Compositions of BI-functional alpha helical peptides and methods thereof for treating TDP-43 proteinopathies

Abstract

Compositions and methods for disrupting pathological aggregation and / or mis-localization of TDP-43 in the brain / CNS have been developed. Compositions including engineered helical polypeptides that bind TDP-43's amyloidogenic core but resist β-sheet conversion are provided. In some forms, the engineered polypeptides include peptide degradation motifs (PDM) to enhance proteolytic degradation of aggregates, and / or targeting motifs to direct the peptides to the brain / CNS. Recombinant constructs including nucleic acids expressing or encoding the polypeptides are also provided. Methods of using the engineered peptides to treat or prevent one or more diseases or disorders associated with pathological aggregation and / or mis-localization of TDP-43 in the brain / CNS are also provided. In some forms, the methods treat or prevent ALS or FTD in a subject in need thereof.

Description

[0001] COMPOSITIONS OF BI-FUNCTIONAL ALPHA HELICAL PEPTIDES AND METHODS THEREOF FOR TREATING TDP-43 PROTEINOPATHIES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 661 ,559 filed June 18, 2024, which is incorporated herein by reference in its entirety

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under NS 122907 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] REFERENCE TO THE SEQUENCE LISTING

[0007] The Sequence Listing XML submitted as a file named “YU_8930PCT_ST26.xml”, created on June 18, 2025, and having a size of 407,557 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0008] FIELD OF THE INVENTION

[0009] The invention is generally related to the fields of structure-based protein design, and more particularly to therapeutic peptides that prevent formation and deposition and / or induce or increase clearance of pathological TDP-43 fibrillar aggregates, and methods of using thereof.

[0010] BACKGROUND OF THE INVENTION

[0011] Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by loss of motor neurons in the brain and spinal cord, leading to progressive muscle weakness, paralysis, and eventually death. An important feature of ALS pathophysiology is the nuclear clearance and misfolding of important RNA-binding proteins, resulting in the formation of insoluble aggregates. Nearly all ALS cases (97%) show pathologic aggregation and mislocalization of TDP-43 proteins within the brain. Although the U.S. Food and Drug Administration has approved several drugs for ALS that may prolong survival, reduce the rate of decline, or help manage the symptoms of ALS, however, there is currently no known treatment that stops or reverses the progression of ALS.

[0012] Aggregation and mis-localization of proteins in the brain is also a hallmark of Frontotemporal dementia (FTD). FTD, sometimes called frontotemporal lobar degeneration (FTLD), results from damage to neurons in the frontal and temporal lobes of the brain. Symptoms of FTD include behavioral and emotional changes, as well as cognitive and neuromuscular impairment. FTD typically occurs at a younger age than other forms of dementia, and roughly 60% of people with FTD are 45 to 64 years old. FTD is progressive, and symptoms worsen as more parts of the brain are affected. There is currently no cure for FTD, nor are there any treatments to slow or stop the progression of the disease, and life expectancy after diagnosis is between 2 and 10 years.

[0013] Aggregation and mis-localization of proteins in the brain is also a hallmark of several other debilitating and untreatable diseases, including Limbic -predominant age-related TDP-43 encephalopathy (LATE), and has been observed in a subset of Alzheimer's disease (AD) cases.

[0014] Despite decades of research to understand the causes and pathology of protein aggregation and mis-localization in the brain, there remain few effective interventions to help treat or prevent multiple diseases associated with this pathological process, and there is a need for therapeutic interventions that can effectively reduce or prevent aggregation and / or mis- localization of proteins within the brain.

[0015] Therefore, it is an object of the invention to provide therapeutic agents that can effectively reduce or prevent pathological aggregation and / or mis-localization and / or induce or increase clearance of proteins within the brain.

[0016] It is another object of the invention to provide compositions and methods for treating or preventing diseases and disorders associated with mis-localization of proteins within the brain.

[0017] SUMMARY OF THE INVENTION

[0018] Compositions that disrupt non-physiological multimers of TAR DNA Binding Protein 43 kDa (TDP-43) and prevent pathological TDP-43 aggregation and / or mis-localization and / or induce or increase its clearance in the brain / CNS have been developed. The compositions and methods thereof effectively prevent aggregation of and / or mis-localization and / or induce or increase clearance of aberrantly-folded TDP-43 proteins within the brain.

[0019] Engineered TAR DNA Binding Protein 43 kDa (TDP-43) polypeptides are provided. The engineered polypeptides typically include residues at positions 321-343 of mature human TDP-43 with one or more mutations of one or more amino acids of the mature human TDP-43 polypeptide, most typically a substitution of the amino acid residue at position 332 of the human TDP-43 polypeptide; and / or substitution of the amino acid residue at position 333 of the human TDP-43 polypeptide. Typically the engineered polypeptides can bind to a TDP-43 aggregate and reduce or eliminate binding of further TDP-43 peptides to the aggregate and / or reduce TDP-43 mis-localization, and / or induce or increase clearance of aberrantly-folded TDP-43.

[0020] In some forms, the substitution at position 332 is alanine, or arginine, or valine. In some forms, the substitution at 333 is tryptophan, or leucine, or valine at position 333 of the human TDP-43 polypeptide. For example, in some forms, the engineered polypeptide includes the amino acid sequence AMMAAAQAALQXXWGMMGMLASQ (SEQ ID NO:51), wherein each X comprises, independently, serine, valine, tryptophan, arginine, alanine, or valine. An exemplary preferred engineered polypeptide includes the amino acid sequence AMMAAAQAALQAWWGMMGMLASQ (SEQ ID NO:52). In some forms, the engineered polypeptide is between 23 and 80 amino acid residues, optionally wherein the engineered TDP- 43 polypeptide consists of 50 amino acid residues, with or without one or more additional heterologous peptide motifs such as a peptide degradation motif (PDM), or a cell penetrating peptide (CPPM), or both a PDM and a CPPM, and / or one or more linker peptides.

[0021] Nucleic acids encoding the polypeptide, including but not limited to, mRNA, plasmids, vectors including viral vectors (e.g. , AAV), and transposons are also provided. Delivery vehicle such as nanoparticle and microparticles for delivery of the engineered polypeptides or nucleic acid encoding the same are also provide.

[0022] Some forms encode a targeting motif that targets the delivery vehicle to the brain and / or the central nervous system (CNS) in vivo and / or a peptide that facilitates passage across the blood-brain barrier (BBB).

[0023] Cells including or expressing the engineered polypeptides and / or nucleic acids encoding them are also provided, as are pharmaceutical compositions including the polypeptides, nucleic acids, and / or cells and a pharmaceutically acceptable buffer, carrier, diluent or excipient.

[0024] Methods of treating a subject having a disease, disorder, or condition are also provided and typically include administering to the subject an effective amount of the pharmaceutical composition.

[0025] The disease, disorder, or condition can be one associated with pathological aggregation and / or mis-localization of TDP-43 aggregates in the brain or central nervous system (CNS). The subject can a human. Exemplary disease include, but are not limited to, Amyotrophic Lateral Sclerosis (ALS), Frontotemporal disorders (FTD), and a genetic disorder.

[0026] The pharmaceutical composition can be administered to the subject via a route selected from the group including intravenous, intramuscular, intracranial, intraosseus, intranasal, intrathecal, intraventricular, intraparenchymal and intracerebroventricular administration. In particular forms, administration to the subject is via intracerebroventricular injection or intrathecal injection.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, explain the principles of the disclosed method and compositions.

[0029] Figure 1A is a schematic representation of TDP-43 ’s amyloidogenic core region, including residues at positions 311-360 having the polypeptide sequence: MNF GAF S INPAMMAAAQAALQS SWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:1), and a corresponding ribbon diagram. In the lower panel is a schematic depicting the residues at positions 321-330 as “Helix-1”, including the amino acid sequence AMMAAAQAAL (SEQ ID NO:2), the variant region including residues at positions 331-334, amino acid sequence QSSW (SEQ ID NO:3) which are involved in misfolding and aggregation of TDP-43, and residues at positions 335-343 as “Helix- 2”, including the amino acid sequence GMMGMLASQ (SEQ ID NO:4). Figure IB is a flowchart of the step-by-step approach used to develop peptides with enhanced helical propensity and analysis methods to assess peptide inhibitor stability and ability to resist beta-strand conversion.

[0030] Figures 2A-2B depict comparative analysis of wild type (Wt) and helical propensity variant peptide structures and intramolecular interaction energies, based on in silico modeling. Figure 2A shows 3D structure representations of Wt and top hit peptides showing enhanced helical propensity. The mutant resides within each of the helices and also total the number of intra-molecular hydrogen bonds in each helical structure are depicted, together within the helical region underscores the structural differences between Wt and the top helical propensity peptides. Figure 2B shows Intra-molecular interaction energy is plotted for Wt, and helical propensity peptides at each residue at positions 311-360. The higher (more negative) differential energy profiles for the top hit peptides offers insight into their stability and illustrates different energetic contributions at specific residues in each mutant, respectively.

[0031] Figures 3A-3B depict data obtained from Molecular Dynamics simulations, used to assess the conformational stability of helical propensity and wild-type peptides. Figure 3A is a graph showing the RMSD values calculated to measure stability and conformational changes of each of the mutant helical propensity peptides and the wild-type model over time (a 100ns simulation period). Figure 3B is a graph showing the relative stability of the helical propensity variants compared to wild-type peptide. The total number of hydrogen bonds formed within the helical domain of each peptide was monitored throughout the 100ns simulation period. The spline plot illustrates the variation in the hydrogen bonds for the helical propensity variants and the wild-type model, providing insights into the strength and stability of the intra-molecular interactions.

[0032] Figures 4A-4B are groups of heatmaps of Predicted Pairwise Interactions of residues 311-360 across Molecular Dynamics Snapshots for each of wild-type (Figure 4A) and S332A / S333W peptide (Figure 4B), respectively. The gradient indicates variation in intramolecular interaction energies (kj / mol). In each panel, the large, dotted boxes highlight inter-residue interactions within the helical domain; the small dotted boxes highlight interactions between helical domain residues and residues in the flanking region (outside of the helical domain).

[0033] Figure 5 is a graph of the analysis performed for wild-type (S332, S333) model and peptide variants with mutated residues S332A / S33W and S333L, showing cumulative contribution of intramolecular interaction energies between residues at 332 and 333 position and neighboring residues (A329, L330, Q331, W334, G335, and M336) observed over the course of 100ns MD. Plotted is Kj over time for each of the WT (■), S332A, S333W (•) and S333L ( A) mutants, respectively.

[0034] Figures 6A-6H are graphs of predicted residue-wise and overall helical or beta-sheet probability for the wild-type peptide and the S332A / S333W helical propensity peptide in two different states. Figures 6A-6D show data obtained from peptide monomer state; Figures 6E- 6H show data obtained from peptide complex state (peptide bound to TDP-43 amyloid- like fibrils). Figures 6A, 6E show residue-wise predicted helical probability and Figures 6A, 6E show beta sheet probability in the monomer and complex state for the wild-type and S332A / S333W peptides. Figures 6B, 6F show overall helical probability for monomer state (Figure 6B), and the complex state (Figure 6F); two-tailed t-test, ****p < 0.0001. Figures 6D, 6H show overall beta sheet probability for monomer state (Figure 6D) and the complex state (Figure 6H); two-tailed t-test, ****p < 0.0001. These figures collectively provide a comprehensive analysis of the helical and beta sheet probabilities for the wild-type and helical propensity peptides in both the monomer and complex states. The data are presented as means with standard error of the mean, from n=2 technical replicates, respectively.

[0035] Figures 7A-7C depict the development and application of in vitro assays to test the efficacy of mutant peptides. Figure 7A is a schematic depicting the systems employed to develop a cellular aggregation assay to test TDP-43 CTD Helical Propensity Peptide. Figure 7B is a schematic depicting the systems employed to develop a cellular aggregation assay to test TDP-43 CTD Helical Propensity Peptide in the presence of the designed mutant stabilizing peptides (S332A / S333W); Figure 7C a graph quantifying the number of aggregates identified according to the assays in Figures 7A and 7B, showing No. of Aggregates observed in samples of GFP + mSC CTD wt, GFP CTD Wt + mSC CTD wt, and in the presence of designed mutant stabilizing peptides (S332A / S333W). Figure 7C is a graph of cytoplasmic aggregation of TDP- 43 amyloidogenic C-terminal domain (CTD) for wild-type and each of the described helical propensity variants, showing TDP 43 aggregates / cell for each of Control (TDP-43 CTD WT); GFP-TDP-43 CTD WT+mScarlet-TDP-43 CTD WT; GFP-helical CTD S332A / S333W+mScarlet- TDP-43 CTD WT; and GFP-helical CTD S332R / S333L+mScarlet- TDP-43 CTD WT, respectively (ANOVA, **p<0.01 and ****p<0.0001). Figures 8A-8B are graphs showing mean fluorescence intensity of endogenous nuclear TDP-43 for HeLa cell groups including each of un-transfected (Control 1); GFP only (Control 2); TDP-43 CTD WT; and TDP-43 CTD S332A / S333W, respectively (Figure 8A; ANOVA, **p-value <0.01); and showing Mean fluorescence intensity of Cy5 peptide, quantified in the nucleus and cytoplasm of cells receiving the Cy5 S332A / S333W peptide, respectively (Figure 8B; two-tailed t-test, p-value <0.0001).

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] The disclosed compositions and methods can be understood more readily by reference to the following detailed description of embodiments and the Examples included therein and to the Figures and their previous and following description.

[0038] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0039] Throughout this specification the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0040] I. Definitions

[0041] “Introduce” in the context of genome modification refers to bringing in to contact. For example, to introduce a gene editing composition to a cell is to provide contact between the cell and the composition. The term encompasses penetration of the contacted composition to the interior of the cell by any suitable means, e.g., via transfection, electroporation, transduction, gene gun, nanoparticle delivery, etc.

[0042] As used herein, “homologous” means derived from a common ancestor. For example, a homologous trait is any characteristic of organisms that is inherited by two or more species from a common ancestor species. Homologous sequences can be orthologous or paralogous. Homologous sequences are orthologous if they were separated by a speciation event: when a species diverges into two separate species, the divergent copies of a single gene in the resulting species are said to be orthologous. Orthologs, or orthologous genes, are genes in different species that are similar to each other because they originated from a common ancestor. Homologous sequences are paralogous if they were separated by a gene duplication event: if a gene in an organism is duplicated to occupy two different positions in the same genome, then the two copies are paralogous. “Heterologous” means having a different relation, relative position, or structure. Thus, unless otherwise specified, heterologous includes joining or linking of two or more amino acid or nucleic acid sequences from that organism (e.g., species) that are not normally found joined or linked (e.g., together) as well as joining or linking of two or more amino acid or nucleic acid sequences from different species.

[0043] “Endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0044] “Exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0045] “Encoding” or “encode” refers to the property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0046] As used herein, the term “locus” is the specific physical location of a DNA sequence (e.g., of a gene) on a chromosome. It is understood that a locus of interest can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e., in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non- limiting examples.

[0047] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non- native environment such as, for example, a host cell. An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes: a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and / or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha- anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methyl phosphonate, and the like.

[0048] In the context of cells, the term “isolated” also refers to a cell altered or removed from its natural state. That is, the cell is in an environment different from that in which the cell naturally occurs, e.g., separated from its natural milieu such as by concentrating to a concentration at which it is not found in nature. “Isolated cell” is meant to include cells that are within samples that are substantially enriched for the cell of interest and / or in which the cell of interest is partially or substantially purified.

[0049] As used herein, “transformed,” “transduced,” and “transfected” encompass the introduction of a nucleic acid or other material into a cell by one of a number of techniques known in the art.

[0050] A “vector” is a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Examples of vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” encompasses an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, and the like.

[0051] As used herein, “subject” includes, but is not limited to, animals, plants, parasites and any other organism or entity. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans). The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some forms, the subject can be any organism in which the disclosed method can be used to genetically modify the organism or cells of the organism.

[0052] The term “inhibit” or other forms of the word such as “inhibiting” or “inhibition” means to decrease, hinder or restrain a particular characteristic such as an activity, response, condition, disease, or other biological parameter. It is understood that this is typically in relation to some standard or expected value, i.e., it is relative, but that it is not always necessary for the standard or relative value to be referred to. “Inhibits” can also mean to hinder or restrain the synthesis, expression or function of a protein relative to a standard or control. Inhibition can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. “Inhibits” can also include, for example, a 10% reduction in the activity, response, condition, disease, or other biological parameter as compared to the native or control level. Thus, the reduction can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

[0053] 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

[0054] 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,

[0055] 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of reduction in between as compared to native or control levels. For example, “inhibits expression” means hindering, interfering with or restraining the expression and / or activity of the gene / gene product pathway relative to a standard or a control.

[0056] “Treatment” or “treating” means to administer a composition to a subject or a system with an undesired condition (e.g., cancer). The condition can include one or more symptoms of a disease, pathological state, or disorder. Treatment includes medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological state, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological state, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological state, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological state, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount. “Prevention” or “preventing” means to administer a composition to a subject or a system at risk for an undesired condition (e.g., cancer). The condition can include one or more symptoms of a disease, pathological state, or disorder. The condition can also be a predisposition to the disease, pathological state, or disorder. The effect of the administration of the composition to the subject can be the cessation of a particular symptom of a condition, a reduction or prevention of the symptoms of a condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or reduction of the chances that a particular event or characteristic will occur.

[0057] As used herein, the terms “effective amount” or “therapeutically effective amount” means a quantity sufficient to alleviate or ameliorate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and / or physiological effect. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise quantity will vary according to a variety of factors such as subjectdependent variables (e.g., age, immune system health, weight, etc.), the disease or disorder being treated, as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.

[0058] By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0059] As used herein, the term “polypeptides” includes proteins and functional fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V). As used herein, the term “functional fragment” or “functional variant” means a fragment or variant of a polypeptide, such as a full-length or native polypeptide, that retains one or more functional properties of the full-length or native polypeptide. For example, in some embodiments, a functional fragment or functional variant of the disclosed engineered peptide inhibitors of TDP-43 aggregates is a fragment or variant that retains the function of binding to a TDP-43 aggregate in vivo.

[0060] As used herein, the terms “variant” or “active variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains one or more functional properties (e.g., functional or biological activity). A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may he a variant that is not known to occur naturally. Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological or functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties (e.g., functional or biological activity).

[0061] Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’ s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

[0062] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (- 1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0063] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0064] Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

[0065] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Vai), (Leu: He, Vai), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Vai: He, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.

[0066] As used herein, “conservative” amino acid substitutions are substitutions wherein the substituted amino acid has similar structural or chemical properties.

[0067] As used herein, “non-conservative” amino acid substitutions are those in which the charge, hydrophobicity, or bulk of the substituted amino acid is significantly altered.

[0068] As used herein, the term “identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can also mean the degree of sequence relatedness of a polypeptide compared to the full-length of a reference polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, W. G., Eds., Humana Press, New lersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988).

[0069] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides of the present disclosure.

[0070] By way of example, a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide.

[0071] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0072] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0073] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0074] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0075] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 2% ; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0076] IL Compositions

[0077] Compositions of peptide inhibitors of TDP-43 aggregates (eTDP-43) are provided. The eTDP-43 include engineered helical polypeptides that bind to TDP-43 ’s amyloidogenic core but resist P-sheet conversion. Tn some forms, the eTDP-43 include engineered helical polypeptides fused with one or more functional polypeptide domains. Exemplary functional domains include protein degradation motifs (PDM). In some forms, a PDM serves to recruit proteasome cellular machinery to degrade TDP-43 aggregates.

[0078] Recombinant constructs including nucleic acids expressing or encoding the polypeptides and fusion proteins thereof are also provided. Viral genomes including the recombinant constructs, recombinant viruses including the constructs, and vaccine formulations formed thereof are also provided. Compositions of eTDP-43, including delivery vehicles, targeting motifs and pharmaceutical formulations including pharmaceutically acceptable excipients are also described.

[0079] A. Engineered peptide inhibitors of TDP-43 aggregates (eTDP-43)

[0080] Engineered peptide inhibitors of TDP-43 aggregates (eTDP-43) have been developed. A panel of polypeptides of approximately 50 residues in length that exhibit enhanced a-helical secondary structure and bind to TDP-43 ’s amyloidogenic core but resist P-sheet conversion is described.

[0081] As demonstrated in the Examples, it has been shown that the peptides effectively cap and arrest the growth of fibrils within a cell, and co-expression of engineered peptides with TDP- 43 ’s aggregation -prone CTD reduced aggregate formation in mammalian cells and biochemical assays. Both the ubiquitin proteasome system and autophago-lysosomal pathway have been implicated in clearance and degradation of soluble and aggregated forms of TDP-43. Therefore, in some forms, a protein degradation motif (PDM) is also added to the engineered helical peptides. In some forms, modification by addition of one or more PDM further enhances the efficacy of the engineered helical peptides, by enabling a clearance mechanism.

[0082] 1. Wild-Type TAR DNA-binding protein 43 (Wt TDP-43)

[0083] TAR DNA-binding protein 43 (TDP-43) is a highly conserved nuclear RNA / DNA- binding protein involved in the regulation of RNA processing. TDP-43 is a ubiquitous protein encoded by the TARDBP gene and belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. In normal cells, TDP-43 is mainly present in the nucleus and plays important roles in RNA regulation, such as transcriptional regulation, alternative splicing, and mRNA stabilization.

