Modified Polypeptides and Uses Thereof

Abstract

Modified cyclin F polypeptides and nucleic acid molecules encoding same are provided herein. In particular, modified cyclin F polypeptides having increased cytoplasmic targeting compared to wild-type cyclin F polypeptides, and functional truncated modified cyclin F polypeptides, and nucleic acid molecules encoding them are provided herein. Also provided herein are uses of modified cyclin F polypeptides and encoding nucleic acid molecules for enhancing motor neuron survival, inhibiting motor neuron degeneration, and treating neurodegenerative conditions, particularly neurodegenerative conditions associated with neuronal TDP-43 proteinopathy.

Description

[Technical field]

[0001] The present disclosure generally relates to modified cyclin F polypeptides and nucleic acid molecules encoding same. In some aspects, the present disclosure relates to modified cyclin F polypeptides having increased cytoplasmic targeting compared to wild-type cyclin F polypeptides. The present disclosure also relates to the use of modified cyclin F polypeptides and encoding nucleic acid molecules to enhance motor neuron survival, inhibit motor neuron degeneration, and treat neurodegenerative conditions. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND) and refers to the selective degeneration of upper and lower motor neurons in the brain and spinal cord, respectively. ALS and frontotemporal dementia (FTD) lie within a spectrum of diseases, with 15% of ALS patients also exhibiting symptoms of FTD, the second most common form of early-onset dementia. Although the pathogenesis of ALS and FTD remains poorly understood, most ALS patients and more than half of all FTD patients share common histopathological features. Postmortem analysis of brain and spinal cord tissue from ALS patients often reveals the presence of tau-negative, ubiquitin-positive aggregates, which most commonly appear as round or skeletal-like inclusions in the cytoplasm of affected neurons and glia. These inclusions are speckled and contain ubiquitin, sqstm1, ubiquilin1, and ubiquilin2, all proteins involved in ubiquitin-mediated protein metabolism, suggesting that defective proteasomal clearance is a contributing factor in ALS / FTD pathogenesis. In 2006, the major component of these inclusions was identified as Transactive Response DNA binding protein of 43 kDa (TDP-43), a primarily nuclear protein found to translocate from the nucleus to the cytoplasm in cases of ALS / FTD. Characterization of sarkosyl-insoluble fractions from patient brain lysates reveals a striking shift in the biochemical profile of TDP-43. In patient lysates, TDP-43 is polyubiquitinated, hyperphosphorylated, and truncated at the C-terminus. TDP-43 proteinopathy is now identified in more than 98% of ALS cases and more than 50% of FTD cases, regardless of familial or sporadic origin, with TDP-43-positive aggregates or inclusions becoming a hallmark of the disease.

[0003] In contrast to its insoluble aggregated pathological form, soluble TDP-43 (sTDP-43) is required for normal cellular function. In this regard, it is involved in several mechanisms of mRNA metabolism, such as pre-mRNA splicing, mRNA stability, mRNA transport, and miRNA processing, and is necessary for neuronal survival. Under normal conditions, the subcellular localization of sTDP-43 is primarily nuclear, but the presence of a nuclear localization sequence (NLS) and nuclear export sequence at the N-terminus of the protein allows sTDP-43 to shuttle between the nucleus and the cytoplasm. sTDP-43 is also known to regulate mRNAs involved in neuronal and embryonic development, and is expressed throughout CNS development into adulthood. It is therefore understood that sTDP-43 is an essential RNA-binding protein, and alterations in its ability to perform its cellular roles are toxic to neuronal cells.

[0004] Familial ALS (fALS) mutations account for 5-10% of all ALS cases, while the remaining cases have no clear cause (sporadic ALS, sALS). Although familial gene mutations account for a minority of ALS cases, they have provided valuable insights into the mechanisms underlying the disease. Thus, mutations have been identified in a number of genes, including SOD1, VCP, TARDBP, FUS, OPTN, SQSTM1, UBQLN2, MATR3, and TBK1. An interesting mutation in TARDBP, the gene that codes for TDP-43, is found in only about 4% of fALS patients and about 1% of sALS cases.

[0005] Strong evidence exists that the intracellular location of TDP-43 within motor neurons is central to the neurodegenerative phenotype. For example, abnormal cytoplasmic accumulation of TDP-43 (insoluble aggregates) is a pathological hallmark of ALS (98% of cases) and FTD (>50%). In 2015, transgenic mice were generated with inducible overexpression of a human TDP-43 variant that specifically mislocalizes in the cytoplasm (the variant is referred to as dNLS-TDP-43). When overexpressed, dNLS-TDP-43 mice develop a rapid ALS-like phenotype, resulting in motor paralysis and death. The dNLS-TDP-43 mouse represents an experimental model of sporadic ALS / FTD because it specifically causes cytoplasmic mislocalization TDP-43 reminiscent of sporadic disease.

[0006] ALS / FTD-associated mutations have been identified in CCNF, which occur with a frequency similar to that found in TARDBP. CCNF is a multiprotein Skp1-Cul1-F box (SCF Cyclin F ) encodes cyclin F, the ligand-binding component of the E3 ligase. Within this SCF complex, cyclin F (an F-box protein) is involved in the recruitment and positioning of substrates for polyubiquitination followed by their proteasomal degradation. To date, cyclin F activity has been largely linked to cell cycle progression and DNA damage, as it mediates the ubiquitination of ribonucleoside diphosphate reductase subunit M2 (RRM2), nucleolus and spindle associated protein 1 (NuSAP), centrosomal coiled-coil protein of 110 kDa (CP110), cell division control protein 6 homolog (CDC6), histone RNA hairpin-binding protein (SLBP) exonuclease 1 (exo1), and fiji-related protein homolog (Fzr1). Cyclin F is also known to bind and modify the mitotic transcription program of myb-related protein B (B-Myb). Importantly, all of these studies report nuclear localization of cyclin F, consistent with its function as a cell cycle regulatory protein.

[0007] Our previous studies showed that (1) TDP-43 binds to SCF Cyclin F We have found that cyclin F is an interacting partner and substrate of the complex, (2) deficiency of cyclin F leads to accumulation of TDP-43 in motor neurons, (3) a subset of patients with neurodegenerative conditions have abnormally low levels or activity of cyclin F in motor neurons, and that increasing cyclin F levels in motor neurons in this subset of patients can reduce the abnormal accumulation of the protein, thereby enhancing survival of the motor neurons, and (4) by supplementing neurons with additional cyclin F, it is possible to enhance survival of neurons, including motor neurons, that have normal levels of endogenous cyclin F (see, e.g., WO2018 / 081878 and PCT / AU2020 / 051133).

[0008] We also identified a serine to glycine substitution at position 621 (S621G) of cyclin F in a multigenerational Australian family with ALS / FTD that causes overactive ubiquitination of TDP-43 and other substrates (Lee et al., 2017). Taken together, this indicates that (i) cyclin F activity is tightly regulated for the maintenance of proper activity of ubiquitination-dependent protein degradation pathways, and that dysregulation leading to low levels or overactive activity of cyclin F impairs these pathways and triggers neurodegenerative diseases such as ALS and FTD, and (ii) that cyclin F can be used as a therapy to enhance neuronal survival, including motor neuron survival, for the treatment of neurodegenerative diseases, including familial and sporadic neurodegenerative diseases favorably associated with TDP-43 proteinopathy, regardless of endogenous cyclin F levels or activity in neurons and / or when neurons do not have reduced levels or activity of endogenous cyclin F compared to controls. Thus, there is a need for cyclin F polypeptides that are optimized for therapeutic use. Summary of the Invention

[0009] The present disclosure arises from the determination that a cyclin F polypeptide can be targeted to the cytoplasm of a cell and function to effectively bind and remove cytoplasmic TDP-43 (the pathogenic form of TDP-43), while leaving the nuclear form of TDP-43 (required for normal cellular function) essentially unaffected. Thus, modified cyclin F polypeptides targeted to the cytoplasm are provided. Also provided are functional truncated modified cyclin F polypeptides. Thus, the present disclosure also provides methods for enhancing neuronal survival, inhibiting neuronal degeneration, inhibiting abnormal protein accumulation in neurons, and / or treating neurodegenerative conditions (e.g., ALS, FTD, AD, etc.), preferably neurodegenerative conditions associated with neuronal TDP-43 proteinopathy, the methods comprising contacting a neuron with a modified cyclin F polypeptide or polynucleotide.

[0010] Thus, in one aspect, the disclosure is directed to a nucleic acid molecule comprising a coding sequence for a modified cyclin F polypeptide, wherein the modified cyclin F polypeptide comprises a heterologous nuclear export signal (NES). In some examples, the NES comprises a sequence of amino acids selected from LPPLERLTL (SEQ ID NO: 8), LQLPPLERLTLD (SEQ ID NO: 9), LALKLAGLDL (SEQ ID NO: 10), PLQLPPLERLTL (SEQ ID NO: 11), ERFEMFRELNEALEL (SEQ ID NO: 12), LSSHFQELSI (SEQ ID NO: 13), ERFEMFRELNEALEL (SEQ ID NO: 14), DHAEKVAEKLEALSV (SEQ ID NO: 15), QLVEELLKIICAFQL (SEQ ID NO: 16), and TNLEALQKKLEELEL (SEQ ID NO: 17). In one embodiment, the NES is at the C-terminus or N-terminus of the modified cyclin F polypeptide.

[0011] In some embodiments, the nucleic acid molecule encodes a modified cyclin F polypeptide that also includes a nuclear localization signal (NLS) inactivating modification at one or both endogenous NLSs. In some examples, the NLS inactivating modification includes a deletion of all or a portion of the endogenous NLS relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2. Thus, in some examples, the modified cyclin F polypeptide includes a deletion of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues from the NLS at amino acid positions 20-28 relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2, using the numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2. In a further example, the modified cyclin F polypeptide includes a deletion of 1, 2, 3, 4, 5, 6, or 7 amino acid residues from the NLS at amino acid positions 568-574 relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2, using the numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2. In certain embodiments, the modified cyclin F polypeptide comprises a sequence of amino acids set forth in SEQ ID NO: 4, or a sequence having at least or about 95% sequence identity thereto. In further embodiments, the NLS-inactivating modification comprises one or more amino acid substitutions of amino acid residues that comprise the endogenous NLS (e.g., one or more amino acid substitutions of K20, R21, R22, R24, R25, R26, and R28, e.g., with a non-basic amino acid, using the numbering relative to wild-type cyclin F set forth in SEQ ID NO: 2, or one or more amino acid substitutions of R568, R569, K571, R572, K574, and R574, e.g., with a non-basic amino acid, using the numbering relative to wild-type cyclin F set forth in SEQ ID NO: 2).

[0012] In further embodiments, the nucleic acid molecule encodes a modified cyclin F polypeptide, which further comprises a deletion of all or a portion of the PEST domain relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2. Thus, in some examples, the modified cyclin F polypeptide comprises a deletion of at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids from the PEST domain at amino acid positions 582-766, using numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2.

[0013] In another aspect, a nucleic acid molecule is provided that encodes a modified cyclin F polypeptide, the modified cyclin F polypeptide comprising a deletion of all or a portion of the PEST domain relative to a wild-type cyclin F polypeptide set forth in SEQ ID NO: 2. Thus, in some examples, the modified cyclin F polypeptide comprises a deletion of at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids from the PEST domain at amino acid positions 582-766, using numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2. In certain embodiments, the modified cyclin F polypeptide comprises a sequence of amino acids set forth in SEQ ID NO: 6, or a sequence having at least or about 95% sequence identity thereto.

[0014] In some examples, the modified cyclin F polypeptide encoded by the nucleic acid molecule further comprises a heterologous nuclear export signal (NES), such as, for example, one comprising a sequence of amino acids selected from LPPLERLTL (SEQ ID NO: 8), LQLPPLERLTLD (SEQ ID NO: 9), LALKLAGLDL (SEQ ID NO: 10), PLQLPPLERLTL (SEQ ID NO: 11), ERFEMFRELNEALEL (SEQ ID NO: 12), LSSHFQELSI (SEQ ID NO: 13), ERFEMFRELNEALEL (SEQ ID NO: 14), DHAEKVAEKLEALSV (SEQ ID NO: 15), QLVEELLKIICAFQL (SEQ ID NO: 16), and TNLEALQKKLEELEL (SEQ ID NO: 17). The NES may be, for example, at the C-terminus or N-terminus of the modified cyclin F polypeptide. In further embodiments, the nucleic acid molecule encodes a modified cyclin F polypeptide that also comprises a nuclear localization signal (NLS) inactivating modification to one or both endogenous NLSs. In some examples, the NLS-inactivating modification comprises a deletion of all or a portion of the endogenous NLS relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2. Thus, in some examples, the modified cyclin F polypeptide comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues from the NLS at amino acid positions 20-28 relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2, using the numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2. In further examples, the modified cyclin F polypeptide comprises a deletion of 1, 2, 3, 4, 5, 6, or 7 amino acid residues from the NLS at amino acid positions 568-574 relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2, using the numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2.In further embodiments, the NLS-inactivating modification comprises one or more amino acid substitutions of amino acid residues that comprise the endogenous NLS (e.g., one or more amino acid substitutions of K20, R21, R22, R24, R25, R26, and R28 with a non-basic amino acid, using the numbering relative to wild-type cyclin F set forth in SEQ ID NO:2, or one or more amino acid substitutions of R568, R569, K571, R572, K574, and R574 with a non-basic amino acid, using the numbering relative to wild-type cyclin F set forth in SEQ ID NO:2).

