating domain is attached to the N-terminus or the C-terminus of the at least one P1.LVM Ref. 102-24WO; 340492

[0154] Aspect A4a. The polymer of aspect A2 or A3, or any preceding aspect, wherein the charge modulating domain is attached to the C-terminus of the at least one P1.

[0155] Aspect A5. The polymer of any one of aspects A1-A4, wherein at least one, optionally all instances of, P2independently comprises a degrading agent.

[0156] Aspect A5a. The polymer of any one of aspects A1-A4, wherein each P2independently comprises a degrading agent.

[0157] Aspect A6. The polymer of aspect A5, or any preceding aspect, wherein the degrading agent comprises an E3 ligase recruiter, optionally wherein the E3 ligase recruiter comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of:(i) a Keapl peptide, optionally SEQ ID NO: 5 (LDPETGEYL);(ii) a cereblon (CRBN) peptide;(iii) a VHL peptide, optionally SEQ ID NO: 6 (ALAPYIP);(iv) a mouse double minute 2 homologue (MDM2) peptide;(v) an inhibitor of apoptosis peptide (IAP), optionally SEQ ID NO: 8 (AVPI); or (vi) a degron peptide, optionally SEQ ID NO: 9 (RRRG);(vii) a KLHL20 peptide, optionally SEQ ID NO: 10 (LPDLV), optionally SEQ ID NO: 11 (LGLPDLVAKYN);(viii) a SOCS / CUL5 peptide, optionally SEQ ID NO: 12 (SLQYLCR)-XXX-SEQ ID NO: 15 (LPLP) wherein XXX is a spacer, optionally wherein the spacer is a spacer of 3-5 amino acids; or(ix) a segment of any one of (i)-(viii) .

[0158] Aspect A7. The polymer of any one of aspects A1-A6, wherein at least one, optionally all instances of, P2independently has a chain length of 3 to 150 amino acids (e.g., 3 to 150, 3 to 100, 3 to 75, 3 to 50, 3 to 25, 3 to 15, 5 to 150, 5 to 100, 5 to 75, 5 to 50, 5 to 25, or 5 to 15).

[0159] Aspect A7a. The polymer of any one of aspects A1-A6, wherein each P2independently has a chain length of 3 to 150 amino acids (e.g., 3 to 150, 3 to 100, 3 to 75, 3 to 50, 3 to 25, 3 to 15, 5 to 150, 5 to 100, 5 to 75, 5 to 50, 5 to 25, or 5 to 15).LVM Ref. 102-24WO; 340492

[0160] Aspect A8. The polymer of any one of aspects A1-A7, wherein at least one, optionally all instances of, P2further comprises a charge modulating domain.

[0161] Aspect A9. The polymer of aspect A8, or any preceding aspect, wherein the charge modulating domain has from 2 to 30 (e.g., from 2 to 30, 2 to 20, 2 to 10, or 2 to 7) amino acid residues that can be the same or different, optionally wherein the amino acid residues are lysines, glycines, serines, or arginines, optionally wherein the amino acid residues are lysines or arginines, optionally wherein the amino acid residues are lysines.

[0162] Aspect A9a. The polymer of aspect A8, or any preceding aspect, wherein the charge modulating domain has from 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues that can be the same or different, optionally wherein the amino acid residues are lysines, glycines, serines, or arginines, optionally wherein the amino acid residues are lysines or arginines, optionally wherein the amino acid residues are lysines.

[0163] Aspect A9b. The polymer of aspect A8, or any preceding aspect, wherein the charge modulating domain has 2 lysine residues or 2 arginine residues or the charge modulating domain has 1 lysine residue and 1 arginine residue.

[0164] Aspect A10. The polymer of any one of aspects A1-A9, wherein each polymer backbone subunit is independently a repeating unit (RU1), (RU2), (RU3), or (RU4):wherein:R2is H or C1-C3 alkyl; andLVM Ref. 102-24WO; 340492X is CH2 or O.

[0165] Aspect A11. The polymer of any one of aspects A1-A10, wherein each of R1, T1, and T2independently is hydrogen, C1-C30 alkyl, C3-C30 cycloalkyl, C5-C30 aryl, C5-C30 heteroaryl, C1-C30 acyl, C1-C30 hydroxyl, C1-C30 alkoxy, C2-C30 alkenyl, C2-C30 alkynyl, C5-C30 alkylaryl, — CO2R4, — CONR5R6, —COR7, — SOR8, — OSR9, — SO2R10, —OR11, —SR12, — NR13R14, — NR15COR16, C1-C30 alkyl halide, phosphonate, phosphonic acid, silane, siloxane, silsesquioxane, C2-C30 halocarbon chain, C2-C30 perfluorocarbon, C2-C30 polyethylene glycol, a metal, or a metal complex, wherein each of R4-R16independently is H, C5-C10 aryl, or C1-C10 alkyl.

[0166] Aspect A12. The polymer of any one of aspects A1-A11, wherein at least one, optionally all instances, of P1, P2, R1, T1, and T2independently further comprises an analytical tag, wherein the analytical tag comprises an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, a fluorescence tag, or any combination thereof.

[0167] Aspect A12a. The polymer of any one of aspects A1-A11, wherein at least one, optionally all instances, of R1, T1, and T2independently further comprises an analytical tag, wherein the analytical tag comprises an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, a fluorescence tag, or any combination thereof.

[0168] Aspect A12b. The polymer of aspect A12, or any preceding aspect, wherein the analytical tag comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of:SEQ ID NO: 25 (GLNDIFEAQKIEWHE);SEQ ID NO: 26 (KRRWKKNFIAVSAANRFKKISSSGAL);SEQ ID NO: 27 (EEEEEE);SEQ ID NO: 28 (GAPVPYPDPLEPR);SEQ ID NO: 29 (DYKDDDDK);SEQ ID NO: 30 (YPYDVPDYA);SEQ ID NO: 31 (HHHHHH);SEQ ID NO: 32 (EQKLISEEDL);SEQ ID NO: 33 (KETAAAKFERQHMDS);SEQ ID NO: 34 (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP); SEQ ID NO: 35 (SLAELLNAGLGGS);LVM Ref. 102-24WO; 340492SEQ ID NO: 36 (TQDPSRVG);SEQ ID NO: 37 (WSHPQFEK);SEQ ID NO: 38 (CCPGCC);SEQ ID NO: 39 (GKPIPNPLLGLDST);SEQ ID NO: 40 (YTDIEMNRLGK);SEQ ID NO: 41 (DLYDDDDK);SEQ ID NO: 42 (TDKDMTITFTNKKDAE); orSEQ ID NO: 43 (AHI MVDAYKPTK).

[0169] Aspect A13. The polymer of any one of aspects A1-A12, wherein each of L1and L2is independently selected from a single bond, an oxygen, and groups having an alkylene group, a heteroalkylene group, an alkenylene group, an arylene group, an alkoxy group, an acyl group, a triazole group, a diazole group, a pyrazole group, and any combination thereof, optionally wherein each of L1and L2is independently selected from a single bond, an oxygen, a Ci-C10 alkyl, a C2-C10 alkenylene, a C3-C10 arylene, a C1-C10 alkoxy, a Ci-C acyl and any combination thereof.

[0170] Aspect A14. The polymer of any one of aspects A1-A13, wherein each of L1and L2is independently a flexible linker, optionally wherein the flexible linker comprises a flexible hydrocarbon linker, optionally wherein the flexible linker comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 2 (PGGGSK) or SEQ ID NO: 13 (GSGSGS).

[0171] Aspect A15. The polymer of any one of aspects A1-A14, wherein the polymer is a block copolymer having a formula (FX2):LVM Ref. 102-24WO; 340492each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV), and wherein each P1further comprises a charge modulating domain, optionally wherein;each P2independently comprises a peptide, and each instance of P2is different from each instance of P1, and wherein each P2independently comprises a degrading agent, optionally wherein the degrading agent is or comprises an E3 ligase recruiter;each AT independently comprises an analytical tag, optionally wherein the analytical tag is or comprises an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, or a fluorescence tag, optionally wherein the analytical tag is or comprises a fluorophore, optionally wherein the fluorophore is a cyanine dye;T2is a polymer backbone terminating group, optionally wherein T2comprises ester vinyl ether (EVE) or biotin;each of L1, L2, and L3is independently a flexible linker, optionally wherein the flexible linker comprises a flexible hydrocarbon linker, optionally wherein the flexible linker comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 2 (PGGGSK) or SEQ ID NO: 13 (GSGSGS);m is an integer from 1 to 49 (e.g., from 1 to 49, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 8);n is an integer from 1 to 49 (e.g., from 1 to 49, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 8); ando is an integer from 0 to 48 (e.g., from 1 to 48, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 8), preferably o is 0 or 1 ;wherein the block copolymer has a degree of polymerization between 2 and 50 (e.g., between 2 and 50, 2 and 30, 2 and 25, 2 and 20, 2 and 15, 5 and 50, 5 and 30, 5 and 25, 5 and 20, 5 and 15, 7 and 30, 7 and 25, 7 and 20, or 7 and 15), optionally between 2 and 30 (e.g., between 2 and 30, 2 and 25, 2 and 20, 2 and 15, 5 and 50, 5 and 30, 5 and 25, 5 and 20, 5 and 15, 7 and 30, 7 and 25, 7 and 20, or 7 and 15), optionally between 2 and 20 (e.g., between 2 and 20, 2 and 15, 5 and 50, 5 and 30, 5 and 25, 5 and 20, 5 and 15, 7 and 30, 7 and 25, 7 and 20, or 7 and 15).LVM Ref. 102-24WO; 340492

[0172] Aspect A16. The polymer of any one of aspects A1-A15, wherein the polymer is a homopolymer, a copolymer, a block copolymer, a brush polymer, or a brush block copolymer.

[0173] Aspect A17. The polymer of any one of aspects A1-A16, wherein the polymer is characterized by a polydispersity index of greater than 0.7, optionally between 0.7 and 1.75, optionally between 0.7 and 1.5, optionally between 0.7 and 1.25, optionally between 0.9 and 1.25, optionally between 1 and 1.2.

[0174] Aspect A17a. The polymer of any one of aspects A1-A17a, wherein the polymer is characterized by a molecular weight of between 25 and 250 kg / mol (e.g., between 25 and 250 kg / mol, 25 and 150 kg / mol, 25 and 100 kg / mol, 45 and 250 kg / mol, 45 and 150 kg / mol, or 45 and 100 kg / mol), optionally between 35 and 100 kg / mol (e.g., between 35 and 100 kg / mol, 35 and 75 kg / mol, 45 and 100 kg / mol, or 45 and 75 kg / mol), optionally between 45 and 60 kg / mol (e.g., between 50 and 60 kg / mol).