[0084] Human “wild-type” TDP-43 includes an N-terminal domain (NTD; residues 1-103), two RNA recognition motifs (RRM1 and RRM2; residues 104-200 and residues 191-262), and a C- terminal domain (CTD; residues 274-413). The NTD region contains a ubiquitin-like fold with one a-helix and six P-sheets and promotes TDP-43 self-oligomerization in a concentrationdependent manner. The C-terminus of TDP-43 is important for solubility and cellular localization of the TDP-43 protein and regulates protein-protein interactions.

[0085] An exemplary amino acid sequence of the TDP-43 ’ s amyloidogenic core is depicted in Figure 1A, including residues at positions 311-360 of the mature protein, including the sequence: MNFGAFS INPAMMAAAQAALQSSWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:1), which is the starting point (i.e., reference or wildtype) for the variants provided herein. Amino acid residues at positions 321-330 are referred to herein as “Helix-1”, including the amino acid sequence AMMAAAQAAL (SEQ ID NO:2). Amino acid residues at positions 331-334 having the amino acid sequence QSSW (SEQ ID NO:3), are involved in misfolding and aggregation of TDP- 43 and are referred to herein as the “variant region”, and residues at positions 335-343 including the amino acid sequence GMMGMLASQ (SEQ ID NO:4) are referred to herein as “Helix-2”. i Misfolded TAR DNA-binding protein 43 (mTDP-43)

[0086] The accumulation of TDP-43 aggregates in the central nervous system is a common feature of many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease (AD), and limbic predominant age-related TDP-43 encephalopathy (LATE).

[0087] TDP-43 fibrillar aggregation is facilitated by destabilization of an a-helical segment within the amyloidogenic core of TDP-43 C-terminal domain (CTD). The TDP-43 protein includes an a-helical region including two helical segments. These residues can, in pathophysiological conditions, interact with neighboring residues and give rise to structural changes that initiate and drive the early stages of aggregation. Polar and aromatic residues (QSSW (SEQ ID NO:3)) that divide the a-helical domain into two helical segments play a key role in this process. The presence of mutations or aberrant posttranslational modification in the CTD leads to the formation of irreversible aggregation via liquid-solid phase separation. Many mutations in the TDP-43 gene have been found to be associated with ALS and FTLD, and most disease-associated mutations are located within the C-terminal domain. In particular, in several models, these mutations in the C-terminal domain of TDP-43 can promote the intrinsic aggregation of TDP-43. The expression of TDP-43 mutations, including Q331K, M337V, Q343R, N345K, R361S, and N390D, leads to increased aggregation and cell toxicity in yeast cells, and other disease-associated mutations, such as G294A, Q331K, M337V, Q343R, N390D, and N390S, enhance protein aggregation when expressed in SH-SY5Y cells. S332 and W334 participate in helix-destabilizing interactions with G357 and N358 residues, a-helical- destabilizing intramolecular contacts between S332, S333 and other residues in the amyloidogenic core contribute to protein misfolding and structural transformation into amyloid- like fragments.

[0088] 2. Engineered Helical TDP-43 peptides

[0089] Engineered helical TDP-43 polypeptides are provided.

[0090] Generally, the engineered helical TDP-43 polypeptides include a sequence of contiguous amino acid residues that corresponds to all or part of the natural amino acid sequence of the “wild-type” human TDP-43 polypeptide (Wt TDP-43), modified to vary the identity (e.g., size, charge, hydrophobicity, etc.) of at least one amino acid at one or more positions within the wildtype sequence that stabilize the structural conformation of the a-helical regions. For example, in some forms, the engineered helical TDP-43 polypeptide includes a substitution of at least one amino acid as compared with the amino acid sequence of the Wt TDP-43 or mTDP-43 that increases the binding efficiency and structural stability as compared with the amino acid sequence of the Wt TDP-43 or mTDP-43 peptides complexed with TDP-43 amyloid-like fibrils.

[0091] Typically, the engineered helical TDP-43 polypeptides include some or all of residues deemed potentially necessary and / or sufficient for TDP-43 aggregate formation in vivo. In some forms, the engineered helical TDP-43 polypeptides include some or all of residues at positions 311-360 of wt TDP-43. In some forms, the engineered helical TDP-43 polypeptides include substitutions of one or more residues that divide the a-helical domain of wt TDP-43 into two helical segments. In some forms, the engineered helical TDP-43 polypeptides include substitutions of one or more residues that play a key role in the conversion of intramolecular helix-helix contacts of wt TDP-43 to helix-beta sheet contacts. In some forms, the engineered helical TDP-43 polypeptides include substitutions of one or more residues that potentially contribute to a lack of helix stabilizing intramolecular contacts between S332, S333 and neighboring residues within the adjacent helical segments. In some forms, the engineered helical TDP-43 polypeptides include substitutions of one or more residues that potentially contribute to protein misfolding and structural transformation into amyloid-like fragments. For example in some forms, the engineered helical TDP-43 polypeptides include substitutions of one or more residues at positions 331-334 (QSSW (SEQ ID NO:3)) of wt TDP-43.

[0092] The engineered helical TDP-43 polypeptides typically include a sequence of about 20 to about 80, or about 30 to about 80, or 30 to about 70, or about 40 to about 70, or about 40 to about 60 contiguous amino acid residues, inclusive. For example, in some forms, the Engineered helical TDP-43 polypeptides include a sequence of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70, contiguous amino acid residues, with or without additional domain(s), e.g., PTD and / or PDM appended thereto. In exemplary forms, the engineered TDP-43 is about 40-60, optional 50 amino acids, before or without additional domain(s), e.g., PTD and / or PDM appended thereto. The engineered helical peptides effectively cap and arrest formation of TDP- 43 aggregates to specifically target pathological TDP-43 aggregates without affecting physiological TDP-43 functions, including splicing, axonal transport, mRNA stability, and localization of mRNA transcripts. In some forms, the engineered helical TDP-43 polypeptides include one or more residues that bind to TDP-43 amyloid-like filaments but resist beta sheet conversion. i. Exemplary engineered helical TDP-43 peptides

[0093] Exemplary engineered helical TDP-43 polypeptides are provided. In some forms an engineered helical TDP-43 polypeptide has the amino acid sequence:

[0094] MNFGAFS INPAMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQ (S332A / S333W;

[0095] SEQ ID NO:5; substituted residue(s) depicted in bold font);

[0096] MNFGAFS INP MMAAAQAALQRLWGMMGMLASQQNQSGP SGNNQNQGNMQ (S332R / S333L;

[0097] SEQ ID NO:6; substituted residue(s) depicted in bold font);

[0098] MNFGAFS INP AMMAAAQAALQASWGMMGMLASQQNQSGP SGNNQNQGNMQ (S332A; SEQ ID

[0099] NO:7; substituted residue(s) depicted in bold font);

[0100] MNFGAFS INP MMAAAQAALQRSWGMMGMLASQQNQSGP SGNNQNQGNMQ (S332R; SEQ ID

[0101] NO: 8; substituted residue(s) depicted in bold font); MNF GAF S INP AMMAAAQAALQSLWGMMGMLASQQNQSGP SGNNQNQGNMQ (S333L; SEQ ID

[0102] NO:9; substituted residue(s) depicted in bold font);

[0103] MNF GAF S INPAMMAAAQAALQWWGMMGMLASQQNQSGP SGNNQNQGNMQ (S332V / S333V;

[0104] SEQ ID NO: 10; substituted residue(s) depicted in bold font); and

[0105] MNF GAF S 1NPAMMAAAQAALQVSWGMMGMLASQQNQSGP SGNNQNQGNMQ (S332V; SEQ ID

[0106] NO:11; substituted residue(s) depicted in bold font).

[0107] In some forms, the engineered helical TDP-43 polypeptides include a sequence having one more additional modifications (including substitutions, additions or deletion of one or more residues within or immediately adjacent to any one of the SEQ ID NOs:5-l l. Typically, when the engineered helical TDP-43 polypeptides include a sequence having one more additional modifications (including substitutions, additions or deletion of one or more residues within or immediately adjacent to any one of the SEQ ID NOs:5-ll, the engineered helical TDP-43 polypeptides maintain the residues within the variant region of SEQ ID NOs:5-l 1 (i.e. , residues at positions 331-334). For example, in some forms, the engineered helical TDP-43 polypeptides include a sequence of any one of the SEQ ID NOs:5-l 1, and having one or more modifications within the Helix-1 region (i.e., at any one or more of positions 321-330); or in some forms, the engineered helical TDP-43 polypeptides include a sequence of any one of the SEQ ID NOs:5-l 1, and having one or more modifications within the Helix-2 (i.e., at any one or more of positions 335-343), or both. In some forms, an engineered helical TDP-43 polypeptides has at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to any one of SEQ ID NOs:5-ll.

[0108] In some forms, the engineered helical TDP-43 polypeptides include one or more additional residues at either end of the polypeptide of any one of SEQ ID NOs:5-l 1. In some forms, the engineered helical TDP-43 polypeptides include one or more of the residues of wtTDP-43 immediately contiguous with the N and / or C terminus of any one of SEQ ID NOs:5- 11. For example, in some forms the engineered helical TDP-43 polypeptides of any one of SEQ ID NOs:5-ll include one or more additional residues at the N terminus, including any of the residues corresponding to positions 300-311 of wt TDP-43. In some forms the engineered helical TDP-43 polypeptides of any one of SEQ ID NOs:5-l l include one or more additional residues at the C terminus, including any of the residues corresponding to positions 360-370 of wt TDP-43. In some forms the engineered helical TDP-43 polypeptides of any one of SEQ ID NOs:5-l l include one or more additional residues at the N terminus, including any of the residues corresponding to positions 300-311 of wt TDP-43 and one or more additional residues at the C terminus, including any of the residues corresponding to positions 360-370 of wt TDP-43. It has been established that the helical domain residues (320-340) are associated with docking of the engineered helical propensity peptides which participate in beta sheet formation in vivo. Therefore, the variant peptides typically include at least 20 residues corresponding to the helical domain residues (320-340), however truncated variants of any one of SEQ ID NOs:5-l 1 having between 20-50 residues are also contemplated. For example, in some forms, the engineered helical TDP-43 polypeptides lack one or more residues at the N terminus of any one of SEQ ID NOs:5-l 1. In other forms, the engineered helical TDP-43 polypeptides lack one or more residues at the C terminus of any one of SEQ ID NOs:5-l 1. In some forms, the engineered helical TDP-43 polypeptides lack one or more residues at the C terminus and one or more residues of the of the C terminus of any one of SEQ ID NOs:5-ll.

[0109] Other exemplary variants are provided in Table 4. In some embodiments, the engineered helical TDP-43 polypeptides includes a non-wildtype Sequence (320-340 Residues) of Table 4. ii. Variants of S332A / S333W

[0110] In some forms, the engineered helical TDP-43 polypeptides are variants of the S332A / S333W peptide.

[0111] In some forms, the engineered helical TDP-43 polypeptides is between about 20 amino acids and about 50 amino acids, inclusive of SEQ ID NO:5, or any subrange thereof, or any specific integer number of amino acids therebetween, including, but not limited to 20, 25, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. Variants can have, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:5, or a functional fragment thereof.

[0112] In some forms, a variant engineered S332A / S333W helical TDP-43 polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO 5. Preferably variants maintain the ability to interact with an amyloid aggregate in vivo. In some forms, a variant engineered S332A / S333W helical TDP-43 polypeptide is considered to be “functional” if it maintains the ability to interact with an amyloid aggregate in vivo, i.e., maintains the residues at positions 20-40 of SEQ ID NO:5. In some forms, variant engineered S332A / S333W helical TDP-43 polypeptide variants are identified as functional if they bind an amyloid aggregate, reduce or arrest the aggregation process, and / or promote clearance, optionally, but preferably in vivo,. Exemplary variants of an engineered S332A / S333W helical TDP-43 polypeptide has the amino acid sequence: NFGAFS 1 NPAMMAAAQAALQAWWGMMGMLASQQNQSGP S GNNQNQGNMQ (SEQ ID NO:12); FGAF S INPAMMAAAQAALQAWWGMMGMLASQQNQS GP SGNNQNQGNMQ (SEQ ID NO:13); GAF S INPAMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:14); AFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO: 15); FS INPAMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:16); SINPAMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:17);

[0113] INP AMMAAAQAALQAWWGMMGMLASQQNQSGPSGNNQNQGNMQ (SEQ ID NO:18);

[0114] MNFGAFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNM (SEQ ID NO:19); MNFGAFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGN (SEQ ID NO:20); MNFGAFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQG (SEQ ID NO:21); MNFGAFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQ (SEQ ID NO:22);

[0115] MNFGAFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQN (SEQ ID NO:23); and MNFGAFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQ (SEQ ID NO:53);

[0116] B. Functional Peptide Motifs

[0117] Engineered peptide inhibitors of TDP-43 aggregates including one or more functional peptide motifs are also provided.

[0118] In some forms, the engineered helical TDP-43 polypeptides are fused to one or more functional peptide motifs designed to perform one or more biological functions at the site of action in vivo.

[0119] In some forms, when an engineered peptide inhibitor of TDP-43 aggregates includes one or more functional peptide motifs, the one or more functional peptide motifs are fused to the amino (N) or carboxyl (C) terminus of the engineered helical TDP-43 polypeptide. Exemplary schematics for the domain structure of a fusion protein include:

[0120] N-[functional peptide motif(s)]- [engineered helical TDP-43 polypeptide]-C; or

[0121] N-[ engineered helical TDP-43 polypeptide]- [functional peptide motifs(s)]-C; or

[0122] N-[functional peptide motifs(s)]-[engineered helical TDP-43 polypeptide] -[functional peptide motifs(s)]-C, where “N” and “C” refer to the amino (NH2) and carboxyl (COOH) termini, respectively.

[0123] In some forms, the functional peptide motif induces, enhances or increases degradation of the peptide(s) with which it is associated.

[0124] An exemplary functional peptide motif includes a peptide degradation motif (PDM).

[0125] 1. Peptide Degradation Motif (PDM)

[0126] In some forms, an engineered peptide inhibitor of TDP-43 aggregates includes an engineered helical TDP-43 polypeptide and an amino acid sequence of a peptide degradation motif (PDM). In some forms, addition of a PDM can recruit the proteasome cellular machinery to degrade the small to medium of TDP-43 aggregates. In other forms, addition of a PDM can target larger TDP-43 aggregates for chaperone-mediated autophago-lysosomal degradation. Typically, a PDM directs degradation of pathological amyloid-like filaments, for example, following binding of the engineered helical TDP-43 polypeptide to an amyloid aggregate. In some forms, a PDM does not only inhibit the aggregation of pathological TDP-43 but also promotes the clearance of aggregates by engaging the proteasome / autophagy pathways.

[0127] Typically, the addition of a PDM to an engineered helical TDP-43 polypeptide enhances efficacy of the engineered helical peptides to remove or reduce amyloid aggregates or otherwise prevent amyloid deposition by adding a clearance mechanism via recruitment of the proteasome or autophago-lysosomal pathways.

[0128] In some forms, a PDM is a Hypoxia-inducible Factorlapha motif. It may be that a hypoxia-inducible Factorlapha motif targets peptides and aggregates associated with these motifs for proteasomal degradation by the VHL E3 ligase Complex. An exemplary hypoxiainducible Factorlapha motif includes the amino acid sequence ALAPYIP (SEQ ID NO:24).

[0129] Therefore, in some forms, the PDM includes the amino acid sequence ALAPY IP (SEQ ID NO:24). In some forms, addition of a PDM (e.g., one of the foregoing SEQ ID Nos) will recruit the proteasome cellular machinery to degrade small to medium sized TDP-43 aggregates.

[0130] In some forms, a PDM is a chaperone-mediated autophagolysosome motif. It may be that a chaperone-mediated autophagolysosome motif functions as a chaperone mediated autophagy targeting motif. An exemplary chaperone-mediated autophagolysosome motif includes the amino acid sequence KFERQKI LDQRFFE (SEQ ID NO:25). Therefore, in some forms, the PDM includes the amino acid sequence KFERQKI LDQRFFE (SEQ ID NO:25). In some forms, a PDM according to SEQ ID NO:25 will target larger TDP-43 aggregates for chaperone-mediated auto-phago-lysosomal degradation.

[0131] Other exemplary PDM include, but are not limited to, LDP ETGEYL (SEQ ID NO:54), RRRG (SEQ ID NO:55), and MDF SGLS LI KLKKQ (SEQ ID NO:56).

[0132] Therefore, in some forms, the PDM includes the amino acid sequence LDPE TGEYL (SEQ ID NO:54), RRRG (SEQ ID NO:55), and MDF SGLSL IKLKKQ (SEQ ID NO:56).

[0133] 2. Cell Penetrating Peptide (CPP) Motif

[0134] In some forms, an engineered peptide inhibitor of TDP-43 aggregates includes an engineered helical TDP-43 polypeptide and an amino acid sequence of a cell penetrating peptide (CPP) motif. It is envisioned that the described engineered peptide inhibitor of TDP-43 aggregates are effective to arrest, prevent or reverse amyloid deposition within one or more areas of the central nervous system and / or brain of a subject. Therefore, in some forms, the described engineered peptide inhibitor of TDP-43 aggregates include one or more moieties that directs or targets the peptides to the CNS or brain of a subject in vivo. It may be that the presence of a CPP motif fused to an engineered helical TDP-43 polypeptide enhances, facilitates or increases the passage of the engineered peptide inhibitor of TDP-43 aggregates across the blood-brain-barrier (BBB). Exemplary CCP peptide sequences that shuttle an associated cargo molecule through the BBB include: ApoE peptide, having an amino acid sequence of LRKLRKRLL (SEQ ID NO:35); ApoB peptide, having an amino acid sequence of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO:36); hApoE peptide, having an amino acid sequence of LRKLRKRLLR (SEQ ID NO:37); RVG-29 peptide, having an amino acid sequence of YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO:38); TAT peptide, having an amino acid sequence of GGGGYGRKKRRQRRR (SEQ ID NO:39); PepH3 peptide, having an amino acid sequence of AGILKRW (SEQ ID NO:40); Apamin peptide, having an amino acid sequence of H-CNCKAPETALCARRCQQH-NH2 (SEQ ID NO:41); MiniAp-4 peptide, having an amino acid sequence of H-DapKAPETALD-NH2 (SEQ ID NO:42); THRre peptide, having an amino acid sequence of PWVP SWMPPRHT (SEQ ID NO:43); TGN peptide, having an amino acid sequence of TGNYKALHPHNG (SEQ ID NO:44); THR peptide, having an amino acid sequence of THRPPMWSPVWP (SEQ ID NO:45); THRre_2f peptide, having an amino acid sequence of (PWVP SWMPPRHT )2KKGK ( CF ) G (SEQ ID NO:46); and KI6AP0E, having an amino acid sequence of HAYED (SEQ ID NO:47). In other forms, the CCP includes a peptide sequence CNSRLHLRC (SEQ ID NO:48); or CENWWGDVC (SEQ ID NO:49); or WRCVLREGPAGGCAWFNRHL (SEQ ID NQ:50). Therefore, in some forms, an engineered peptide inhibitor of TDP-43 aggregates includes one or more CPP motif having an amino acid sequence of any one of SEQ ID NOS:35-50.

[0135] In other forms, the cell-penetrating and / or homing component is a protein or polypeptide that functions to bind or activate an extracellular molecule, such as a receptor or component of an intracellular transport system or pathway. An exemplary protein or polypeptide is an immunoglobulin Fc molecule which facilitates receptor- mediated transcytosis across the BBB. An exemplary Fc molecule includes a modified Fc domain that binds human transferrin receptor (huTfR), a protein highly expressed at the BBB, for enhanced CNS biodistribution. Therefore, in some forms, the described engineered peptide inhibitor of TDP-43 aggregates includes an engineered helical TDP-43 polypeptide fused to an engineered Fc molecule that binds to huTfR and enhances BBB passage / CNS penetrance.

[0136] 3. Other Protein Motifs

[0137] Any of the disclosed engineered peptide inhibitors of TDP-43 aggregates can include one or more additional domains. For example, any of the disclosed engineered peptide inhibitors of TDP-43 aggregates can include one or more linkers or spacers. The term “linker” as used herein includes, without limitation, peptide linkers. The peptide linker can be any size provided it does not interfere with the function of the engineered peptide inhibitors of TDP-43 aggregates.

[0138] In some forms, the linker includes one or more glycine and / or serine amino acid residues. In some forms, the linker includes a glycine-glutamic acid di-amino acid sequence. For example, a linker can include 4-8 amino acids. In a particular embodiment, a linker includes the amino acid sequence GQSSRSS (SEQ ID NO:28). In another embodiment, a linker includes 15- 20 amino acids, for example 18 amino acids. Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO:29), Ala-Ser, Gly-Gly-Gly- Ser (SEQ ID NO:30), GGGGSGGGGS (SEQ ID NO:31) and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:32), (Gly-Gly-Gly-Ser) (SEQ ID NO:33) and GGGGSGGGGSGGGGS (SEQ ID NO:34).

[0139] The linkers can be used to link or connect two domains, regions, or sequences of an engineered peptide inhibitors of TDP-43 aggregates. Molecular biology techniques have developed so that therapeutic proteins can be genetically engineered to be expressed by microorganisms. The gram negative bacterium, Escherichia coli, is a versatile and valuable organism for the expression of therapeutic proteins. Although many proteins with therapeutic or commercial uses can be produced by recombinant organisms, the yield and quality of the expressed protein are variable due to many factors. For example, heterologous protein expression by genetically engineered organisms can be affected by the size and source of the protein to be expressed, the presence of an affinity tag linked to the protein to be expressed, codon biasing, the strain of the microorganism, the culture conditions of microorganism, and the in vivo degradation of the expressed protein. Some of these problems can be mitigated by fusing the protein of interest to an expression or solubility enhancing amino acid sequence. Exemplary expression or solubility enhancing amino acid sequences include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and a small ubiquitin-related modifier (SUMO).