[0015] In one embodiment, the modified cyclin F polypeptide encoded by the nucleic acid molecule of the present disclosure binds TDF-43. In some examples, the modified cyclin F polypeptide retains at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the TDF-43 binding ability of the wild-type cyclin F polypeptide set forth in SEQ ID NO:2.

[0016] In certain embodiments, a modified cyclin F polypeptide encoded by a nucleic acid molecule of the present disclosure comprises at least or about 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 amino acid residues of a cyclin domain at positions 292-405 of a wild-type cyclin F polypeptide set forth in SEQ ID NO: 2. In further examples, the modified cyclin F polypeptide forms a Skp1-Cul1-F box (SCF) E3 ubiquitin-protein ligase complex (e.g., the modified cyclin F polypeptide retains at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the ability of a wild-type cyclin F polypeptide set forth in SEQ ID NO: 2 to form a Skp1-Cul1-F box (SCF) E3 ubiquitin-protein ligase complex). In further embodiments, the modified cyclin F polypeptide encoded by the nucleic acid molecule of the present disclosure comprises at least or about 15, 20, 25, 30, 35, 40, or 45 amino acid residues of the F box at positions 29-76 of the wild-type cyclin F polypeptide set forth in SEQ ID NO:2.

[0017] In other embodiments, a modified cyclin F polypeptide encoded by a nucleic acid molecule of the present disclosure, when expressed in or delivered to a neuron, accumulates in, is localized to, is introduced into, and / or is targeted to the cytoplasm of the neuron.

[0018] In certain embodiments, the nucleic acid molecule comprises an expression construct comprising a promoter operably linked to a coding sequence for the modified cyclin F polypeptide.

[0019] In another aspect, there is provided a modified cyclin F polypeptide encoded by a nucleic acid molecule as described above and herein.

[0020] Also provided is a delivery vehicle comprising the nucleic acid molecule or modified cyclin F polypeptide described above and herein. In one embodiment, the delivery vehicle is a viral vector (e.g., an adeno-associated viral vector (AAV) (e.g., rAAV2 / 1, rAAV2 / 8, or rAAV2 / 9), a lentiviral vector, an adenoviral vector, or a herpes simplex viral vector) comprising the nucleic acid molecule of the present disclosure. In one example, the viral vector is a neurotropic viral vector. In further embodiments, the delivery vehicle is a non-viral vector (e.g., a macromolecular complex, a nanocapsule, a microsphere, a bead, or a lipid-based system such as an oil-in-water emulsion, a micelle, a mixed micelle, or a liposome) comprising the nucleic acid molecule or modified cyclin F polypeptide of the present disclosure.

[0021] In a further aspect, there is provided a method for enhancing neuronal survival, inhibiting neuronal degeneration, inhibiting abnormal protein accumulation in neurons, inhibiting aggregated or insoluble TDP-43 accumulation in neurons, the method comprising, consisting of, or consisting essentially of exposing a neuron to a nucleic acid molecule, modified cyclin F polypeptide, or delivery vehicle described above and herein. In some examples, the neuron is a motor neuron.

[0022] In another aspect, a method is provided for treating a subject having a neurodegenerative condition or at risk of developing a neurodegenerative condition, the method comprising, consisting of, or consisting essentially of administering to the subject a nucleic acid molecule, modified cyclin F polypeptide, or delivery vehicle as described above and herein. In one embodiment, the neurodegenerative condition is associated with neuronal TDP-43 proteinopathy. In some examples, the subject has a familial neurodegenerative condition (e.g., familial ALS, familial FTD, or familial AD). In other examples, the subject has a sporadic neurodegenerative condition (e.g., sporadic ALS, sporadic FTD, or sporadic AD).

[0023] Also provided is the use of a nucleic acid molecule, modified cyclin F polypeptide, or delivery vehicle as described above and herein in the manufacture of a medicament for treating or inhibiting the onset of a neurodegenerative condition associated with neuronal TDP-43 proteinopathy.

[0024] In another aspect, a kit is provided that includes a nucleic acid molecule, modified cyclin F polypeptide, or delivery vehicle as described above and herein for use in a method for treating or inhibiting the onset of a neurodegenerative condition associated with neuronal TDP-43 proteinopathy. In some examples, the kit includes instructional materials for carrying out the method. [Brief description of the drawings]

[0025] [Figure 1]FIG. 1 is a schematic diagram of cyclin F polypeptides produced for experimental studies, including wild-type cyclin F, cyclin F(CT), and cyclin F(deltaPEST, also referred to herein as dPEST). The locations of the nuclear localization signal (NLS), F box, cyclin box (or domain), PEST domain, and nuclear export signal (NES) are shown, with numbering relative to the respective amino acid sequences of the polypeptides shown in SEQ ID NO: 2 for wild-type cyclin F, SEQ ID NO: 4 for cyclin F(CT), and SEQ ID NO: 6 for cyclin F(deltaPEST). [Diagram 2] The wild-type cyclin F nucleotide and amino acid sequences are presented, annotated to show the NLS, F-box, cyclin domain, and PEST domain. [Diagram 3] The cyclin F(CT) nucleotide and amino acid sequences are presented, annotated to show the F-box, cyclin domain, PEST domain, and NES. [Figure 4] The cyclin F (deltaPEST) nucleotide and amino acid sequences are presented, annotated to show the NLS, F box, and cyclin domain. [Diagram 5] Schematic and photographic representation of the production and characterization of cyclin polypeptides. A) Production of wild-type cyclin F and modified cytoplasmic cyclin F (CT-cyclin F, also called cyclin F (CT)). B) Expression of mCherry-cyclin F (WT) in HEK293 cells. C) Expression of mCherry-CT-cyclin F in HEK293 cells. [Figure 6]Figure 1 is a graphical representation of clearance of dNLS-TDP-43 by CT-cyclin F in cultured HEK cells. Cells were either co-transfected with DNA constructs encoding dNLS TDP-43 and (CT)cyclin F (CT-CCNF) or transfected with dNLS TDP-43 alone. Urea-soluble dNLS TDP-43 was then assessed 24 hours after transfection. One-way ANOVA statistical tests were performed with post-hoc Dunnett's multiple comparisons n=3. [Figure 7] Graphical representation of clearance of dNLS-TDP-43 by CT-cyclin F in transgenic zebrafish, represented by whole-cell TDP-43 fluorescence intensity. mRNA encoding human CT-cyclin F was transfected into stable transgenic zebrafish overexpressing human dNLS-TDP-43-GFP in zebrafish spinal motor neurons. After 48 h, the fluorescence intensity of GFP in three randomly selected motor neurons per spinal cord image (three images per animal) was measured in at least three animals per treatment group. CT-cyclin F caused a significant reduction in dNLS-TDP-43 levels in spinal motor neurons. [Figure 8] FIG. 1 is a photographic representation of the cellular localization of mCherry-wild-type cyclin F, mCherry-cytoplasmic cyclin F (CT-cyclin F), and mCherry-cyclin F (deltaPEST) in HEK293 cells. [Figure 9] Figure 1 is a graphical representation of clearance of dNLS-TDP-43 by cyclin F (deltaPEST) in cultured HEK cells. Cells were co-transfected with DNA constructs encoding dNLS TDP-43 and cyclin F (deltaPEST) or transfected with dNLS TDP-43 alone. Urea-soluble dNLS TDP-43 was then assessed 24 hours after transfection. One-way ANOVA statistical tests were performed with post-hoc Dunnett's multiple comparisons n=3. [Figure 10]FIG. 1 is a graphical representation of clearance of wild-type TDP-43 by CT-cyclin F and deltaPEST-cyclin F in transgenic zebrafish, as represented by whole-cell TDP-43 fluorescence intensity. mRNA encoding human CT-cyclin F or human deltaPEST-cyclin F was transfected into stable transgenic zebrafish overexpressing human wild-type TDP-43-GFP in zebrafish spinal motor neurons. After 48 hours, the fluorescence intensity of GFP in three randomly selected motor neurons per spinal cord image (three images per animal) was measured in at least three animals per treatment group. Neither CT-cyclin F nor deltaPEST-cyclin F had any effect on wild-type TDP-43 levels in spinal motor neurons. [Figure 11] Graphical representation of clearance of wild-type TDP-43 by LP / AA-cyclin F in transgenic zebrafish, as represented by whole-cell TDP-43 fluorescence intensity. mRNA encoding human wild-type cyclin F or human LP / AA cyclin F was transfected into stable transgenic zebrafish overexpressing human wild-type TDP-43-GFP in zebrafish spinal motor neurons. After 48 h, the fluorescence intensity of GFP in three randomly selected motor neurons per spinal cord image (three images per animal) was measured in at least three animals per treatment group. WT-cyclin F causes a significant reduction in WT-TDP-43 levels in spinal motor neurons, whereas the inactive LP / AA variant (IA) has no effect on WT-TDP-43 clearance. [Figure 12]Graphical representation of clearance of wild-type TDP-43 (A) and dNLS-TDP-43 (B) by cyclin F variants in transgenic zebrafish, as represented by whole-cell TDP-43 fluorescence intensity. mRNA encoding deltaPEST-cyclin F, CT-cyclin F, human wild-type cyclin F (WT), or human LP / AA cyclin F (IA) was transfected into stable transgenic zebrafish overexpressing either human wild-type TDP-43-GFP (A) or dNLS-TDP-43-GFP (B) in zebrafish spinal motor neurons. After 48 hours, the fluorescence intensity of GFP in three randomly selected motor neurons per spinal cord image (three images per animal) was measured in at least three animals per treatment group. The results confirm those illustrated in Figures 7, 10, and 11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] [Table A-1] [Table A-2] [Table A-3] [Table A-4]

[0027] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described. For purposes of this disclosure, the following terms are defined below.

[0028] The articles "a," "an," and "the" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context clearly indicates otherwise.

[0029] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0030] Additionally, the terms "about" and "approximately," when used herein when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, location, length, and the like, are meant to encompass a variation of ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, location, length, and the like.

[0031] As used herein, the term "activity" should be understood as a measure of the ability of a transcription or translation product to produce a biological effect, or a measure for the level of a biologically active molecule. Thus, in the context of cyclin F, the term "activity" refers to any one or more of the following activities, as disclosed herein: (1) Associating with other subunits to form the Skp1-Cul1-F box (SCF) E3 ubiquitin-protein ligase complex (SCF Cyclin F (2) inhibiting B-Myb activity and promoting cell cycle checkpoint control; (3) interacting with substrates (e.g., CDC6, RRM2, CP110, and SLBP, as well as TDP-43) and promoting their ubiquitination and degradation; and (4) directly binding to TDP-43.

[0032] As used herein, the term "administration" refers to the placement of an agent described herein into a subject by a method or route that results in at least partial localization of the compound at a desired site. The agents described herein may be administered by any suitable route that results in effective treatment in the subject, i.e., administration results in delivery to a desired location in the subject where at least a portion of the composition is delivered. Exemplary modes of administration include, but are not limited to, injection, infusion, infusion, or ingestion. "Injection" includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

[0033] Terms such as "concurrent administration" or "administered simultaneously" or "co-administration" refer to administration of a single composition containing two or more actives, or each active agent delivered as a separate composition and / or by a separate route, either simultaneously, simultaneously, or sequentially within a sufficiently short period of time that the effective results are comparable to those obtained when all such active agents are administered as a single composition. "Concurrently" means that the active agents are administered at substantially the same time, desirably together in the same formulation. "Concurrently" means that the active agents are administered precisely in time, e.g., one agent is administered within about one minute to about one day before or after another agent. Any time of simultaneous administration is useful. However, when not administered simultaneously, the agents are often administered within about one minute to about eight hours, preferably within about one to about four hours. When administered simultaneously, the agents are preferably administered at the same site on the subject. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably within about 0.5 to about 5 centimeters. As used herein, the term "separately" means that the agents are administered at intervals, for example, from about one day to several weeks or months. The active agents may be administered in any order. As used herein, the term "sequentially" means that the agents are administered one after the other, for example, with intervals of minutes, hours, days, or weeks between each. If desired, the active agents may be administered in a regular repeat cycle.

[0034] The term "drug" includes compounds that induce a desired pharmacological and / or physiological effect. The term also encompasses pharma- ceutically acceptable and pharmacologically active components of those compounds specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs, and the like. When the above term is used, it should be understood to include the active drug itself, as well as pharma- ceutically acceptable and pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, and the like. The term "drug" should not be narrowly construed, but extends to small molecules, proteinaceous molecules such as peptides, polypeptides, and proteins, compositions containing them, genetic molecules such as RNA, DNA, and mimetics, chemical analogs thereof, and cellular agents. The term "drug" includes cells capable of producing and secreting the polypeptides referred to herein, as well as polynucleotides containing nucleotide sequences encoding the polypeptides. Thus, the term "drug" extends to nucleic acid constructs, including vectors such as viral or non-viral vectors, expression vectors, and plasmids for expression and secretion in a range of cells. Exemplary agents include modified cyclin F polypeptides and nucleic acids encoding modified cyclin F polypeptides.

[0035] The terms "cis-acting element," "cis-acting sequence," or "cis-regulatory region" are used interchangeably herein and refer to any sequence of nucleotides that regulates the transcriptional activity of an operably linked promoter and / or the expression of an operably linked nucleotide sequence. One of skill in the art will recognize that a cis sequence may be capable of activating, silencing, enhancing, suppressing, or otherwise altering the level of expression and / or cell type specificity and / or developmental specificity of any nucleotide sequence, including coding and non-coding sequences.