[0175] Aspect A18. The polymer of any one of aspects A1-A17, wherein each of L1, L2, and / or L3is independently an enzymatically degradable linker, optionally wherein the enzymatically degradable linker is a matrix metalloproteinase (MMP) cleavage sequence, cathepsin B cleavage sequence, ester bond, reductive sensitive bond- disulfide bond, pH sensitive bond- imine bond or any combinations of these.

[0176] Aspect A18a. The polymer of any one of aspects A1-A17, wherein each of L1, L2, and / or L3is independently a linker comprising a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of:SEQ ID NO: 2 (PGGGSK);SEQ ID NO: 13 (GSGSGS);SEQ ID NO: 18 (GSGSG);SEQ ID NO: 19 (RGSGSG);SEQ ID NO: 20 (RRGSGSG);SEQ ID NO: 21 (GSGSGR);SEQ ID NO: 22 (GSGSGRR);SEQ ID NO: 23 (GSGSGK); orSEQ ID NO: 24 (GSGSGKK).LVM Ref. 102-24WO; 340492

[0177] Aspect B1. A method for inhibiting tau fibril formation and / or tau fibril elongation in a tissue, the method comprising administering to the tissue a polymer, or a pharmaceutical composition comprising said polymer, having a formula (FX1):wherein:each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater (e.g., 75% or greater, 85% or greater, 95% or greater, 99% or greater, or 100%) sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV);each P2independently comprises a peptide, and each instance of P2is different from each instance of P1;T1and T2are each independently polymer backbone terminating groups that can be the same or different;B1, B2, and B3are each independently a polymer backbone subunit;L1and L2are each independently a linking group;R1is independently a substituent;m is an integer from 2 to 1000 (e.g., from 2 to 1000, 2 to 500, 2 to 250, 2 to 100, 2 to 50, 2 to 30, 5 to 1000, 5 to 500, 5 to 250, 5 to 100, 5 to 50, or 5 to 30);n is an integer from 0 to 1000 (e.g., from 0 to 1000, 0 to 500, 0 to 250, 0 to 100, 0 to 50, 0 to 30, 2 to 1000, 2 to 500, 2 to 250, 2 to 100, 2 to 50, 2 to 30, 5 to 1000, 5 to 500, 5 to 250, 5 to 100, 5 to 50, or 5 to 30);o is an integer from 0 to 1000 (e.g., from 0 to 1000, 0 to 500, 0 to 250, 0 to 100, 0 to 50, 0 to 30, 2 to 1000, 2 to 500, 2 to 250, 2 to 100, 2 to 50, 2 to 30, 5 to 998, 5 to 500, 5 to 250, 5 to 100, 5 to 50, or 5 to 30); andeach connecting line in the formula (FX1) represents a covalent linkage comprising at least one of a single bond, a double bond, one or more atoms, or any combination thereof, optionally wherein the one or more atoms comprise carbon, nitrogen, and / or oxygen atoms.LVM Ref. 102-24WO; 340492

[0178] Aspect B2. The method of aspect B1, or any preceding aspect, wherein the method further comprises at least one additional administration step comprising administering the polymer to the tissue within 24 and 72 hours of the initial administration.

[0179] Aspect C1. A method for inhibiting tau fibril formation and / or tau fibril elongation in a tissue, the method comprising administering to the tissue the polymer of any one of aspects A1-A18, or any preceding aspect, or the pharmaceutical composition of aspect F1.

[0180] Aspect D1. A method for treating a condition in a subject, the method comprising administering the polymer of any one of aspects 1-18, or any preceding aspect, to the subject, optionally wherein the condition is a 4R tauopathy, optionally wherein the 4R tauopathy is CBD, PSP, or PS19, optionally wherein the 4R tauopathy comprises a familial frontotemporal dementia (FTD) having a tau gene mutation optionally wherein the tau gene mutation comprises P301L, P301S, N279K, S305I, S305N, S305S, or a combination thereof.

[0181] Aspect E1. A method for modeling tau fibril seeding in a human nervous system, the method comprising:fusing human iPSC-derived ventral organoids with human iPSC-derived dorsal-directed organoids to generate assembloids;treating the assembloids with fractions comprising active tau seeds;dividing the assembloids into a treatment group of assembloids and a control group of assembloids;administering a tau seeding inhibitor to the treatment group of assembloids; staining the assembloids with an antibody, optionally a conformational antibody configured to detect misfolded tau; andquantifying the amount of the antibody in the treatment group of assembloids and the control group of assembloids with a machine learning model,thereby modeling tau fibril seeding in a human nervous system.

[0182] Aspect F1. A pharmaceutical composition comprising the polymer of any one of aspects A1-A18, or any preceding aspect, and a pharmaceutically acceptable excipient.

[0183] The invention can be further understood by the following non-limiting examples. In each of the following non-limiting examples, jR2R3 P301L refers to an exemplary peptide sequence used to created fibrils. In the following non-limiting examples, an exemplary modified jR2R3 P301L peptide sequence is discussed in the context of a PLP and is intended to refer toLVM Ref. 102-24WO; 340492an exemplary jR2R3 P301L sequence (SEQ ID NO: 1) that has been augmented with an exemplary linker (SEQ ID NO: 2) and a cationic residue domain (two lysine residues), referred to herein as jR2R3-P301L-PLP. While efforts have been made to distinguish between the exemplary peptide sequence and the exemplary PLP (i.e. , use of “jR2R3-P301L” for a peptide sequence and use of “jR2R3-P301L-PLP” for a PLP), there may be instances where the recitation of “jR2R3-P301 L” is intended to refer to the peptide sequence in the context of a PLP. Those of skill in the art will understand howto distinguish between a peptide sequence and a peptide sequence incorporated into a PLP in view of the surrounding context and descriptions herein.

[0184] Example 1: Overview of Experimental Examples

[0185] Protein misfolding and aggregation are hallmarks of numerous neurodegenerative and systemic diseases, where aggregates spread through a prion-like mechanism, recruiting healthy proteins to adopt pathological conformations (1-6). Diseases such as Alzheimer’s (7, 8), Parkinson’s (3), amyotrophic lateral sclerosis (ALS) (9) and systemic amyloidosis (6) exhibit this pattern of progressive protein aggregation. As these misfolded proteins accumulate, they impose an increasing burden on cellular degradation pathways, such as the ubiquitin-proteasome system and autophagy, which are already compromised due to aging. The inability of these systems to effectively clear aggregates allows their unchecked growth, resulting in cellular dysfunction, toxicity, and ultimately cell death. A therapeutic approach aimed at targeting the growing face of these fibrils, thus blocking the further conversion of unaffected protein, holds promise in halting the spread of aggregates by preventing their elongation, thereby mitigating disease progression.

[0186] Tauopathies represent specific neurodegenerative disorders characterized by the prion-like spread of pathological tau aggregates through interconnected neuronal networks(2, 4, 10). Each tauopathy displays unique cryo-EM determined structural folds and distinct anatomical distributions (11-18). Given tau’s central role in disease progression, targeting misfolded tau spread and seeding is a promising strategy for disrupting disease progression. We recently reported that a 19-residue probe peptide containing a P301L mutation and spanning the R2 / R3 splice junction of tau folds and stacks into seeding-competent fibrils capable of specifically inducing aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations (19). The peptide adopts a U-shaped fold, closely aligning with the structures that define GPT, CBD, and PSP 4R tauopathiesLVM Ref. 102-24WO; 340492(20). Notably, the peptide acts as a mini-prion, accurately mimicking 4R isoform-specific spread of tau in cellular models.

[0187] Small peptides have been identified which are capable of acting as capping agents and preventing the elongation and propagation of tau aggregates (21-29). In the disease process, tau fibrils facilitate misfolded transitions by adding naive tau monomers. These fibrils can then fragment, exit the originating cells, and spread to neighboring cells, thereby advancing disease. To prevent this templating and spread, efforts have focused on designing peptides that effectively bind to and “cap” the growing fibril ends, hindering further aggregation (21-29). While this technique has shown promise, it remains in its nascent stages.

[0188] A significant challenge with peptides is their rapid degradation and limited cellular penetration, not to mention their inability to traverse the blood-brain barrier (30). Consequently, there has been a push towards developing antibodies and nanobodies that inhibit tau spread. Some designs incorporate capping peptides directly into the variable CD3 domain of nanobodies (31). Our previous work demonstrated that the VHH-Z70 nanobody significantly reduced the seeding of naive tau in culture systems (19).

[0189] In aspects, to overcome the obstacles of peptide delivery, a synthetic proteomimetic strategy was used wherein peptide side chains are incorporated as high-density brush polymers, termed protein-like polymers (PLPs) (see, e.g., FIG. 1C) (32-37). PLPs incorporating a more hydrophobic polymer backbone adopt a globular conformation, which has been shown to enhance their resistance to proteolytic degradation while retaining the bioactivity of the conjugated peptides (32, 35). The features of transient amphiphilicity (metaphilicity), facile peptide modification, and multivalency combine to make PLPs suitable to enter cells and engage intracellular target proteins like tau (38-40).

[0190] In the following examples, we introduce a novel approach to target tau spread using protein-like-polymers (PLPs), functionalized with peptides designed to target tau fibrils. These functionalized PLPs halt the growth of tau fibrils in vitro and effectively prevent the seeding of naive tau by preformed tau fibrils from both disease and synthetic sources in cells. Our findings have substantial implications for tau-based therapeutics, as these functionalized PLPs are not only multi-functional but also exhibit favorable pharmacokinetic properties (40).

[0191] A 19-amino acid peptide that could recapitulate 4R tauopathy disease folds was identified, as confirmed by its cryo-EM analysis (FIG. 1A) (19). These results suggested anLVM Ref. 102-24WO; 340492opportunity for tau isoform-specific therapeutic interventions. The published work on the jR2R3 P301L fibril, which mimics 4R conformation-specific tauopathies such as CBD and PSP, led to the hypothesis that a monomer of the jR2R3 P301 L peptide — containing the aggregation-prone PHF6 motif — could bind and cap a propagating fibril when presented on a PLP scaffold (FIG. 1B). While a monomer of jR2R3 P301L can readily add to a growing end of a preformed fibril seed, we hypothesized that when this monomer is presented on a large hydrophobic polymer backbone that occludes further tau monomer additions, given its globular structure it should act as an effective capping agent.