[0140] In some embodiments, the engineered peptide inhibitors of TDP-43 aggregates disclosed herein include expression or solubility enhancing amino acid sequence(s). In some embodiments, the expression or solubility enhancing amino acid sequence is cleaved prior to administration of the composition to a subject in need thereof. The expression or solubility enhancing amino acid sequence can be cleaved in the recombinant expression system, or after the expressed protein is purified.

[0141] 4. Exemplary Engineered peptide inhibitors of TDP-43 aggregates

[0142] Exemplary engineered peptide inhibitors of TDP-43 aggregates including a PDM are provided. In some forms, the engineered peptide inhibitors of TDP-43 aggregates includes an amino acid sequence of any one of SEQ ID NOs:l-23 or 53 contiguous with an amino acid sequence of any one or more of SEQ ID NOs:24-52 and 54-56. For example, in some forms, an engineered peptide inhibitor of TDP-43 aggregates includes an amino acid sequence of: MNFGAFS INPAMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQALAPYIP (SEQ ID NO:26). In other forms, an engineered peptide inhibitor of TDP-43 aggregates includes an amino acid sequence of:

[0143] MNFGAFS INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQKFERQKI LDQRFFE (SEQ ID NO:27).

[0144] C. Nucleic Acids

[0145] Nucleic acids and vectors encoding or expressing the disclosed engineered peptide inhibitors of TDP-43 aggregates are also described.

[0146] 1. Isolated Nucleic Acid Molecules of engineered peptide inhibitors of TDP-43 aggregates

[0147] Isolated nucleic acid sequences encoding the engineered peptide inhibitors of TDP-43 aggregates are disclosed. In some embodiments, the isolated nucleic acid sequences encode an engineered peptide inhibitors of TDP-43 aggregates including an amino acid sequence of any one of SEQ ID NOs:l-23 or 53. In some forms, the isolated nucleic acid sequences encode an engineered peptide inhibitors of TDP-43 aggregate including an amino acid sequence of any one or more of SEQ ID NOs:24-52 and 54-56.

[0148] The term “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome. An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid. Nucleic acids can be in sense or antisense orientation or can be complementary to a reference sequence encoding a disclosed engineered peptide inhibitor of TDP-43 aggregates. Thus, nucleic acids encoding SEQ ID NOS: 1-23, 53, 26, or 27 optionally further including a nucleic acid sequence encoding one or more of SEQ ID NOS:24, 25, 28-52, and 54-56, or fragments and variants thereof, in sense and antisense, and in single stranded and double stranded forms, are provided.

[0149] The nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2’-deoxycytidine or 5 -bromo-2’ -deoxycytidine for deoxy cytidine. Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-0-methyl or 2’-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

[0150] 2. Vectors Expressing or Encoding engineered peptide inhibitors of TDP-43 aggregates

[0151] In some embodiments, nucleic acids encoding the disclosed engineered peptide inhibitors of TDP-43 aggregates are present within vectors. In some embodiments, the vectors encode or express a disclosed engineered peptide inhibitor of TDP-43 aggregates including one or more PDM motifs. In some forms, a vector encodes or expresses the disclosed engineered peptide inhibitor of TDP-43 aggregates set forth by any one or more of SEQ ID NOS: 1-23, 53, 26, or 27 optionally further including one or more of SEQ ID NOS:24, 25, 28-52, and 54-56, or fragments or variants thereof.

[0152] Vectors including an isolated polynucleotide encoding a nucleic acid encoding SEQ ID NOS:l-23, 53, 26, or 27 optionally further including one or more of SEQ ID NOS:24, 25, 28-52, and 54-56, or fragments and variants thereof for the expression of an engineered peptide inhibitor of TDP-43 aggregates within a host cell are described.

[0153] The term “vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and / or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). A vector can be a DNA vector or an RNA vector. In some embodiments, a vector is a DNA plasmid. One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.

[0154] In some forms, the vector including nucleic acids encoding an engineered peptide inhibitor of TDP-43 aggregates is an expression vector. The term “expression vector’’ refers to any type of genetic construct including a nucleic acid coding for an RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.

[0155] In some embodiments, vectors contain one or more regulatory sequences. The term “regulatory sequence” refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and / or transport of the nucleic acid or one of its derivative (i.e. mRNA) into the host cell or organism. In the context of the disclosure, this term encompasses promoters, enhancers and other expression control elements e.g., polyadenylation signals and elements that affect mRNA stability).

[0156] In some embodiments, the vector is a non-viral vector. Examples of non- viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Examples of non-viral vectors include, but are not limited to, RNA replicon, mRNA replicon, modified mRNA replicon or self-amplifying mRNA, closed linear deoxyribonucleic acid, e.g., a linear covalently closed DNA, e.g., a linear covalently closed double stranded DNA molecule. Preferably, a non-viral vector is a DNA plasmid. A “DNA plasmid”, which is used interchangeably with “DNA plasmid vector,” “plasmid DNA” or “plasmid DNA vector,” refers to a double- stranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell. DNA plasmids used for expression of an encoded polynucleotide typically include an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene. Examples of suitable DNA plasmids that can be used include, but are not limited to, commercially available expression vectors for use in well-known expression systems (including both prokaryotic and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif.), which can be used for production and / or expression of protein in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used for production and / or expression in Saccharomyces cerevisiae strains of yeast; MAXBAC®. complete baculovirus expression system (Thermo Fisher Scientific), which can be used for production and / or expression in insect cells; pcDNA™. or pcDNA3™ (Life Technologies, Thermo Fisher Scientific), which can be used for high level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of a protein of interest in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified to optimize protein expression in the host cell, such as to reverse the orientation of certain elements (e.g., origin of replication and / or antibiotic resistance cassette), replace a promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and / or replace the polynucleotide sequence encoding transcribed proteins (e.g., the coding sequence of the antibiotic resistance gene), by using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).

[0157] In some forms, a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNA™, pcDNA3™, pVAX, pVAX-1, ADVAX, NTC8454, etc. In some embodiments, an expression vector is based on pVAX-1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is a commonly used plasmid in DNA vaccines, and contains a strong human immediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH)-derived polyadenylation sequence (pA). pVAX-1 further contains a pUC origin of replication and a kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.

[0158] In some embodiments the vector is a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi-segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which include an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding a PC1-CTT polypeptide or another therapeutic polypeptide as described herein), arenavirus such as lymphocytic chori omeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain, etc.

[0159] In some embodiments, the viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or S AdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53. In some embodiments, a recombinant viral vector is prepared using methods known in the art in view of the present disclosure. For example, in view of the degeneracy of the genetic code, several nucleic acid sequences can be designed that encode the same polypeptide. In some embodiments, a polynucleotide encoding an engineered peptide inhibitor of TDP-43 aggregates is codon- optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon- optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure.

[0160] In some embodiments, the vectors, e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), include any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the engineered peptide inhibitor of TDP-43 aggregates encoded by the polynucleotide sequence of the vector.

[0161] 3. Regulatory Elements

[0162] In some embodiments, the disclosed nucleic acids, including RNAs and DNAs such as DNA vectors expressing or encoding an engineered peptide inhibitor of TDP-43 aggregates include one or more regulatory elements.

[0163] Regulatory elements include, but are not limited to, a promoter, an enhancer, a polyadenylation signal, translation stop codon, a ribosome binding element, a transcription terminator, selection markers, origin of replication, etc. An isolated nucleic acid can be, and a vector can include, one or more expression cassettes. An “expression cassette” is part of a nucleic acid such as a vector that directs the cellular machinery to make RNA and protein. An expression cassette typically includes three components: a promoter sequence, an open reading frame, and a 3 '-untranslated region (UTR) optionally including a poly adenylation signal. An open reading frame (ORF) is a reading frame that contains a coding sequence of the disclosed engineered peptide inhibitors of TDP-43 aggregates from a start codon to a stop codon. Regulatory elements of the expression cassette can be operably linked to a polynucleotide sequence encoding a PC1-CTT polypeptide or other therapeutic polypeptide.

[0164] As used herein, the term “operably linked” is to be taken in its broadest reasonable context, and refers to a linkage of polynucleotide (or polypeptide, etc.) elements in a functional relationship. A polynucleotide is “operably linked” when it is placed into a functional relationship with another polynucleotide. For instance, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Any components suitable for use in an expression cassette described herein can be used in any combination and in any order to prepare vectors of the application. i. Promotors

[0165] The disclosed nucleic acids, including vectors, can include a promoter sequence, preferably within an expression cassette, to control expression of an engineered peptide inhibitor of TDP-43 aggregates. The term “promoter” is used in its conventional sense and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. A promoter is located on the same strand near the nucleotide sequence it transcribes. Promoters can be a constitutive, inducible, or repressible. Promoters can be naturally occurring or synthetic. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be employed is a DNA plasmid, the promoter can be endogenous to the plasmid (homologous) or derived from other sources (heterologous). Preferably, the promoter is located upstream of the polynucleotide encoding an engineered peptide inhibitor of TDP-43 aggregates within an expression cassette.

[0166] Examples of promoters that can be used include, but are not limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. A promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein.

[0167] A promoter can also be a tissue specific promoter, such as a kidney specific promoter, preferably a kidney epithelial cell promoter, which can be natural or synthetic. Examples include, but are not limited to, the CDH 16 promoter, which is mostly kidney specific (it is also expressed in the thyroid) (Igarashi, et al., Am J Physiol., 277(4):F599-610 (1999). doi: 10.1152 / ajprenal.l999.277.4.F599. PMID: 10516285.); the Pax-8 promoter, which is also expressed primarily in the kidney as well as in the thyroid (Dehbi, et al., EMBO J., 15( 16):4297- 306 (1996) PMID: 8861958); the aquaporin 2 promoter, which drives expression specifically in principal cells of the renal collecting duct (which are the target of Tolvaptan) (Stricklett, et al., Exp Nephrol., 7(l):67-74 (1999). doi: 10.1159 / 000020587. PMID: 9892817.), and kidney tubule-specific promoters in association with gene delivery viral vectors (Watanabe, et al., PloS one, vol. 12,3 eO168638 (2017), doi: 10.1371 / joumal.pone.0168638).

[0168] In some embodiments, the promoter is a strong eukaryotic promoter, such as cytomegalovirus immediate early (CMV-IE) promoter. ii. Other Expression Control Elements

[0169] In some embodiments, the nucleic acids, including vectors, include additional polynucleotide sequences that stabilize the expressed transcript, enhance nuclear export of the RNA transcript, and / or improve transcriptional-translational coupling. Examples of such sequences include polyadenylation signals and enhancer sequences. A poly adenylation signal is typically located downstream of the coding sequence for an engineered peptide inhibitor of TDP- 43 aggregates within an expression cassette of the vector. Enhancer sequences are regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene. An enhancer sequence is preferably located upstream of the polynucleotide sequence encoding an engineered peptide inhibitor of TDP-43 aggregates, but downstream of a promoter sequence within an expression cassette of the vector.

[0170] Any polyadenylation signal known to those skilled in the art in view of the present disclosure can be used. For example, the polyadenylation signal can be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human beta-globin polyadenylation signal. Preferably, a polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or a SV40 polyadenylation signal.

[0171] Any enhancer sequence known to those skilled in the art in view of the present disclosure can be used. For example, an enhancer sequence can be a human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV. Examples of particular enhancers include, but are not limited to, Woodchuck HBV Post- transcriptional regulatory element (WPRE), intron / exon sequence derived from human apolipoprotein Al precursor (ApoAI), untranslated R-U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR), a splicing enhancer, a synthetic rabbit betaglobin intron, or any combination thereof. Preferably, an enhancer sequence is a composite sequence of three consecutive elements of the untranslated R-U5 domain of HTLV-1 LTR, rabbit beta-globin intron, and a splicing enhancer, which is referred to herein as “a triple enhancer sequence.”

[0172] A vector can include a polynucleotide sequence encoding a signal peptide sequence. Preferably, the polynucleotide sequence encoding the signal peptide sequence is located upstream of the polynucleotide sequence encoding an engineered peptide inhibitor of TDP-43 aggregates. Signal peptides typically direct localization of a protein, facilitate secretion of the protein from the cell in which it is produced, and / or improve expression the therapeutic polypeptide when expressed from the vector, but is cleaved off by signal peptidase, e.g., upon secretion from the cell. An expressed protein in which a signal peptide has been cleaved is often referred to as the “mature protein.” Any signal peptide known in the art in view of the present disclosure can be used. For example, a signal peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion signal, such as the Ig heavy chain gamma signal peptide SPIgG or the Ig heavy chain epsilon signal peptide SPIgE.

[0173] A vector, such as a DNA plasmid, can also include a bacterial origin of replication and an antibiotic resistance expression cassette for selection and maintenance of the plasmid in bacterial cells, e.g., E. coli. Bacterial origins of replication and antibiotic resistance cassettes can be located in a vector in the same orientation as the expression cassette encoding an engineered peptide inhibitor of TDP-43 aggregates, or in the opposite (reverse) orientation. An origin of replication (ORI) is a sequence at which replication is initiated, enabling a plasmid to reproduce and survive within cells. Examples of ORIs suitable for use in the application include, but are not limited to ColEl, pMBl, pUC, pSClOl, R6K, and 15A, preferably pUC.

[0174] Expression cassettes for selection and maintenance in bacterial cells typically include a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to an antibiotic resistance gene differs from the promoter sequence operably linked to a polynucleotide sequence encoding a protein of interest, e.g. ,an engineered peptide inhibitor of TDP-43 aggregates. The antibiotic resistance gene can be codon optimized, and the sequence composition of the antibiotic resistance gene is normally adjusted to bacterial, e.g., E. coli, codon usage. Any antibiotic resistance gene known to those skilled in the art in view of the present disclosure can be used, including, but not limited to, kanamycin resistance gene (Kanr), ampicillin resistance gene (Amr), and tetracycline resistance gene (Tetr), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.

[0175] An expression vector can include a tag sequence, such as those discussed above.

[0176] SUBSTITUTE SHEET (RULE 26) D. Delivery Vehicles

[0177] Any of the disclosed compositions including, but not limited to an engineered peptide inhibitor of TDP-43 aggregates and / or nucleic acids encoding an engineered peptide inhibitor of TDP-43 aggregates, can be delivered to target cells using a delivery vehicle.

[0178] The delivery vehicles can be, for example, polymeric particles, inorganic particles, silica particles, liposomes, micelles, multilamellar vesicles, etc.

[0179] Delivery vehicles may be microparticles or nanoparticles. Nanoparticles are often utilized for inter-tissue application, penetration of cells, and certain routes of administration. The nanoparticles may have any desired size for the intended use. The nanoparticles may have any diameter from 10 nm up to about 1,000 nm. The nanoparticle can have a diameter from 10 nm to 900 nm, from 10 nm to 800 nm, from 10 nm to 700 nm, from 10 nm to 600 nm, from 10 nm to 500 nm, from 20 nm from 500 nm, from 30 nm to 500 nm, from 40 nm to 500 nm, from 50 nm to 500 nm, from 50 nm to 400 nm, from 50 nm to 350 nm, from 50 nm to 300 nm, or from 50 nm to 200 nm. In some embodiments the nanoparticles can have a diameter less than 400 nm, less than 300 nm, or less than 200 nm. The range can be between 50 nm and 300 nm.

[0180] Thus, in some embodiments, the delivery vehicles are nanoscale compositions, for example, 10 nm up to, but not including, about 1 micron. However, it will be appreciated that in some embodiments, and for some uses, the particles can be smaller, or larger (e.g., microparticles, etc.). Although many of the compositions disclosed herein are referred to as nanoparticle or nanocarrier compositions, it will be appreciated that in some embodiments and for some uses the carrier can be somewhat larger than nanoparticles. Such compositions can be referred to as microparticulate compositions. For example, a nanocarriers according to the present disclosure may be a microparticle. Microparticles can a diameter between, for example, 0.1 and 100 pm in size.

[0181] 1. Viral Capsid Proteins

[0182] In some forms, the delivery vehicle is a viral capsid, or a virus-like particle formed from partly or entirely of a multiplicity of viral capsid proteins. Generally, virus capsids are stable toward thermal denaturation at temperatures up to 80-100°C, chaotropic agents, and to extremes of pH. Exemplary viral-like particles that are stable toward thermal denaturation at temperatures up to 80-100°C, chaotropic agents, and to extremes of pH include bacteriophage capsids and phage particles.

[0183] In some forms, the delivery vehicle includes a viral-like particle (VLP), or vesicle, composed of a bacteriophage capsid protein.

[0184] The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. In some forms, the icosahedral capsid of a bacteriophage is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes, providing enhanced stability to VLPs formed from capsid proteins. In some forms, the capsid is a modified capsid, for example, modified by attachment of a peptide, carbohydrate, small molecule or nucleic acid to the viral capsid.

[0185] 2. Polymeric Particles

[0186] In some forms, the delivery vehicle is or includes one or more polymers, such as polymeric nanoparticles or microparticles. Exemplary polymers include biocompatible polymers. In some forms, the biocompatible polymer(s) is biodegradable or bioabsorbable. In other forms, the polymer is non-degradable. In some forms, the particles are a mixture of degradable and non- degradable particles.

[0187] In some forms, the delivery vehicle is a particles that includes one or more biocompatible polymer(s) including, but not limited to, polyamino acids; cyclodextrin-containing polymers, in particular cationic cyclodextrin-containing polymers, such as those described in U.S. Patent No. 6,509,323; polymers prepared from lactones such as poly (caprolactone) (PCL); polyhydroxy acids and copolymers thereof such as poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co- glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and blends thereof, polyalkyl cyanoacralate, polyurethanes, poly(valeric acid), and poly-L-glutamic acid; hydroxypropyl methacrylate (HPMA); poly anhydrides; other polyesters; poly orthoesters; poly(ester amides); polyamides; poly(ester ethers); polycarbonates; polyalkylenes such as polyethylene and polypropylene; polyalkylene glycols such as poly(ethylene glycol) (PEG) and polyalkylene oxides (PEO), and block copolymers thereof such as polyoxyalkylene oxide (“PLURONICS®” or block copolymers containing PEG where PEG has a molecular weight of any values within the range of 300 Daltons to 1 MDa); polyalkylene terephthalates such as poly (ethylene terephthalate); ethylene vinyl acetate polymer (EVA); polyvinyl alcohols (PVA); polyvinyl ethers; polyvinyl esters such as poly( vinyl acetate); polyvinyl halides such as poly( vinyl chloride) (PVC), polyvinylpyrrolidone; poly siloxanes; polystyrene (PS); and celluloses including alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, and carboxymethylcellulose; polymers of acrylic acids including poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"); polydioxanone and its copolymers; polyhydroxyalkanoates; polypropylene fumarate; polyoxymethylene; poloxamers; poly(butyric acid); trimethylene carbonate; and polyphosphazenes.

[0188] Examples of preferred natural polymers include proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate. Copolymers of the above, such as random, block, or graft copolymers, or blends of the polymers listed above can also be used.

[0189] Functional groups on the polymer can be capped to alter the properties of the polymer and / or modify (e.g., decrease or increase) the reactivity of the functional group. For example, the carboxyl termini of carboxylic acid contain polymers, such as lactide- and glycolide-containing polymers, may optionally be capped, e.g., by esterification, and the hydroxyl termini may optionally be capped, e.g. by etherification or esterification.

[0190] The weight average molecular weight can vary for a given polymer but is generally from about 1000 Daltons to 1,000,000 Daltons, 1000 Daltons to 500,000 Dalton, 1000 Daltons to 250,000 Daltons, 1000 Daltons to 100,000 Daltons, 5,000 Daltons to 100,000 Daltons, 5,000 Daltons to 75,000 Daltons, 5,000 Daltons to 50,000 Daltons, or 5,000 Daltons to 25,000 Daltons.

[0191] In some forms, the delivery vehicles are particles modified with one or more surfactants. Examples of surfactants include, but are not limited to, L-a-phosphatidylcholine (PC), 1 ,2- dipalmitoylphosphatidy choline (DPPC), oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cotton seed oil, and sunflower seed oil, lecithin, oleic acid, and sorbitan trioleate.

[0192] In some forms where polyalkylene glycol (e.g., PEG) is used in a composition of polymers to modify the particles, PEG surface density may be controlled by varying the amount of PEG in the polymer composition or by mixing a blend of pegylated polymer component and non-pegylated polymer component. The density of PEG or polyalkylene glycol on the surface of formed particles may be evaluated using several techniques.

[0193] In some forms, the delivery vehicles are modified by the addition of one or more polymers to possess a specific ^-potential. For example, in some forms, the delivery vehicles are modified by the attachment of PEG and / or other polymers to the surface to possess a ^-potential of between about 20 mV and about -20 mV, preferably between about 10 mV and about -10 mV, more preferably between about 2 mV and about -2 mV.

[0194] 3. Liposomes, Micelles and Lipidic Particles

[0195] In some forms, the particles are lipidic particles, such as liposomes, or micelles. Lipidic particles include unilamellar phospholipid vesicles, liposomes, or lipoprotein particles. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes, Chapter 10, 1979.

[0196] Formulations of liposomes and methods of making such formulations are well known to one of ordinary skill in the art. Liposomes are formed from commercially available phospholipids supplied by a variety of vendors including Avanti Polar Lipids, Inc. (Birmingham, Ala.).