[0036] By "coding sequence" is meant any nucleic acid sequence that contributes to the coding for the polypeptide product of a gene or the final mRNA product of a gene (e.g., the mRNA product of a gene after splicing). In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the coding for the polypeptide product of a gene or the final mRNA product of a gene.

[0037] Throughout this specification, unless the context requires otherwise, the terms "comprise, comprise, and comprising" will be understood to imply the inclusion of a recited step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements. Thus, use of the term "comprise" and similar terms indicates that the recited elements are required or essential, while other elements are optional and may or may not be present. "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and other elements may not be present. "Consisting essentially of" means including any elements recited after the phrase, and limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0038] As used herein, the term "condition" includes anatomical and physiological deviations from the normal that constitute a disturbance of the normal state of a living animal or one of its parts and that interfere with or modify the performance of bodily functions.

[0039] The terms "conditional expression," "conditionally expressed," "conditionally expressed," and the like refer to the ability to activate or repress expression of a gene of interest depending on the presence or absence of a stimulus or other signal (e.g., chemical, light, hormone, stress, or pathogen). In certain embodiments, the conditional expression of a nucleic acid sequence of interest is dependent on the presence of an inducer or the absence of an inhibitor.

[0040] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be broadly subclassified as follows: [Table 1]

[0041] Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonably expected that the substitution of leucine with isoleucine or valine, the substitution of aspartic acid with glutamic acid, the substitution of threonine with serine, or similar substitution of amino acids with structurally related amino acids will not have a significant effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can be easily determined by assaying its activity. Conservative substitutions are shown in Table 2 under the headings of exemplary and preferred substitutions. Amino acid substitutions that fall within the scope of the present disclosure are generally achieved by selecting substitutions that do not differ significantly in their effect on (a) the structure of the peptide backbone in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. [Table 2]

[0042] As used herein in reference to contacting a motor neuron or a substitute cell for a motor neuron, the term "contacting" or "contacting" includes subjecting the motor neuron or substitute cell to an appropriate culture medium containing the indicated compound and / or agent. If the motor neuron or substitute cell is in vivo, "contacting" or "contacting" includes administering the compound and / or agent in a pharmaceutical composition to a subject via an appropriate route of administration such that the compound and / or agent contacts the motor neuron or substitute cell in vivo. In certain embodiments, the contacted motor neuron or substitute cell is assayed for cell viability. The measurement of cell viability may be based on the number of viable cells after a period of time has elapsed after contacting the cells with the compound or agent. For example, the number of viable cells may be counted after at least about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days or more and compared to the number of viable cells in an untreated control.

[0043] The term "construct" refers to a recombinant genetic molecule that contains one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule, including any construct that contains (1) a nucleic acid sequence that contains regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of the other nucleotide sequences), or (2) a sequence that encodes a functional RNA molecule or protein site that is not naturally adjacent, or (3) a promoter site that is not naturally adjacent. Representative constructs include any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source and capable of genomic integration or autonomous replication, including a nucleic acid molecule to which one or more nucleic acid molecules are operably linked. The constructs of the present disclosure will generally include the elements necessary to induce expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a regulator nucleic acid sequence. Such elements may include control elements, such as a promoter (to induce transcription) operably linked to the nucleic acid sequence of interest, and often also include a polyadenylation sequence. In certain embodiments of the present disclosure, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of a host cell. The two or more constructs may be contained within a single nucleic acid molecule, such as a single vector, or may be contained within two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to the nucleotide sequence of interest. Thus, for example, a promoter in operably linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in an organism or part thereof, including a host cell.Conventional compositions and methods for preparing and using the constructs and host cells for the practice of the methods of the present disclosure are well known to those of skill in the art and can be found, for example, in Molecular Cloning: A Laboratory Manual, 3. rd See, editions Volumes 1, 2, and 3. JF Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0044] As used herein, the term "control neuron" refers to a neuron (e.g., a motor neuron) from one or more healthy subjects or a subject that does not have a neurodegenerative condition and / or does not have a TDP-43 proteinopathy (e.g., a control subject).

[0045] "Corresponding to" or "corresponding to" refers to an amino acid sequence that exhibits substantial sequence similarity or identity with a reference amino acid sequence. Generally, the amino acid sequence will exhibit at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or even up to 100% sequence similarity or identity with at least a portion of the reference amino acid sequence.

[0046] The terms "reduction", "reduction" or "inhibition" and their grammatical equivalents are all used herein to generally mean a statistically significant amount of reduction. However, for the avoidance of doubt, the terms "reduction", "reduction" or "inhibition" and their grammatical equivalents mean a reduction of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, where the reduction is less than 100%. In one embodiment, the reduction includes a 100% reduction (e.g., an absent level compared to a reference sample), or any reduction between 10 and 100% compared to a reference level.

[0047] As used herein, "dosage unit" refers to physically discrete units suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0048] As used herein, the term "effective amount" refers to an amount of a compound and / or agent that is effective for promoting survival of motor neuron cells or inhibiting or delaying the death of such cells. The determination of an effective amount is well within the capabilities of a person skilled in the art. Generally, an effective amount may vary depending on the subject's medical history, age, condition, sex, and the severity and type of medical condition in the subject, and the administration of other agents that inhibit pathological processes in neurodegenerative conditions.

[0049] As used herein, the terms "encode", "encoding" and the like refer to the ability of a nucleic acid to provide another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and / or translated to produce the polypeptide, or if it can be processed into a form that can be transcribed and / or translated to produce the polypeptide. Such a nucleic acid sequence can include coding sequences, or both coding and non-coding sequences. Thus, the terms "encode", "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and subsequent translation of the RNA product, or a protein resulting from translation of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA), and subsequent translation of the processed RNA product.

[0050] As used herein, the phrase "enhancing motor neuron survival" refers to an increase in survival of motor neuron cells compared to a control. In some embodiments, contacting a motor neuron with an agent described herein results in at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more increase in survival of the motor neuron compared to a non-treated control. Survival of motor neurons can be assessed, for example, by (i) increased survival time of motor neurons in culture, (ii) increased production of neuron-associated molecules in culture or in vivo, such as choline acetyltransferase, acetylcholinesterase, and cyclin F, (iii) reduced abnormal accumulation of proteins including TDP-43 in culture or in vivo, or (iv) decreased symptoms of motor neuron dysfunction in vivo. Such effects can be measured by any method known in the art. In one non-limiting example, increased survival of motor neurons can be measured by the method described by Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased production of neuron-associated molecules can be measured by bioassays, enzyme assays, antibody binding, Northern blot assays, etc., depending on the molecule being measured; reduced abnormal accumulation of proteins can be assayed, for example, through detection of aggregated proteins in aggresomes and inclusion bodies, as described by Shen et al. (2011, Cell Biochem Biophys 60:173-185); and motor neuron dysfunction can be measured by assessing physical signs of motor neuron injury. In one embodiment, increased survival of motor neurons can be assessed by measuring increased levels of cyclin F. Cell survival can also be measured by uptake of calcein AM, an analog of the viability dye fluorescein diacetate. Calcein is cleaved intracellularly into a fluorescent salt that is taken up by viable cells and retained by intact membranes of viable cells. Microscopic counts of viable neurons directly correlate with relative fluorescence values ​​obtained using the fluorescent viability assay.Thus, this method provides a reliable and quantitative measurement of cell survival in the total cell population of a given culture (Bozyczko-Coyne et al., J.Neur.Meth.50:205-216,1993). Other methods of assessing cell survival are described in U.S. Patent Nos. 5,972,639, 6,077,684, and 6417,160, the contents of which are incorporated herein by reference. In vivo motor neuron survival can be assessed by an increase in motor neuron, neuromotor, or neuromuscular function in a subject. In one non-limiting example, motor neuron survival in a subject can be assessed by amelioration, alleviation, improvement, inhibition, slowing down, or halting the progression, worsening, or severity of a condition associated with motor neuron dysfunction or death in a subject, such as ALS or FTD.

[0051] The term "endogenous" refers to a molecule (e.g., a nucleic acid, carbohydrate, lipid, or polypeptide) present and / or naturally expressed within a host organism or a cell thereof, or a domain or region that occurs naturally within a gene, nucleic acid, or protein. For example, "endogenous cyclin F" refers to a cyclin F polypeptide that is naturally expressed in a cell (e.g., a motor neuron), and "endogenous nuclear localization signal" refers to a nuclear localization signal that occurs naturally in a protein such as cyclin F.

[0052] As used herein, the term "exogenous" refers to a molecule (e.g., a nucleic acid, carbohydrate, lipid, or polypeptide) that is introduced into a host cell. In certain embodiments, an exogenous polypeptide refers to a polypeptide expressed from a polynucleotide that is foreign to the cell into which it is introduced, or a polypeptide that is homologous to a sequence in the cell into which it is introduced, but is expressed at a location within the host cell nucleic acid where the polynucleotide is not normally found.

[0053] The term "expression" with respect to a gene sequence refers to the transcription of the gene to produce an RNA transcript (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.) and, if necessary, the translation of the resulting mRNA transcript into a protein. Thus, as will be clear from the context, expression of a coding sequence results from the transcription and translation of the coding sequence. Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence.

[0054] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA, etc., and in some embodiments, a polypeptide. A gene may or may not be used to produce a functional protein. A gene may include both coding and non-coding regions (e.g., regulatory elements including introns, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). In certain embodiments, the term "gene" includes within its scope an open reading frame that codes for a particular polypeptide, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in regulating expression. In this regard, a gene may further include control sequences, such as promoters, enhancers, termination signals, and / or polyadenylation signals, naturally associated with a given gene, or heterologous control sequences. A gene sequence may be cDNA or genomic DNA, or a fragment thereof. A gene may be introduced into a suitable vector for extrachromosomal maintenance or introduction into a host.

[0055] The term "heterologous," for purposes of this disclosure, refers to a molecule (e.g., a nucleic acid or polypeptide) that is not naturally occurring and / or not naturally expressed in a host organism or cells thereof, or a domain or region that does not naturally occur within a gene, nucleic acid, or protein. For example, a "heterologous NES" refers to an NES that is present in a modified polypeptide (e.g., a modified cyclin F polypeptide) but is not present (or is not present at the location) in the corresponding wild-type polypeptide (e.g., a wild-type cyclin F polypeptide).

[0056] The terms "increase," "enhance," or "activate," and their grammatical equivalents are all used herein generally to mean an increase by a statistically significant amount, and for the avoidance of doubt, the terms "increase," "enhance," or "activate," and their grammatical equivalents mean an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to 100% or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0057] As used herein, the phrase "inhibiting motor neuron degeneration" refers to reducing the loss of motor neuron viability, reducing the loss of motor neuron function, and / or reducing the loss of motor neuron number. In some embodiments, contacting a motor neuron with an agent described herein results in at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or greater reduction in motor neuron degeneration compared to a non-treated control. Motor neuron degeneration can be assessed, for example, by assaying oxidative or endoplasmic reticulum stress, or apoptosis or neuronal death in general.

[0058] As used herein, the term "modulation" means to cause or facilitate a qualitative or quantitative change, alteration, or modification in a molecule, process, pathway, or phenomenon of interest. Without limitation, such a change can be an increase, decrease, change in the binding properties, or a change in the relative strength or activity of different components or branches of the process, pathway, or phenomenon.

[0059] As used herein, the phrase "motor neuron degeneration" or "degeneration of motor neurons" refers to a state of deterioration of motor neurons, in which the neurons die or change to a lesser or less functionally active form.

[0060] The term "neurodegenerative condition" is an umbrella term that encompasses acute and chronic conditions, disorders, or diseases of the central or peripheral nervous system, and generally is caused by or associated with the deterioration of cells or tissues of the nervous system. Neurodegenerative conditions may be age-related, or may result from injury or trauma, or may be associated with a specific disease or disorder. Acute neurodegenerative conditions include conditions associated with neuronal cell death or failure, including, but not limited to, cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain injury, spinal cord injury, or peripheral nerve trauma, for example, resulting from physical or chemical burns, deep cuts, or limb amputation. Examples of acute neurodegenerative disorders are cerebral ischemia or infarction, including embolic and thrombotic occlusion, reperfusion after acute ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, and any type of intracranial hemorrhage (such as epidural, subdural, subarachnoid, and intracerebral), intracranial and intravertebral lesions (such as contusion, penetration, shear, compression, and laceration), as well as whiplash and shaken baby syndrome.Chronic neurodegenerative conditions include, but are not limited to, Alzheimer's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multiple system degeneration (Shy-Drager syndrome), chronic epileptic conditions associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), degenerative ataxias, corticobasal degeneration, Guam ALS-Parkinson-Dementia Complex, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease / Spinocerebellar degeneration type 3 and olivopontocerebellar degeneration, Gilles de Lamarque syndrome, and others. Tourette's syndrome, bulbar and pseudobulbar palsy, spinal and bulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff disease, familial spastic disorders, Wohlfart-Kugelberg-Welander disease, spastic paraplegia, progressive multifocal leukoencephalopathy, familial dysautonomia (Riley-Day syndrome), and prion diseases (including, but not limited to, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, rickets, and fatal familial insomnia), demyelinating diseases and disorders including multiple sclerosis, and genetic diseases such as leukodystrophies. In certain embodiments, the neurodegenerative condition is selected from ALS and FTD.