[0192] Example 2: Synthesis and Characterization of Tau PLPs and Control PLPs

[0193] The peptide sequence for the jR2R3 P301 L-PLP was optimized based on design considerations to permit cellular uptake, improve water solubility, and enable flexibility from the backbone that allows the key residues to freely interact with tau. To address both cell penetration and water solubility, the original jR2R3 P301L sequence SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV) was augmented with cationic charge residues (FIG. 1A).Specifically, lysines which have been shown in previous studies to enhance cell penetration when incorporated into a PLP (33), were selected over arginines. This choice was based on the natural presence of lysines, but not arginines, in the jR2R3 P301 L peptide sequence. While lysines were selected in this example, it is expressly noted that other charge modifiers are hypothesized to enhance cell penetration, including, for example, non-natural cationic charge residues, such as arginine, histidine, or a combination thereof. The addition of two lysine residues at the carboxy terminus was strategically implemented to confer a net positive charge (+2) to the peptide monomer, enhancing its hydrophilicity and water solubility. While the peptide monomer already incorporates a flexible hydrocarbon linker to connect the norbornene polymerizable unit to the peptide, additional SEQ ID NO: 2 (PGGGSK) residues were appended to the DNIK portion of SEQ ID NO: 1 of thejR2R3 P301L variant (FIG. 1A, FIG. 1C). The modification was designed to increase conformational flexibility and ensure optimal accessibility of the binding region to tau protein targets, and mimic part of the native tau sequence. The final peptide used in this example and conjugated to the PLP is shown in FIG. 1A. While SEQ ID NO: 2 was used as the flexible linker in this example, other suitable flexible linkers are expressly contemplated, including, but not limited to peptide sequences that are unstructured and hydrophilic, peptide sequences that are unstructured and hydrophobic, GS type linkers (e.g., SEQ ID NO: 13 (GSGSGS)), simple hydrocarbon linkers, PEG linkers, and / or neutral noncanonical amino acid sequences. In embodiments, the linker is a non-peptide moiety, forLVM Ref. 102-24WO; 340492example, oligoethylene glycol (OEG), polyethylene glycol) 5000 (PEG 5000), or poly(ethylene glycol) 2000 (PEG 2000).

[0194] Detailed PLP synthetic procedures have been described previously, for example, in Carrow et al., Advanced Materials 36, 2311467 (2024); Wang et al., J. Am. Chem. Soc. 146, 14959-14971 (2024); Choi et al., Science Advances 10, eado8307 (2024); Blum et al., J. Am. Chem. Soc. 136, 15422-15437 (2014); Blum et al., Chem. Sci. 7, 989-994 (2016); Callmann et al., Acc. Chem. Res. 53, 400-413 (2020); Sun et al., ACS Cent. Sci. 7, 2063-2072 (2021); and Sun et al., Angewandte Chemie Int’l Ed. 58, 17359-17364 (2019), each of which is incorporated by reference in its entirety for all purposes to the extent not inconsistent with the present description, and specifically for its discussion of PLP synthesis. Briefly, peptides were synthesized via solid-phase peptide synthesis wherein the addition of a norbornene polymerizable unit (N-(hexanoic acid)-exo-5-norbornene-2,3-dicarboximide) was conjugated as a non-natural amino acid to the N terminus of the peptide (FIG. 1C). See, e.g., Coin et al., Nat Protoc 2, 3247-3256 (2007) and Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide, J. of Am. Chem. Soc’y, available at: pubs-acs-org.proxy.library.ucsb.edu / doi / abs / 10.1021 / ja00897a025, each of which is incorporated by reference in its entirety for all purposes to the extent not inconsistent with the present description, and specifically for its discussion of solid-phase peptide synthesis. Peptide monomers were cleaved from resin as described in “Exemplary Experimental Aspects for Examples 1-11”, below, and purified via preparative high-performance liquid chromatography (Prep HPLC), and further characterized by electrospray ionization mass spectrometry (ESI-MS) and analytical HPLC for molecular weight determination and purity (FIGs. 9A-9D) (41, 42).

[0195] Peptide monomers were polymerized using ring-opening metathesis polymerization (ROMP), and the kinetics of polymerization was monitored for completion via nuclear magnetic resonance spectrometry (NMR, FIG. 10). Upon consumption of the peptide monomer, a norbornene-conjugated cyanine 5.5 (Cy5.5) dye label was incorporated to the polymer backbone before termination if a dye labeled version of the PLP was desired (FIGs. 11A-11C).For some studies, jR2R3301 L PLP was additionally biotinylated with a biotin-terminating using synthesis previously reported by our group (FIGs. 12A-12C; FIGs. 13A-13C) (43).

[0196] Polymers were terminated with ethyl vinyl ether (EVE), and immediately purified on Prep HPLC as prolonged static resting of the polymer solution in DMF resulted in gelation in the vial within 6 hours. The resulting pure fractions were lyophilized to obtain the PLPs in powderLVM Ref. 102-24WO; 340492form. PLPs were characterized to determine molecular weight, and thus degree of polymerization, using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE, as shown in FIGs. 14A-14E) and size exclusion chromatography multi-angle light scattering (SEC-MALS, as shown in FIGs. 15A-15D). Polymers (except the PROTACs) were designed to contain 15 repeats of thejR2R3 P301L modified peptide, and actual degrees of polymerization are reported in Table A, below. Physical characterization with dynamic light scattering (DLS) (FIGs. 16A-16B), Circular Dichroism (CD) (FIG. 17), and small angle x-ray scattering (SAXS) (FIGs. 18A-18C) showed monomeric PLPs under native conditions. The CD results (FIG. 17) suggest the secondary structure of the PLP does not exhibit alpha helical or beta sheet behavior, and beta sheet behavior is absent as the fibrils are structured in beta sheets in tau. Similar to DLS and SAXS, the PLP structure does not reflect a higher order assembly and shows aggregate formation when sitting at 37°C after more than a few days, according to DLS. These characterization results suggest that the PLPs are not diffusing as aggregates in solution, but rather as individual polymer chains, i.e. , they are active as globular PLP entities, not as higher order assemblies.Table A. Chart of the different PLPs used in these Examples. “DP” refers to degrees of polymerization.

[0197] Example 3: Tau IR2R3 P301L-PLP Prevents Tau Seeding in vitro

[0198] After synthesizing and characterizing the jR2R3 P301L-PLP, its efficacy in inhibiting tau fibril formation in vitro was tested. Previous work demonstrated that jR2R3 P301L fibrils can seed the formation of new fibrils from jR2R3 P301L monomers when fibrils were introduced at low concentrations (19). To quantify this in the presence of PLPs, 25 pM jR2R3 P301L monomer was added to 25 pM jR2R3 P301L fibrils (with fibril concentration based on the concentration of individual peptides in the fibril solution) and fibril formation was monitored viaLVM Ref. 102-24WO; 340492transmission electron microscopy (TEM) and Thioflavin T (ThT) fluorescence assays, evaluating the process across jR2R3 P301L-PLP concentrations ranging from 3.125 to 25 pM (FIGs. 2A-2B).

[0199] In the absence of jR2R3 P301L-PLP, fibril formation occurred rapidly, reaching a gradually increasing plateau by 4 hours, as indicated by a sharp rise in ThT fluorescence. In contrast, when jR2R3 P301L-PLP was introduced at concentrations as low as 6.25 pM, there was a near-complete suppression of ThT fluorescence, indicating strong inhibition of fibril formation. All concentrations above 6.25 pM led to complete inhibition of fibril formation as monitored by both TEM (FIG. 2A) and ThT fluorescence (FIG. 2B).

[0200] TEM images comparing reactions at 0 pM and 6.25 pM jR2R3 P301 L-PLP showed the formation of long paired-helical fibrils in the absence of the PLP (FIG. 2A, top). In contrast, samples treated with jR2R3 P301 L-PLP displayed a notable absence of fibrils, and those present appeared clumped at the thin filamentous ends (FIG. 2A, bottom). These results suggest thatjR2R3 P301L-PLP prevents fibril elongation through a steric hindrance mechanism, wherein the PLP binds to the fibril ends, blocking further addition of monomeric tau.

[0201] Example 4: jR2R3 P301 L-PLP Interacts Strongly with IR2R3 P301L Monomers and Fibrils

[0202] The TEM data suggested that jR2R3 P301 L-PLP acts by binding directly to fibril ends, preventing further elongation. To further characterize this interaction, we used a combination of dot-blot assays and isothermal titration calorimetry (ITC) to directly measure the binding of jR2R3 P301 L-PLP to monomeric and fibrillar tau species.

[0203] We first biotin-tagged the jR2R3 P301L monomer, fibril, and jR2R3 P301 L-PLP constructs. In parallel, fluorescently labeled jR2R3 P301L monomer and fibril stocks were prepared, maintaining a 1:10 ratio of labeled to unlabeled species. This 1:10 ratio was selected, in part, to prevent / minimize perturbations to the fibrillar structures of the material used.However, other suitable ratios of labeled to unlabeled species are expressly contemplated, including, but not limited to, 1:1, 1:2, 1:5, 1:10, 1:20, 1:50, and all sub-ratios thereof. Equimolar concentrations (10 pM) of the biotin-labeled species and fluorescently-labeled species were incubated together for 1 hour at 37°C. The mixtures were then incubated with streptavidin-coated magnetic beads for an additional 30 minutes at 37°C. Following stringent washing, bound species were eluted and detected via dot-blot assays based on their fluorescent signals.LVM Ref. 102-24WO; 340492

[0204] The results suggested that biotin-tagged jR2R3 P301 L-PLP bound robustly to both fluorescently labeled monomers and fibrils, with a stronger signal observed from fibrils. This enhanced signal could be in part explained by the fact that a single fibril can contain multiple fluorophores, whereas a monomer can only contain one. Consequently, the binding of a single fibril leads to a greater overall fluorescent signal. In contrast, biotin-tagged fibrils showed minimal interaction with other fluorescently labeled fibrils or monomers. Biotin-tagged monomers, however, pulled down fluorescently labeled fibrils, but showed weaker interaction with monomers (FIG. 2C). Control experiments, where fluorescently-labeled species were incubated with streptavidin beads in the absence of a biotin-labeled species, produced no detectable signal in the elution (FIG. 18A).