[0197] Suitable methods, materials and lipids for making liposomes are known in the art. Liposome delivery vehicles are commercially available from multiple sources. The liposome may be formed from a single lipid; however, in some embodiments, the liposome is formed from a combination of more than one lipid. The lipids can be neutral, anionic or cationic at physiologic pH. In some forms, the liposomes incorporate PEG, or PEGylated lipid derivatives. Incorporation of one or more PEGylated lipid derivatives can result in a liposome which displays polyethylene glycol chains on its surface. The resulting liposomes may possess increased stability and circulation time in vivo as compared to liposomes lacking PEG chains on their surfaces. Liposomes are formed from one or more lipids, which can be neutral, anionic, or cationic at physiologic pH. Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including, but limited to, 1 ,2-diacyL glycero-3-phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2- dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and / or synthetic e.g., saturated and unsaturated l,2-diacyl-5 / i-glycero-3-phosphocholines, l-acyl-2-acyl-sn-glycero-3- phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids. In some forms, the liposomes contain a phosphaditylcholine (PC) head group, and preferably sphingomyelin. In another form, the liposomes contain DPPC. In a further form, the liposomes contain a neutral lipid, preferably 1 ,2-dioleoylphosphatidylcholine (DOPC).

[0198] In certain forms, the liposomes are generated from a single type of phospholipid. In such forms, preferably the phospholipid has a phosphaditylcholine head group, and, most preferably is sphingomyelin. The liposomes may include a sphingomyelin metabolite. Sphingomyelin metabolites used to formulate the liposomes include, without limitation, ceramide, sphingosine, or sphingosine 1 -phosphate. The concentration of the sphingomyelin metabolites included in the lipids used to formulate the liposomes can range from about 0.1 mol % to about 10 mol %. Preferably from about 2.0 mol % to about 5.0 mol %, and more preferably can be in a concentration of about 1.0 mol %.

[0199] Suitable cationic lipids in the liposomes include, but are not limited to, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, also references as TAP lipids, for example methylsulfate salt. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids in the liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3-trimethylammonium propanes, N-[l-(2,3- dioloyloxy)propyl]-N,N-dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2- dialkyloxy-3 -dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 -[N- (N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2- (sperminecarboxamido)-ethyl)-N,N-dimethyl-l-propanaminium trifluoro- acetate (DOSPA), - alanyl cholesterol, cetyl trimethyl ammonium bromide (CTAB), diCi 4- amidine, N-ferf-butyl-N'- tetradecyl-3-tetradecylamino-propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D- glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N , N , N' , N'- tetramethyl- , N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-l ,4-butanediammonium iodide. In one form, the cationic lipids can be l-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)- imidazolinium chloride derivatives, for example, l-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)- heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and l-[2- (hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM). In one embodiment, the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl- hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl- hydroxybutyl ammonium bromide (DORTE-HB), 1 ,2-dioleyloxypropyl-3-dimethyl- hydroxypentyl ammonium bromide (DORIE- Hpe), 1 ,2-dimyristyloxypropyl-3 -dimethylhydroxylethyl ammonium bromide (DMRIE), 1 ,2-dipalmityloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DPRIE), and 1 ,2-disteryloxypropyl-3-dimethyl- hydroxy ethyl ammonium bromide (DSRIE).

[0200] The lipids may be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH. Non-ionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleolylgly ceryl phosphatidylethanolamine), with cholesterol being most preferred. The molar ratio of a first phospholipid, such as sphingomyelin, to second lipid can range from about 5 : 1 to about 1 : 1 or 3:1 to about 1: 1, more preferably from about 1.5:1 to about 1:1, and most preferably, the molar ratio is about 1:1.

[0201] 4. Targeting Moieties and Compositions

[0202] Any of the disclosed compositions including a delivery vehicle, such as a nanoparticle, encapsulating an engineered peptide inhibitor of TDP-43 aggregates or a nucleic acid encoding an engineered peptide inhibitor of TDP-43 aggregates can include a targeting moiety.

[0203] It is envisioned that the described engineered peptide inhibitor of TDP-43 aggregates are effective to arrest, prevent or reverse amyloid deposition within one or more areas of the central nervous system and / or brain of a subject. Therefore, in some forms, the described compositions of engineered peptide inhibitor of TDP-43 aggregates include one or more moieties that directs or targets the compositions to the CNS or brain of a subject in vivo. For example, in some forms, the compositions include one or more moieties conjugated to, complexed with or otherwise associated with the engineered peptide inhibitor of TDP-43 aggregates that facilitate or enhance passage of the compositions across the blood-brain barrier. For example, in some forms, the targeting moiety is a peptide, a lipid, a polymer, a small molecule, a carbohydrate, or combinations thereof. i. Peptide Targeting Moieties

[0204] In some forms, the targeting moiety is a peptide, such as a cell-penetrating peptide (CPP). CPPs are described, for example, in Ghorai, el al., Pharmaceutics 2023, 15(7), 1999, the contents of which are incorporated herein in their entirety. CPPs are known in the art and typically include a 15-25 long amino-acid sequence of amphipathic molecules rich in positively charged amino acids, primarily arginine. Arginine is preferred over lysine owing to the extra H- bond of the guanidium group. Naturally, all characteristic features of CPPs are primarily aimed at improving internalization into the cells. The Pep- and MPG families of small peptides are instances of such amphipathic cellpenetrating molecules that can form conjugates with proteins and nucleic acids, respectively, and can aid in obtaining the desired results. CPPs are designed to successfully deliver macromolecules into the cytosol; thus, they are used as delivery systems rather than therapeutic agents. CPPs may be transported directly across the cellular membrane or by entrapment as peptides / cargo within the endosomes. Endocytic pathways usually involve one of the energy-dependent mechanisms such as phagocytosis, caveolae-mediated endocytosis (CvME), clathrin-mediated endocytosis (CME), or cholesterol-dependent endocytosis.

[0205] In exemplary forms, CPPS are conjugated directly to a delivery vehicle, such as a nanoparticle, or they may be conjugated via one or more spacers or linkers.

[0206] Exemplary CCP peptide sequences active in shuttling an associated cargo molecule through the BBB include: ApoE peptide, having an amino acid sequence of LRKLRKRLL (SEQ ID NO:35); ApoB peptide, having an amino acid sequence of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO:36); hApoE peptide, having an amino acid sequence of LRKLRKRLLR (SEQ ID NO:37); RVG-29 peptide, having an amino acid sequence of YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO:38); TAT peptide, having an amino acid sequence of GGGGYGRKKRRQRRR (SEQ ID NO:39); PepH3 peptide, having an amino acid sequence of AGILKRW (SEQ ID NO:40); Apamin peptide, having an amino acid sequence of H-CNCKAPETALCARRCQQH-NH2 (SEQ ID NO:41); MiniAp-4 peptide, having an amino acid sequence of H-DapKAPETALD-NH2 (SEQ ID NO:42); THRre peptide, having an amino acid sequence of PWVP SWMPPRHT (SEQ ID NO:43); TGN peptide, having an amino acid sequence of TGNYKALHPHNG (SEQ ID NO:44); THR peptide, having an amino acid sequence of THRPPMWSPV P (SEQ ID NO:45); THRre_2f peptide, having an amino acid sequence of (PWVP SWMPPRHT ) 2KKGK ( GF ) G (SEQ ID NO:46); and KI6AP0E, having an amino acid sequence of HAYED (SEQ ID NO:47). In other forms, the CCP includes a peptide sequence CNSRLHLRC (SEQ ID NO:48); or CENWWGDVC (SEQ ID NO:49); or WRCVLREGPAGGCAWFNRHL (SEQ ID NO: 50). Therefore, in some forms, a delivery vehicle includes or is conjugated to one or more CPP having an amino acid sequence of any one of SEQ ID NOS:35-50. ii. Formulations for Targeting to the Brain / CNS

[0207] In some forms, compositions including or encoding the engineered peptide inhibitor of TDP-43 aggregates are formulated with or mixed within a specific excipient or carrier that is designed to enhance delivery to the brain and / or CNS. Systems and compositions for targeting of molecules to the brain are described, for example in Sousa, Pharmaceutics. 2022 Sep; 14(9): 1835.

[0208] In some forms, nucleic acids encoding an engineered peptide inhibitor of TDP-43 aggregates are formulated into a hyaluronidase-coated glycol chitosan-DNA polyplexes. In other forms, compositions including or encoding the engineered peptide inhibitor of TDP-43 aggregates are encapsulated within a nanostructured lipid carrier (NLC) with thymoquinone (TQ) oil. In other forms, compositions including or encoding the engineered peptide inhibitor of TDP-43 aggregates are formulated within a pomegranate seed oil (PSO) phospholipid oil gel. In some forms, the compositions including or encoding the engineered peptide inhibitor of TDP-43 aggregates are loaded into hollow-gold nanoparticles tethered to liposomes (HGN-liposomes) for intravenous administration and subsequent release of the compositions from the nanoparticles by laser or ultrasound stimulation.

[0209] E. Pharmaceutical Compositions

[0210] Pharmaceutical compositions containing an engineered peptide inhibitor of TDP-43 aggregates, or nucleic acids encoding an engineered peptide inhibitor of TDP-43 aggregates, or a delivery vehicle, such as a nanoparticle, encapsulating an engineered peptide inhibitor of TDP- 43 aggregates or a nucleic acid encoding an engineered peptide inhibitor of TDP-43 aggregates, are also described.

[0211] In some embodiments, the pharmaceutical compositions include one or more of a pharmaceutically acceptable buffer, carrier, diluent or excipients.

[0212] The term “pharmaceutically acceptable carrier” describes a pharmaceutically acceptable material, or composition, that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, in some forms the carrier is a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0213] In some embodiments, pharmaceutical compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical compositions can be formulated for delivery via any route of administration. The term “route of administration” can refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and / or injections. The pharmaceutical compositions are preferably formulated for intravenous administration.

[0214] Typically, the disclosed pharmaceutical compositions are administered in a manner appropriate to a disease to be treated (or prevented). The quantity and frequency of administration is typically determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0215] The disclosed pharmaceutical compositions can be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavail ability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0216] III. Methods

[0217] Methods of using the disclosed compositions including engineered peptide inhibitors of TDP-43 aggregates are also provided. Typically, the methods include administering a therapeutic composition including or encoding the described engineered peptide inhibitors of TDP-43 aggregates to a subject in need thereof in an amount effective to arrest, reduce or prevent aggregation of TDP-43 aggregates in the subject.

[0218] The therapeutic compositions administered according to the methods typically include a pharmaceutically acceptable formulation including an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates, and / or nucleic acids encoding engineered peptide inhibitors of TDP-43 aggregates, and / or a delivery vehicle, such as a nanoparticle, encapsulating an engineered peptide inhibitor of TDP-43 aggregates or a nucleic acid(s) encoding an engineered peptide inhibitor of TDP-43 aggregates. Therefore, methods of treating or preventing a disease or disorder associated with mislocalization and / or aggregation of TDP-43 in the brain and / or CNS of a subject are provided. In some forms, the methods treat or prevent a disease or disorder such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) in a subject. In some forms, the methods deliver an amount of the therapeutic compositions in an amount and / or for a time effective to treat or prevent one or more symptoms of a disease or disorder associated with mis-localization and / or aggregation of TDP-43 in the brain and / or CNS of a subject.

[0219] Typically, the methods include one or more steps of administering the described therapeutic composition including or encoding the described engineered peptide inhibitors of TDP-43 aggregates to a subject. The administering typically delivers the therapeutic compositions across to the brain and / or CNS of a subject in vivo. For example, in some forms, the methods administer the therapeutic compositions directly into the brain and / or CNS. For example, in some forms, the methods deliver the therapeutic compositions locally to the site of action. In other forms, the methods deliver the therapeutic compositions indirectly into the body, for example, via systemic administration. Therefore, in some, forms, the methods administer the therapeutic compositions via a route such as intravenous (iv) administration, such that the therapeutic compositions pass across the BBB to access the CNS and / or brain of the subject from the circulation.

[0220] Any of the described methods can include one or more steps of identifying and selecting a subject, such as a subject in need of therapeutic intervention according to the described methods. For example in some forms the methods select a subject who has been identified as having or who is at risk of having mis-localization and / or aggregation of TDP-43 in the brain and / or CNS. In some forms, the methods select a subject who has been diagnosed as having or who is suspected as having a disease or disorder such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).

[0221] Controls are also described. Any of the methods can include one or more steps of comparing an effect or a subject or a data point with a control. Suitable controls include negative controls or positive controls, for example, having a known or determined characteristic, or lack thereof. In some forms a control is an untreated cell or subject.

[0222] A. Methods of Treatment

[0223] Methods of using the described engineered peptide inhibitors of TDP-43 aggregates, to treat a disease or disorder are provided. Typically the methods reduce TDP-43 aggregates in the CNS or brain of the subject, for example, halting or limiting the on-going building and / or new formation of aggregates, and / or by directing proteolytic degradation of the aggregates. Therefore, in some forms, the methods reduce or prevent one or more pathophysiological processes associated with TDP-43 aggregates in the CNS or brain of the subject.

[0224] For example, in some forms, the methods treat a subject having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including the described engineered peptide inhibitors of TDP-43 aggregates.

[0225] As set forth in the Examples below, therapeutic composition including or encoding the described engineered peptide inhibitors of TDP-43 aggregates substantially inhibit the mislocalization and / or aggregation of TDP-43 in cells without cellular toxicity or adverse effects. Therefore, the described therapeutic composition including or encoding the described engineered peptide inhibitors of TDP-43 aggregates can be applied to a broad range of therapy and can be used in combination with other therapeutic approaches.

[0226] Typically, the methods prevent mis-localization and / or aggregation of TDP-43 in cells, and / or reduce or halt the progression of mis-localization and / or aggregation of the same. Generally, the methods reduce, ablate or prevent one or more symptoms associated with mis- localization and / or aggregation of TDP-43 in cells. The described engineered peptide inhibitors of TDP-43 aggregates can cap and prevent aggregation or fibril -like TDP-43 aggregates in the brain or CNS. In some forms, the compositions additionally or alternatively direct the capped aggregates for proteolytic degradation. Thus, the methods can not only stop and prevent the development of disease and disorders resulting from mis-localization and / or aggregation of TDP-43, but in some forms the methods may also reverse the pathological effects associated with mis-localization and / or aggregation of TDP-43. For example, in some forms, the methods can reverse one or more symptoms associated with ALS or FTD in a subject having ALS or FTD. In some forms, when a subject has suffered a neurological deficit associated with a disease or disorder resulting from the mis-localization and / or aggregation of TDP-43, the methods can restore some or all of the neurological ability of the subject to a level similar to that prior to the onset of mis-localization and / or aggregation of TDP-43.

[0227] An exemplary method involves treating a subject (e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including the described engineered peptide inhibitors of TDP-43 aggregates. In some forms, the methods administer the described engineered peptide inhibitors of TDP-43 aggregates to a subject (e.g., a human) having a disease, disorder, or condition in an amount effective to treat the disease, disorder, or condition.

[0228] 1. Diseases to be Treated

[0229] Methods of treating diseases and / or disorders in a subject in need thereof are provided. The subject to be treated can have a disease, disorder, or condition such as but not limited to, amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), or combinations thereof. The disease, disorder, or condition can be associated with mis-localization and / or aggregation of TDP-43 in the brain and / or CNS.

[0230] In some forms the methods include administering a therapeutic composition including or encoding the described engineered peptide inhibitors of TDP-43 aggregates to treat one or more disease or disorder in a subject in need thereof. For example, in some forms the methods treat one or more genetic disease or disorders in a subject, such as a hereditary genetic disease or disorder, or a somatic genetic disease or disorder in a subject.

[0231] Any of the methods can include treating a subject having an underlying disease or disorder. For example, in some forms, the methods treat a disease or disorder, such as a amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), in a patient having another disease or disorder, such as diabetes, a bacterial infection (e.g., Tuberculosis), viral infection (e.g., Hepatitis, HIV, HPV infection, etc.), or a drug-associated disease or disorder. In some forms, the methods treat an immunocompromised subject. In some forms, the methods treat a subject having a disease of the kidney, liver, heart, lung, brain, bladder, reproductive system, bowel / intestines, stomach, bones or skin. i. Amyotrophic Lateral Sclerosis (ALS)

[0232] In some forms, the methods treat or prevent amyotrophic lateral sclerosis (ALS). Methods for ameliorating, preventing, reducing or limiting the progression of, or reversing one or more symptoms of ALS in a subject in need thereof include administering to the subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates.

[0233] ALS is a nervous system disease that affects nerve cells in the brain and spinal cord. ALS causes progressive loss of muscle control, leading to disability and death. The exact cause of the disease is still not known, however a small number of cases are inherited. In some forms, the methods treat or prevent one or more symptoms associated with ALS in a subject in need thereof. Symptoms of ALS include trouble coordinating the movement of muscles, such as walking; excessive tripping and falling; weakness and muscle fatigue, e.g., in the legs, feet or ankles; hand weakness or “clumsiness”; slurred speech; trouble swallowing; muscle cramps and twitching in the arms, shoulders and tongue; untimely crying, laughing or yawning; and thought or behavioral changes.

[0234] ALS develops as nerve cells die in and around the CNS, including in the brain, spinal cord, and neuromuscular junctions. Symptoms often start in the hands, feet, arms or legs, then spread to other parts of the body, as muscles get weaker as more nerve cells die. This atrophy eventually affects chewing, swallowing, speaking and breathing. In some forms, a subject with ALS does not experience ALS-related pain; loss of bladder control, or loss of the ability to taste, smell, touch and / or hear. In some forms, the methods include administering to a subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates to ameliorate, prevent or reverse one or more symptoms of ALS in the subject. ii. Frontotemporal Disorders

[0235] In some forms, the methods treat or prevent Frontotemporal Disorders. Frontotemporal Disorders is a broad term used to encompass a range of recognized cognitive and / or neurological diseases, including Frontotemporal Dementia (FTD); behavioral variant Frontotemporal Dementia (bvFTD); Primary Progressive Aphasia (PPA) including Semantic PPA, Agrammatic PPA and Logopenic PPA; FTD with motor neuron disease (FTD-ALS), and in rare cases movement disorders such as Corticobasal syndrome.

[0236] FTD results from damage to neurons in the frontal and temporal lobes of the brain, resulting in symptoms including unusual behaviors, emotional problems, trouble communicating, difficulty with work, or difficulty with walking. FTD is tends to occur at a younger age than other forms of dementia and approximately 60% of people with FTD are 45 to 64 years old.

[0237] Individuals with family history of Frontotemporal Disorders are more likely to develop such a disorder. Frontotemporal Disorders that runs in a family is often related to variants in certain genes, including the Tau / MAPT gene; the GRN gene; and the C9ORF72 gene. Approximately 10 to 30% of bvFTD is due to specific genetic causes. Approximately 50% of bvFTD patients have TDP-43 pathology.

[0238] Methods for ameliorating, preventing, reducing or limiting the progression of, or reversing one or more symptoms of FTD in a subject in need thereof include administering to the subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates. Therefore, in some forms, the methods ameliorate, prevent, reducing or limiting the progression of, or reverse one or more symptoms including unusual behavior, emotional problems, trouble communicating, difficulty with work, or difficulty with walking in a subject with FTD.

[0239] In some forms, the methods treat or prevent Behavioral variant FTD (bvFTD) in a subject in need thereof. bvFTD involves changes in personality, behavior, and judgment. People with this disorder may have problems with cognition, but their memory may stay relatively intact. Symptoms of bvFTD include problems planning and sequencing (thinking through which steps come first, second, and so on), difficulty prioritizing tasks or activities, repeating the same activity or saying the same word, acting impulsively or saying or doing inappropriate things without considering how others perceive the behavior. In some forms, the methods include administering to a subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates to ameliorate, prevent, reducing or limiting the progression of, or reverse one or more symptoms of bvFTD in the subject.

[0240] In some forms, the methods treat or prevent Primary Progressive Aphasia (PPA) in a subject in need thereof. PPA involves changes in the ability to communicate, e.g., to use language to speak, read, write, and understand what others are saying. This includes difficulty using or understanding words (aphasia) and difficulty speaking properly e.g., slurred speech). Subjects with PPA may have one or both of these symptoms and in some forms, may become mute or unable to speak. In addition, subjects with PPA may develop other symptoms of dementia, including problems with memory, reasoning, and judgment. In some forms, the secondary symptoms of dementia are not apparent at first but develop over time. In some forms, subjects with PPA experience significant behavioral changes, similar to those seen in bvFTD, as the disease progresses. There are three types of PPA, categorized by the kind of language problems that appear first, including Semantic PPA, Agrammatic PPA and Logopenic PPA.

[0241] In some forms, the methods treat or prevent Semantic PPA in a subject in need thereof. Symptoms of semantic PPA include slow loss of the ability to understand single words and / or to recognize faces of familiar people and / or common objects. In some forms, the methods include administering to a subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates to ameliorate, prevent, reducing or limiting the progression of, or reverse one or more symptoms of semantic PPA in the subject.

[0242] In some forms, the methods treat or prevent Agrammatic PPA in a subject in need thereof. Symptoms of Agrammatic PPA include difficulty speaking, including omitting words that link nouns and verbs (such as to, from, the); in some forms, a subject with Agrammatic PPA is unable to speak sensibly and in some forms the subject will develop movement symptoms similar to those seen in corticobasal syndrome. In some forms, the methods include administering to a subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates to ameliorate, prevent, reducing or limiting the progression of, or reverse one or more symptoms of agrammatic PPA in the subject.