[0061] As used herein, the term "neuron" includes neurons and parts thereof (e.g., neuronal cell body, axon, or dendrites). As used herein, the term "neuron" refers to a nervous system cell that includes a central cell body or soma and two types of extensions or processes: dendrites, which generally transmit the majority of neuronal signals to the cell body, and axons, which generally transmit the majority of neuronal signals from the cell body to effector cells such as target neurons or muscles. Neurons can transmit information from tissues and organs to the central nervous system (parent neurons or sensory neurons) and send signals from the central nervous system to effector cells (effector or motor neurons). Other neurons, called interneurons, connect neurons within the central nervous system (brain and spinal cord). Neurons can be any neuron, including, without limitation, sensory, sympathetic, parasympathetic, or enteric, e.g., dorsal root ganglion neurons, motor neurons, and central neurons, e.g., neurons from the spinal cord. Certain specific examples of neuronal types that may be subject to treatment or methods according to the present disclosure include cerebellar granule neurons, dorsal root ganglion neurons, and cortical neurons. In some embodiments, the neurons are sensory neurons. In some embodiments, the neurons are motor neurons.

[0062] The terms "neurodegeneration" and "neuronal degeneration" are used interchangeably herein and refer to any pathological change in a neuronal cell, including, without limitation, death or loss of a neuronal cell, any changes preceding cell death, and any reduction or loss of activity or function of a neuronal cell. The pathological change may be spontaneous or induced by any event, including, for example, pathological changes associated with apoptosis. The neuron may be any neuron, including, without limitation, sensory, sympathetic, parasympathetic, or enteric, e.g., dorsal root ganglion neurons, motor neurons, and central neurons, e.g., neurons from the spinal cord. Neurodegeneration or cell loss is a feature of various neurological diseases or disorders, e.g., neurodegenerative diseases or disorders. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a motor neuron.

[0063] The term "neurotropic viral vector" refers to a viral vector that selectively infects neuronal cells, including motor neurons.

[0064] "Obtained" means in possession of. Samples so obtained include, for example, nucleic acid extracts or polypeptide extracts isolated or derived from a particular source. For example, an extract can be isolated directly from a biological fluid or tissue of a subject.

[0065] As used herein, the term "operably connected" or "operably linked" refers to a juxtaposition in which the components so described are in a relationship that allows them to function in their intended manner. For example, a regulatory sequence (e.g., a promoter) "operably linked" to a nucleotide sequence of interest (e.g., a coding and / or non-coding sequence) refers to the positioning and / or orientation of the control sequence relative to the nucleotide sequence of interest to permit expression of that sequence under conditions compatible with the control sequence. Control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to induce its expression. Thus, for example, intervening non-coding sequences (e.g., non-translated but transcribed sequences) can be present between the promoter and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0066] The terms "patient," "subject," "host," or "individual," as used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly, a mammalian subject for whom therapy or prevention is desired. Suitable vertebrates falling within the scope of the present disclosure include, but are not limited to, primates (e.g., humans, monkeys and apes, Macaca (e.g., cynomolgus monkeys, such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (species from the genus Saguinus), and chimpanzees (Pan pantheons). Examples of the species of apes that may be used include: apes (such as primates, primates, and primates of the genus troglodytes), rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cows), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, canaries, companion birds such as budgerigars, and the like), marine mammals (e.g., dolphins, whales, and the like), reptiles (snakes, frogs, lizards, and the like), and fish. A preferred subject is a human in need of increasing the level or activity of cyclin F and / or treating a neurodegenerative condition. However, it will be understood that the above terms do not imply that symptoms are present.

[0067] As used herein, the term "pharmacologically acceptable" refers to those compounds, agents, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0068] As used herein, the term "pharmaceutical acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting a subject drug from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient.

[0069] The term "polynucleotide" is used interchangeably herein with "nucleic acid" to refer to a polymer of nucleosides. Typically, polynucleotides of the present disclosure are composed of nucleosides naturally occurring in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term encompasses molecules that contain nucleosides or nucleoside analogs that contain chemically or biologically modified bases, modified backbones, and the like, whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the present application refers to polynucleotides, it is understood that both DNA, RNA, and in each case, both single-stranded and double-stranded forms (as well as the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or to sequence information that biochemically characterizes a particular nucleic acid (e.g., a sequence of letters used as an abbreviation for bases). Polynucleotide sequences presented herein are presented in the 5' to 3' orientation unless otherwise indicated.

[0070] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2-60 amino acids in length. A polypeptide as used herein typically contains amino acids, such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art may be used. One or more of the amino acids in a polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, conjugation, linkers for functionalization, etc. A polypeptide having a non-polypeptide moiety covalently or non-covalently associated therewith is still considered to be a polypeptide. Exemplary modifications include glycosylation and palmitoylation. Polypeptides may be purified from natural sources, produced using recombinant DNA technology, or synthesized through chemical means such as conventional solid-phase peptide synthesis. As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or to sequence information that biochemically characterizes the polypeptide (e.g., the sequence of letters or three-letter code used as an abbreviation for the amino acid name). Polypeptide sequences presented herein are presented in an N-terminal to C-terminal orientation unless otherwise indicated.

[0071] The term "promoter" refers to a nucleotide sequence, usually upstream (5') of a transcribable sequence, that controls the expression of the transcribable sequence by providing recognition for RNA polymerase and other factors necessary for proper transcription. "Promoter" includes minimal promoters, which are short nucleic acid sequences composed of a TATA box and other sequences that serve to specify the transcription start site where regulatory elements (e.g., cis-acting elements) are added to control expression. "Promoter" also refers to a nucleotide sequence that contains a minimal promoter plus regulatory elements (e.g., cis-acting elements) that can control the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often called enhancers. Thus, an "enhancer" is a nucleic acid sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. It can operate in both orientations (normal or inverted) and can function when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific nucleic acid binding proteins that mediate their effects. A promoter may be derived entirely from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even be composed of synthetic nucleic acid segments. A promoter may also contain nucleic acid sequences involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. Promoter elements, particularly TATA elements, that are inactive or have greatly reduced promoter activity in the absence of upstream activation are called "minimal or core promoters". In the presence of suitable transcription factors, a minimal promoter functions to allow transcription. Thus, a "minimal or core promoter" consists only of all the basic elements necessary for transcription initiation, e.g., the TATA box and / or initiator. The term "regulated promoter" refers to a promoter that induces gene expression in a non-constitutive, but temporally and / or spatially regulated manner, and includes both tissue-specific and inducible promoters.It includes natural and synthetic sequences, as well as sequences that may be a combination of synthetic and natural sequences. Different promoters may induce the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. New promoters of various types useful for host cells are constantly being discovered. In most cases, the exact boundaries of regulatory sequences have not been fully defined, so that nucleic acid fragments of different lengths may have the same promoter activity. Exemplary regulated promoters include, but are not limited to, safer inducible promoters, promoters from tetracycline-inducible systems, promoters from salicylic acid-inducible systems, promoters from alcohol-inducible systems, promoters from glucocorticoid-inducible systems, promoters from pathogen-inducible systems, promoters from carbohydrate-inducible systems, promoters from hormone-inducible systems, promoters from antibiotic-inducible systems, promoters from metal-inducible systems, promoters from heat shock-inducible systems, and promoters from ecdysone-inducible systems.

[0072] "Regulatory sequence," "regulatory element," and the like refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that directly or indirectly influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory elements include enhancers, promoters, translation leader sequences, introns, Rep recognition elements, intergenic regions, and polyadenylation signal sequences. They include natural and synthetic sequences, as well as sequences that may be a combination of synthetic and natural sequences.

[0073] As used herein, the term "recombinant polynucleotide" refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature. For example, a recombinant polynucleotide may be in the form of an expression vector. Generally, such expression vectors include transcriptional and translational regulatory nucleic acid operably linked to a nucleotide sequence.

[0074] By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant polynucleotide.

[0075] As used herein, the term "sample" includes any biological sample that may be extracted, untreated, treated, diluted, or concentrated from a subject. Samples may include biological fluids, such as, without limitation, whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, stool (i.e., feces), tears, sweat, sebum, nipple aspirate, ductal lavage, tumor exudate, synovial fluid, peritoneal fluid, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymphatic fluid, fine needle aspirate, amniotic fluid, other bodily fluids, cell lysates, cell secretions, inflammatory fluid, semen, and vaginal fluid. Samples may include tissue samples and biopsies, tissue homogenates, and the like. In certain embodiments, the sample contains tissue, and in representative examples of this type, the sample is from a resection, biopsy, or core needle biopsy. In addition, fine needle aspirate samples may be used. Samples may include paraffin-embedded tissue and frozen tissue. In certain embodiments, the sample includes neuronal tissue, including motor neurons. In other embodiments, the sample includes cells that are surrogates for motor neurons, non-limiting examples of which include fibroblasts, as disclosed, for example, by Yang et al. (2015, Neurotox Res 28:138-146), and blood cells, as disclosed, for example, at www.sciencedaily.com / releases / 2014 / 04 / 140408121918.htm. The term "sample" also includes untreated or pretreated (or pre-processed) samples. In some embodiments, the sample is an untreated biological sample. The sample may be obtained by removing a sample of cells from a subject, but may also be accomplished by using previously isolated cells (e.g., isolated at a previous time point, by the same or a different person).

[0076] As used herein, the term "sequence identity" refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, the "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences resulting in a number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The present disclosure contemplates the use of full-length cyclin F polypeptides as well as biologically active fragments thereof in the methods disclosed herein. Typically, biologically active fragments of a full-length cyclin F polypeptide are capable of participating in an interaction, for example an intra- or intermolecular interaction.

[0077] "Similarity" refers to the percentage of amino acids that are identical or that constitute conservative substitutions, as defined in Tables 1 and 2 above. Similarity can be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12:387-395). In this way, sequences of similar or substantially different lengths to those cited herein can be compared by inserting gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0078] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" (e.g., wild-type cyclin F) is at least 12, but often 15-18, and often at least 25 monomer units in length, including nucleotides and amino acid residues. Two polynucleotides or polypeptides each contain (1) sequences that are similar between the two polynucleotides or polypeptides (i.e., only a portion of the complete polynucleotide or polypeptide sequence) and (2) sequences that are mismatched between the two polynucleotides or polypeptides, and sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 consecutive positions, usually about 50 to about 100, more usually about 100 to about 150, in which a sequence is compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. The comparison window may include about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window may be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by any of a variety of selected methods. See, for example, the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Unit 19.3 of Chapter 15. In one example, when the sequence of a modified cyclin F polypeptide aligns with a wild-type cyclin F polypeptide, the comparison window includes the full length of the modified cyclin F polypeptide. As will be appreciated, when a modified cyclin F polypeptide is truncated and compared to a wild-type cyclin F polypeptide, the comparison window will be less than the full length of the wild-type cyclin F polypeptide (e.g., 40%, 50%, 60%, 70%, 80%, or 90% of the length of the wild-type cyclin F polypeptide).

[0079] The term "statistically significant" or "significant" refers to statistical significance, generally meaning a concentration of a marker that is two standard deviations (2SD) below the normal value or lower. The term refers to statistical evidence that a difference exists. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using a p-value.

[0080] As used herein, the term "TDP-43 proteinopathy" is used to describe neurodegenerative conditions associated with accumulation of TDP-43, including, but not limited to, amyotrophic lateral sclerosis (ALS), argyrophilic granulosis, frontotemporal dementia (e.g., FTD-TDP-43 and FTD-tau), Guam ALS-Parkinsonism complex, corticobasal degeneration, dementia with Lewy bodies, Huntington's disease (HD), Lewy body disease, motor neuron disease, frontotemporal lobar degeneration (FTLD), frontotemporal dementia, frontotemporal lobar degeneration with ubiquitin-positive inclusions, hippocampal sclerosis, inclusion body myopathy, inclusion body myositis, Parkinson's disease (PD), Parkinson's disease dementia, Parkinsonism-dementia complex in Kii Peninsula, Pick's disease, Machado-Joseph disease, and the like. Further details of TDP-43 proteinopathy are described in Gendron et al., 2010, Neuropathol. Appl. Neurobiol. 36:97-112, and Lagier-Tourenne et al., 2010, Hum. Mol. Gen. 19(1):R46-R64, the disclosures of which are incorporated herein by reference. In certain embodiments, TDP-43 proteinopathy is associated with TDP-43 accumulation in neurons, which is referred to herein as "neuronal TDP-43 proteinopathy."

[0081] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in that a disease or its symptoms are completely or partially prevented, and / or may be therapeutic in that a partial or complete cure is achieved for the condition and / or side effects resulting from the condition. As used herein, "treatment" encompasses any treatment of a condition in a mammal, particularly a human, including (a) inhibiting the onset of the condition in a subject susceptible to, but not yet diagnosed as having, the condition, (b) inhibiting the condition, i.e., halting its onset, and (c) alleviating the condition, i.e., causing regression of the condition. Thus, "treatment of a neurodegenerative condition" includes within its scope the delaying or preventing the onset of such a condition (e.g., death of motor neurons), the reversing, alleviating, ameliorating, inhibiting, slowing down, or halting the progression, worsening, or deterioration of such a condition, the progression or severity of such a condition. In one embodiment, the symptoms of a neurodegenerative condition are alleviated by at least 20%, at least 30%, at least 40%, or at least 50%. In one embodiment, the symptoms of the neurodegenerative condition are alleviated by more than 50%. In one embodiment, the symptoms of the neurodegenerative condition are alleviated by 80%, 90% or more. Treatment also includes improving neuromuscular function. In some embodiments, neuromuscular function is improved by at least about 10%, 20%, 30%, 40%, 50% or more.