[0205] To further quantify the interaction of jR2R3 P301 L-PLP with monomers, we used ITC. In these experiments, 500 nM jR2R3 P301 L-PLP (titrate) was titrated with 5 pM jR2R3 P301L monomer (titrant). The interaction generated an exothermic reaction, and fitting the data to an independent binding model yielded a dissociation constant (KD) of 17.8 ± 22.7 nM, indicating strong nanomolar binding (FIG. 2D). However, attempts to derive a binding constant for jR2R3 P301L fibrils were unsuccessful due to the complexity of the interaction. Although we know the monomer concentration of the fibrils and that each fibril has two ends, the length of the fibrils is highly variable. Because we cannot determine how many monomers each fibril contains, it is impossible to accurately calculate the fibril end concentration, making it challenging to derive a KD for this interaction.

[0206] Example 5: IR2R3 P301 L-PLP Directly Enters Cells in Culture Models

[0207] To further characterize the mechanism of jR2R3 P301 L-PLP uptake, we quantified the fluorescence intensity over time following PLP administration, normalizing to total cellular area to account for differences in cell density and size across trials. Concentrations ranging from 0.25 to 2 pM were tested, and results indicated that the cellular uptake of PLP increased with concentration until a plateau was observed at approximately six hours post-administration at all concentration ranges (FIG. 3A). This plateau suggests a saturation point in the uptake mechanism, which is consistent with direct cellular penetration rather than receptor-mediated endocytosis, as the latter would likely display a more gradual, sigmoidal uptake pattern due to the involvement of cellular machinery like receptors and vesicle formation. This is consistent with behavior seen by other PLPs, where the uptake is proportional to concentration within theLVM Ref. 102-24WO; 340492same time frame (38, 39), and when a singular concentration produces the same uptake signals between 2 hours and 24 hours, suggesting saturation.

[0208] Additionally, a linear correlation between PLP concentration and uptake at the 6-hour timepoint (R2=0.95, FIG. 3A, inset) further supports the hypothesis of passive entry.Endocytosis, which is an active and regulated process, typically involves vesicle formation and trafficking, which would not scale linearly with increasing ligand concentration. This deviation from expected endocytic kinetics reinforces the idea that PLPs penetrate the cell membrane directly, bypassing these more regulated pathways. Imaging cells treated with fluorescently labeled PLP at six hours, showed a population of cells in which a clear PLP signal is associate with the cell membrane before becoming a cytoplasmic signal (FIG. 3B).

[0209] To further assess whether endocytosis plays a role in PLP uptake, we next examined the colocalization of the labeled PLPs with markers for key endocytic compartments: Rab5 for endosomes and Lampl for lysosomes (FIG. 3C). Cells were treated with labeled PLPs for six hours, fixed, and stained with antibodies specific to these endosomal markers. As shown in FIG.3C, no significant colocalization was observed between the PLPs and any of these markers, suggesting that the PLPs do not enter the canonical endocytic pathway. The lack of colocalization with Rab5 also suggests that PLPs do not undergo vesicular trafficking via endosomes, further supporting the hypothesis of a non-endocytic mode of entry.

[0210] To test the independence of PLP uptake from traditional endocytic pathways, the cells were treated with inhibitors of key components of endocytosis. Cytochalasin D, which disrupts actin polymerization, was used to inhibit clathrin-mediated endocytosis, while filipin III, which sequesters cholesterol, was employed to block caveolae-mediated uptake. Dynasore, a dynamin inhibitor, was used to prevent scission of endocytic vesicles. Despite these treatments, no reduction in PLP uptake was observed after four hours (FIG. 3D), persistence of robust PLP internalization under these conditions reinforces the conclusion that the peptides penetrate cells through a mechanism independent of the endocytic machinery. This characteristic appears to be primarily influenced by the peptide sequence rather than other properties of PLPs. Evidence from two additional cases (33, 38) indicates that PLPs can undergo endocytosis, highlighting the significant role that sequence plays in determining the functional behavior of PLPs. It was hypothesized this may be possible due to “metaphilic tunneling” (44, 45), a phenomenon describing transient amphiphilicity of the PLP, driven by the hydrophobic backbone and the hydrophilic terminal lysines.LVM Ref. 102-24WO; 340492

[0211] These results collectively support our hypothesis that jR2R3 P301 L-PLP directly penetrates cell membranes in a concentration-dependent manner, bypassing the endocytic pathway. This cell-penetrating ability opens up potential therapeutic applications, as these peptides could be designed to deliver bioactive molecules directly into cells, circumventing the limitations of endocytic trafficking that often leads to degradation in lysosomes.

[0212] Example 6: Tau PLPs Prevent Seeding of jR2R3 P301L Fibrils in Cellular Culture System

[0213] A cell line expressing a fluorophore-tagged tau construct, mClover3-Tau187 P301 L, was previously constructed to monitor the seeding activity of exogenous tau fibrils. Using this model, jR2R3 P301L fibrils strongly induced tau aggregation in a 4R isoform-specific manner, as described in Longhini, et al., Proceedings of the Nat’ I Acad, of Seis. 121, e2320456121 (2024), which is hereby incorporated by reference in its entirety for all purposes, and specifically for its description of jR2R3 P301L fibrils. Extending the in vitro ThT findings described above, it was hypothesized thatjR2R3 P301 L-PLP could effectively inhibit the seeding of JR2R3 P301L fibrils in the cell culture models. In this assay, jR2R3 P301 L-PLP was added for six hours, then washed out of the media before the addition of jR2R3 P301L fibrils (FIG. 4A). It was reasoned that removing the jR2R3 P301 L-PLP prior to fibril addition would allow only the internalized PLP to interact with the fibrils, thereby preventing seeding while still facilitating cellular uptake.

[0214] 1 pM FITC-labeled jR2R3 P301L fibrils and 2 pM Cy5.5jR2R3 P301 L-PLP were added to wild-type cells not expressing mClover3-Tau187-P301L, and their colocalization was monitored (FIG. 4B). Robust colocalization of fibrils and PLP was observed, with nearly all fibrils colocalized with PLP (Manders' M1 = 0.99 ± 0.004). This high M1 value indicates a strong overlap of PLP with the fibrils. However, there was a significant amount of PLP that did not colocalize with the fibrils, as suggested by the M2 value (Manders' M2 = 0.46 ± 0.10), indicating that approximately 54% of the PLP signal was not associated with the fibrils. Sixteen biological replicates were included in the colocalization analysis.

[0215] For inhibition studies, cells were treated with jR2R3 P301 L-PLP at concentrations ranging from 62.5 nM to 4 pM for six hours, followed by a media change to prevent extracellular interactions with the subsequently added fibrils. Imaging conducted 24 hours after fibril addition revealed a potent, sub-pM inhibitory effect in the presence of jR2R3 P301 L-PLP, with an EC50 of 105 ± 14 nM (R2= 0.91) (FIG. 4C, FIG. 20A). In contrast, a control protein-like polymer with aLVM Ref. 102-24WO; 340492previously designed capping peptide (21), SEQ ID NO: 3 (VQPINK), failed to block seeding activity, underscoring the effectiveness of the rationally-designed construct against 4R mimetic mini-prion fibrils (FIGs. 20E-20F).

[0216] Further exploring the efficacy of the multivalent PLPs, monomers of the jR2R3 P301L cell-penetrating peptides — enriched with multiple lysines for enhanced cellular uptake — were introduced into the cell cultures (FIGs. 20C-20D). These monomers, which were identical to those conjugated to the PLP, did not prevent tau aggregation, suggesting that the steric hindrance provided by linking the jR2R3 P301L motif to a multivalent scaffold plays a role in effectively blocking further aggregate growth in this cellular model.

[0217] To assess long-term inhibition, cells were imaged 48 hours post-fibril exposure. After an initial six-hour incubation with PLPs, cells were rinsed and exposed to fibrils. At 48 hours, aggregate formation increased compared to the 24-hour mark, with the percentage of cells containing aggregates rising from 1.2 ± 0.8% at 4 pM PLP to 22.9 ± 7.3% (FIG. 4C, FIG. 20B).Although there was a decrease in the fit to a dose-response model (R2= 0.67), the EC50 was still 479 ± 100 nM compared to 105 ± 14 nM at 24 hours. It was hypothesized that at these extended time points, either the peptides conjugated to the PLP are being degraded, or the introduced fibrils are being degraded, leading to the release of newly available fibril ends that are free to seed naive tau aggregates.

[0218] Example 7: IR2R3 P301L-PLP is Stable Over Time

[0219] To emphasize the remarkable stability of jR2R3 P301L-PLP and to rule out the hypothesis that jR2R3 P301L-PLP moieties were being degraded at the 48-hour time point, the peptide was subjected to a range of in vitro conditions, designed to mimic physiological environments and test its stability. In the initial assessment, the PLP’s ability to inhibit tau aggregation was evaluated using a ThT fluorescence assay. As shown in FIG. 5B, the addition of jR2R3 P301L-PLP to a mixture of fibril seeds and monomers significantly reduced the ThT signal, indicating strong inhibition of tau fibril formation. Notably, this inhibitory effect was sustained over a seven-day period, without any observable loss of efficacy (FIG. 5B). These data suggest that the PLP retains its function and integrity in vitro over extended periods and implies that capping of fibrils by jR2R3 P301 L-PLP is thermodynamically stable. Other in vitro investigations into PLPs support these findings of extended stability in solution (33, 39, 40).LVM Ref. 102-24WO; 340492

[0220] To further assess jR2R3 P301 L-PLP stability under more physiologically relevant conditions, the jR2R3 P301 L-PLP was exposed to human serum and mouse striatal cell lysate at 37°C for 7 days (FIG. 5A). Following incubation, the samples were analyzed using SDS-PAGE and fluorescent imaging. Impressively, no degradation of the Cy5.5-labeled PLP was detected under these conditions (FIG. 5A). The absence of any observable breakdown in both serum and cell lysate indicates the peptide’s strong resistance to extracellular proteases, suggesting that any potential degradation would likely occur in an intracellular, rather than extracellular, environment and is consistent with robust performance by other PLPs investigated (38, 43). This underscores the PLP’s robust stability in biologically relevant environments.