[0243] In some forms, the methods treat or prevent Logopenic PPA in a subject in need thereof. Symptoms of Logopenic PPA include trouble finding the right words during a conversation. In some forms, the subject can understand words and sentences and does not have problems with understanding grammar. In some forms, the methods include administering to a subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates to ameliorate, prevent, reducing or limiting the progression of, or reverse one or more symptoms of logopenic PPA in the subject. In some forms, the methods treat or prevent a movement disorder associated with Frontotemporal Disorders in a subject in need thereof. Exemplary movement disorders associated with frontotemporal disorders include corticobasal syndrome and progressive supranuclear palsy. Movement disorders typically occur when the parts of the brain that control movement are affected, and in some forms these disorders also affect thinking and language abilities in a subject.

[0244] In some forms, the methods treat or prevent Corticobasal syndrome in a subject in need thereof. Symptoms of Corticobasal syndrome include corticobasal degeneration, i.e., gradual atrophy and loss of nerve cells in specific parts of the brain, causing progressive loss of the ability to control movement, including apraxia, (i.e., inability to use the hands or arms to perform a movement despite normal strength), muscle rigidity and difficulty swallowing. In some forms, symptoms appear first on one side of the body, and eventually both sides are affected. In some forms, a subject with corticobasal syndrome first has language problems or trouble orienting objects in space and later develops movement symptoms. In some forms, a subject having corticobasal syndrome has no problems with memory, cognition, language, or behavior. In some forms, the methods include administering to a subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates to ameliorate, prevent, reducing or limiting the progression of, or reverse one or more symptoms of Corticobasal syndrome in the subject.

[0245] In some forms, the methods treat or prevent FTD with motor neuron disease (FTD-ALS) in a subject in need thereof. FTD-ALS is a combination of bvFTD and ALS, which includes, in addition to the behavioral and / or language changes seen in bvFTD, development of a progressive muscle weakness similar to that seen in ALS, including fine jerks and / or wiggling in muscles. In some forms, symptoms of either ALS or FTD disease may appear first, with other symptoms developing over time. Changes in certain genes have been found in some people with FTD-ALS, though most cases are not hereditary. In some forms, the methods include administering to a subject an effective amount of the described engineered peptide inhibitors of TDP-43 aggregates to ameliorate, prevent, reducing or limiting the progression of, or reverse one or more symptoms of FTD-ALS in the subject. iii. Other Diseases associated with TDP-43 Pathology

[0246] In some forms, the methods treat or prevent one or more other diseases associated with TDP-43 pathology in a subject in need thereof. TDP-43 pathology has also been associated with Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE), Lewy body disease (LBD), Huntington’s disease, argyrophilic grain disease (AGD), hippocampal sclerosis, and limbic predominant age-related TDP-43 encephalopathy (LATE) (Nikaver and Urbanski, Front Aging Neurosci., 15: 1142617 (2023). Other disease include Inclusion body myositis (IBM), Multiple system atrophy (MSA), Corticobasal degeneration (CBD), Basophilic inclusion body disease (BIBD), Posterior cortical atrophy (PCA), cardiac diseases such as Cardiomyopathy and heart failure and metabolic disorders such as obesity and diabetes. a. Symptoms to be Treated

[0247] In some forms the methods administer therapeutic composition including or encoding the described engineered peptide inhibitors of TDP-43 aggregates to treat one or more symptoms of a disease or disorder in a subject.

[0248] In some forms, the described methods treat or prevent one or more symptoms of neurodegenerative disorders such as Alzheimer’s disease (AD), Chronic traumatic encephalopathy (CTE), Lewy body disease (LBD), Huntington’s disease, argyrophilic grain disease (AGD), and hippocampal sclerosis by targeting TDP-43 pathology include mitigating cognitive decline by preventing neuronal dysfunction and synaptic loss associated with TDP-43 aggregation in affected brain regions, preserving motor function by preventing neuronal loss and dysfunction in motor control regions, preserving neuronal integrity and reducing neuroinflammation in brain regions implicated in impulse control and reward processing. Other symptoms to be addressed include improving mood, behavior changes, and sleep-wake regulation.

[0249] Symptoms to be treated for limbic predominant age-related TDP-43 encephalopathy (LATE) can include preserving memory and language function by preventing neuronal loss and dysfunction in hippocampus, amygdala, and other brain regions.

[0250] Symptoms to be treated for Inclusion body myositis (IBM) can include improving muscle strength by preventing muscle fiber dysfunction and inflammation in motor neurons and muscle fibers affected by TDP-43 aggregation, mitigating muscle atrophy, and improving swallowing function by preserving muscle strength.

[0251] Symptoms to be treated for Corticobasal degeneration (CBD) can include improving motor function by preventing neuronal dysfunction, preserving language function by preventing neuronal loss and dysfunction in brain regions implicated in language processing, and improving sensory function by preserving neuronal integrity and reducing neuroinflammation in sensory processing regions.

[0252] Symptoms to be treated for Basophilic inclusion body disease (BIBD) can include preserving neuronal integrity and reducing neuroinflammation in brain regions implicated in psychosis and cognitive disturbances along with improving motor functions, emotional regulation, and behavioral control.

[0253] Symptoms to be treated for Posterior cortical atrophy (PCA) can include improving visual function, alleviating cognitive deficits by preventing neuronal dysfunction and synaptic loss, and preventing neuronal loss and dysfunction in brain regions implicated in language processing.

[0254] =Symptoms to be treated for cardiomyopathy and heart failure can include improving respiratory function, alleviating fatigue by reducing neuronal damage and inflammation in brain regions involved in energy regulation and metabolism, improving fluid balance and reducing edema by preserving neuronal integrity in brain regions involved in fluid regulation and electrolyte balance, alleviating chest pain by preventing neuronal dysfunction and inflammation in brain regions involved in pain perception and modulation, reducing palpitations by affecting brain regions involved in cardiac conduction and rhythm control, improving blood pressure regulation and cerebral perfusion by affecting brain regions involved in autonomic control and blood pressure regulation.

[0255] Symptoms to be treated for obesity and diabetes can include modulating insulin signaling pathways and glucose metabolism in peripheral tissues, helping regulate blood glucose levels by modulating insulin secretion and glucose uptake in pancreatic beta cells and peripheral tissues, mitigating obesity-related complications by modulating metabolic pathways involved in lipid metabolism, inflammation, and energy homeostasis, attenuating inflammation by modulating inflammatory signaling pathways and cytokine production in adipose tissue and other metabolic organs, protecting against diabetes-related neurological complications by preserving neuronal integrity and reducing neuroinflammation in the central and peripheral nervous systems.

[0256] B. Effective Amounts

[0257] In some forms the methods administer the described engineered peptide inhibitors of TDP-43 aggregates in an effective amount. The effective amount or therapeutically effective amount of a pharmaceutical compositions including the described engineered peptide inhibitors of TDP-43 aggregates can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, such as ALS or FTD or to otherwise provide a desired pharmacologic and / or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying a disease or disorder, such as ALS or FTD.

[0258] In some forms, when administration of the pharmaceutical compositions including administer the described engineered peptide inhibitors of TDP-43 aggregates elicits an effective therapeutic response, the amount administered can be expressed as the amount effective to achieve a desired effect in the recipient. For example, in some forms, the amount of the pharmaceutical compositions including the described engineered peptide inhibitors of TDP-43 aggregates, is effective to reduce the amount, concentration, size and / or distribution of TDP-43 aggregates in the brain and / or CNS of the recipient. In some forms, the amount of the pharmaceutical composition including the described engineered peptide inhibitors of TDP-43 aggregates is effective to reduce the symptoms of a disease in the recipient, or prevent or reduce a neurological deficit or decline, and combinations thereof. In other forms, the amount of the pharmaceutical compositions including the described engineered peptide inhibitors of TDP-43 aggregates is effective to reduce one or more symptoms or signs of ALS in an ALS patient, or signs of FTD in a patient having FTD. Signs of ALS or FTD can include biochemical markers, such as levels of a biomarker detectable in the blood of a patient.

[0259] The effective amount of the pharmaceutical compositions including the described engineered peptide inhibitors of TDP-43 aggregates that is required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, and its mode of administration. Thus, it is not possible to specify an exact amount for every pharmaceutical composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the pharmaceutical compositions including the described engineered peptide inhibitors of TDP-43 aggregates can be determined empirically, and making such determinations is within the skill in the art. In some forms, the dosage ranges for the administration of the compositions including the described engineered peptide inhibitors of TDP-43 aggregates are those large enough to effect reduction in TDP-43 aggregation and / or a symptom thereof, for example.

[0260] The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, and sex of the patient, route of administration, whether other drugs are included in the regimen, and the type, stage, and location of the disease to be treated. The dosage can be adjusted by the individual physician in the event of any counter-indications. It will also be appreciated that the effective dosage of the composition including the described engineered peptide inhibitors of TDP-43 aggregates used for treatment can increase or decrease over the course of a particular treatment. Changes in dosage can result and become apparent from the results of diagnostic assays.

[0261] Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject or patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual pharmaceutical compositions, and can generally be estimated based on ECsos found to be effective in in vitro and in vivo animal models.

[0262] It can generally be stated that a pharmaceutical composition containing the described engineered peptide inhibitors of TDP-43 aggregates can be administered at a dosage of 10’4mg / kg body weight to 100 mg / kg body weight, inclusive, preferably 0.1 to 10.0 mg / kg body weight, including all integer values within those ranges. In some forms, patients can be treated by infusing a disclosed pharmaceutical composition containing the described engineered peptide inhibitors of TDP-43 aggregates in the range of about 0.1 to 10.0 or more mg / kg body weight of the subject.

[0263] The infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved. Compositions including the described engineered peptide inhibitors of TDP-43 aggregates can also be administered once or multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg el al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. In some forms, the unit dosage is in a unit dosage form for intravenous injection. In some forms, the unit dosage is in a unit dosage form for oral administration. In some forms, the unit dosage is in a unit dosage form for inhalation. In some forms, the unit dosage is in a unit dosage form for intra-cranial injection.

[0264] Treatment can be continued for an amount of time sufficient to achieve one or more desired therapeutic goals, for example, a reduction of the amount of TDP-43 aggregates in the brain and / or CNS relative to the start of treatment, or complete absence of TDP-43 aggregates in the brain and / or CNS in the recipient. Treatment can be continued for a desired period of time, and the progression of treatment can be monitored using any means known for monitoring the presence of TDP-43 aggregates in the brain and / or CNS in a patient. In some forms, administration is carried out every day of treatment, or every week, or every fraction of a week. In some forms, treatment regimens are carried out over the course of up to two, three, four or five days, weeks, or months, or for up to 6 months, or for more than 6 months, for example, up to one year, two years, three years, or up to five years.

[0265] The efficacy of administration of a particular dose of the pharmaceutical compositions including modified cells, such as the described engineered peptide inhibitors of TDP-43 aggregates, according to the methods described herein can be determined by evaluating the aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of ALS or FTD or other diseases and / or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and / or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g., a diseases state has partially or fully regressed), (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.

[0266] C. Modes of Administration

[0267] In some embodiments the methods administer the described engineered peptide inhibitors of TDP-43 aggregates via any route that enables access of the compositions to the site of action in vivo, i.e., to the brain and / or CNS. For example, in some forms, the route of administration is tailored depending upon the form of the composition, to provide the greatest and / or deepest penetration of agents into the brain parenchyma. Circumventing the BBB via cerebral spinal fluid (CSF) microcirculation is one strategy for the delivery of the described peptides into the brain, achieving deep penetration and distribution. For example, in some forms, the methods administer the described engineered peptide inhibitors of TDP-43 aggregates as peptides directly to the CNS / brain via cerebral spinal fluid (CSF) administration. In some forms, when the methods administer the described engineered peptide inhibitors of TDP-43 aggregates via the CSF, the amount of peptide inhibitors required to achieve a therapeutic effect is less than that required when using other routes of administration, such as IV administration.

[0268] In some forms, the methods include administering the described engineered peptide inhibitors of TDP-43 aggregates directly into cerebral spinal fluid (CSF), for example, via lumbar puncture. Therefore, in some forms, the methods include administering the described engineered peptide inhibitors of TDP-43 aggregates via injection into cerebral spinal fluid (CSF) in combination with a pharmaceutically acceptable carrier suitable for administration to the CSF. In some forms, when the methods include administering the described engineered peptide inhibitors of TDP-43 aggregates as peptides including a CPP motif, the methods include administering the engineered peptide inhibitors into the bloodstream, to traverse the BBB, or via injection directly into CSF, e.g., in combination with a pharmaceutically acceptable carrier suitable for administration to the CSF. In some forms, when the methods include administering the described engineered peptide inhibitors of TDP-43 aggregates as nucleic acids encoding the engineered peptides, the methods include administering the nucleic acids in an expression vector, for example, encapsulated within a nanoparticle. A nanoparticle can be delivered into the bloodstream to traverse the BBB, or via injection directly into CSF in combination with a pharmaceutically acceptable carrier suitable for administration to the CSF.

[0269] The compositions described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington’s Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the therapeutics described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, for humans and non-humans, these include solutions such as sterile water, saline, and buffered solutions at physiological pH. Other therapeutics can be administered according to standard procedures used by those skilled in the art.

[0270] Another administration route that successfully reaches the brain is the intranasal route. Intranasal administration has recently been explored by researchers because it reaches the brain, bypassing the BBB through the olfactory bulb.

[0271] The pharmaceutical compositions including the described engineered peptide inhibitors of TDP-43 aggregates can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the therapeutic(s) of choice.

[0272] Pharmaceutical compositions containing the described engineered peptide inhibitors of TDP-43 aggregates, and optionally one or more additional therapeutic agents can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, for example, a pharmaceutical composition including the described engineered peptide inhibitors of TDP-43 aggregates, can be administered as an intravenous infusion, or directly injected into a specific site, for example, into or surrounding target aggregates. Moreover, a pharmaceutical composition can be administered to a subject as an ophthalmic solution and / or ointment to the surface of the eye, vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. In some forms, the compositions are administered directly into a tumor or tissue, e.g., stereotactically.

[0273] Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. Suitable parenteral administration routes include intravascular administration (e.g. , intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intratissue injection (e.g., intraocular injection, intra-retinal injection, or sub-retinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application by a catheter or other placement device (e.g., an implant including a porous, non-porous, or gelatinous material).

[0274] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0275] Administration of the pharmaceutical compositions containing the described engineered peptide inhibitors of TDP-43 aggregates can be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic.

[0276] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0277] D. Combination therapy

[0278] In some embodiments the methods administer the described engineered peptide inhibitors of TDP-43 aggregates in combination with other therapeutic agents or treatment modalities. Any of the disclosed pharmaceutical compositions including the described engineered peptide inhibitors of TDP-43 aggregates, can be used alone, or in combination with other therapeutic agents or treatment modalities, for example, conventional medications. As used herein, “combination” or “combined” refer to either concomitant, simultaneous, or sequential administration of the therapeutics.

[0279] In some forms, the pharmaceutical compositions and other therapeutic agents are administered separately through the same route of administration. In other forms, the pharmaceutical compositions and other therapeutic agents are administered separately through different routes of administration. The combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject; one agent is given orally while the other agent is given by infusion or injection, etc.,), or sequentially (e.g., one agent is given first followed by the second).

[0280] Examples of preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder or condition. In some forms, the therapeutic agent is one or more other targeted therapies.

[0281] The compositions and methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio- frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.

[0282] The disclosed pharmaceutical compositions and / or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of remission or less active disease. The pharmaceutical compositions can be administered before the additional treatment, concurrently with the treatment, post-treatment, or during remission of the disease or disorder. When administered in combination, the disclosed pharmaceutical compositions and the additional therapeutic agents (e.g. , second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain forms, the administered amount or dosage of the disclosed pharmaceutical composition, the additional therapeutic agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy (e.g., required to achieve the same therapeutic effect).

[0283] 1. Additional Therapeutic Agents

[0284] In some embodiments, the methods administer one or more additional therapeutic agents to a subject.

[0285] Treatment strategies for ALS can include treatment with Riluzole (Rilutek), Edaravone (Radicava), Baclofen, Diazepam, Gabapentin, Trihexyphenidyl, or Amitriptyline. Other treatment strategies for ALS include gene therapy, stem cell therapy, neuromuscular electrical stimulation (NMES), anti-inflammatory agents, or respiratory support. Therefore, in some forms, methods for treatment of ALS include administering to a subject the described engineered peptide inhibitors of TDP-43 aggregates in combination with one or more therapeutic agents or treatment modalities such as Riluzole (Rilutek), Edaravone (Radicava), Baclofen, Diazepam, Gabapentin, Trihexyphenidyl, Amitriptyline, Qalsody (tofersen), Nuedexta, QRL-201 (QurAlis), BI1B105, ABBV-CLS-7262, and / or ANX005, and / or gene therapy and / or stem cell therapy and / or neuromuscular electrical stimulation (NMES) and / or anti-inflammatory agents, or respiratory support.

[0286] Treatment strategies for FTD can include treatment with selective serotonin reuptake inhibitors (SSRIs) such as sertraline and citalopram, antipsychotic medications such as risperidone and quetiapine, cholinesterase inhibitors such as donepezil, rivastigmine, and galantamine, gene therapy, stem cell therapy, neurotrophic factors, occupational therapy, speech and language therapy, or physiotherapy. Therefore, in some forms, for the methods for treating FTD include administering to a subject the described engineered peptide inhibitors of TDP-43 aggregates in combination with one or more therapeutic agents or treatment modalities such as selective serotonin reuptake inhibitors (SSRIs), such as sertraline and citalopram, or antipsychotic medications, such as risperidone and quetiapine, or cholinesterase inhibitors such as donepezil, rivastigmine, and galantamine, or gene therapy, or stem cell therapy, or neurotrophic factors, or occupational therapy, or speech and language therapy, or physiotherapy.

[0287] Treatment strategies for Alzheimer's disease include using cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) or NMDA receptor antagonists or amyloid-P targeting therapies, or tau protein targeting therapies, or anti-inflammatory agents (cytokine inhibitors and microglial modulators), or antioxidants (vitamins C and E, coenzyme Q10, and resveratrol), or stem cell therapy, or gene therapies. Therefore, in some forms, methods for treatment of Alzheimer's disease include administering to a subject the described engineered peptide inhibitors of TDP-43 aggregates in combination with one or more therapeutic agents or treatment modalities such as cholinesterase inhibitors (donepezil, rivastigmine, and galantamine) or NMDA receptor antagonists or amyloid-P Targeting Therapies, or tau protein targeting therapies, or anti-inflammatory agents (cytokine inhibitors and microglial modulators), or antioxidants (vitamins C and E, coenzyme Q10, and resveratrol), or Stem cell therapy, or gene therapies.

[0288] E. Envisioned Experimental Methods and Data

[0289] Docking and molecular dynamics simulations confirmed strong binding to patient- derived TDP-43 cryo-EM structures, and overexpression of top helical variants reduced formation of TDP-43 aggregates induced by TDP-43 ’s C-terminal domain in cellular and biochemical assays.

[0290] The following envisioned experimental methods and data can provide further evidence of efficacy and target engagement of the engineered peptides in preclinical models. 1. Evaluation of engineered helical peptides for target engagement and efficacy in reducing TDP-43 aggregates

[0291] To validate the in silic results, the engineered helical peptides can be tested for target engagement and ability to reduce TDP-43 aggregation. A series of biophysical assays can be used to analyze secondary structure, binding affinity, and target engagement between the topranked engineered peptides (shown in Fig. 2) and TDP-43 fibrils. Winners, e.g., the top 2-3 engineered peptides displaying robust helical secondary structure and target engagement, can be selected for ThioflavinT time course and cellular aggregation assays to examine their efficacy for reducing TDP-43 aggregation kinetics and for clearing cellular aggregates.

[0292] Circular Dichroism (CD) Measurements

[0293] As described in the Examples, below, Wild type peptide and the 5 top-ranked engineered peptides have been synthesized (Genscript) to >95% purity and verified via HPLC and mass spectrometry. CD can be used to validate the secondary structure properties of top-ranked engineered peptides in isolation and when bound to TDP-43 amyloid-like fibrils. far-UV CD spectra (190 to 250 nm) of each peptide in isolation can be recorded. Residue-wise and overall predicted helical probability and beta sheet probability of the wild-type amyloidogenic core peptide or engineered S332A / S333W peptide bound to TDP-43 amyloid-like fibrils; two-tailed t- test, ****p < 0.0001. As described in the Examples, below, snapshots from 300 nanosecond molecular dynamics simulations showed conformational stability of S332 / S333W peptide bound to TDP-43 amyloid-like fibril structure, compared to the wild type peptide where characteristic absorbance patterns arise from the interactions of peptide bonds and secondary structural elements. Quantitative analysis can be performed using CD deconvolution software to determine the percentage of each secondary structural component. In addition, full-length TDP-43 filaments can be generated and their fibrillation induced. CD can be performed at successive timepoints during TDP-43 fibrillation, e.g., in the presence of either TDP-43 wild type amyloidogenic core peptide, the top-ranked engineered peptides, or buffer alone. This experiment will allow us to determine whether the presence of our engineered helical peptides slows beta-sheet formation during TDP-43 fibrillation.

[0294] Assessment of target engagement and efficacy in vitro

[0295] As described in the Examples, below, the docking analysis and molecular dynamics simulations suggest that the S332A / S333W engineered peptide exhibits strong binding affinity to TDP-43 fibrils and maintains a stable helical conformation, in contrast to the wild type amyloidogenic core peptide which binds and transitions to a beta- sheet.