[0082] By "vector" is meant a polynucleotide molecule, preferably a DNA molecule derived from a plasmid, bacteriophage, yeast, or virus, into which a polynucleotide can be inserted or cloned. A vector may contain one or more unique restriction sites and may be capable of autonomously replicating in a defined host cell, including a target cell or tissue, or a progenitor cell or tissue thereof, or may be capable of integrating with the genome of a defined host such that the cloned sequence is reproducible. Thus, a vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example, a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. A vector may contain any means for ensuring self-replication. Alternatively, a vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicates together with the chromosome into which it is integrated. A vector system may contain a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of vector will typically depend on the compatibility of the vector with the host cell into which it is introduced. In this case, the vector is preferably a virus or virus-derived vector that operably functions in animals and preferably mammalian cells. Non-limiting viral vectors useful in implementing the present disclosure include adeno-associated virus vectors (AAV), lentivirus vectors, adenovirus vectors, and herpes simplex virus vectors. The vector may also include a selection marker, such as an antibiotic resistance gene, that can be used to select suitable transformants. Examples of such resistance genes are known to those skilled in the art and include the nptII gene, which confers resistance to the antibiotics kanamycin and G418 (Geneticin®), and the hph gene, which confers resistance to the antibiotic hygromycin B.

[0083] The terms "wild-type," "native," and "naturally-occurring" are used interchangeably herein to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a natural source. A wild-type, native, or naturally-occurring gene or gene product (e.g., a polypeptide) is one that is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene or gene product.

[0084] As used herein, the underlining or italics of a gene name shall indicate the gene designated by the gene name in the absence of any underlining or italics, as opposed to its protein product. For example, "cyclin F" shall mean the cyclin F gene, while "cyclin F" shall indicate the protein product generated from transcription and translation, and / or alternative splicing of the "cyclin F" gene.

[0085] Each embodiment described in this specification should be applied mutatis mutandis to each and every embodiment unless otherwise specified.

[0086] 2. Abbreviation The following abbreviations are used throughout this application: [Table B]

[0087] 3. Modified Cyclin F Polypeptides Modified cyclin F polypeptides and encoding nucleic acid molecules are provided. The modified cyclin F polypeptides of the present disclosure contain one or more modifications (e.g., amino acid substitutions, deletions, and / or insertions) compared to a wild-type cyclin F polypeptide, such as the wild-type cyclin F polypeptide set forth in SEQ ID NO:2. Exemplary modifications include the inclusion or addition of one or more heterologous nuclear export signals (NES), a modification that inactivates an endogenous nuclear localization signal (i.e., an NLS-inactivating modification), and the deletion of one or more amino acid residues or domains that are not required for the therapeutic activity of cyclin F (e.g., all or a portion of the PEST domain). In some examples, the modified cyclin F polypeptide comprises at least one heterologous NES and at least one NLS-inactivating modification (e.g., deletion of all or a portion of the NLS and / or one or more amino acid substitutions in the NLS that reduce the ability of the NLS to target the polypeptide to the nucleus). In further examples, the modified cyclin F polypeptide comprises at least one NES, at least one NLS-inactivating modification, and deletion of the PEST domain.

[0088] Modifications can be made to any cyclin F polypeptide, i.e., any cyclin F polypeptide can represent an "unmodified cyclin F polypeptide" in which a modification has been made, thereby resulting in a modified cyclin F polypeptide. In some examples, an unmodified cyclin F polypeptide is a wild-type cyclin F as set forth in SEQ ID NO:2, or a cyclin F polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, or 99% sequence identity to SEQ ID NO:2. Thus, a modified cyclin F polypeptide of the disclosure may include one or more modifications described herein, with the remainder of the polypeptide (i.e., the portion of the polypeptide that does not include the modifications) having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, or 99% sequence identity to the wild-type cyclin F set forth in SEQ ID NO: 2. In some examples, a modified cyclin F polypeptide has at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, or 99% sequence identity to the wild-type cyclin F set forth in SEQ ID NO: 2.

[0089] Typically, the modified cyclin F polypeptide has increased targeting to the cytoplasm compared to wild-type cyclin F, i.e., targeting of the modified cyclin F polypeptide to the cytoplasm or accumulation of the modified cyclin F polypeptide in the cytoplasm is increased by at least or about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% or more compared to targeting to or accumulation in the cytoplasm as observed with wild-type cyclin F.

[0090] The modified cyclin F polypeptides of the present disclosure retain cyclin F activity (e.g., the ability to bind to TDP-43 and form a SKP1-CUL1-F box protein (SCF) ubiquitin ligase complex, as described in further detail below), and thus have therapeutic utility when delivered to neurons as described herein. More specifically, when delivered to neurons and introduced into the cytoplasm, the modified cyclin F polypeptides bind to and remove pathological TDP-43 that is preferentially and primarily cytoplasmically localized, without significantly interfering with nuclear-localized TDP-43.

[0091] 3.1 Nuclear export signals In some examples, the modified cyclin F polypeptides of the present disclosure comprise a heterologous nuclear export signal (NES). NESs are naturally present in many proteins that are exported from the nucleus to the cytoplasm. Nuclear export of proteins is primarily regulated through the exportin / CRM1 pathway, which involves the specific recognition of NESs in proteins by CRM1. NESs are usually 8-15 amino acids long and contain four or five hydrophobic (φ) residues characteristically spaced in diverse patterns, resulting in 11 classes of NES classes (classes 1a-d, 2, 3, 4, and 1a-d inversions) (Kosugi et al., 2008, Traffic9, 2053-2062; Fung et al., 2015, eLife4, e10034.). NESs are generally classified as Φ1-X 2,3 -Φ2-X 2,3 -A consensus sequence of Φ3-XΦ4 is identified, where Φn represents Leu, Val, Ile, Phe, or Met, and X can be any amino acid (Xu et al., 2012, Mol Biol Cell. 23:3677-3693). As demonstrated herein, cyclin F can be modified to contain a heterologous NES, resulting in a modified cyclin F polypeptide that is targeted primarily to the cytoplasm.

[0092] The modified cyclin F polypeptide may contain one, two, three or more NES. Additionally, any NES that directs a polypeptide into the cytoplasm may be utilized in the modified polypeptides of the present disclosure, and such NES are well known to those of skill in the art. Non-limiting examples of NES include those containing the sequence LPPLERLTL (SEQ ID NO: 8), LQLPPLERLTLD (SEQ ID NO: 9), LALKLAGLDL (SEQ ID NO: 10), PLQLPPLERLTL (SEQ ID NO: 11), ERFEMFRELNEALEL (SEQ ID NO: 12), LSSHFQELSI (SEQ ID NO: 13), ERFEMFRELNEALEL (SEQ ID NO: 14), DHAEKVAEKLEALSV (SEQ ID NO: 15), QLVEELLKIICAFQL (SEQ ID NO: 16), or TNLEALQKKLEELEL (SEQ ID NO: 17). Other NES sequences described in the art can be utilized in the context of the present disclosure, including those described by Xu et al. (2012, Mol Biol Cell. 23:3677-3693).

[0093] One or more NES may be included at any position in a cyclin F polypeptide, provided that it does not significantly adversely affect the activity of the cyclin F polypeptide (e.g., TDF binding activity, or SCF (Skp1-Cul1-F box protein) ubiquitin ligase complex formation activity) and is available for binding to CRM1 (e.g., not embedded within the three-dimensional structure of the modified cyclin F polypeptide). For example, an NES may be present at the N- or C-terminus of a modified cyclin F polypeptide (e.g., fused to the N- or C-terminus of an unmodified cyclin F polypeptide) or may be present within a modified cyclin F polypeptide. In a particular example, an NES is present at the C-terminus.

[0094] 3.2 Nuclear localization signal modification The modified cyclin F polypeptides of the present disclosure also include those having a nuclear localization signal (NLS) inactivating modification, resulting in inactivation of the endogenous NLS and thus reduced localization of the modified cyclin F polypeptide to the nucleus compared to wild-type cyclin F.

[0095] Wild-type cyclin F polypeptides have two endogenous NLSs. The first NLS (NLS1) is near the N-terminus and has been variously reported to be at positions 20-28 or 20-29, using the numbering for wild-type cyclin F as set forth in SEQ ID NO:2, while the second NLS (NLS2) is at positions 568-574 of wild-type cyclin F as set forth in SEQ ID NO:2. Inactivation of the NLS due to the presence of an NLS-inactivating modification may be partial or complete. As a result, the NLS-inactivating modification results in a modified cyclin F polypeptide that exhibits at least or about a 15%, 20%, 30%, 35%, 40%, 45%, or 50% reduction in nuclear targeting or localization compared to wild-type cyclin F when the modified cyclin F polypeptide is present. As will be understood, where a modified cyclin F polypeptide contains NLS-inactivating modifications at both endogenous NLSs, there may be a reduction in targeting or localization of the modified cyclin F polypeptide to the nucleus of at least or about 30%, 35%, 40%, 45% or 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% compared to wild-type cyclin F. Thus, in some cases, cytoplasmic localization of modified cyclin F polypeptides having an NLS-inactivating modification in one or both NLSs is increased by at least or about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400% or more compared to wild-type cyclin F.

[0096] In some instances, the NLS-inactivating modification comprises a deletion of all or a portion of the endogenous NLS. Thus, in some embodiments, the modified polypeptide comprises a deletion of all or a portion of NLS1, e.g., a deletion of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues from positions 20-29 relative to wild-type cyclin F as set forth in SEQ ID NO:2. In some examples, there is an amino acid deletion at positions 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 21-29, 21-28, 21-27, 21-26, 21-25, 22-29, 22-28, 22-27, 22-26, 22-29, 23-28, 23-27, 24-29, or 24-28, using numbering relative to wild-type cyclin F set forth in SEQ ID NO: 2. In further embodiments, the modified polypeptide comprises a deletion of all or a portion of NLS2, for example a deletion of 1, 2, 3, 4, 5, 6, or 7 amino acid residues from positions 568-574 relative to wild-type cyclin F set forth in SEQ ID NO: 2. Thus, in some embodiments, there is an amino acid deletion at positions 568-574, 568-573, 568-572, 568-571, 569-574, 569-573, 569-572, 569-571, 570-574, 570-573, 570-572, 571-574, 571-573, or 570-572, using the numbering relative to wild-type cyclin F set forth in SEQ ID NO: 2. As will be appreciated, if only a portion of NLS1 and / or NLS2 is deleted, a sufficient number of amino acids in the NLS (i.e. a sufficient portion of the NLS) is deleted such that localization of the modified cyclin F polypeptide to the nucleus is reduced compared to wild-type cyclin F.

[0097] In one embodiment, the modified cyclin F polypeptide comprises a deletion of both NLS1 and NLS2 and further comprises a heterologous NES. In particular examples, the modified cyclin F polypeptide comprises a sequence set forth in SEQ ID NO:4, or a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, or 99% sequence identity thereto (provided that the polypeptide comprises a deletion of both NLS1 and NLS2 and further comprises a heterologous NES).

[0098] In other examples, the NLS-inactivating modification comprises one or more amino acid substitutions of amino acid residues comprising NLS1 (i.e., positions 20-28 or 20-29, using the numbering relative to the wild-type cyclin F set forth in SEQ ID NO:2) or NLS2 (i.e., positions 568-574 of the wild-type cyclin F set forth in SEQ ID NO:2), such that the ability of the NLS to target the polypeptide to the nucleus is reduced compared to the wild-type NLS set forth as amino acids 20-28 or 20-29 (i.e., the ability of the modified cyclin F polypeptide comprising one or more amino acid substitutions to target or localize to the nucleus is reduced by at least or about 15%, 20%, 30%, 35%, 40%, 45%, or 50%, etc., compared to the wild-type cyclin F).

[0099] NLS motifs vary substantially in length and characteristics, but almost all share the simple feature of a short stretch of primarily basic amino acids, such as lysine (K) and arginine (R). Thus, in some instances, the NLS-inactivating modification comprises an amino acid substitution of one or more of K20, R21, R22, R24, R25, R26, and R28 (using the numbering relative to wild-type cyclin F set forth in SEQ ID NO:2) with a different amino acid residue. In some instances, the substitution is with a non-basic amino acid residue (e.g., aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), glycine (G), proline (P), alanine (A), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), or valine (V)). In one example, one or more of K20, R21, R22, R24, R25, R26, and R28 are substituted with alanine. In another example, the NLS-inactivating modification comprises an amino acid substitution of one or more of R568, R569, K571, R572, K574, and R574 (using numbering relative to wild-type cyclin F set forth in SEQ ID NO:2) with a different amino acid residue. In some examples, the substitution is with a non-basic amino acid residue (e.g., aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine ​​(C), glycine (G), proline (P), alanine (A), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), or valine (V)). In one example, one or more of R568, R569, K571, R572, K574, and R574 are substituted with alanine.

[0100] In particular examples, the NLS inactivating modification comprises a deletion of a portion of NLS1 and an amino acid substitution of one or more amino acid residues from NLS1. In other examples, the NLS inactivating modification comprises a deletion of a portion of NLS2 and an amino acid substitution of one or more amino acid residues from NLS2.