[0221] To truly push the boundaries of the PLP’s stability, a controlled degradation study was conducted using trypsin, a potent proteolytic enzyme. The jR2R3 P301 L-PLP was incubated with trypsin for 60 minutes at 37°C to force degradation, followed by the addition of TLCK to inhibit further proteolysis. After trypsin exposure, the ability of the PLP to inhibit tau aggregation was significantly compromised, as evidenced by the substantial increase in ThT fluorescence, which indicates the loss of PLP’s inhibitory function (FIG. 5C). This outcome was expected, as trypsin specifically cleaves at the PLP’s internal lysine residues. However, when trypsin was pre-incubated with TLCK, preventing proteolytic activity, the PLP maintained its inhibitory effect, with no significant increase in ThT signal observed. Imaging the resulting fibrils from the trypsin experiments by TEM showed that in the presence of trypsin no PLP was bound to fibrils, however in the absence of trypsin, biotin-tagged PLP was seen localized to fibrils in streptavidin immuno-gold imaging (FIG. 5D). This phenomenon of proteolytic stability was studied extensively in PLPs, revealing that increased hydrophobicity of the polymer backbone enhances proteolytic stability by promoting the condensation of pendant peptides via the hydrophobic effect, thereby reducing surface exposure of cleavage sites and decreasing interactions with proteases (35).

[0222] These results suggest that while jR2R3 P301 L-PLP can be degraded, this only occurs in the presence of protease activity, specifically high levels of trypsin. Under normal physiological conditions, the PLP remains highly stable and functional. This resilience makes the PLP a promising candidate for therapeutic applications where long-term stability is often preferred.

[0223] Example 8: Prolonged Exposure and Multiple Doses of IR2R3 P301 L-PLP Prevent 48-hour AggregationLVM Ref. 102-24WO; 340492

[0224] Based on the stability of jR2R3 P301L-PLP to a variety of conditions, we believe that the reduced efficacy in preventing seeding at48-hours is due to the fragmentation of tau fibrils leading to too many fibril ends to cap. We speculated that maintaining a constant intracellular concentration of the PLP could enhance long-term aggregation prevention. To test this, we implemented two dosing strategies that differed from our initial washout protocol.

[0225] In the first approach, jR2R3 P301 L-PLP was introduced to the cells for six hours before fibril addition, as in our previous experiments. However, unlike earlier procedures, the PLP was not removed from the media prior to adding the fibrils. This strategy was based on our observation that, although a significant amount of PLP uptake occurred within the first six hours — as demonstrated by the inflection point in FIG. 3A — accumulation continued beyond this point. Employing this method, we noted a substantial reduction in aggregate formation at 48 hours, achieving a percentage of cells with aggregates of 15.4 ± 2.1% at 4 pM PLP, with a EC50 of 628 ± 113 nM (R2=0.79), compared to 19.5 ± 6.2% and 479 ± 100 nM from previous experiments (FIG. 6A, FIGs. 21 A & 21 B).

[0226] Building on this approach, we added a second dose of PLP at 24 hours. The results were markedly improved, with an EC50 of 381 ± 45 nM (R2=0.67) and cells exhibiting puncta decreased to ~4% at 4 pM PLP (FIG. 6A). These findings indicate that enhancing intracellular retention and concentrations of PLPs could be an effective strategy for long-term prevention of tau aggregation and spread. This dual-dosing regimen maintains the PLP concentration and caps newly fragmented tau fibrils seeding naive monomeric tau.

[0227] Example 9: Bifunctional PLP for Increased Clearance of Exogenous Tau Fibrils

[0228] Since multiple additions of jR2R3 P301 L-PLP dramatically increased the effectiveness of aggregate prevention at 48 hours, we hypothesized that directing the PLP-bound fibrils to appropriate degradation machinery could further enhance its ability to prevent tau aggregation. To test this, we engineered a bifunctional PLP containing eight copies of jR2R3 P301L and seven copies of a Keapl-binding peptide, termed jR2R3 P301L Keap1-PLP. Keapl, when engaged by a binding partner, facilitates the ubiquitination of the binder, thus targeting it for proteasomal degradation.

[0229] Previous studies have incorporated a Keapl peptide sequence successfully into a tau-targeting PROteolysis TArgeting Chimeras (PROTACs) (46). Keapl (Kelch-like ECH-associated protein 1) acts as an adaptor protein that facilitates the recruitment of its substrate,LVM Ref. 102-24WO; 340492Nrf2 (nuclear factor erythroid 2-related factor 2), to a Cullin-RI NG E3 ubiquitin ligase complex (CRL) that marks the protein for ubiquitination. Given the high affinity of the jR2R3 P301 L sequence for tau, we employed it as the foundation for designing a PLP-based PROTAC.Notably, Keapl has also been explored as a therapeutic in our lab (38). The optimized Keapl-binding sequence, SEQ ID NO: 4 (LDPETGEFLRRRR), which carries a net charge of +1, was used in conjunction with the jR2R3 P301 L sequence to construct a heterobifunctional PLP PROTAC. This Keapl-binding sequence mimics the binding site of its endogenous partner, Nrf2, enabling selective engagement with Keapl. While Keapl was used in this example, other ligase recruiting peptide sequences may be employed. In aspects, an intracellular E3 ubiquitin ligase peptide sequence is used. In aspects, a cereblon (CRBN) peptide sequence is used. In aspects, a Von Hippel-Lindau (VHL) peptide sequence is used.

[0230] In this experiment, both PLPs were washed out after six hours to assess their lasting effects on tau aggregate prevention. Despite having fewer jR2R3 P301L peptides than the original jR2R3 P301L-PLP, thejR2R3 P301L Keapl -PLP reduced the percentage of cells with puncta to 5.9% ± 6.0%, compared to around 19.5 ± 6.2% for the PLP without the Keapl peptide (FIG. 6B, FIG. 13C). These results suggest that the addition of the Keapl-binding domain effectively enhances the clearance of tau aggregates by directing them to the proteasomal degradation pathway.

[0231] Taken together, these findings demonstrate that multifunctional PLPs, such as the PROTAC-like jR2R3 P301L Keap1-PLP, represent a promising strategy for improving the efficacy of aggregate prevention, providing a dual mechanism of action by both inhibiting aggregation and promoting the degradation of tau fibrils.

[0232] Example 10: Prevention of Seeding by 4R Tauopathy Patient-Derived and Mouse-Derived Fibrils

[0233] The jR2R3 P301 L peptide was designed to structurally mimic the fibrils associated with 4R tauopathies. Comparative analysis of the Cryo-EM structures from patient-derived CBD and PSP, as-well-as PS19 mouse models, against our own jR2R3 P301 L fibrils, revealed a common strand-loop-strand architecture (FIG. 7A). This motif was absent in mixed tauopathies such as Alzheimer’s disease (AD), suggesting a structural divergence (FIG. 7A). Further, replica exchange molecular dynamics (REMD) simulations of the jR2R3 P301 L monomer showed a sampling of various conformations that we predict are capable of binding to disease fibrils (FIG.LVM Ref. 102-24WO; 3404927B). Based on these observations, we hypothesized that jR2R3 P301L-PLP would effectively inhibit seeding by CBD, PSP, and PS19 fibrils, but show reduced efficacy against AD fibrils.

[0234] To test this hypothesis, we extracted sarkosyl-insoluble material from the brains of patients with CBD, PSP, and AD — as well as material from 9-month-old PS19 mouse brain — and were able to observe fibrils by TEM (FIG. 22). Dot-blot assays using biotin-labeled jR2R3 P301L-PLP pulled down with magnetic-streptavidin beads confirmed specific interactions with the fibrils from CBD, PSP, PS19, and unexpectedly with AD fibrils (FIG. 7C and FIG. 22). Fibrils bound selectively to jR2R3 P301L-PLP, with no interaction observed between the fibrils and the streptavidin beads alone. This selective binding supports our hypothesis and underscores the potential of jR2R3 P301L-PLP as a targeted therapeutic approach in distinct tauopathies.

[0235] In our cell-based aggregation assay, we optimized experimental conditions to observe a significant proportion of cells with tau aggregation after fibril addition, with PS19 and AD showing aggregation after 24 hours and CBD and PSP requiring 48 hours. These values were determined empirically and will vary based on different fibril preparations from different patients. Our experimental protocol involved treating cells with PLP for six hours before fibril addition without a subsequent washout, to maintain consistent conditions between the 24- and 48-hour time points.

[0236] For each of the 4R tauopathies (CBD, PSP, and PS19), a robust prevention of aggregation, characterized by a classic dose-response curve was observed (FIG. 7D, top, FIGs.21D-21F). The EC50s forCBD, PSP, and PS19 were 771 ± 403 nM (R2=0.61), 186 ± 40 nM (R2=0.73), and 523 ± 114 nM (R2=0.75), respectively. In contrast, the dose-response curve for AD did not exhibit a typical logarithmic trend but was instead linear, with significant inhibition only observed at concentrations around 2 pM (FIG. 7D, Bottom, FIG. 21 G). This deviation in the AD fibrils is notable, as we predicted that our 4R selective jR2R3 P301L-PLP should not be as effective at inhibiting a mixed 3R / 4R tauopathy such as AD.

[0237] Example 11: jR2R3 P301L-PLP Blocks Tau Aggregation in Human iPSC-Derived Cortical Organoid Model

[0238] To determine if our PLPs could prevent tau seeding in a model more representative of the human brain, we utilized human forebrain assembloids. These were generated by fusing ventral and dorsal-directed organoids into assembloids, which contain a variety of neuronal subtypes as well as glial cells, including astrocytes (47-49).LVM Ref. 102-24WO; 340492

[0239] 6-month-old organoids were treated with sarkosyl-insoluble fractions derived from 9-month-old PS19 mice. These fractions contain highly active tau seeds, which had previously been shown to induce seeding in certain planar cell culture biosensor lines. Treatments were administered either with or without 4 pM jR2R3-P301L-PLP. For PLP-treated organoids, the PLP was maintained during media changes.

[0240] After seven days, the organoids were fixed, sectioned, and stained for MC1 , a conformational antibody that specifically detects misfolded tau. In the untreated organoids, strong MC1 signal was observed, particularly along the long, linear neuronal processes, predominantly at the outer layer of the organoid (FIG. 8A). This localization is consistent with the reliance on diffusion for molecular penetration into these organoids, suggesting that the PS19 tau seeds likely did not reach deeper layers.

[0241] In contrast, organoids treated with PLP showed a significant reduction in the linear MC1 staining, indicating that tau misfolding was effectively inhibited in the neuronal processes (FIGs. 8B-8D). Notably, the tau detected in this model is likely the endogenously expressed wild-type tau within the neurons.