[0296] Surface plasmon resonance (SPR) can be used to analyze the binding kinetics of the tophit engineered peptides with the target, TDP-43 fibrils. To determine the binding kinetics between TDP-43 fibrils and each engineered peptide (or the wild type amyloidogenic core peptide), purified TDP-43 fibrils can be immobilized on a sensor chip and purified peptides can be flowed over them. Using a suitable buffer, the binding and dissociation process can be recorded and the data analyzed to derive association and dissociation rates. This experiment can provide insights into the binding affinity and interaction strength between TDP-43 fibrils and the engineered peptides. The engineered peptides are expected to bind TDP-43 fibrils with similar binding affinity as the wild type amyloidogenic core peptide.

[0297] As a complementary approach, smFRET can be employed to analyze the interaction between TDP-43 fibrils and Cy5-tagged wild type or engineered peptides at the single-molecule level. Ensemble FRET is a well established approach used to measure time-averaged and often static distances, however smFRET is a much more sensitive technique capable of differentiating individual oligomers within an ensemble. By labeling the TDP-43 fibrils and engineered peptides with appropriate fluorophores, FRET efficiency can be measured to indicate close proximity (z.e., binding) between individual molecules within oligomeric mixtures. This method will provide a dynamic view of target engagement interactions. For smFRET experiments and analyses, intensity-based FRET efficiency (ETeff) requires correction factors for background signal from donor and acceptor channels as well as for spectral crosstalk. Corrected ETeff events can be plotted as a histogram and fitted with a Gaussian to represent the distance between FRET fluorophores, averaged over time. In order to convert FRET efficiency to distance between single molecules, methods can be used taking into account dye position and rates of protein reconfiguration.

[0298] Thioflavin-T (ThT) aggregation assay

[0299] To determine whether the top 2-3 engineered peptides (selected based on CD, SPR, and smFRET results) can effectively compete with TDP-43 wild type monomers to slow fibrillation, aggregation kinetics of TDP-43 monomers can be measured in the presence of 2.5% full length TDP-43 filament seeds and varying concentrations of purified engineered helical peptides. ThT fluorescence can be measured every 5 minutes for 8 hours using 440nm / 480nm excitation / emission filters.

[0300] To correct for autofluorescence, ThT can be incubated alone with the buffer as a control. This assay can provide an in vitro readout of our engineered peptide’s efficacy for reducing TDP-43 aggregation. The assay can demonstrate that ThT fluorescence, as a measure of aggregation kinetics, can be slowed (right shifted) and have a longer lag phase in the presence of the engineered peptides. 2. Assay of efficacy of engineered helical peptides for clearing TDP-43 aggregates in vivo and evaluate endogenous TDP-43 function.

[0301] To examine in vivo efficacy and potential toxicity of engineered helical peptides. The objective is to provide an in vivo proof-of-concept, testing the efficacy of the described engineered peptide for reducing TDP- 43 aggregates and evaluating potential toxicity.

[0302] Exemplary Methods

[0303] To establish whether the engineered helical peptides effectively clear TDP-43, aggregates in transgenic Caenorhabditis elegans strains that express yellow fluorescent protein (YFP) or YFP-tagged human TDP-43 (either full length or CTD) under a pan-neuronal promoter (snb-1) can be developed as a model system. The TDP-43 transgenic strains develop cytoplasmic TDP- 43 aggregates associated with severe locomotor defects. Importantly, the C. elegans model can also determine if the engineered helical peptides induce locomotor defects in YFP transgenic (control) or mis-localization of nuclear TDP-43 in YFP-TDP-43 full length strains, respectively. Exemplary methods can use the C. elegans strains IW62 [Psnb-l::YFP(iwIs25)]; IW63 [Psnb- l::TDP-43-YFP WT(iwIs26)]; IW33 [Psnb-l::TDP-C25-YFP(iwIs22)]. Synthesized helical peptides (Genescript) can be dissolved in lx PBS to final concentrations ranging from (0.1 to 100 pM). Synchronized LI worms can be cultured on nematode growth media (NGM) plates seeded with heat-treated OP50 E. coli and divided into the following treatment groups:

[0304] (i) Controls: Worms grown on NGM plates without any additional treatment (lx PBS as solvent control); and

[0305] (ii) Experimental: Worms grown on NGM plates treated with engineered peptides at a final concentrations of 0.1, 1, 10, 50, 100 pM.

[0306] Worms in all treatment groups can be grown for 7 days at 17 °C, and worms from both the control and experimental groups can be collected and prepared for biochemical, imaging, and locomotor analysis.

[0307] For biochemical analysis, worms can be homogenized by sonication in ice cold lysis buffer. Soluble and insoluble fractions can be prepared and analyzed by Western blot. If reduction of insoluble TDP-43 is observed in worms receiving engineered peptides with functional PDMs, the worms can also be treated with MG132, hydroxychloroquine (or DMSO control) to verify whether clearance is dependent on proteasome and / or autophago-lysosome pathways. To examine clearance of TDP-43 aggregates by confocal imaging, worms can be mounted onto 5% agar pads on glass slides, immobilized with 1% NaN3 solution, and imaged using a spinning disk confocal microscope to visualize and quantify number and size of YFP- TDP-43 aggregates in ventral cord motor neurons. Envisioned Results

[0308] Worms receiving the engineered peptides can have reduced number of YFP-TDP-43 cytoplasmic aggregates, compared to control conditions. In addition, whether treatment with the engineered peptides affects the localization of nuclear TDP-43 in C. elegans transgenic expressing YFP-TDP-43 (full length) can be assessed. Locomotor behavior of control and experimental groups can be analyzed by analyzing thrashing rate (number of body bends in liquid medium per unit time).

[0309] Treatment with the engineered peptides can significantly ameliorate locomotor behavior of TDP-43 transgenic strains, compared to controls. In some forms, the control YFP transgenic worms do not show significant locomotor alterations, compared to YFP control worms that are grown / treated with control NGM.

[0310] 3. Assessment of disruption of physiological function and / or localization of endogenous TDP-43.

[0311] Given that the engineered helical peptides possess sequence similarity to the CTD of wild type TDP-43, the objective is to determine whether the top hit engineered peptides adversely impact localization of endogenous TDP-43 or one or more of its important RNA regulatory functions.

[0312] To test whether the top hit engineered peptides have functional implications for TDP-43 splicing in the nucleus and for mRNA localization in the cytoplasm, respectively three functional readouts can be examined:

[0313] (i) TDP-43 cryptic exon splicing;

[0314] (ii) nuclear-to-cytoplasmic (N:C) ratio of endogenous TDP-43; and

[0315] (iii) axonal transport and mRNA localization.

[0316] To assess (i) splicing, and (ii) N:C ratio of endogenous TDP-43, the impact of the top 2-3 engineered peptides can be investigated on cryptic exon inclusion / exclusion splicing patterns and RNA stability of key neuronal transcripts known to be affected in TDP-43 proteinopathies. Cy5- tagged engineered peptide can be delivered with functional PDMs or control non-functional PDMs (via BioPORTER) to SH-SY5Y cells and human Dendra-TDP-43 iPSC-derived cortical i3-neurons. The primary focus will be on detecting aberrant splicing events and stability of STMN2 and UNC13A transcripts, as these are important neuronal genes known to be affected in neurodegenerative diseases with TDP-43 mis-localization and aggregation, quantitative real-time PCR (qRT-PCR) can be employed for precise quantification of STMN2 and UNC13A transcripts levels, and reverse transcription PCR (RT-PCR) can be used to detect inclusion of cryptic exons (e.g., exon 2a of STMN2). Samples can be collected at 24, 48, and 96 hours following peptide delivery to monitor temporal changes in gene expression and splicing patterns. In addition, Dendra-TDP-43 iPSC-derived cortical i3 -neurons can be fixed and confocal imaging will be performed to quantify N:C fluorescence intensity ratio of endogenously tagged Dendra-TDP-43. This approach can enable determination of whether the engineered peptide can disrupt endogenous nuclear TDP-43 localization or splicing pattern.

[0317] To assess (iii) axonal transport and mRNA localization, smFISH can be used to examine endogenous TDP-43 and target mRNAs along the axon, and molecular beacons can be used to examine axonal mRNAs in real time in neurons treated with engineered helical peptides.

[0318] Axonal transport is an important cellular mechanism for neurons to maintain cytoskeletal and synaptic homeostasis. TDP-43, as a component of neuronal RNA granules, is actively transported in the dendrites and axon and exhibits dynamic biophysical properties (10-13). TDP- 43 binds the 3’UTR of at least 1000 mRNAs, including those encoding synaptic, axonal, and cytoskeletal proteins (e.g., App, Kif5a, Nefl), suggesting a key role in cytoskeletal and synaptic maintenance (7, 27, 64).

[0319] Rodent primary cortical neurons can be treated with top 2-3 engineered peptides or the wild type TDP-43 amyloidogenic core peptide, immunofluorescence for endogenous TDP-43 can be combined with smFISH to examine:

[0320] 1) distribution of endogenous TDP-43 along the axon, and

[0321] 2) axonal mRNAs known to bind to TDP-43 (based on CLIP-seq data) along the axon.

[0322] TDP-43 localization, size and number of granules can be assessed in the axons with treatment of engineered peptides. To validate the effect of engineered peptide on the mRNA localization, selected TDP-43 target mRNAs (e.g., Nefl, Kif5) important for axonal maintenance can be visualized.

[0323] Appropriate control mRNAs, such as beta-actin, which is abundant in the axon but not regulated by TDP-43, can also be analyzed. The position of TDP-43 and each mRNA along the axon can be analyzed using the “Analyze particles” function in Fiji / ImageJ, and the distribution of each mRNA can be plotted in a histogram. In addition, the density of TDP-43 and target mRNAs along the axon in proximal and distal regions of the axon can be quantified (calculated as no. of TDP-43 or mRNA per unit length).

[0324] These experiments can determine if there are adverse effects of the engineered peptides on TDP-43 localization (nuclear and axonal), target mRNA localization, and splicing. The data can confirm that trafficking and spatial localization of mRNAs important for axonal and cytoskeletal maintenance is not impaired by the engineered peptides. IV. Kits

[0325] Compositions including the described engineered peptide inhibitors of TDP-43 aggregates reagents, and other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the methods. It is useful if the components in a given kit are designed and adapted for use together in the method. For example, kits with one or more compositions for administration to a subject, may include a pre-measured dosage of the composition in a sterile needle, ampule, tube, container, or other suitable vessel. The kits may include instructions for dosages and dosing regimens.

[0326] Provided are kits containing the described engineered peptide inhibitors of TDP-43 aggregates as proteins. Also provided are kits containing the described engineered peptide inhibitors of TDP-43 aggregates as proteins encapsulated or complexed with one or more carriers, such as a nanocarrier, such as a nanoparticle. Also provided are kits containing nucleic acids encoding the described engineered peptide inhibitors of TDP-43 aggregates within a vector (e.g., a viral vector) and / or mRNA encoding the described engineered peptide inhibitors of TDP- 43 aggregates, and instructional material for use thereof. In preferred forms, the kit includes a plurality of vectors, where each vector independently contains a described engineered peptide inhibitor of TDP-43 aggregates for insertion into a host cell genome, such as an expression cassette. In some forms, the kit contains a population of cells containing nucleic acids encoding described engineered peptide inhibitors of TDP-43 aggregates. The instructional material can include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the kit. For example, the instructional material may provide instructions for methods using the kit components, such as performing transfections, transductions, infections, and conducting screens. In some forms, kits include a transposon that includes a promoter and / or polyadenylation signal operationally linked to a reporter gene expressed with the described engineered peptide inhibitors of TDP-43 aggregates.

[0327] In some embodiments, the kit includes a cell or vector including the described engineered peptide inhibitors of TDP-43 aggregates. In exemplary embodiments, the kits include a nucleic acid and / or a vector expressing or encoding the described engineered peptide inhibitors of TDP- 43 aggregates and / or cells.

[0328] The disclosed compositions and methods can be further understood through reference to the following numbered paragraphs.

[0329] 1. An engineered TAR DNA Binding Protein 43 kDa (TDP-43) polypeptide including residues at positions 321-343 of mature human TDP-43, wherein the engineered TDP-43 polypeptide includes one or more mutations of one or more amino acids of the mature human TDP-43 polypeptide, wherein

[0330] (a) one mutation includes substitution of the amino acid residue at position 332 of the human TDP-43 polypeptide; and / or

[0331] (b) one mutation includes substitution of the amino acid residue at position 333 of the human TDP-43 polypeptide, wherein the engineered binds to a TDP-43 aggregate; and wherein the engineered peptide precludes binding of further TDP-43 peptides to the aggregate.

[0332] 2. The engineered TDP-43 polypeptide of paragraph 1, including alanine, or arginine, or valine at position 332 of the human TDP-43 polypeptide.

[0333] 3. The engineered TDP-43 polypeptide of paragraph 1 or 2, including tryptophan, or leucine, or valine at position 333 of the human TDP-43 polypeptide.

[0334] 4. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including leucine at position 333 of the human TDP-43 polypeptide.

[0335] 5. The engineered TDP-43 polypeptide of any one of paragraphs 1 -3, including valine at position 333 of the human TDP-43 polypeptide.

[0336] 6. The engineered TDP-43 polypeptide of any one of paragraphs 1-5, including alanine at position 332 of the human TDP-43 polypeptide.

[0337] 7. The engineered TDP-43 polypeptide of any one of paragraphs 1-5, including valine at position 332 of the human TDP-43 polypeptide.

[0338] 8. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including alanine at position 332 and tryptophan at position 333 of the human TDP-43 polypeptide.

[0339] 9. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including arginine at position 332 and leucine at position 333 of the human TDP-43 polypeptide.

[0340] 10. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including valine at position 332 and valine at position 333 of the human TDP-43 polypeptide.

[0341] 11. The engineered TDP-43 polypeptide of any one of paragraphs 1-10, including the amino acid sequence AMMAAAQAALQXXWGMMGMLASQ (SEQ ID NO:51), wherein each X includes, independently, serine, valine, tryptophan, arginine, alanine, or valine.

[0342] 12. The engineered TDP-43 polypeptide of any one of paragraphs 1-11, including the amino acid sequence AMMAAAQAALQAWWGMMGMLASQ (SEQ ID NO:52).

[0343] 13. The engineered TDP-43 polypeptide of any one of paragraphs 1-12, including between 23 and 80 amino acid residues, optionally wherein the engineered TDP-43 polypeptide consists of 50 amino acid residues.

[0344] 14. The engineered TDP-43 polypeptide of any one of paragraphs 1 -3, including the amino acid sequence MNF GAF S INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:5), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:5.

[0345] 15. The engineered TDP-43 polypeptide of paragraph 14, wherein the variant or fragment consists of an amino acid sequence set forth in any one of SEQ ID NOs.12-23 or 53.

[0346] 16. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including the amino acid sequence

[0347] MNF GAF S INP AMMAAAQAALQRLWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:6), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:6.

[0348] 17. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including the amino acid sequence

[0349] MNF GAF S INPAMMAAAQAALQASWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:7), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:7.

[0350] 18. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including the amino acid sequence

[0351] MNF GAF S INP AMMAAAQAALQRSWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:8), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:8.

[0352] 19. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including the amino acid sequence

[0353] MNF GAF S INP AMMAAAQAALQWWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:9), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:9.

[0354] 20. The engineered TDP-43 polypeptide of any one of paragraphs 1-3, including the amino acid sequence

[0355] MNFGAF S INP AMMAAAQAALQSVWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:10), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO: 10.

[0356] 21. The engineered TDP-43 polypeptide of any one of paragraphs 1-20, further including one or more additional function peptide motifs.

[0357] 22. The engineered TDP-43 polypeptide of paragraph 21, wherein the additional function peptide motif includes a peptide degradation motif (PDM), or a cell penetrating peptide (CPPM), or both a PDM and a CPPM. 23. The engineered TDP-43 polypeptide of paragraph 22, wherein the PDM includes the amino acid sequence ALAP YI P (SEQ ID NO:24), KFERQKI LDQRFFE (SEQ ID NO:25), LDPETGEYL (SEQ ID NO:54), RRRG (SEQ ID NO:55), MDF SGLS LIKLKKQ (SEQ ID NO:56) or a combination thereof.

[0358] 24. The engineered TDP-43 polypeptide of paragraph 22, wherein the peptide includes an amino acid sequence:

[0359] MNF GAF S INPAMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQALAPY IP (SEQ ID NO:26).

[0360] 25. The engineered TDP-43 polypeptide of paragraph 22, wherein the peptide includes an amino acid sequence:

[0361] MNF GAF S INP AMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQKFERQKI LDQRFFE (SEQ ID NO:27).

[0362] 26. The engineered TDP-43 polypeptide of any one of paragraphs 23-25, wherein the CPPM includes an amino acid sequence selected from the group including LRKLRKRLL (SEQ ID NO:35); SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO:36); LRKLRKRLLR (SEQ ID NO:37); YT IWMPENPRP GTP CD IFTNSRGKRASNG (SEQ ID NO:38); GGGGYGRKKRRQRRR (SEQ ID NO:39); AGI LKRW (SEQ ID NO:40); H- CNCKAPE TALCARRCQQH-NH2 (SEQ ID NO:41); H-DapKAPETALD-NH2 (SEQ ID NO:42); PWVP SWMPPRHT (SEQ ID NO:43); TGNYKALHP HNG (SEQ ID NO:44); THRP PMWSPVWP (SEQ ID NO:45); ( PWVP SWMP PRHT ) 2 KKGK ( CF ) G (SEQ ID NO:46); HAYED (SEQ ID NO:47); CNSRLHLRC (SEQ ID NO:48); CENWWGDVC (SEQ ID NO:49); and WRCVLREGPAGGCAWFNRHL (SEQ ID NO:50), or combinations of two or more thereof.

[0363] 27. A nucleic acid including a nucleic acid sequence encoding the polypeptide of any one of paragraphs 1-26.

[0364] 28. The nucleic acid of paragraph 27, wherein the nucleic acid is RNA or DNA.

[0365] 29. The nucleic acid of paragraph 27, wherein the nucleic acid is mRNA.

[0366] 30. The nucleic acid of paragraph 28, wherein the nucleic acid is, or is encoded by a vector or a transposon.

[0367] 31. The nucleic acid of paragraph 30, wherein the vector is a viral vector.

[0368] 32. The nucleic acid of paragraph 31, wherein the viral vector is selected from the group including a lentiviral vector, an Adeno-associated virus (AAV) vector, or an adenovirus vector, or a Herpes Simplex virus (HSV) vector, or a vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV), or a chimeric vector including a combination of any two or more of a Adeno-associated virus (AAV) vector, Herpes Simplex virus (HSV) vector, vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV).

[0369] 33. The nucleic acid of paragraph 31 , wherein the vector is a nucleic acid expression vector selected from the group including a plasmid, a cosmid, and a replicon.

[0370] 34. The nucleic acid of any one of paragraphs 27-33, including one or more of a promotor, a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto.

[0371] 35. A delivery vehicle including the polypeptide of any one of paragraphs 1-26, or the nucleic acid of any one of paragraphs 27-34.

[0372] 36. The delivery vehicle of paragraph 35, wherein the delivery vehicle includes a nanoparticle, or a microparticle.

[0373] 37. The delivery vehicle of paragraph 36, wherein the polypeptide of any one of paragraphs 1-26, or the nucleic acid of any one of paragraphs 27-34 is encapsulated within or conjugated to the nanoparticle or microparticle.

[0374] 38. The delivery vehicle of any one of paragraph 35-37, wherein the delivery vehicle is selected from the group including a polymeric particle, a viral particle, a liposome, a nucleic acid conjugate, and a metallic particle, or a combination thereof.

[0375] 39. The delivery vehicle of any one of paragraphs 35-38, further including a targeting motif that targets the delivery vehicle to the brain and / or the central nervous system (CNS) in vivo.

[0376] 40. The delivery vehicle of paragraph 39, wherein the targeting motif includes a peptide that facilitates passage across the blood-brain barrier (BBB).

[0377] 41. The delivery vehicle of paragraph 39 or 40, wherein the targeting motif includes a peptide having an amino acid sequence set forth in any one of SEQ ID NOs.35-50.

[0378] 42. A cell including the polypeptide of any one of paragraphs 1-26, or the nucleic acid of any one of paragraphs 27-34, or the delivery vehicle of any one of paragraphs 35-41.

[0379] 43. A pharmaceutical composition including the polypeptide of any one of paragraphs 1-26, or the nucleic acid of any one of paragraphs 27-34, or the delivery vehicle of any one of paragraphs 35-41, or the cell of paragraph 42, and a pharmaceutically acceptable buffer, carrier, diluent or excipient.

[0380] 44. A method of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of the pharmaceutical composition of paragraph 43.

[0381] 45. A method of treating a subject having a disease, disorder, or condition associated with pathological aggregation and / or mis-localization of TDP-43 aggregates in the brain or central nervous system (CNS) of the subject including administering to the subject the pharmaceutical composition of paragraph 43. 46. The method of any one of paragraphs 44-45, wherein the subject is a human.

[0382] 47. The method of any one of paragraphs 44-46, wherein the subject has a disease selected from the group including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal disorders (FTD), and a genetic disorder.

[0383] 48. The method of any one of paragraphs 44-46, wherein the pharmaceutical composition is administered to the subject via a route selected from the group including intravenous, intramuscular, intracranial, intraosseus, intranasal, intrathecal, intraventricular, intraparenchymal and intracerebroventricular administration.

[0384] 49. The method of paragraph 48, wherein the pharmaceutical composition is administered to the subject via intracerebroventricular injection.

[0385] 50. The method of paragraph 48, wherein the pharmaceutical composition is administered to the subject via intrathecal injection.

[0386] 51. An engineered TDP-43 polypeptide including the amino acid sequence of a non-wildtype “Sequence(320-340 Residues)” polypeptide of Table 4.

[0387] The disclosed compositions and methods can be further understood through the following Examples.