[0101] 3.3 Other deletions Modified cyclin F polypeptides of the present disclosure may have one or more additional amino acid deletions, thereby resulting in a truncated cyclin F polypeptide compared to wild-type cyclin F. Truncated cyclin F polypeptides and encoding polynucleotides may have advantages compared to wild-type polypeptides and polynucleotides, such as in the production of polypeptides or polynucleotides, where shorter polypeptides or polynucleotides are typically easier to produce in large quantities and high quality than longer polypeptides or polynucleotides, and in the delivery of polypeptides or polynucleotides, where shorter polypeptides or polynucleotides are typically easier to deliver to cells, such as neurons, than longer polypeptides or polynucleotides. This is particularly true in the case of viral vector delivery of polynucleotides, where viral vectors have an upper limit to the length of nucleic acid they can carry in their genome.

[0102] As will be appreciated, the modified cyclin F polypeptides inhibit TDP-43 binding and the SCF (Skp1-Cul1-F box protein) ubiquitin ligase complex, SCF cyclin F Thus, it contains at least the TDP-43 binding domain and the SCF cyclin F Modified cyclin F polypeptides are provided that contain a structural domain of SCF. cyclin F The formation domain and the TDP-binding domain can be linked by one or more endogenous cyclin F amino acids or regions, or by other amino acid or peptide linkers, such that the resulting modified cyclin F polypeptide binds to TDP-43 and inhibits SCF cyclin FIt has the necessary conformation to form a complex. Amino acid and peptide linkers are well known in the art. Suitable linkers include, by way of example only, amino acids such as aminohexanoic acid, glycine, and serine, and stretches of two or more amino acids such as glycine and serine. The linker can be of any length, but is typically at least 20 amino acids long. Thus, in one embodiment, the linker is at least 20, 30, 40, or 50 amino acids long.

[0103] The TDP-43 binding domain is the minimal amino acid region required to facilitate the binding of cyclin F to TDP-43. For purposes of this disclosure, binding of a modified cyclin F polypeptide comprising a TDP-43 binding domain to TDP-43 means at least 50% of the binding observed for wild-type cyclin F as set forth in SEQ ID NO:2. Methods for assessing the binding of a polypeptide comprising a TDP-43 binding domain to TDP-43 are well known in the art and include, for example, pull-down assays, microscale thermophoresis (MST), surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC). In one example, an immunoprecipitation assay (e.g., using an antibody or GFP / RFP trap) is utilized to co-immunoprecipitate cyclin F polypeptide with TDP-43. The resulting eluate is analyzed, for example, by immunoblotting or mass spectrometry. In another example, immunofluorescence microscopy and / or proximity ligation-based methods are utilized. As previously determined by the inventors, the TDP-43 binding domain is within the cyclin box of cyclin F (i.e., within the region spanning amino acid positions 292-405 of wild-type cyclin F as set forth in SEQ ID NO:2). Thus, in some embodiments, modified cyclin F polypeptides include those spanning positions 292-405, 300-400, 310-390, 320-370, 330-360, 300-390, 310-390, 320-370, 330-360, 330-390, 340-345, 340-346, 340-347, 340-348, 340-349, 340-350, 340-351, 340-352, 340-353, 340-354, 340-355, 340-356, 340-357, 340-358, 340-359, 341-359, 342-359, 343-359, 344-359, 345-359, 346-359, 347-359, 348-359, 349-350, 349-350, 349-351, 349-352, 349-353, 349-354, 349-355, 349-356, 349-357, 349-358, 349-359, 350-350, 351-352, 352-353, 35 or 320 to 380, 310 to 370, 310 to 360, 310 to 350, 300 to 340, 310 to 380, 310 to 370, 310 to 360, 310 to 350, 320 to 380, or 320 to 260, of the cyclin domain at positions 292 to 405 of the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2.

[0104] The modified cyclin F polypeptide also binds to the SCF (Skp1-Cul1-F box protein) ubiquitin ligase complex, SCF cyclin FThus, the modified cyclin F polypeptide retains the domains and sequences necessary to facilitate the formation of SCF cyclin F Contains the structural domain. SCF cyclin F Methods for assessing the ability of a polypeptide to form a complex are well known in the art and include, for example, immunoprecipitation and ubiquitination assays. In one example, the ability of a cyclin F polypeptide to bind to Skp1, Rbx1, and Cul1 is assessed by immunoprecipitating the cyclin F polypeptide from cells (e.g., using a tagged polypeptide and an antibody that recognizes the tag e), and then assessing the eluates of Skp1, Rbx1, and Cul1 by immunoblotting or mass spectrometry. Once Skp1-Cul1-Rbx1 E3 ligase is confirmed to be intact, an in vitro ubiquitination assay is performed to determine the ability of the immunoprecipitated SCF cyclin F The assay can be used to measure the activity of the complex. In this assay, all the components required for ubiquitination (e.g., ATP, ubiquitin, substrate (recombinant TDP-43), E1, E2, and immunoprecipitated SCF cyclin F The modified cyclin F polypeptides (complexes) are mixed and ubiquitination is assessed and compared to background ubiquitination (e.g., assessed using an enzymatically inactive cyclin F variant, such as a cyclin F(LP / AA) variant). In some embodiments, the modified cyclin F polypeptide comprises all or a portion of the F-box present at positions 29-76 of a wild-type cyclin F polypeptide set forth in SEQ ID NO:2, such as at least or about at least or about 15, 20, 25, 30, 35, 40, or 45 amino acid residues of the F-box. In some examples, the modified cyclin F polypeptide comprises, or at least comprises, positions 29-76, 29-70, 29-65, 29-60, 29-55, 29-50, 35-76, 35-70, 35-65, 35-60, 35-55, 40-79, 40-70, 40-65, 40-60, 45-76, 45-70, or 45-65 of the wild-type cyclin F polypeptide set forth in SEQ ID NO:2.

[0105] In some examples, modified cyclin F polypeptides of the disclosure include a deletion of all or a portion of the endogenous PEST domain (amino acids 582-766 of wild-type cyclin F as set forth in SEQ ID NO:2). As demonstrated for the first time herein, deletion of all or a portion of the endogenous PEST domain surprisingly results in a modified cyclin F polypeptide that is directed to and localized in the cytoplasm. In some examples, modified cyclin F polypeptides include a deletion of at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids from the PEST domain at amino acid positions 582-766, using numbering relative to the wild-type cyclin F polypeptide as set forth in SEQ ID NO:2. Thus, in some examples, the modified cyclin F polypeptide has a structure similar to that of a wild-type cyclin F polypeptide set forth in SEQ ID NO:2, but which is selected from the group consisting of positions 582-766, 582-760, 582-750, 582-740, 582-730, 582-720, 582-710, 582-700, 590-766, 590-760, 590-750, 590-740, 590-730, 590-720, 590-730, 590-740, 590-750, 590-740, 590-730, 590-720, 590-750, 590-740, 590-730, 590-720, 590-730, 590-74 ... The sequence comprises or at least comprises a deletion of amino acids at positions 90 to 710, 590 to 700, 600 to 766, 600 to 760, 600 to 750, 600 to 740, 600 to 730, 600 to 720, 600 to 710, 600 to 700, 610 to 766, 610 to 760, 610 to 750, 610 to 740, 610 to 730, 610 to 720, 610 to 710, or 610 to 700. In one example, the modified cyclin F polypeptide comprises a sequence set forth in SEQ ID NO:6, or a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 95%, or 99% sequence identity thereto (provided that the polypeptide includes a deletion of all or a portion of the PEST domain).

[0106] 3.4 Nucleic acid constructs Also provided are nucleic acid molecules that encode modified cyclin F polypeptides described herein, i.e., that include a coding sequence for a modified cyclin F polypeptide described herein. Thus, the nucleic acid molecule includes a modified CCNF polynucleotide that includes one or more modifications (e.g., nucleotide deletions, insertions, or substitutions) relative to a wild-type CCNF polynucleotide (as set forth in SEQ ID NO:1) or to a polynucleotide encoding a wild-type cyclin F. In certain embodiments, the nucleic acid molecule includes a coding sequence for a modified cyclin F polypeptide, the coding sequence being operably linked to a promoter. Thus, non-limiting examples of CCNF polynucleotides of the present disclosure include those that encode modified cyclin F polypeptides as set forth in SEQ ID NO:4 or 6, or modified cyclin F polypeptides having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 or 6. In some embodiments, the nucleic acid molecule comprises a sequence set forth in SEQ ID NO:3 or 5, a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3 or 5, e.g., as determined by a sequence alignment program described herein using default parameters.

[0107] The present disclosure also contemplates polynucleotides that hybridize to the CCNF nucleotide sequences set forth in SEQ ID NO: 3 or 5, or their complements, under stringent conditions as described below. As used herein, the term "hybridizes under moderate stringency, high stringency, or very high stringency conditions" describes hybridization and wash conditions. Guidance for performing hybridization reactions can be found in Ausubel et al., (1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference, and either can be used. References herein to moderate stringency conditions include and encompass at least about 16% v / v to at least about 30% v / v formamide, and at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C., and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C. Moderate stringency conditions may also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS, or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 60-65° C. One embodiment of medium stringency conditions includes hybridizing in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. High stringency conditions include and encompass at least about 31% v / v to at least about 50% v / v formamide, and about 0.01 M to about 0.15 M salt for hybridization at 42° C., and about 0.01 M to about 0.02 M salt for washing at 55° C. High stringency conditions can also include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS, or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at temperatures greater than 65° C.One embodiment of high stringency conditions includes hybridizing in 6xSSC at about 45°C, followed by one or more washes in 0.2xSSC, 0.1% SDS at 65°C.

[0108] In certain embodiments, the cyclin F polypeptide is encoded by a polynucleotide that hybridizes to the disclosed nucleotide sequence under very high stringency conditions. One embodiment of very high stringency conditions includes hybridizing at 65° C. in 0.5 M sodium phosphate, 7% SDS, followed by one or more washes at 65° C. in 0.2×SSC, 0.1% SDS.

[0109] Other stringency conditions are well known in the art, and one of skill in the art will recognize that various factors can be manipulated to optimize hybridization specificity. Optimization of the stringency of the final wash can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al., (1998, supra), pages 2.10.1-2.10.16, and Sambrook et al., (1989, supra), sections 1.101-1.104.

[0110] Stringent washes are typically performed at temperatures between about 42°C and 68°C, although one of skill in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rates typically occur at temperatures between about 20°C and 25°C below the Tm for the formation of DNA-DNA hybrids. Tm is well known in the art to be the melting temperature, or the temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating Tm are well known in the art (see Ausubel et al., (1994, supra), p. 2.10.8). In general, the Tm of a perfectly matched DNA duplex can be predicted as an approximation by the following formula: Tm = 81.5 + 16.6 (log10M) + 0.41 (% G + C) - 0.63 (% formamide) - (600 / length)

[0111] where M is the concentration of Na+, preferably in the range of 0.01 molar to 0.4 molar, %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases in the range of 30% to 75% G+C, %formamide is the volume percent formamide concentration, and length is the number of base pairs in the DNA duplex. The Tm of a double-stranded DNA decreases by about 1° C. for every 1% increase in the number of randomly mismatched base pairs. Washing is generally performed at Tm-15° C. for high stringency or Tm-30° C. for medium stringency.

[0112] In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose or nylon membrane) containing immobilized DNA is hybridized overnight at 42° C. in hybridization buffer (50% deionized formamide, 5×SSC, 5×Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% bovine serum albumin), 0.1% SDS, and 200 mg / mL denatured salmon sperm DNA) containing labeled probe. The membrane is then subjected to two successive moderate stringency washes (i.e., 2x SSC, 0.1% SDS at 45°C for 15 minutes, followed by 2x SSC, 0.1% SDS at 50°C for 15 minutes), followed by two successive higher stringency washes (i.e., 0.2x SSC, 0.1% SDS at 55°C for 12 minutes, followed by 0.2x SSC and 0.1% SDS solution at 65-68°C for 12 minutes).

[0113] 4. Delivery Vehicle The present disclosure also contemplates a delivery vehicle for delivering the CCNF polynucleotides (i.e., nucleic acid molecules encoding modified cyclin F polypeptides) of the present disclosure or the modified cyclin F polypeptides of the present disclosure to neurons (e.g., motor neurons). Delivery vehicles suitable for nucleic acid molecules and / or polypeptides are well known in the art and can be utilized in methods for delivering CCNF polynucleotides or modified cyclin F polypeptides to neurons. For example, viral vectors can be used to deliver the CCNF polynucleotides of the present disclosure, while non-viral vectors (e.g., liposomes, exosomes, polymers, nanoparticles, etc.) can be used to deliver the modified cyclin F polypeptides of the present disclosure. Thus, delivery vehicles, such as any of those described below, that include the modified cyclin F polypeptides described herein or the nucleic acid molecules encoding the modified cyclin F polypeptides described herein are also provided.

[0114] 4.1 Viral vectors Viral vectors suitable for delivery of nucleic acid molecules encoding the modified cyclin F polypeptides disclosed herein include, but are not limited to, adeno-associated viral vectors (AAV), lentiviral vectors, adenoviral vectors, and herpes simplex viral vectors, and in certain embodiments, are neurotropic viral vectors.

[0115] 4.1.1 Adeno-associated viruses CCNF polynucleotide can be delivered to cells of the central nervous system, including neurons (e.g., motor neurons) by using adeno-associated virus vector (AAV vector).The use of AAV vector to deliver genes into the brain is well known in the art (see, for example, U.S. Patent Nos. 8,198,257 and 7,534,613, U.S. Patent Application No. 13 / 881,956, each of which is incorporated by reference).