[0242] A machine learning model was trained to identify and partition signal that was present in these linear tracts and applied the model to images of multiple sections from four PLP treated and four untreated biological replicates. The area occupied by this signal was normalized against the perimeter of the organoids, since most of the signal was localized on the periphery of the organoids. An independent T-Test on the normally distributed data was significant with a p-value of 0.017 (FIG. 8E).

[0243] Discussion Corresponding to Examples 1-11.

[0244] Tauopathies, characterized by the accumulation and spread of misfolded tau fibrils, pose a significant health challenge due to their association with neurodegeneration and cognitive decline. The hypothesis that preventing the spread of tau aggregates could halt disease progression is relevant to our research. To address this, we have developed innovative tools and models to study tau fibril formation, which can vary among different tauopathies. Recent advancements in our lab include the resolution of the structure of a minimal tau peptide that selectively seeds 4-repeat (4R) tau isoforms in cellular models (19, 20). We posited that this potent mini-prion could play a dual role when incorporated into a multivalent protein-like polymerLVM Ref. 102-24WO; 340492(PLP) scaffold, effectively occluding fibril ends and preventing further monomer addition after initial binding.

[0245] PLPs are an attractive drug modality due to their tunable properties for cellular penetration and blood-brain barrier (BBB) permeability (38, 40). Their multifunctionality also enables their potential as PROTAC molecules. Our findings suggest that the 4R-directed jR2R3 P301L-PLP not only inhibits the spread of our model peptide in vitro and in cell culture but also is capable of effectively blocking the propagation of patient-derived tau fibrils. Importantly, in a human induced pluripotent stem cell (iPSC)-derived brain organoid model, we robustly prevented tau fibril seeding as indicated by the well-characterized MC1 conformational tau antibody. This represents the first application of a bona fide cortical organoid for modeling tau fibril seeding and highlights the potential of this advanced model to further investigate tau pathologies.

[0246] Mechanistically, our results suggest that jR2R3 P301 L-PLP directly interacts with tau fibrils both in vitro and in vivo. Transmission electron microscopy (TEM) images indicate that the PLP binds to and aggregates at the ends of fibrils (see, e.g., FIGs. 23A-23D), effectively halting the addition of further tau monomers. Immuno-gold labeling confirms that these aggregates contain PLP, while ThT assays demonstrate that fibril formation is inhibited for at least seven days in vitro. Furthermore, our studies reveal that fluorescently labeled PLP enters cells via direct diffusion and colocalizes with seeded fibrils, preventing the recruitment of additional tau monomers. In each disease sample extraction, Alzheimer’s (FIG. 23A), PSP (FIG. 23B), PS19 Mouse (FIG. 23C), and corticobasal degeneration (FIG. 23D), straight long fibrillar species were observed. This confirmed the nature of the species used correspond to that of FIGs. 7A-7D, which supports the claim that these species are bona fide disease fibrils and that their prion-like spread can be prevented / slowed in our assay system with PLPs.

[0247] A potential challenge in our work was the potential decreased efficacy of PLP in preventing aggregate formation over extended time points that was observed in at least some experiments. Initial experiments involved washing out PLP from the media, which inadvertently reduced its interaction with seeding fibrils. However, by maintaining PLP in the media or adding a second dose at 48 hours, we significantly reduced the number of seeded cells to approximately 5%. Additionally, the introduction of a Keapl PROTAC multifunctional PLP demonstrated the potential to prevent tau aggregation even when washed out at six hours postadministration.LVM Ref. 102-24WO; 340492

[0248] Together, our findings on reduced efficacy at longer time points, the stability of the PLP, and the effectiveness of increased doses or PROTAC PLPs suggest a mechanism whereby jR2R3 P301L-PLP binds to introduced fibrils, preventing the addition of naive monomers. As time progresses, the fibrils are ultimately degraded, but this can lead to an increase in fibril ends. This phenomenon has been previously reported in studies where overexpression of VCP cleared fibrils but resulted in increased seeding due to many smaller fibril fragments. In our system, PROTACs not only facilitated the clearance of these introduced seeds but also allowed for the incorporation of additional jR2R3 P301L-PLP to sponge up the produced fibril ends.

[0249] Many therapeutics developed to treat neurodegenerative diseases, particularly those targeting tauopathies, have failed to show significant efficacy in clinical trials. A key issue is the poor translatability of treatments developed in mouse models of dementia to human patients. This discrepancy arises from fundamental differences between murine and human nervous systems, including the necessity to overexpress tau in mice, which does not accurately reflect human disease pathology. Additionally, the isoform ratios of tau and expression levels differ between species, further complicating the development of effective therapies. To address these challenges, we developed a novel model system using six-month-old human-induced pluripotent stem cell (iPSC)-derived forebrain assembloids seeded with tau fibrils. Staining with conformational antibodies revealed the presence of aggregated tau in these assembloids. In this more relevant human model, which may offer a better platform for developing drugs with clinical efficacy, we observed that our protein-like polymers (PLPs) effectively blocked MC1 staining, indicating inhibition of tau aggregation. This approach may represent a promising step forward in advancing therapies that translate more successfully from bench to bedside.

[0250] Exemplary Experimental Aspects for Examples 1-11:

[0251] Materials’. All materials and reagents, unless otherwise noted, were purchased from commercial sources and used without further purification. / V-(hexanoic acid)-c / s-5-norbornene-exo-dicarboximide and initiator (IMesH2)(C5H5N)2(CI)2Ru=CHPh were synthesized according to previous reports. (53, 54).

[0252] Instrumentation:1H NMR spectra were recorded on a Varian Inova spectrometer (500 MHz) in DMF-d7. Analytical HPLC analysis of peptides was performed on a Jupiter 4 Proteo 90A Phenomenex column (150 x 4.60 mm) using a Hitachi-Elite LaChrom L-2130 pumpLVM Ref. 102-24WO; 340492equipped with UV-Vis detector (Hitachi-Elite LaChrom L2420). The solvent system consists of (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile. For preparative HPLC, a Gilson PLC 2050 purification system was used to purify all peptides. The solvent system consisted of (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile. ESI-MS spectra of peptides were collected using a Bruker Amazon-SL spectrometer configured with an ESI source in both negative and positive ionization mode. Aqueous Phase Gel Permeation Chromatography (Aqueous GPC): Aqueous phase GPC measurements were performed with a Tosoh Bioscience TSK-GEL®PWxl-CP column 7.8 mm ID x 30 cm, 10 mm, using 0.05% NaN3as the mobile phase. Detection consisted of a Wyatt Optilab T-rEX refractive index detector operating at 658 nm and a Wyatt DAWN® H ELEOS® II light scattering detector operating at 659 nm.

[0253] Peptide Monomer Synthesis. Peptide monomers were synthesized on either Rink Amide Protide™ resin or Rink Amide Protide™ resin (LL) using standard Fmoc SPPS procedures on a Liberty Blue Automated Microwave Synthesizer. N-(hexanoic acid)-cis-5-norbornene-exo-dicarboximide was added to the end of the peptide using the same Liberty Blue procedures used for the other amino acids. The peptide monomers were cleaved off the resin by treating the resin with TFA / H2O / TIPS (95:2.5:2.5 v / v) for 4 h. The crude products were obtained by precipitation in cold diethyl ether, mass confirmed by Bruker Amazon SL and further purified via preparative HPLC.

[0254] Polymer Synthesis. PLPs were achieved by ring-opening metathesis polymerization (ROMP) under nitrogen gas in a glove box. Norbornene conjugated peptide monomers (15.0 equiv., 30 mM) were dissolved in degassed DMF with 1M LiCI. Next, the olefin metathesis initiator (IMesH2)(C5H5N)2(CI)2Ru=CHPh stock solution (1.0 equiv., 20 mg / mL in DMF) was quickly added into the monomer solution. For reaction kinetics, deuterated DMF with LiCI with hexamethyldisilane standard was used, and monomer and polymer olefin peaks were tracked via nuclear magnetic resonance spectroscopy on 400 MHz Bruker Avance III HD Nanobay. The solution was left to stir until the full consumption of monomers. In the case of dye tagged polymers, this was achieved by the addition of 1 eq (30 mM) of a rhodamine or Cy5.5 linked to norbornene via a six-carbon chain linker with an amide bond. After the polymerization, the polymer solution was purified via preparative HPLC. Finally, the polymer product was obtained by lyophilization.

[0255] Cell Culture and Maintenance. HEK293T and H4 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1%LVM Ref. 102-24WO; 340492Penicillin / Streptomyci n at 37° C in a 5% CO2 environment. Cells were passaged using trypsin equal to 1 / 10 the volume of the culture vessel and kept under 10 passages to ensure the reproducibility of the data. We have previously described the construction and characterization of the H4 mClover3-Tau187-P301L line used for the majority of the cell-based seeding assays(19).

[0256] Cell-Based Puncta Seeding Assay. H4 mClover3-Tau187-P301L cells were plated at 20,000 cells / well in a 96-well plate. For all conditions 5 replicates were performed. The following day, varying concentrations of PLP constructs were added to the media for 6 hours unless otherwise noted. After 6 hours, the media was replaced and 2 pM jR2R3-P301 L fibrils (or disease-derived fibrils at optimized dilutions) were introduced with lipofectamine 2000. 2 pM fibril brought to a total volume of 10 pL with optiMEM was combined with 1.25 pL lipofectamine 2000 in 8.75 pL of optiMEM, incubated for 15 minutes at room temperature, added to 1 well. After 24 hours, unless otherwise stated, a minimum of 100 cells were imaged per replicate with confocal microscopy.

[0257] ThT Assay. Thioflavin (ThT) experiments were conducted using a TECAN fluorescent plate reader. Each well of a 384-well plate (Corning, low volume non-binding surface, black with clear flat bottom) contained 25 pM of jR2R3 P301L monomer, 20 pM ThT, 25 pM jR2R3 P301L fibril in a 20 mM HEPES buffer (pH 7.4), making a total volume of 30 pL per well. Varying amounts PLP were added before the reaction was initiated.

[0258] For degradation assays, 12.5 pM PLP was added to 20 mM HEPES pH 7.4, 5 pM Trypsin (from porcine pancreas, Sigma Aldrich, CAS: 9002-07-7) was added and placed into an incubator at 37°C for 30 min. 500 pM Inhibitor (Na-Tosyl-L-lysine chloromethyl ketone hydrochloride, Sigma Aldrich) was then added and allowed to incubate for an additional 30 min.25 pM jR2R3 P301L monomer, 20 pM ThT was added to the mixture. Mixture was then distributed into 5 wells, and 25 pM jR2R3 P301L fibril was added to individual wells for a total volume of 30 pL per well.