[0388] EXAMPLES

[0389] Example 1: Computational Design of TDP-43 Derived ( / -Helical Peptide Inhibitors

[0390] Considering the important role of the amyloidogenic core and a-helical domain residues in initiating TDP-43 aggregation, an in silico approach was used to investigate whether peptide inhibitors with enhanced a-helical propensity are a potential therapeutic strategy to inhibit the growth of pathological TDP-43 filaments.

[0391] Methods

[0392] Protein Dataset Preparation:

[0393] Starting structures for docking analysis and molecular dynamics simulations were extracted from the Protein Data Bank. Three TDP-43 crystal structures were retrieved PDB ID: 7KWZ, 6N37, 7PY2). These include structures of ALS / FTLD patient-derived TDP-43 aggregates from the frontal and motor cortices, as well as two structures elucidated through crystallographic analysis. The selected PDB structures were prepared for computational approaches using the Preparation wizard of Schrodinger.

[0394] The preparation of proteins includes adding hydrogens, ionizing metals, and fixing missing residues and atoms. To mimic the neutral pH conditions of the physiological environment, modifications were made such that the N-terminus was positively charged (NH3+) and the C-terminus was negatively charged (C00-). The prepared structures were used for docking and molecular dynamics analysis, trogocytosis.

[0395] Peptide Library Design

[0396] A library of approximately 400 unique peptides (Table 4), derived from the helical domain of TDP-43 was generated, with a length of 50 residues each, using the wild-type sequence [Uniprot (Q13148)]. Mutations were introduced at residue S332 and / or S333 in this region to generate 400 unique peptides. RaptorX was utilized (http: / / raptorx.uchicago.edu / StructurePropertyPred / predict / ) to predict the secondary structure properties of the peptides. RaptorX Property is an advanced web server that can predict structural properties of a protein sequence without relying on any template information. This server excels in performance, particularly for proteins that lack closely related sequences in the Protein Data Bank (PDB) or have limited sequence profile information available. The peptides were then ranked based on their helical propensity, and this list was refined further based on helical penalty score and disorder prediction (Tables 3 and 4). The five top-ranked peptides with the highest helical propensity were selected for further analysis.

[0397] Ab initio 3D Structure Prediction and Preparations:

[0398] To validate sequence based secondary structural properties, the top five peptide hits with highest helical propensity were selected for 3D-structure prediction and further analysis. Peptide structures were predicted using comparative modeling with Rosetta comparative modeling (RosettaCM method). Wild- type TDP-43 amyloidogenic core region solution structure (PDB ID: 2N3X)42 was used as a template for the comparative modeling. This region spans 50 amino acids and encompasses residues 311-360 of the TDP-43 C-terminal LCD. The residues from 311-320 (MNFGAFSINP (SEQ ID NO:57)) and 344-360 (QNQSGPSGNNQNQGNMQ (SEQ ID NO:58)) are disorder coils, residues 321-330 (AMMAAAQAAL (SEQ ID NO:59) and 335- 343 (GMMGMLASQ (SEQ ID NO:60)) are known as conserved helical regions. The predicted peptide structures were prepared for computational approaches using the Preparation wizard of Schrodinger as described.

[0399] Docking Studies

[0400] Docking analysis was undertaken with the top five peptide candidates and amyloid-like filament structures (PDB ID: 7KWZ, 7PY2, 6N37). This study was facilitated using the locally installed HDOCK software package, following standard procedures. Compared to other molecular docking tools, the HDOCK server is a comprehensive tool for protein-protein docking that integrates homology search, template-based modeling, structure prediction, and macromolecular docking functions. It uniquely allows for amino acid sequences as inputs and employs a hybrid docking algorithm combining template-based and template-free docking. This hybrid approach integrates experimental data about protein-protein binding sites and small-angle X-ray scattering data. Compared to other docking tools, such as HADDOCK and Patch dock, HDOCK is adept at handling novel peptides and mutated sequences where limited structural information is available. Therefore, HDOCK was well-suited for the chosen experimental approach. Based on the positions of peptides, docking scores, and favorable interactions with amyloid-like fibrils, the top complex poses were selected for molecular dynamics simulation.

[0401] Molecular Dynamics Simulation

[0402] Molecular dynamics simulations were performed utilizing GROMACS 2021.3, complemented by PLUMED 2 for advanced analysis. Numerous studies comparing the effects of different force fields on simulating monomeric and fibrillar forms of aggregation prone proteins were considered. Among the primary force fields frequently used in disordered protein and fibril experiments AMBER99SB- ILDN, CHARMM22, and GROMOS43al the GROMOS43al force field was adopted based on several factors: (i) GROMOS43al, similar to AMBER99SBILDN, has demonstrated exceptional stability in maintaining fibril structures, a crucial aspect for our study. This stability is in contrast to force fields like CHARMM36M, which have shown tendencies to allow dissociation of edge chains in fibril systems, (ii) The capability of GROMOS43al to accurately simulate intramolecular CH -71 interactions was a significant factor in its selection. These interactions are vital in the transition from monomeric to fibrillar forms, which is a key process in understanding helical propensity peptide conformation, (iii) GROMOS43al shows a propensity for supporting P-strand-like structures. Taking into account these factors, the GROMOS43al force field was selected for our protein modeling and simulation work. Water molecules were represented using the SPC model. The simulations focused on both individual monomers and complexes, including fibrils and monomers. Each was centrally placed in a truncated octahedral box, maintaining a minimum distance of 1.2 nm from the protein to the box wall. The box, with its narrowest cross-section being 8.09 nm, was populated with water molecules; Na+ ions and NaCl were introduced to replicate physiological ionic conditions at 150 mM. The systems underwent initial relaxation through the steepest descent method. This was followed by NVT and NPT equilibrations maintained at 310 K and 1 bar, employing velocity-rescaling and the Parrinello-Rahman methods.78 Bond lengths were constrained using LINCS79 for proteins and SETTLE80 for water molecules, applying a 2 fs integration step. Both electrostatic and van der Waals interactions were computed with a 1.2 nm cutoff, utilizing the PME method. For comprehensive analysis, two sets of simulations were conducted for the monomer and the complex, each spanning durations of 100 ns and 300 ns.

[0403] Analysis Methods

[0404] The Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and secondary structural properties were calculated from trajectory data. To analyze the free energy landscape (FEL) and generate 3D figures, the following steps were employed. First, the structural coordinates, including RMSD, and Radius of Gyration (RG), helical probability and beta sheet probability, were extracted from trajectory data. Next, input files for Sham analysis were generated using the "sham.pl" Perl script. The Sham analysis was then performed using the "gmx sham" command, which produced the free energy landscape stored in the "free-energy- landscape.xpm" file. To further analyze, the "FEL.xpm" file was converted to a text format using the "xpm2txt.py" Python script. Finally, Mathematica 12.1, graphpad prism software were used to visualize the FEL, allowing for the generation of informative 3D figures. These methods provided a robust approach for investigating the free energy landscape and visualizing the results for comprehensive analysis. To predict the intramolecular interaction energy, Interaction energy matrix tools were used. Multiple structural conformations from various snapshots were retrieved from molecular dynamics trajectory data and used for predicting the intramolecular interaction energies. Statistical tests were performed in GraphPad Prism. A two-tailed t test (for normally distributed data) or Mann-Whitney U test (for non-normally distributed data) was used to compare two groups. One-way analysis of variation with Tukey’s post hoc test was used to compare multiple groups of normally distributed data. The Kruskal-Wallis test with Dunn’s correction was performed to compare multiple groups of non-normally distributed data

[0405] Results

[0406] Computational Design Pipeline

[0407] The wild-type sequence of TDP-43 (residues 311-360) was retrieved from the Uniprot database (QI 3148) and generated a library of unique peptides each with a length of 50 residues. Given the helix destabilizing effects of serine residues 332 and 333,50 peptides with modifications of these polar residues were focused on. Helical secondary structure is characterized by robust intramolecular interactions; therefore, the substitutions at S332 and / or S333 could potentially increase interactions that contribute to the stability of the helical domain. This led to concentrate on 400 distinct mutant peptide variants with diverse secondary structural properties. RaptorX, an ultra-deep convolutional residual neural network that makes structure predictions from the primary sequence, was used to predict the secondary structure of each variant in our peptide library. Using this approach, a ranked list of peptides was generated according to their helical propensity; this list was refined further based on helical penalty scores, disorder prediction, and amino acid biochemical properties peptides with substitutions of S332A / S333W, S332R / S333L, S332A, S332R, and S333L which exhibit enhanced helical propensity compared to the wild-type amyloidogenic core peptide sequence and low helical penalty scores were selected. Considering that the TDP-43 C- terminal domain is predominantly disordered, a few variants, such as S332V / S333V and S332V, that are more ordered compared to wild-type but have only slightly increased helical propensity scores were also included. These top peptide candidates were analyzed further with 3D- modeling, docking analysis, and molecular dynamics.

[0408] Increased intramolecular interactions stabilize the helical structure in top hit peptide variants

[0409] To gain a deeper understanding of the structural characteristics of the top peptide hits, Rosetta comparative modeling (RosettaCM) was used to generate 3D-models of backbone and side-chain atom topologies. The predicted 3D structures were used to analyze the number and stability of intramolecular interactions, structural behavior, and differential energy profiles at each residue (Figures 2A-2B). Fig. 2A depicts the 3D structural coordinates of the top peptide hits, highlighting their increased helical propensity compared to the wild-type model. In order to understand the basis of enhanced helical structure of top peptide hits, the number of hydrogen bonds and intramolecular interaction energies for each peptide were analyzed. Notably, the helical propensity variants displayed an increased number of intramolecular hydrogen bonds compared to the wild-type peptide (Fig. 2A). Overall, 22 hydrogen bonds were maintained in the wild-type helical domain, whereas >30 hydrogen bonds (range 30-37) were maintained in the helical propensity peptides’ helical domain (Table 1).

[0410] Beyond the analysis of hydrogen bonds, the investigation sought to comprehensively assess the various energy interaction types within these peptides by utilizing an interaction energy matrix. This encompassed a comprehensive prediction of intramolecular interaction energies, including all categories of covalent, polar, and non-polar interactions. This analysis demonstrated distinct values within the helical domain (residues 320-340) for the helical propensity variants and the wild-type peptide. Specifically, the helical propensity variants exhibit an average energy of -96.119 kJ / mol, while the wild-type counterpart displays an average energy of -57.8 kJ / mol (Fig. 2B). These findings suggest increased intramolecular energy observed in the helical propensity variants plays an important role in maintaining the stability of the helical structure. Upon closer examination, it was observed that the substitutions at S332 and / or S333 residues contributed a marked increase in the intramolecular interaction energy. Specifically, mutations to S332A / S332W yielded an intramolecular interaction energy of -90. 14 kj / mol, whereas the wild-type model exhibited less intramolecular interactions at S332 and S333 (-33.2 kj / mol) (Fig. 2B). These data indicate that alterations in the serine residues significantly influence the intramolecular interaction characteristics in the helical region (residues 320-340). These observations also suggest the mutated serine residues in the top peptide hits demonstrate a more favorable intramolecular interaction energy profile with neighboring residues, thus contributing robust interactions that stabilize their helical secondary structure.

[0411] Table 1: No. of hydrogen bonds and contributing atoms in helical domain for Wt and helical propensity variants. Analysis of Helical Propensity peptide inhibitor stability through Molecular Dynamics Comprehensive molecular dynamics (MD) simulations were performed to validate the folding and conformational stability of top hit peptides and to gain insight on their dynamic behavior and interplay among constituent residues, compared to the wild-type peptide. MD simulations were conducted by using GROMACS (version 2021). For the initial equilibrium system, Optimized GROMOS43al force field was employed in conjunction with the Simple Point-Charge (SPCE) water model. The wild-type and top helical propensity monomer structures were analyzed for up to 100 ns to understand the time evolution of their structural characteristics (Figs. 3A-3B), intramolecular interactions (Fig. 4), and free energy properties (Fig. 5). Root mean square deviation (RMSD) analysis was employed to assess the stability and conformational changes of the proteins. Generally, the lowest RMSD values indicate a more stable conformation, while higher or fluctuating RMSD values suggest structural changes. Notably, the helical propensity variants exhibited lower RMSD values compared to the wild-type model throughout the simulation period (Fig.3A), suggesting their enhanced stability. Hydrogen bonds are an important source of intramolecular interactions that maintain stable helical structure. Therefore, the total number of hydrogen bonds in the helical domain (320-340) were also examined for both the wild-type and helical propensity variants. The wild-type model exhibited an average of 10 to 15 hydrogen bonds, whereas the helical propensity variants maintained 15 to 20 hydrogen bonds over the simulation period (Fig 3B). Furthermore, the secondary structure properties were determined for the helical propensity peptides throughout the simulation period. Secondary structural analysis of the wild-type model revealed an unstable helical segment spanning residues 327 to 340, that transitioned into loop structures (Fig 3C). In contrast, the variants displaying enhanced propensity for helical formation, specifically S332A / S333W, S332R / S333L, and S333L, maintained stable secondary structures characterized by well-defined helical segments throughout the simulation. S332V / S333V and S332V variants that were more ordered but had slightly lower helical propensity scores, showed some fluctuations to coil and turn structures but still displayed more helical stability than the wild type-model. Combining all of these results, MD data indicate that substitutions of serine residues in our top peptide hits substantially enhance helical conformational stability, in part by contributing robust intramolecular hydrogen bonds that stabilize their helical secondary structure, pairwise interactions between the residues including TDP-43’s amyloidogenic core region were next examined (Figs. 4A-4B). In the wild-type peptide, 25 distinct interactions between helical domain residues and residues outside of the helical region were observed during the 100ns simulation. In particular, S332 and W334 residues within the helical domain interact with G357 and N358 residues outside of the helical domain (Fig. 4A, small dotted box). These observations underscore the relative lack of helix-stabilizing interactions between S332 and neighboring residues within the helical domain. These findings are also consistent with prior studies which show that S332 and W334 participate instead in helix- destabilizing interactions that initiate helix to beta sheet conversion.42,49 In contrast, pairwise interactions for the S332A / S333W helical propensity peptide are most robust within the helical regions and contribute to stable intramolecular interaction energies (Fig. 4B, larger dotted box). Thus, relative to S332A / S333W peptide, the wild-type peptide exhibits an elevated frequency of crosstalk interactions between helical domain residues and residues outside of this region, which contributes to destabilization of a-helical secondary structure. Next, thermodynamic stability of the wild-type and top two helical propensity peptides was predicted over the simulation period. From the MD simulations, Gibbs free energy heatmaps were generated (Fig. 5). distinct clusters were found representing different conformational states of the peptides, with cooler blue colors indicating relatively stable states. The helical propensity variants S332A / S333W and S333L, exhibited stable helical conformations throughout the simulation, as indicated by the favorable Gibbs free energy values within their respective clusters. In contrast, the wild-type model showed structural changes at 1.5 A RMSD, at which point it reached an unstable state with reduced free energy. This instability was mainly attributed to the presence of polar residues, such as S332 and S333, in the helical domain, resulting in fewer hydrogen bonds and unstable intramolecular interaction energy. Conversely, the helical propensity variants maintained stable conformations with stable intramolecular interaction energies (Fig. 2B;)

[0412] The Role of Serine 332,333 Residues in TDP-43 Fibrillization: Insights into the Importance of Neighbor Interactions in the Helical Domain

[0413] The data (Figs. 3A-3B, 4A-4B) agree with previous investigations pinpointing interresidue interactions, particularly interactions involving S332 and W334, that contribute to structural conformational changes of TDP-43 ’s amyloidogenic core and subsequent fibril formation. It has been suggested that the lack of intramolecular interactions between S332 and neighboring residues has a potential impact on fibril initiation.49 SSNMR with proton detection also revealed a close interaction between the indole Nel-He I of W334 and the side-chain carbonyl of Q343. Given the fundamental requirement of 3 to 4 residues for sustaining a single turn within the helical structural motif, how substitutions of S332 and / or S333 impact the cumulative intramolecular interaction energies between those residues and their three neighboring residues in the helical domain (e.g. S332 / 3 with A329, L330, Q331, W334, G335, and M336) were investigated. Over the course of a 100ns simulation period, it became evident that S332A / S333W and S333L substitutions resulted in more favorable (more negative) cumulative intramolecular interaction energies compared to the wild-type model (Fig. 5). These findings support the view that robust intramolecular neighbor residue interactions seen with S332A / S333W and S333L substitutions likely contribute to strong helix-stabilizing interactions in our top hit helical propensity peptides. To test this idea further, the probability of helical secondary structure was analyzed for wild-type and the helical propensity peptide monomers throughout the simulation period. It was found that the wild-type model has reduced helical probability compared to our top hit peptides, the residue- wise probability (i.e., helical probability at each residue) of the wild-type and helical propensity peptides were also plotted (Fig. 6A). It was found that S332A / S333W, S333L, and S332V / S333V peptides display higher helical probability at S332 / 3 neighbor residues (331 to 335) compared to the wild-type model. Therefore, this probability data provides additional evidence that serine residues in the helical domain play a major role in helix destabilization and / or initiating misfolding. Based on these findings, the S332A / S333W helical propensity variant was focused on for further analysis, including docking studies and complex simulations with amyloid-like fibrils.

[0414] Efficiency of Helical Propensity Peptide Binding with TDP-43 Amyloid-like Structures

[0415] Binding efficacy of the S332A / S333W helical propensity peptide bound to TDP-43 amyloid- like fibrils was evaluated using three distinct amyloid-like filament crystal structures of TDP-43. The first structure was derived from aggregated TDP-43 located in the frontal and motor cortices of a patient diagnosed with ALS / FTLD. Two additional structures were elucidated through crystallographic analysis. Each of these structures displays unique beta sheet conformations. The patient-derived structure 7PY2 contains ten short beta sheets, with the longest one extending from serine 332 to leucine 340. Meanwhile, structures 7KWZ and 6N37 exhibit ten and three beta sheets, respectively, each adopting distinct beta sheet conformations. Interestingly, the helical domain within these structures forms a lengthybeta sheet conformation. As indicated by prior studies, the transition step of the helical domain serves as the initial phase of aggregate formation. When taking into account these previous findings and the patient- derived and crystal-solved structures, it suggests that helical domains participate in forming the most elongated, stable beta sheets within these three structures. Therefore, when these reference TDP-43 fibrillar structures are complexed with our top hit helical propensity peptides, it was predicted that the S332A / S333W peptide inhibitor will bind amyloid-like fibrils but resist structural transformation, compared to the wild-type peptide; furthermore, it should prevent monomer addition of TDP-43 CTD fragments to amyloid-like fibrils. Previous findings have suggested that TDP-43 CTD needs interacting partners (additional C-terminal fragment) to undergo fibrillation. As a result, in our MD simulation results with only monomers, stable beta sheet formation was not observed (Figs. 3A-3B). Therefore, top helical propensity peptides and the wild-type model bound to several crystal structures of C-terminal TDP-43 fibrils, including 7KWZ, 7PY2 and 6N37 were tested. The amyloid-like fibrils and peptides were docked using HDOCK, a protein-protein docking tool that allows amino acid sequences to be used as inputs and combines homology search, template-based modeling, structure prediction, and macromolecular docking functions. The docking analysis revealed that the helical propensity variants and wild-type peptide achieved similar scores (range: -685.51 to -759.83 kcal / mol) (Table 2). Importantly, for each TDP-43 fibril structures that were tested, the S332A / S333W helical propensity peptide binds to the helical domain residues (320-340), which participate in beta sheet formation. These data suggest the S332A / S333W peptide has favorable binding affinity with TDP-43 amyloid-like fibrils, and has potential to act as a TDP-43 C-terminal fragment mimetic. To further investigate stability and structural changes between the complexes, top poses with favorable interactions were selected for the analysis.

[0416] Table 2: Docking scores of higher helksl propensity variants with Amyloid like fibrils of TDP-43

[0417] Helical propensity peptides maintain helical structure and resist beta-sheet transformation when bound to TDP-43 fibrils

[0418] As a next step in testing our top peptide inhibitors, it was decided to assess the structural stability of helical propensity peptides after binding to TDP-43 amyloid-like fibrils. From this analysis, it was intended to find the probability of helix to beta sheet transformation of (i) peptide monomers and (ii) peptides bound to TDP-43 amyloid-like fibrils. To elucidate the molecular interaction between the complexes, top poses from the docking analysis were used for MD simulation. To minimize bias of the starting position, simulations were carried out for 100 ns (monomer) and 300ns technical replicates were performed for increased reliability. The simulation results indicate that the peptide / fibril complex formed a stable structure and maintained favorable interactions with TDP-43 fibrils. In the monomer state, the S332A / S333W helical propensity showed a greater than two-fold increase in helical probability compared to the wild-type peptide (Figs. 6A, 6B). As mentioned previously, monomers do not readily fibrillize, but it was still found that the S332A / S333W peptide shows a small decrease in beta sheet probability compared to the wild-type monomer state (Figs. 6C, 6D). Consistent with previous studies of the known amyloidogenic core, the wild-type peptide bound fibril model showed a significant increase in beta sheet probabilities and a corresponding reduction in helical probability (Figs. 6E-6H). Residue-wise analysis showed that S332 and neighbor residues, such as 334 to AA’s, had a marked increase in beta sheet probability and decreased helical probability (Figs. 6E, 6G). On the other hand, the helical propensity peptide (S332A / S333W) maintains stable interaction with neighbor residues, and very low values for beta sheet probability were found in both monomer and complex states (Figs. 6D, 6H). To gain a structural view, snapshots of different time points from the MD simulations were examined for complexes of the wild-type peptide or S332A / S333W peptide with 7KWZ (peptide-7KWZ complexes). The data indicates that the wild-type model bound with amyloid like fibrils is unable to maintain helical conformations, and most of the residues from the helical domain transition to loops or beta sheets. In contrast, the helical propensity peptides maintained the helical conformation throughout the simulation period, some conformational changes in the helical propensity peptides were also observed during the simulation, suggesting their flexibility and potential for adopting different conformations.