[0116] AAV vectors for delivering CCNF polynucleotides to motor neurons are known in the art (see U.S. Patent No. 7,335,636, incorporated by reference). AAV vectors can be constructed using known techniques to provide at least operably connected components of control elements, including a transcription initiation region (e.g., promoter), a transcription termination region, and optionally at least one posttranscriptional regulatory sequence. The control elements are selected to be functional in targeted cells. The resulting constructs containing the operably connected components are typically flanked at the 5' and 3' regions by functional AAV inverted terminal repeats (ITRs).

[0117] The nucleotide sequence of the AAV ITR region is known. The ITR sequence of AAV-2 is described, for example, by Kotin et al. Human Gene Therapy, 5:793-01 (1994); Fields & Knipe, Fundamental Virology, "Parvoviridae and their Replication" (2d ed. 1986). Those skilled in the art will understand that AAV ITRs can be modified using standard molecular biology techniques (e.g., Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)). Thus, the AAV ITRs used in the vectors of the present disclosure do not need to have a wild-type nucleotide sequence, but can be modified, for example, by insertion, deletion, or substitution of nucleotides. In addition, the AAV ITRs can be derived from any of several AAV serotypes, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, AAV7, AAV8, and AAV9. Furthermore, the 5' and 3' ITRs flanking a selected nucleotide sequence in an AAV expression vector do not necessarily need to be identical or derived from the same AAV serotype or isolate, so long as the intended ITRs are functional, i.e., allow for removal and replication of the linked nucleotide sequence of interest when the AAV rep gene product is present in a cell.

[0118] Those skilled in the art will appreciate that regulatory sequences can often be provided from commonly used promoters derived from viruses such as polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. The use of viral regulatory elements to induce protein expression can allow high-level constitutive expression of the protein in a variety of host cells. Ubiquitously expressed promoters that can be used similarly also include, for example, the early cytomegalovirus promoter (Boshart et al., Cell, 41:521-30 (1985)), herpes virus thymidine kinase promoter (McKnight et al. Cell, 37:253-62 (1984)), β-actin promoter (e.g., human β-actin promoter, Ng et al., Molecular Cell Biology, 5:2720-32 (1985)), and colony-stimulating factor-1 promoter (Ladner et al., EMBO J., 6:2693-98 (1987)).

[0119] Alternatively, the regulatory sequence of the AAV vector can preferentially direct the expression of a gene in a particular cell type, i.e., tissue-specific regulatory elements can be used. Non-limiting examples of tissue-specific promoters that can be used include central nervous system (CNS) specific promoters, such as neuron-specific promoters (e.g., neurofilament promoter, Byrne and Ruddle, Proc. Natl. Acad. Sci. USA, 86:5473-77 (1989)) and glia-specific promoters (Morii et al., Biochemical & Biophysical Research Communications, 175:185-91 (1991)). In certain embodiments, the promoter is tissue-specific and is essentially not active outside the central nervous system, or the activity of the promoter is higher in the central nervous system than in other systems. For example, promoters may be selected that are specific to the spinal cord, brainstem, (medulla, corpus callosum, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (striatum, cerebral cortex, or occipital, temporal, parietal, or frontal lobes in the cerebral cortex), or combinations thereof. The promoter may be specific to a particular cell type, such as neurons or glial cells in the CNS. If active in glial cells, it may be specific to astrocytes, oligodendrocytes, ependymal cells, Schwann cells, or microglia. If active in neurons, it may be specific to a particular type of neuron, such as motor neurons, sensory neurons, or interneurons. In addition, it may be specific to neurons with a particular phenotype, such as dopaminergic neurons, serotonergic neurons, and the like. In certain embodiments, the promoter is specific to cells in a particular region of the brain, such as the cortex, striatum, substantia nigra, and hippocampus.

[0120] Suitable neuron-specific promoters include, but are not limited to, the synapsin promoter, neuron-specific enolase (NSE) (Olivia et al., Genomics, 10:157-65 (1991). GenBank Accession No. X51956), and the human neurofilament light chain promoter (NEFL) (Rogaev et al., Human Molecular Genetics, 1:781 (1992), GenBank Accession No. L04147). Glia-specific promoters include, but are not limited to, the glial fibrillary acidic protein (GFAP) promoter (Morii et al., Biochemical & Biophysical Research Communications, 175:185-91 (1991), GenBank Accession No. M65210), the S100 promoter (Morii et al., Biochemical & Biophysical Research Communications, 175:185-91 (1991), GenBank Accession No. M65210), and the glutamine synthase promoter (Van den et al., Biochimica Biophysica Acta, 2:249-51 (1991), GenBank Accession No. X59834). In a preferred embodiment, the gene is flanked upstream (i.e., 5') by the neuron-specific enolase (NSE) promoter. In another preferred embodiment, the gene of interest is flanked upstream (i.e., 5') by the elongation factor 1 alpha (EF) promoter. Suitable phenotype-specific promoters include, but are not limited to, the tyrosine hydroxylase promoter, the dopamine beta hydroxylase promoter, the acetylcholinesterase promoter, the choline acetyltransferase promoter, the dopamine receptor I and II promoter, the dopamine transporter promoter, the vesicular monoamine transporter promoter, the neopsin promoter, and the vesicular acetylcholine transporter promoter.

[0121] The nucleic acid construct capable of expressing CCNF polynucleotide and AAV vector carrying post-transcriptional regulatory sequence (PRE) adjacent to AAV ITR can be constructed by directly inserting the nucleotide sequence of interest and PRE into the AAV genome, which has the main AAV open reading frame (ORF) excised therefrom.Other parts of the AAV genome can also be deleted, as long as sufficient parts of ITR remain to allow replication and packaging functions.These constructs can be designed using techniques well known in the art. (For example, Lebkowski et al., Molecular & Cellular Biology, 8:3988-96 (1988), Vincent et al., Vaccines90 (Cold Spring Harbor Laboratory Press, 1990), Carter, Current Opinion Biotechnology, 3:533-39 (1992), Muzyczka, Current Topics Microbiology & Immunology, 158:97-29 (1992), Kotin, Human Gene Therapy, 5:793-01 (1994), Shelling et al., Gene Therapy, 1:165-69 (1994), and Zhou et al., J Experimental Medicine, 179:1867-75 (1994)). Alternatively, the AAV ITRs can be excised from the viral genome or from an AAV vector containing them and fused 5' and 3' to a selected nucleic acid construct present in another vector using standard ligation techniques, such as those described in Green & Sambrook (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)). Several AAV vectors are available from the American Type Culture Collection (ATCC) under accession numbers 53222, 53223, 53224, 53225, and 53226.

[0122] To produce recombinant AAV particles, the AAV vector can be introduced into a suitable host cell using known techniques such as transfection. Several transfection techniques are commonly known in the art (see, for example, Graham et al., Virology, 52:456 (1973); Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012); Davis et al., Basic Methods Molecular Biology, (Elsevier, 1986); and Chu et al., Gene, 13:197 (1981)). Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al., Virology, 52:456-67 (1973)), direct microinjection into cultured cells (Capecchi, Cell, 22:479-88 (1980)), electroporation (Shigekawa et al., BioTechniques, 6:742-51 (1988)), liposome-mediated gene transfer (Mannino et al., BioTechniques, 6:682-90 (1988)), lipid-mediated transduction (Feigner et al., Proceedings Nat'l Acad. Sci. USA, 84:7413-17 (1987)), and nucleic acid delivery using high-velocity microprojectiles (Klein et al., Nature 327:70-73 (1987)).

[0123] Suitable host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of exogenous nucleic acid molecules. Thus, as used herein, "host cells" generally refer to cells that are transfected with exogenous nucleic acid molecules. Host cells include any eukaryotic cell or cell line, so long as the cell or cell line is not incompatible with the protein to be expressed, the selection system selected, or the fermentation system used. Non-limiting examples include CHO DHFR minus cells (Urlaub and Chasin Proceedings Nat'l Acad. Sci. USA, 77:4216-420 (1980)), 293 cells (Graham et al., J. General Virology 36:59-72 (1977)), or myeloma cells such as SP2 or NSO (Galfre & Milstein, Methods Enzymology, 73:3-46 (1981)).

[0124] In some embodiments, the host cells are cells from a stable human cell line, 293 (readily available through the ATCC under accession number ATCC CRL1573), which is a human embryonic kidney cell line that has been transformed with adenovirus type 5 DNA fragments (Graham et al., J. General Virology, 36:59-72 (1977)) and expresses the adenovirus E1a and E1b genes (Aiello et al., Virology, 94:460-69 (1979)). The 293 cell line is easily transfected and provides a particularly convenient platform for producing AAV virions.

[0125] The host cell containing the AAV vector described above must be made capable of providing AAV helper functions to replicate and encapsidate the expression cassette flanked by AAV ITRs to produce recombinant AAV particles. AAV helper functions are generally AAV-derived coding sequences that can be expressed to provide AAV gene products that then function in trans for productive AAV replication. AAV helper functions are used herein to complement the necessary AAV functions that are missing from AAV vectors. Thus, AAV helper functions include one or both of the major AAV open reading frames (ORFs), i.e., the rep and cap coding regions, or functional homologues thereof.

[0126] The AAV rep coding region of the AAV genome encodes the replication proteins Rep78, Rep68, Rep52, and Rep40. These Rep expression products have been shown to have many functions, including recognition, binding, and nicking of the AAV origin of DNA replication, DNA helicase activity, and regulation of transcription from AAV (or other exogenous) promoters. The Rep expression products are collectively required to replicate the AAV genome. The AAV cap coding region of the AAV genome encodes the capsid proteins VP1, VP2, and VP3, or functional homologs thereof. AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct prior to or simultaneously with transfection of the AAV vector containing the expression cassette, and thus the AAV helper construct is used to provide at least transient expression of the AAV rep and / or cap genes to complement missing AAV functions required for productive AAV infection. AAV helper constructs lack AAV ITRs and cannot replicate or package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. Several AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and plM29+45, which code for both Rep and Cap expression products (see, e.g., Samulski et al., J. Virology, 63:3822-28 (1989); McCarty et al., J. Virology, 65:2936-45 (1991)). Several other vectors have been described that code for Rep and / or Cap expression products (see, e.g., U.S. Patent No. 5,139,941, incorporated by reference).

[0127] As a result of the infection of host cells by helper virus, AAV Rep and / or Cap proteins are produced. Rep protein also plays a role in replicating the AAV genome. The expressed Cap protein assembles into a capsid, and the AAV genome is packaged into the capsid. This results in AAV being packaged into recombinant AAV particles that contain the expression cassette. After recombinant AAV replication, the recombinant AAV particles can be purified from the host cells using various conventional purification methods, such as CsCl gradient. The resulting recombinant AAV particles are then ready to be used for gene delivery into various cell types.

[0128] In some embodiments, the number of viral vectors and / or virion particles administered to a subject is greater than or equal to 10 3 ~10 15 on the order of magnitude of particles / mL, or, for example, about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some embodiments, the amount of the saturation agent may be 10 particles / mL or any value therebetween. 13 More than 1000 particles / mL of vector and / or virion particles are administered. Volumes between 1 μL and 10 mL are used to administer 10 ... 2 ~10 16 Thus, in some embodiments, the total vector and / or virion particles may be administered to receive about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 14 , or 10 16of vectors and / or virion particles are administered.

[0129] In the practice of the method of the present disclosure, any serotype of AAV can be used. The serotype of the viral vector used in certain embodiments of the present invention is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 (see, for example, Gao et al., 2002, PNAS99:11854-11859, and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Other serotypes than those listed herein may also be used. Furthermore, pseudotyped AAV vectors can also be utilized in the methods described herein. A pseudotyped AAV vector is a vector that contains the genome of one AAV serotype in the capsid of a second AAV serotype, for example, an AAV vector containing an AAV2 capsid and an AAV1 genome, or an AAV5 capsid and an AAV2 genome (Auricchio et al., 2001. Hum. Mol. Genet. 10(26):3075-81). AAV vectors are derived from single-stranded (ss) DNA parvoviruses that are non-pathogenic to mammals (reviewed in Muzyscka, 1992. Curr. Top. Microb. Immunol. 158:97-129). Briefly, recombinant AAV-based vectors have the rep and cap viral genes, which make up 96% of the viral genome, removed, leaving two adjacent 145 base pair (bp) inverted terminal repeats (ITRs) that are used to initiate viral DNA replication, packaging, and integration. In the absence of helper virus, wild-type AAV can integrate into the human host cell genome with preferential site specificity at chromosome 19q 13.3 or be maintained episomally. A single AAV particle can accommodate up to 5 kb of ssDNA, thus leaving about 4.5 kb for transgenes and regulatory elements, which is typically sufficient. However, the trans-splicing system described, for example, in U.S. Patent No. 6,544,785, can nearly double this limit.

[0130] In certain cases, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, and CSp3.

[0131] AAV-based gene therapy vectors targeted to cells of the CNS are described, for example, in U.S. Patent Nos. 6,180,613 and 6,503,888. Additional exemplary AAV vectors are recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 7, AAV2 / 8, and AAV2 / 9 serotype vectors encoding human proteins. In certain embodiments, the AAV is a neurotropic AAV selected from rAAV2 / 1, rAAV2 / 8, and rAAV2 / 9, for example, as described in Ayers et al. (2015, Mol Ther. 23(1):53-62).