[0259] The plate reader was set to a temperature of 37°C and allowed time to equilibrate. Subsequently, ThT fluorescence intensity was recorded at excitation and emission wavelengths of 440 nm and 485 nm, respectively. Measurements were taken every 2 minutes until a plateau in fluorescence intensity was observed. Each experiment was performed with five replicates andLVM Ref. 102-24WO; 340492repeated three times using independent samples, typically spanning a total time of 24 hours, unless otherwise noted.

[0260] TEM Analysis. For transmission electron microscopy (TEM) analysis, 5 pL of recombinant tau fibril samples were placed onto a glow-discharged copper grid (Electron Microscopy Science, FCF-200-Cu) for 20 seconds before blotting dry with filter paper. The samples were then stained by applying 5 pL of 1.5% (w / v) uranyl acetate solution, immediately blotting dry. An additional 5 pL of uranyl acetate solution was added for 60 seconds, followed by blotting dry. The samples were examined using a Thermo Scientific Tales G2200X TEM / STEM microscope operating at 200 kV and room temperature. The grids were imaged using a Ceta II CMOS 4k x 4k camera at the specified magnifications.

[0261] For biotin labeled PLP TEM analysis, the above conditions were used, one without trypsin and one with trypsin. After 24 hours, 10% streptavidin conjugated to gold nanoparticle (Electron Microscopy Sciences, Particle size 6 nm) by volume was added and incubated at 4°C overnight. The resulting solution was added to a grid and rinsed 3 times with mill iQ water to remove excess gold nanoparticles. The samples were then stained by applying 5 pL of 1.5% (w / v) uranyl acetate solution, immediately blotting dry. An additional 5 pL of uranyl acetate solution was added for 60 seconds, followed by blotting dry.

[0262] In Vitro Dot Blot Assays. Prior to experiment, all stocks were diluted in 1x Phosphate Buffer Saline (PBS) containing 137 mM NaCI, 2.7mM KCI, 10 mM phosphate (pH 7.4). 50uL of 10uM jR2R3 P301L Fibril and 2uM R2R3 P301L Monomer labeled with FITC fluorophore were incubated with 50uL of 10uM of Biotin labeled JR2R3 P301L species rotating 1 hour at 37C. 50uL of Thermo Scientific™ Pierce™ Streptavidin Magnetic Beads were added to each solution and incubated rotating for 1 hour at room temperature. The beads were washed 3 times with Tris Buffered Saline with 0.1% Tween (TBST) containing 20 mM Tris, 150 mM NaCI, Tween® 20 detergent: 0.1% (w / v) and eluted with 1mM Glycine pH 2.2. The eluate was subsequently dot-blotted on PVDF membrane and imaged with BIO RAD ChemiDoc MP Imaging System.

[0263] Fibrils were extracted from PSP, PS19, CBD, and AD patients' brain tissue using a sarkosyl treatment and ultracentrifugation. Stocks were diluted with PBS. 20 pL of diseased fibrils isolated from human patients were incubated with 20 pL of 10 pM Biotin labeled JR2R3 P301L species at 37°C rotating for 1 hour. 20 pL of Thermo Scientific™ Pierce™ Streptavidin Magnetic Beads were added to each solution and incubated rotating for 1 hour at roomLVM Ref. 102-24WO; 340492temperature. The beads were washed 3 times with Tris Buffered Saline with 0.1% Tween (TBST) containing 20 mM Tris, 150 mM NaCI, Tween® 20 detergent: 0.1% (w / v) and eluted with 20 pL of 1 mM Glycine pH 2.2. 10 pL of eluate was subsequently dot-blotted on PVDF membrane along with unincubated diseased fibril and TBST washes. Tau Monoclonal Antibody (TAU-5; Thermofischer: AHB0042) was incubated at 1 to 1000 dilution in Intercept (PBS) Blocking Buffer with the dot-blotted PVDF membrane overnight shaking at4°C. Primary incubation was followed by three washes with TBST. Subsequently, 680 nm anti-mouse secondary antibody was used at 1 to 10,000 dilution and was incubated for 1.5 hours and four washes were performed with TBST. The membrane was imaged with BIO RAD Chemi Doc MP Imaging System.

[0264] ICC Experiments. H4 mClover3-Tau187-P301L cells were treated with fluorophore labeled PLPs and after either 6 or 24 hours were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Cells were washed 3x with PBS and placed in blocking buffer (4% BSA, 1% NGS, 0.2% Triton-X100, in PBS pH 7.4) for 1 hour and room temperature. Anti-Rab5 (1:500) or anti Rab7 antibody (1:500) in antibody dilution buffer (2% BSA, 0.5% NGS, 0.2% Triton-X100, in PBS pH 7.4) was added overnight at 4°C. Cells were washed 3x with PBS and secondary antibody (1:500) in antibody dilution buffer was incubated at room temperature for 2 hours. Cells were washed 3x in PBS and imaged.

[0265] ITC Experiments. All isothermal titration calorimetry (ITC) experiments were performed using a Nano ITC (TA Instruments). Prior to the experiments, all buffers were degassed for at least 30 minutes. The experiments were conducted in 1x Phosphate Buffer Saline (PBS) containing 137 mM NaCI, 2.7 mM KCI, 10 mM phosphate (pH 7.4).

[0266] Before initiating the experiments, the components were loaded into the Nano ITC and stirred for at least one hour to achieve a stable baseline. A total of 950 pL of 500 nM jR2R3 P301L-PLP was added to the gold cylindrical cell of the Nano ITC. Following this, 30 injections of 7.5 pL of 5 pM jR2R3 P301 L monomer were performed, with a 500-second interval between each injection. Identical experiments were performed with jR2R3 P301 L fibril samples replacing monomers.

[0267] Data analysis was conducted using a combination of NanoAnalyze software (TA Instruments) and custom Python scripts. Control experiments, in which one or both componentsLVM Ref. 102-24WO; 340492were replaced with 1X PBS, were performed to determine baseline heats of dilution. These baseline heats were found to be well below those observed in the experimental conditions.

[0268] Human Forebrain Assembloids Generation. Human induced pluripotent stem cells (iPSCs) were donated by Karch (50). The iPSC line F12442.4 was cultured in mTeSR Plus medium (STEMCELL Technologies) in 6-well plates (Corning) coated with Matrigel (Corning). Medium was changed daily, and cells were passed with ReleSR (STEMCELL Technologies) when 70-80% confluent.

[0269] For the generation of human brain organoids, we followed previously described protocols (47, 48, 51) with some modifications (49). On day 0, hiPSC were dissociated into single cells with Accutase (STEMCELL Technologies), then seeded into 96 slit-well plates (S-Bio). 104cells were seeded per well in mTeSR Plus medium plus 10 nM ROCK inhibitor. After 24h, media was replaced with Neural Induction Medium (NIM), containing Essential 6 (Gibco) as the base, plus 1x Antibiotic-Antimycotic (Gibco), 2.5 uM dorsomorphin (Tocris), 10 uM SB-431542 (Tocris), and 2.5 uM XAV-939 (Tocris). Human cortical organoids (hCO) and human ventral organoids (hVO) were fed daily with NIM from days 1-5, and 5 M IWP-2 was added to medium on days 4-6 only for hVO. On day 6, organoids were transferred to Neural Differentiation Medium (NM), containing Neurobasal-A (Gibco), B-27 supplement without vitamin A (Gibco), 1x GlutaMax, and 1x Antibiotic-Antimycotic, supplemented with 20 ng / ml EGF (Shenandoah) and 20 ng / ml FGF2 (Shenandoah). hCO organoids were fed daily with NM plus FGF2 and EGF from days 6-15, and every other day from days 16-24. For hVO organoids, NM was also supplemented with 5 uM IWP-2 (Selleckchem) from days 6-11; 5 uM IWP-2, 100 nM SAG (Selleckchem) and 100 nM RA (Sigma) from days 12-14; 5 uM IWP-2, 100 nM SAG, 100 nM RA and 100 nM AlloP (Selleckchem) on day 15; and 5 uM IWP-2, 100 nM SAG, and 100 nM AlloP from days 16-24. Medium was changed daily for hVO from days 6-24. For both hCO and hVO, NM was supplemented with 20 ng / ml BDNF (Shenandoah) and 20 ng / ml NT3 (Shenandoah) from days 25-43, with medium changes every other day. From day 43 on, hCO and hVO organoids were maintained in NM without additional supplements and fed every 4 days. hCO and hVO were fused at D60 and the fused assembloids used at around D180.

[0270] Immunofluorescence of Organoid Cryosections. Whole organoids were fixed in PFA 4% for 24h at 4°C, washed 3x with PBS, and then cryopreserved by 48h incubation at 4°C in a solution of 30% sucrose in PBS. Cryopreserved organoids where then embedded in Neg-50 Frozen Section Medium (Epredia), flash frozen in dry ice, and stored at -80° C. FrozenLVM Ref. 102-24WO; 340492organoids were sectioned in 20pm slices in a Leica Cryostat CM 1850. For immunofluorescence, fixed organoid cryoslices were incubated in blocking buffer containing PBTA (0.5% BSA and 0.1% Triton X-100 in PBS) plus 5% normal goat serum for 1h at RT. Samples were then incubated with primary antibodies in blocking buffer overnight at 4°C, washed three times with PBTA, then incubated with secondary antibodies in blocking buffer for 1h at RT. Samples were washed three times with PBTA, then mounted with Prolong Diamond Antifade Mountant (Invitrogen) with DAPI for nuclei staining. The following primary antibodies were used: mouse anti-MC1, chicken anti-GFAP (Abeam ab4674, 1:500). The following secondary antibodies were used: goat anti-chicken Alexa Fluor 647 (ThermoFisher A-21449), goat anti-mouse Alexa Fluor 647 (ThermoFisher A-21235). Secondary antibodies were used at a dilution of 1:1,000.

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[0327] All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0328] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the product, product components, methods steps set forth in the present description. As will be obvious to one ofLVM Ref. 102-24WO; 340492skill in the art, products and methods useful for the present invention can include a large number of optional composition and processing elements and steps.

[0329] As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. The expression “of any of embodiments XX- YY” (wherein XX and YY refer to embodiment numbers) is intended to provide a multiple dependencies in the alternative form, and in some embodiments is interchangeable with the expression “as in any one of embodiments XX- YY.”