[0419] Promising Capping of TDP-43 Amyloid-Like Fibrils by Helical Propensity Peptides Finally, the thermodynamic stability of complexes of either wild-type or helical propensity peptide bound to TDP-43 amyloid-like fibrils were tested. To perform this analysis, the free energy landscape was predicted as a function of time on the x-axis and plotted helical or beta sheet probabilities on the y-axis. To understand whether helical propensity peptides bound to TDP-43 fibrils display more thermodynamically stable helical secondary structure than the wild-type peptide / fibril complex, it was found that the S332A / S333W peptide exhibits a higher, more stable helical probability in the complex state (45-50%), with more favorable (dark blue) free energy clusters, compared to the wild-type peptide / fibril complex. Moreover, beta sheet probability of the S332A / S333W peptide / fibril complex is extremely low throughout the simulation period, with minimal favorable free energy clusters. As was expected, the wild-type peptide / fibril complex displays increasing beta sheet probability as the simulation progresses, and reaches -20% beta sheet probability within favorable free energy clusters by 250-300 ns. Overall, the data suggest that the S332A / S333W helical propensity peptide has strong binding efficiency with amyloid like fibrils, forming a thermodynamically stable complex structure, while still maintaining stable helical propensity. Thus, this rationally designed helical propensity peptide displays promising features, as it has potential to bind and cap TDP-43 amyloid like fibrils and act as a mimetic of TDP-43 ’s amyloidogenic core region but resists beta sheet transformation. Overall, the study provides insights into the binding mechanism and stability of TDP-43 C-terminal fragment peptide mimetics, which could be useful for developing new therapies combating TDP-43 proteinopathies.

[0420] Conclusion

[0421] By utilizing advanced in-silico techniques a new direction for the development of therapeutic strategies against TDP-43 aggregation-related disorders, such as ALS and FTD was uncovered. This work specifically targets the destabilization of the a-helical domain in the C- terminal region of TDP-43, a process that is important to the formation of pathological amyloid- like filaments. These helical domains act as crucial sentinels, effectively safeguarding against the formation of amyloid-like fibrils. Whether the intrinsic flexibility of TDP-43 ’s a-helical region, which is split into two short helices, plays a physiological role is likely, and to ensure that the described helical propensity peptides do not disrupt endogenous TDP-43 splicing function, mRNA transport, or hinder important protein-protein interactions should be considered.

[0422] The helical peptides highlighted in these computational analyses, notable for their binding affinity and improved stability when complexed with TDP-43 amyloid-like fibrils, show potential to bind and cap the growing tips of filaments. Validated through investigations of target engagement, efficacy, and specificity in vitro and in vivo, these peptides will bring the field closer to developing novel therapeutic strategies for TDP-43 aggregation-related disorders. The potential of in silico methodologies to accelerate the discovery of therapeutic leads, represents a significant contribution to the field of drug discovery for neurodegenerative diseases. These findings underscore the prospect of targeting the a-helical domain in TDP-43 to prevent the formation of pathological filaments, a strategy that could potentially revolutionize the treatment of conditions such as ALS and FTD. Example 2: Validation of target engagement, efficacy, and specificity of TDP-43 Derived a-Helical Peptide Inhibitors in vitro.

[0423] Methods

[0424] Cellular TDP-43 aggregation assay

[0425] To test whether a helical variant C-terminal TDP-43 is able to effectively reduce TDP-43 aggregates in mammalian cells, a TDP-43 aggregation assay was developed. mScarlet-tagged wild type TDP-43 C-terminal plasmid was co-transfected with either (1) GFP-control plasmid, (2) GFP-tagged wild type TDP-43 C-terminal TDP-43, or (3) GFP-tagged helical variant C- terminal TDP-43 plasmid into Hela TDP-43 KO Cells. Post 16 hours of transfection, HeLa cells were fixed 4% PFA in PBS for 12-15 minutes, washed three times in PBS, Samples were then mounted in ProLong Gold (Life Technologies) and imaged on an ZEISS LSM900 63X to quantify the number and size of TDP-43 aggregates. See Figures 7A and 7B.

[0426] Results

[0427] HeLa TDP-43 knockout (KO) cells co-expressing either: GFP (control) and mScarlet- TDP-43 CTD wild-type (WT) (274-414 AA); GFP-TDP-43 CTD WT and mScarlet-TDP-43 CTD WT; or GFP-tagged helical variant TDP-43 CTD (either S332A / S333W or S332R / S333L) and mScarlet-TDP-43 CTD WT were imaged with a Zeiss LSM 900 confocal, 60X objective lens, and the number of TDP-43 cytoplasmic aggregates per cell was quantified using an automated script in ImageJ / Fiji with reference to the number of TDP-43 aggregates per cell (n>20 cells) for each condition.

[0428] Robust colocalization of Cy5-tagged peptides and pTDP-43 aggregates was observed (Figure 7C and 7D).

[0429] After verifying target engagement, a similar experiment in which eGFP-TDP-43 ANLS cells were transduced (per protocol described above) with full length TDP-43 monomers, full length TDP-43 filaments, or proteinase K-treated full-length TDP-43 filaments + Cy 5 -tagged engineered peptide with functional PDMs (ALAPYIP (SEQ ID NO:24) or KFERQKILDQRFFE SEQ ID NO:25)) or control non-functional PDMs.

[0430] The size and number of pTDP43 aggregates in cells receiving top hit engineered peptides were quantified. Treatment with engineered peptides having functional PDMs resulted in fewer phospho-TDP-43 cytoplasmic aggregates, compared to engineered peptides with non-functional PDMs, and also compared to cells receiving the wild type amyloidogenic core peptide.

[0431] Next, the effect of expression of helical propensity variant S332A / S333W on endogenous nuclear TDP-43 expression was examined. To this end, wild type HeLa cells expressing GFP- tagged TDP-43 WT C-terminal domain (CTD) (residues 274-414), or helical propensity variant CTD (S332A / S333W) were fixed and immunostained for endogenous TDP-43 using an N- terminal TDP-43 antibody (10782-2-AP, 1:500), and the Mean fluorescence intensity of endogenous nuclear TDP-43 for each condition is shown; ANOVA, **p-value <0.01. Results are depicted in Figure 8A. The data indicated that, unlike expression of the wild type TDP-43 amyloidogenic C-terminal domain (CTD), which aberrantly recruits endogenous TDP-43 (N- TDP-43) to the cytoplasm, expression of helical propensity variant S332A / S333W does not impact endogenous nuclear TDP-43 expression.

[0432] Finally, to determine whether the engineered helical peptides can be delivered successfully to the cytoplasm of cells, Cy5-tagged WT or helical peptide (1 pg, GenScript), was delivered into wild-type HeLa cells using BioPorter delivery agent, and the Cy5 fluorescence intensity was quantified in the nucleus and cytoplasm of cells receiving the Cy5 S332A / S333W peptide. Results are depicted in Figure 8B. The data indicated that engineered helical peptides can indeed be delivered successfully to the cytoplasm of cells and do not enter nucleus where the majority of endogenous physiologic TDP-43 resides.

[0433] Additional Experimental Concepts

[0434] To further validate the role of the PDMs in reducing / clearing cytoplasmic aggregates, follow-up experiments can be conducted in the presence or absence of proteasome or autophagolysosome inhibition. Cells can be treated with DMSO, MG132 (IpM), or pepstatin A (10 pM) for 16 hours. The inhibition of proteasome or autophago-lysosome activity may increase the number of pTDP-43 aggregates compared to DMSO controls. These data will demonstrate target engagement and the efficacy of the described engineered peptides and PDMs for reducing phospho-TDP-43 levels. These experiments will also determine the relative contribution of different mechanisms of action (i.e., whether the designed peptides reduce de novo formation of aggregates and / or the role of the proteasome vs autophago-lysosome pathways for clearing TDP- 43 aggregates).

[0435] Table 3: Final ranked list of Serine 332 and Serine 333 top peptide variants, based on helical propensity and penalty scores, biochemical properties, and disorder prediction

[0436]

[0437] Table 4: Predicted sequence based secondary structural properties for Serine 332 and

[0438] Serine 333 mutant variants at Helical domain 320-340 TDP-43 Salaikumaran and Gopal, “Rational Design of TDP-43 Derived a-Helical Peptide Inhibitors: an In-Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders”, bioRxiv 2023.10.26.564235; doi.org / 10.1101 / 2023. 10.26.564235 and

[0439] Salaikumaran and Gopal, “Rational Design of TDP-43 Derived a-Helical Peptide Inhibitors: An In Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders” ACS Chemical Neuroscience 15 (6) (2024), 1096-1109, DOI: 10.1021 / acschemneuro.3c00659, are specifically incorporated by reference herein in their entireties, including all supplementary and supporting data associated therewith.

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[0505] All references cited herein are specifically incorporated by reference herein in their entireties, including all supplementary and supporting data associated therewith.

[0506] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Although the description of materials, compositions, components, steps, techniques, etc. can include numerous options and alternatives, this should not be construed as, and is not an admission that, such options and alternatives are equivalent to each other or, in particular, are obvious alternatives. Thus, for example, a list of different gene targets does not indicate that the listed gene targets are obvious one to the other, nor is it an admission of equivalence or obviousness.

[0507] Every component disclosed herein is intended to be and should be considered to be specifically disclosed herein. Further, every subgroup that can be identified within this disclosure is intended to be and should be considered to be specifically disclosed herein. As a result, it is specifically contemplated that any component, or subgroup of components can be either specifically included for or excluded from use or included in or excluded from a list of components.

[0508] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:1 . An engineered TAR DNA Binding Protein 43 kDa (TDP-43) polypeptide comprising residues at positions 321-343 of mature human TDP-43, wherein the engineered TDP-43 polypeptide comprises one or more mutations of one or more amino acids of the mature human TDP-43 polypeptide, wherein(a) one mutation comprises substitution of the amino acid residue at position 332 of the human TDP-43 polypeptide; and / or(b) one mutation comprises substitution of the amino acid residue at position 333 of the human TDP-43 polypeptide, wherein the engineered binds to a TDP-43 aggregate; and wherein the engineered peptide precludes binding of further TDP-43 peptides to the aggregate.

2. The engineered TDP-43 polypeptide of claim 1 comprising alanine, or arginine, or valine at position 332 of the human TDP-43 polypeptide.

3. The engineered TDP-43 polypeptide of claim 1 or 2, comprising tryptophan, or leucine, or valine at position 333 of the human TDP-43 polypeptide.

4. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising leucine at position 333 of the human TDP-43 polypeptide.

5. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising valine at position 333 of the human TDP-43 polypeptide.

6. The engineered TDP-43 polypeptide of any one of claims 1-5, comprising alanine at position 332 of the human TDP-43 polypeptide.

7. The engineered TDP-43 polypeptide of any one of claims 1-5, comprising valine at position 332 of the human TDP-43 polypeptide.

8. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising alanine at position 332 and tryptophan at position 333 of the human TDP-43 polypeptide.

9. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising arginine at position 332 and leucine at position 333 of the human TDP-43 polypeptide.

10. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising valine at position 332 and valine at position 333 of the human TDP-43 polypeptide.

11. The engineered TDP-43 polypeptide of any one of claims 1-10, comprising the amino acid sequence AMMAAAQAALQXXWGMMGMLASQ (SEQ ID NO:51), wherein each X comprises, independently, serine, valine, tryptophan, arginine, alanine, or valine.

12. The engineered TDP-43 polypeptide of any one of claims 1-11, comprising the amino acid sequence AMMAAAQAALQAWWGMMGMLASQ (SEQ ID NO:52).

13. The engineered TDP-43 polypeptide of any one of claims 1-12, consisting of between 23 and 80 amino acid residues, optionally wherein the engineered TDP-43 polypeptide consists of 50 amino acid residues.

14. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising the amino acid sequence MNFGAF S INPAMMAAAQAALQAWWGMMGMLASQQNQS GP SGNNQNQGNMQ (SEQ ID NO:5), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:5.

15. The engineered TDP-43 polypeptide of claim 14, wherein the variant or fragment consists of an amino acid sequence set forth in any one of SEQ ID NOs.12-23 or 53.

16. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising the amino acid sequenceMNFGAF S INPAMMAAAQAALQRLWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:6), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:6.

17. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising the amino acid sequenceMNFGAF S INP AMMAAAQAALQASWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:7), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:7.

18. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising the amino acid sequenceMNFGAF S INP AMMAAAQAALQRSWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:8), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:8.

19. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising the amino acid sequenceMNFGAF S INP MMAAAQAALQWWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:9), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NO:9.

20. The engineered TDP-43 polypeptide of any one of claims 1-3, comprising the amino acid sequenceMNFGAF S INP MMAAAQAALQSVWGMMGMLASQQNQSGP SGNNQNQGNMQ (SEQ ID NO:10), or a variant or fragment thereof having at least 90% sequence identity to SEQ ID NOTO.

21. The engineered TDP-43 polypeptide of any one of claims 1-20, further comprising one or more additional function peptide motifs.

22. The engineered TDP-43 polypeptide of claim 21, wherein the additional function peptide motif comprises a peptide degradation motif (PDM), or a cell penetrating peptide(CPPM), or both a PDM and a CPPM.

23. The engineered TDP-43 polypeptide of claim 22, wherein the PDM comprises the amino acid sequence ALAPYIP (SEQ ID NO:24), KFERQKILDQRFFE (SEQ ID NO:25), LDPETGEYL (SEQ ID NO:54), RRRG (SEQ ID NO:55), MDFSGLSLIKLKKQ (SEQ ID NO:56) or a combination thereof.

24. The engineered TDP-43 polypeptide of claim 22, wherein the peptide comprises an amino acid sequence: MNFGAFS INPAMMAAAQAALQAWWGMMGMLASQQNQSGP SGNNQNQGNMQALAPYIP (SEQ ID NO:26).

25. The engineered TDP-43 polypeptide of claim 22, wherein the peptide comprises an amino acid sequence: MNFGAFSINPAMMAAAQAALQAWWGMMGMLASQQNQSGPSGNNQNQGNMQ KFERQKI LDQRFFE (SEQ ID NO:27).

26. The engineered TDP-43 polypeptide of any one of claims 23-25, wherein the CPPM comprises an amino acid sequence selected from the group consisting of LRKLRKRLL (SEQ ID NO:35); S SVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO:36);LRKLRKRLLR (SEQ ID NO:37); YTIWMPENPRPGTPCD IFTNSRGKRASNG (SEQ ID NO:38); GGGGYGRKKRRQRRR (SEQ ID NO:39); AGILKRW (SEQ ID NQ:40); H- CNCKAPETALCARRCQQH-NH2 (SEQ ID NO:41); H-DapKAPETALD-NH2 (SEQ ID NO:42); PWVP SWMPPRHT (SEQ ID NO:43); TGNYKALHPHNG (SEQ ID NO:44); THRPPMWSPVWP (SEQ ID NO:45); (PWVP SWMPPRHT ) 2 KKGK ( CF ) G (SEQ ID NO:46); HAYED (SEQ ID NO:47); CNSRLHLRC (SEQ ID NO:48); CENWWGDVC (SEQ ID NO:49); and WRCVLREGPAGGCAWFNRHL (SEQ ID NQ:50), or combinations of two or more thereof.

27. A nucleic acid comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1-26.

28. The nucleic acid of claim 27, wherein the nucleic acid is RNA or DNA.

29. The nucleic acid of claim 27, wherein the nucleic acid is mRNA.

30. The nucleic acid of claim 28, wherein the nucleic acid is, or is encoded by a vector or a transposon.

31. The nucleic acid of claim 30, wherein the vector is a viral vector.

32. The nucleic acid of claim 31, wherein the viral vector is selected from the group consisting of a lentiviral vector, an Adeno-associated virus (AAV) vector, or an adenovirus vector, or a Herpes Simplex virus (HSV) vector, or a vesicular stomatitis (VSV) vector, or ahuman Bocavirus vector (hBoV), or a chimeric vector comprising a combination of any two or more of a Adeno-associated virus (AAV) vector, Herpes Simplex virus (HSV) vector, vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV).

33. The nucleic acid of claim 31, wherein the vector is a nucleic acid expression vector selected from the group consisting of a plasmid, a cosmid, and a replicon.

34. The nucleic acid of any one of claims 27-33, comprising one or more of a promotor, a protein transduction domain, fusogenic polypeptide, or targeting signal conjugated thereto.

35. A delivery vehicle comprising the polypeptide of any one of claims 1-26, or the nucleic acid of any one of claims 27-34.

36. The delivery vehicle of claim 35, wherein the delivery vehicle comprises a nanoparticle, or a microparticle.

37. The delivery vehicle of claim 36, wherein the polypeptide of any one of claims 1-26, or the nucleic acid of any one of claims 27-34 is encapsulated within or conjugated to the nanoparticle or microparticle.

38. The delivery vehicle of any one of claim 35-37, wherein the delivery vehicle is selected from the group consisting of a polymeric particle, a viral particle, a liposome, a nucleic acid conjugate, and a metallic particle, or a combination thereof.

39. The delivery vehicle of any one of claims 35-38, further comprising a targeting motif that targets the delivery vehicle to the brain and / or the central nervous system (CNS) in vivo.

40. The delivery vehicle of claim 39, wherein the targeting motif comprises a peptide that facilitates passage across the blood-brain barrier (BBB).

41. The delivery vehicle of claim 39 or 40, wherein the targeting motif comprises a peptide having an amino acid sequence set forth in any one of SEQ ID NOs.35-50.

42. A cell comprising the polypeptide of any one of claims 1-26, or the nucleic acid of any one of claims 27-34, or the delivery vehicle of any one of claims 35-41.

43. A pharmaceutical composition comprising the polypeptide of any one of claims 1-26, or the nucleic acid of any one of claims 27-34, or the delivery vehicle of any one of claims 35- 41, or the cell of claim 42, and a pharmaceutically acceptable buffer, carrier, diluent or excipient.

44. A method of treating a subject having a disease, disorder, or condition comprising administering to the subject an effective amount of the pharmaceutical composition of claim 43.

45. A method of treating a subject having a disease, disorder, or condition associated with pathological aggregation and / or mis-localization of TDP-43 aggregates in the brain or central nervous system (CNS) of the subject comprising administering to the subject the pharmaceutical composition of claim 43.

46. The method of any one of claims 44-45, wherein the subject is a human.

47. The method of any one of claims 44-46, wherein the subject has a disease selected from the group consisting of Amyotrophic Lateral Sclerosis (ALS), Frontotemporal disorders (FID), and a genetic disorder.

48. The method of any one of claims 44-46, wherein the pharmaceutical composition is administered to the subject via a route selected from the group consisting of intravenous, intramuscular, intracranial, intraosseus, intranasal, intrathecal, intraventricular, intraparenchymal and intracerebroventricular administration.

49. The method of claim 48, wherein the pharmaceutical composition is administered to the subject via intracerebroventricular injection.

50. The method of claim 48, wherein the pharmaceutical composition is administered to the subject via intrathecal injection.

51. A method of treating Amyotrophic Lateral Sclerosis (ALS) in a subject, comprising administering to the subject a pharmaceutical composition comprising(i) an engineered TDP-43 polypeptide comprising an amino acid sequencePAMMAAAQAALQAWWGMMGML (SEQ ID NO:61 ), and / or PAMMAAAQAALQRLWGMMGML (SEQ ID NO:63); or(ii) a nucleic acid encoding a polypeptide comprising an amino acid sequence comprising SEQ ID NO:61, and / or SEQ ID NO:63, in an amount effective to reduce or prevent one or more symptom of ALS in the subject.

52. A method of treating Frontotemporal disorders (FTD) in a subject, comprising administering to the subject a pharmaceutical composition comprising(i) an engineered TDP-43 polypeptide comprising an amino acid sequencePAMMAAAQAALQAWWGMMGML (SEQ ID NO:61), and / or PAMMAAAQAALQRLWGMMGML (SEQ ID NO:63); or(ii) a nucleic acid encoding a polypeptide comprising an amino acid sequence comprising SEQ ID NO:61, and / or SEQ ID NO:63, in an amount effective to reduce or prevent one or more symptom of FTD in the subject.

53. A method of reducing, delaying or preventing pathological aggregation and / or mislocalization of TDP-43 aggregates in the brain or central nervous system (CNS) of a subject, comprising administering to the subject a pharmaceutical composition comprising(i) an engineered TDP-43 polypeptide comprising an amino acid sequencePAMMAAAQAALQAWWGMMGML (SEQ ID NO:61), and / or PAMMAAAQAALQRLWGMMGML(SEQ ID NO:63), or(ii) a nucleic acid encoding a polypeptide comprising an amino acid sequence comprising SEQ ID NO:61, and / or SEQ ID NO:63, in an amount effective to reduce or prevent pathological aggregation and / or mis-localization of TDP-43 aggregates in the brain or central nervous system (CNS) of a subject in the subject.

54. The method of any one of claims 44-53, wherein the polypeptide, or nucleic acid encoding the polypeptide, is delivered to the cytoplasm of cells of the subject.

55. The method of any one of claims 44-54, wherein, wherein the engineered (TDP-43) polypeptide does not alter the nuclear expression of endogenous TDP-43 in the subject.

56. An engineered TDP-43 polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:61-369 or 371-462.

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