[0330] When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. When a compound is described herein such that a particular isomer, enantiomer or diastereomer of the compound is not specified, for example, in a formula or in a chemical name, that description is intended to include each isomers and enantiomer of the compound described individual or in any combination.

[0331] Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the embodiments herein.

[0332] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art.

[0333] As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecitedLVM Ref. 102-24WO; 340492elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the said embodiment. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the embodiment. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0334] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by embodiments herein.

[0335] Without wishing to be bound by any particular theory, there may be discussion herein of beliefs or understandings of underlying principles relating to the products and methods disclosed herein. It is recognized that regardless of the ultimate correctness of any mechanistic explanation or hypothesis, an embodiment of the invention can nonetheless be operative and useful.

[0336] In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art.

Claims

LVM Ref. 102-24WO; 340492WHAT IS CLAIMED IS:

1. A polymer having a formula (FX1):wherein:each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV);each P2independently comprises a peptide, and each instance of P2is different from each instance of P1;at least one P1independently, or in combination with other instances of P1inhibits tau fibril formation and / or tau fibril elongation;T1and T2are each independently polymer backbone terminating groups that can be the same or different;B1, B2, and B3are each independently a polymer backbone subunit;L1and L2are each independently a linking group;R1is independently a substituent;m is an integer from 2 to 1000;n is an integer from 0 to 1000;o is an integer from 0 to 1000; andeach connecting line in the formula (FX1) represents a covalent linkage comprising at least one of a single bond, a double bond, one or more atoms, or any combination thereof, optionally wherein the one or more atoms comprise carbon, nitrogen, and / or oxygen atoms.

2. The polymer of claim 1 , wherein at least one P1further comprises a charge modulating domain.

3. The polymer of claim 2, wherein the charge modulating domain has from 2 to 7 amino acid residues that can be the same or different, optionally wherein the amino acid residues are lysines, glycines, serines, or arginines, optionally wherein the amino acid residues are lysines.LVM Ref. 102-24WO; 3404924. The polymer of claim 2 or 3, wherein the charge modulating domain is attached to the N-terminus or the C-terminus of the at least one P1.

5. The polymer of any one of claims 1-4, wherein each P2independently comprises a degrading agent.

6. The polymer of claim 5, wherein the degrading agent comprises an E3 ligase recruiter, optionally wherein the E3 ligase recruiter comprises a sequence having 75% or greater sequence identity of:(i) a Keapl peptide, optionally SEQ ID NO: 5 (LDPETGEYL);(ii) a cereblon (CRBN) peptide;(iii) a VHL peptide, optionally SEQ ID NO: 6 (ALAPYIP);(iv) a mouse double minute 2 homologue (MDM2) peptide;(v) an inhibitor of apoptosis peptide (IAP), optionally SEQ ID NO: 8 (AVPI); or(vi) a degron peptide, optionally SEQ ID NO: 9 (RRRG);(vii) a KLHL20 peptide, optionally SEQ ID NO: 10 (LPDLV), optionally SEQ ID NO: 11 (LGLPDLVAKYN);(viii) a SOCS / CUL5 peptide, optionally SEQ ID NO: 12 (SLQYLCR)-XXX-SEQ ID NO: 15 (LPLP) wherein XXX is a spacer, optionally wherein the spacer is a spacer of 3-5 amino acids; or(ix) a segment of any one of (i)-(ix).

7. The polymer of any one of claim 1-6, wherein each P2independently has a chain length of 3 to 150 amino acids.

8. The polymer of any one of claims 1-7, wherein at least one P2further comprises a charge modulating domain.

9. The polymer of claim 8, wherein the charge modulating domain has from 2 to 7 amino acid residues that can be the same or different, optionally wherein the amino acid residues are lysines, glycines, serines, or arginines, optionally wherein the amino acid residues are lysines.

10. The polymer of any one of claims 1-9, wherein each polymer backbone subunit is independently a repeating unit (RU1), (RU2), (RU3), or (RU4):LVM Ref. 102-24WO; 340492wherein:R2is H or C1-C3 alkyl; andX is CH2 or O.

11. The polymer of any one of claims 1-10, wherein each of R1, T1, and T2independently is hydrogen, C1-C30 alkyl, C3-C30 cycloalkyl, C5-C30 aryl, C5-C30 heteroaryl, C1-C30 acyl, C1-C30 hydroxyl, C1-C30 alkoxy, C2-C30 alkenyl, C2-C30 alkynyl, C5-C30 alkylaryl, — CO2R4, — CONR5R6, —COR7, — SOR8, — OSR9, — SO2R10, —OR11, —SR12, — NR13R14, — NR15COR16, C1-C30 alkyl halide, phosphonate, phosphonic acid, silane, siloxane, silsesquioxane, C2-C30 halocarbon chain, C2-C30 perfluorocarbon, C2-C30 polyethylene glycol, a metal, or a metal complex, wherein each of R4-R16independently is H, C5-C10 aryl, or C1-C10 alkyl.

12. The polymer of any one of claims 1-11, wherein at least one of R1, T1, and T2independently further comprises an analytical tag, wherein the analytical tag comprises an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, a fluorescence tag, or any combination thereof.

13. The polymer of any one of claims 1-12, wherein each of L1and L2is independently selected from a single bond, an oxygen, and groups having an alkylene group, a heteroalkylene group, an alkenylene group, an arylene group, an alkoxy group, an acyl group, a triazole group, a diazole group, a pyrazole group, and any combination thereof, optionally wherein each of L1and L2is independently selected from a single bond, an oxygen, a C1-C10 alkyl, a C2- Cw alkenylene, a C3-C10 arylene, a C1-C10 alkoxy, a Ci-C acyl and any combination thereof.LVM Ref. 102-24WO; 34049214. The polymer of any one of claims 1-13, wherein each of L1and L2is independently a flexible linker, optionally wherein the flexible linker comprises a flexible hydrocarbon linker, optionally wherein the flexible linker comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 2 (PGGGSK) or SEQ ID NO: 13 (GSGSGS).

15. The polymer of any one of claims 1-14, wherein the polymer is a block copolymer having a formula (FX2):" <"<wherein:each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV), and wherein each P1further comprises a charge modulating domain;each P2independently comprises a peptide, and each instance of P2is different from each instance of P1, and wherein each P2independently comprises a degrading agent, optionally wherein the degrading agent is or comprises an E3 ligase recruiter;each AT independently comprises an analytical tag, optionally wherein the analytical tag is or comprises an affinity tag, a solubilization tag, a chromatography tag, an epitope tag, or a fluorescence tag, optionally wherein the analytical tag is or comprises a fluorophore, optionally wherein the fluorophore is a cyanine dye;T2is a polymer backbone terminating group, optionally wherein T2comprises ester vinyl ether (EVE) or biotin;each of L1, L2, and L3is independently a flexible linker, optionally wherein the flexible linker comprises a flexible hydrocarbon linker, optionally wherein the flexible linker comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 2 (PGGGSK) or SEQ ID NO: 13 (GSGSGS);LVM Ref. 102-24WO; 340492each m is an integer from 1 to 49;n is an integer from 1 to 49; ando is an integer from 0 to 48;wherein the block copolymer has a degree of polymerization between 2 and 50, optionally between 2 and 20.

16. The polymer of any one of claims 1-15, wherein the polymer is a homopolymer, a copolymer, a block copolymer, a brush polymer, or a brush block copolymer.

17. The polymer of any one of claims 1-16, wherein the polymer is characterized by a polydispersity index of greater than 0.7, optionally between 0.7 and 1.75, optionally between 0.7 and 1.5, optionally between 0.7 and 1.25, optionally between 0.9 and 1.25.

18. The polymer of any one of claims 1-17, wherein each of L1, L2, and / or L3is independently an enzymatically degradable linker, optionally wherein the enzymatically degradable linker is a matrix metalloproteinase (MMP) cleavage sequence, cathepsin B cleavage sequence, ester bond, reductive sensitive bond- disulfide bond, pH sensitive bond-imine bond or any combinations of these.

19. A method for inhibiting tau fibril formation and / or tau fibril elongation in a tissue, the method comprising administering to the tissue a polymer having a formula (FX1):wherein:each P1independently comprises a tau-binding peptide, wherein the tau-binding peptide comprises a sequence having 75% or greater sequence identity of SEQ ID NO: 1 (DNIKHVLGGGSVQIVYKPV);each P2independently comprises a peptide, and each instance of P2is different from each instance of P1;T1and T2are each independently polymer backbone terminating groups that can be the same or different;LVM Ref. 102-24WO; 340492B1, B2, and B3are each independently a polymer backbone subunit;L1and L2are each independently a linking group;R1is independently a substituent;m is an integer from 2 to 1000;n is an integer from 0 to 1000;o is an integer from 0 to 1000; andeach connecting line in the formula (FX1) represents a covalent linkage comprising at least one of a single bond, a double bond, one or more atoms, or any combination thereof, optionally wherein the one or more atoms comprise carbon, nitrogen, and / or oxygen atoms.

20. The method of claim 19, wherein the method further comprises at least one additional administration step comprising administering the polymer to the tissue within 24 and 72 hours of the initial administration.

21. A method for inhibiting tau fibril formation and / or tau fibril elongation in a tissue, the method comprising administering to the tissue the polymer of any one of claims 1-18.

22. A method for treating a condition in a subject, the method comprising administering the polymer of any one of claims 1-18 to the subject, optionally wherein the condition is a 4R tauopathy, optionally wherein the 4R tauopathy is CBD, PSP, or PS19, optionally wherein the 4R tauopathy comprises a familial frontotemporal dementia (FTD) having a tau gene mutation optionally wherein the tau gene mutation comprises P301 L, P301S, N279K, S305I, S305N, S305S, or a combination thereof.

23. A method for modeling tau fibril seeding in a human nervous system, the method comprising:fusing human iPSC-derived ventral organoids with human iPSC-derived dorsal-directed organoids to generate assembloids;treating the assembloids with fractions comprising active tau seeds;dividing the assembloids into a treatment group of assembloids and a control group of assembloids;administering a tau seeding inhibitor to the treatment group of assembloids;LVM Ref. 102-24WO; 340492staining the assembloids with an antibody, optionally a conformational antibody configured to detect misfolded tau; andquantifying the amount of the antibody in the treatment group of assembloids and the control group of assembloids with a machine learning model,thereby modeling tau fibril seeding in a human nervous system.

24. A pharmaceutical composition comprising the polymer of any one of claims 1-18 and a pharmaceutically acceptable excipient.

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