[0132] Alternatively, the vector of the present disclosure may be a virus other than adeno-associated virus, or a portion thereof, that allows expression of the CCNF nucleic acid molecule introduced into the viral nucleic acid. For example, replication-defective retroviruses, adenoviruses, herpes simplex viruses, and lentiviruses may be used. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel et al., Current Protocols in Molecular Biology §§9.10-9.14 (Greene Publishing Associates, 1989) and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are well known to those skilled in the art. Examples of suitable packaging virus lines include Crip, Cre, 2, and Am. The genome of adenovirus can be engineered to encode and express a protein of interest but be inactivated in terms of its ability to replicate in the normal lytic viral life cycle (see, e.g., Berkner et al., BioTechniques, 6:616-29 (1988); Rosenfeld et al., Science, 252:431-34 (1991); Rosenfeld et al., Cell 68:143-55 (1992)). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art.

[0133] 4.1.2 Lentiviruses Lentiviral vectors can be utilized to express CCNF polynucleotides in cells of the nervous system, including neurons (e.g., motor neurons), and the production of suitable lentiviral vectors is well known in the art (see, e.g., U.S. Patent Application No. 13 / 893,920, incorporated by reference). Lentiviral vectors according to the present disclosure can be derived from or derived from any suitable lentivirus. Recombinant lentiviral particles can transduce target cells with nucleotides of interest. Once inside the cell, the vector particles are reverse transcribed into DNA and integrated into the genome of the target cell from the RNA genome.

[0134] Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin et al., Retroviruses 758-763 (Cold Spring Harbor Laboratory Press, 1997). Briefly, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SrV). The non-primate lentivirus group includes the prototypic "slow virus" visnaimaedi virus (VMV) and the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[0135] Lentiviruses differ from other members of the Retroviridae family in that they have the ability to infect both dividing and non-dividing cells (Lewis et al., EMBO J., 11:3053-58 (1992); Lewis & Emerman, J Virology, 68:510-16 (1994)). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as the cells that make up muscle, brain, lung, and liver tissue.

[0136] A lentiviral vector, as used herein, is a vector that contains at least one component part that can be derived from a lentivirus. The component part can be involved in the biological mechanism by which the vector infects cells, expresses genes, or is replicated. The basic structure of retrovirus and lentivirus genomes share many common features, such as 5'LTR and 3'LTR, between or among which are located packaging signals that allow the genome to be packaged, primer binding sites, integration sites that allow integration into the host cell genome, and gag, pol, and env genes that code for packaging components that are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as rev and rev response element (RRE) sequences, which allow efficient export of RNA transcripts of integrated proviruses from the nucleus to the cytoplasm of infected target cells. In proviruses, viral genes are flanked on both sides by regions called long terminal repeats (LTRs). LTRs are involved in proviral integration and transcription. LTRs can also function as enhancer promoter sequences and control the expression of viral genes. The LTRs themselves are identical sequences that can be divided into three elements called "U3", "R" and "U5". U3 is derived from a sequence unique to the 3' end of the RNA, R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably between different viruses.

[0137] In defective lentiviral vector genomes, gag, pol, and env may be absent or non-functional. The R regions at both ends of the RNA are repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome, respectively.

[0138] In the exemplary lentiviral vector of the present disclosure, at least a portion of one or more protein coding regions essential for replication can be removed from the virus, which makes the viral vector replication-deficient. A portion of the viral genome can also be replaced by a nucleic acid to generate a vector that can transduce a target non-dividing host cell and / or that contains a nucleic acid that can integrate its genome into the host genome. In one embodiment, the lentiviral vector is a non-integrating vector as described in U.S. Patent Application No. 12 / 138,993 (herein incorporated by reference).

[0139] In further embodiments, the vector has the ability to deliver sequences that are entirely free of or devoid of viral RNA. A heterologous binding domain (heterologous to gag) can be located on the RNA to be delivered, and a cognate binding domain on gag or pol can be used to ensure packaging of the RNA to be delivered. Both of these vectors are described in U.S. Patent Application Serial No. 12 / 139,035, which is incorporated herein by reference. Lentiviral vectors may be "non-primate" vectors, i.e., derived from viruses that do not primarily infect primates, especially humans.

[0140] Examples of non-primate lentiviruses can be any member of the Lentiviridae family that does not naturally infect primates and can include feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), Maedi-visna virus (MW), or equine infectious anemia virus (EIAV).

[0141] In some embodiments, the viral vector is derived from EIAV. EIAV has the simplest genome structure of lentiviruses. In addition to gag, pol, and env genes, EIAV encodes three other genes: tat, rev, and S2. tat acts as a transcriptional activator of the viral LTR (Derse & Newbold, Virology, 194:530-36 (1993); Maury et al., Virology, 200:632-42 (1994)). rev regulates and modulates the expression of viral genes through the rev response element (RRE) (Martarano et al., J. Virology, 68:3102-11 (1994)). The mechanism of action of these two proteins is thought to be broadly similar to the similar mechanism in primate viruses (Martarano et al., J. Virology, 68:3102-11 (1994)). The function of S2 is unknown. In addition, an EIAV protein, Ttm, has been identified that is encoded by the first exon of tat spliced ​​into the env coding sequence at the start of the transmembrane protein.

[0142] Viral vectors can be engineered to remove non-essential elements and retain essential elements to provide the functions necessary to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell (see, e.g., U.S. Pat. No. 6,669,936, incorporated by reference). In some embodiments, the genome is limited to sufficient lentiviral genetic information to allow packaging of the RNA genome into viral particles capable of infecting a target cell in the presence of packaging components. Infection of a target cell can include reverse transcription and integration into the target cell genome. Lentiviral vectors carry non-viral coding sequences that are delivered by the vector to the target cell. In some embodiments, the vector is incapable of independent replication to produce infectious lentiviral particles in the final target cell. Typically, recombinant lentiviral vectors lack functional gag-pol and / or env genes, and / or other genes essential for replication. The vectors of the present disclosure can be configured as split intron vectors (see, e.g., U.S. Pat. No. 7,303,910, incorporated by reference).

[0143] The vector may be a self-inactivating vector. A self-inactivating retroviral vector can be constructed by deleting the transcription enhancer or enhancer and promoter in the U3 region of the 3'LTR. After a round of vector reverse transcription and integration, these changes are copied into both the 5' and 3' LTRs producing a transcriptionally inactive provirus (Yu et al., Proceedings Nat'l Acad. Sci. USA, 83:3194-98 (1986); Dougherty and Temin et al., Proceedings Nat'l Acad. Sci. USA, 84:1197-01 (1987); Hawley, Proceedings Nat'l Acad. Sci. USA, 84:2406-10 (1987); Yee et al., Proceedings Nat'l Acad. Sci. USA, 91:9564-68 (1994)). However, any promoters within the LTRs in such vectors will still be transcriptionally active. This strategy has been employed to eliminate the effect of enhancers and promoters in viral LTRs on transcription from internally placed genes. Such effects include increased transcription (Jolly et al., Nucleic Acids Research, 11:1855-72 (1983)) or repression of transcription (Emerman & Temin, Cell, 39:449-67 (1984)). This strategy can also be used to eliminate downstream transcription from the 3'LTR into genomic DNA (Herman & Coffin, Science, 236:845-48 (1987)). This is of particular concern in human gene therapy, where it is crucial to prevent the accidental activation of endogenous oncogenes.

[0144] The plasmid vector used to produce the viral genome in the host cell / packaging cell will also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the host cell / packaging cell. These regulatory sequences may be the native sequences associated with the transcribed lentiviral sequence, i.e., the native sequences associated with the 5'U3 region, or they may be another viral promoter, for example a heterologous promoter such as the CMV promoter. Some lentiviral genomes require additional sequences for efficient virus production. For example, in the case of HIV, rev and RRE sequences are preferably included, although the requirement for rev and RRE can be reduced or eliminated by codon optimization (see U.S. Patent Application Serial No. 12 / 587,236, incorporated by reference). Alternative sequences that perform the same function as the rev / RRE system are also known. For example, a functional analogue of the revIRRE system is found in the Mason-Pfizer monkey virus. It contains an RRE-type sequence in its genome known as a constitutive transport element (CTE) that is believed to interact with factors of the infected cell. The cellular factors can be considered as rev analogues. Therefore, CTE can be used as a substitute for the reviRRE system. Any other functional equivalents that are known or become available can be relevant to the methods of the present disclosure. For example, the Rex protein of HTLV-1 can functionally replace the Rev protein of HIV-1. Rev and Rex are also known to have similar effects as IRE-BP.

[0145] In certain embodiments, the lentiviral vector is a self-inactivating minimal lentiviral vector derived from Equine Infectious Anemia Virus (EIAV) capable of expressing CCNF polynucleotides. The vector can be produced by transient transfection of cells (e.g., HEK293T cells) with three plasmids encoding (1) recombinant EIAV PROSAVIN (Oxford BioMedica pic, Oxford UK) vector genome (Farley et al., J. Gen. Med., 9:345-56 (2007), U.S. Patent No. 7,259,015, incorporated by reference), (2) synthetic EIAV gag / pol expression vector (pESGPK, U.S. Patent Application Nos. 13 / 893,920 and 12 / 587,236, incorporated by reference), and (3) VSV-G envelope expression vector (pHGK).

[0146] 4.1.3 Herpes simplex virus Herpes simplex virus (HSV) vectors can also be utilized to deliver and express CCNF polynucleotides in cells of the nervous system, including neurons (e.g., motor neurons). The genome of type 1 (HSV-1) is a linear, double-stranded DNA of about 150 kb, characterized by about 70 genes. Many viral genes can be deleted without the virus losing its ability to grow. The "immediate early" (IE) genes are transcribed first. They code for trans-acting factors that regulate the expression of other viral genes. The "early" (E) gene products are involved in the replication of viral DNA. The late genes code for structural components of the virion and proteins that turn on the transcription of the IE and E genes or disrupt host cell protein translation.

[0147] HSV vectors can be a plasmid-based system, whereby a plasmid vector (called an amplicon) is generated that contains a gene-encoding nucleotide sequence and two cis-acting HSV recognition signals. The recognition signal is an origin of DNA replication and a cleaved packaging signal that does not code for an HSV gene product. Thus, a helper virus is required to replicate the amplicon and package it into an HSV coat. Thus, the vector does not express viral gene products in the recipient cell, and recombination or reactivation of latent virus by the vector is limited due to the minimal amount of HSV DNA sequences present in the defective HSV vector genome.

[0148] Examples of HSV-mediated gene therapy are well known in the art (Breakefield & DeLuca. New Biologist, 3:203-18 (1991); Ho & Mocarski, Virology, 167:279-93 (1988); Palella, et al., Molecular & Cellular Biology, 8:457-60 (1988); Palella et al., Gene, 80:137-44 (1988); Andersen et al., Human Gene Therapy, 3:487-99 (1992); Kaplitt et al., Current Topics Neuroendocrinology, 11:169-91 (1993); Spade & Frenkel, Cell, 30:295-04 (1982); Kaplitt et al., Molecular & Cellular Neuroscience, 2:320-30 (1991), Federoff et al., Proceedings Nat. Acad. Sci. USA, 89:1636-40 (1992)).

[0149] 4.1.4 Adenovirus Adenoviral vectors can be utilized to deliver and express CCNF polynucleotides in cells of the nervous system, including neurons (e.g., motor neurons). The adenoviral genome consists of approximately 36 kb of double-stranded DNA. Adenoviruses target airway epithelial cells, but can also infect neurons. Recombinant adenoviral vectors have been used as gene transfer vehicles for non-dividing cells. These vectors are similar to recombinant HSV vectors, in that the adenoviral E1a immediate early gene is deleted, but most viral genes are retained. The Ela gene is small (approximately 1.5 kb) and the adenoviral genome is approximately one-third the size of the HSV genome, so other non-essential adenoviral genes are deleted in order to insert foreign genes into the adenoviral genome.

[0150] Examples of adenovirus-mediated gene therapy are well known in the art (Akli et al., Nature Genetics, 3:224-28 (1993); La Salle et al., Science, 259:988-90 (1993); La Salle, Nature Genetics, 3:1-2 (1993); Neve, Trends Biochemical Sci., 16:251-53 (1993)).

[0151] 4.2 Non-viral delivery systems The modified cyclin F polypeptide and the nucleic acid molecule encoding the modified cyclin F polypeptide can be delivered using a non-viral delivery system. Any delivery method or system for delivery of nucleic acid molecules and / or proteins known in the art can be used to deliver the modified cyclin F polypeptide or the nucleic acid molecule encoding the modified cyclin F polypeptide. This includes, for example, delivery of the modified cyclin F polypeptide or the nucleic acid molecule encoding the modified cyclin F polypeptide to the desired tissue in colloidal dispersion systems, including macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Other delivery systems include exosomes, virosomes, nanoparticles (including gold or silica nanoparticles), polymers (e.g., dendrimers, polymer nanogels, etc.). Suitable delivery reagents for nucleic acids include, but are not limited to, Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, polycations (e.g., polylysine), atelocollagen, nanoplexes, and liposomes. The use of atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al. Nucleic Acids Research, 32:e109 (2004), Hanai et al. Annals NY Acad. Sci., 1082:9-17 (2006), and Kawata et al. Molecular Cancer Therapeutics, 7:2904-12 (2008).

[0152] In one example, liposomes are used as delivery vehicles. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. For liposomes to be an efficient transfer vehicle, the following properties should be present: (1) high efficiency encapsulation of genetic material or protein without impairing biological activity, (2) preferential and substantial binding to target cells compared to non-target cells, (3) high efficiency delivery of the aqueous contents of the vesicle to the target cell cytoplasm, and when relevant, (4) accurate and effective expression of genetic information (Mannino et al., BioTechniques, 6:682-90 (1988)).

[0153] Liposomes suitable for delivery of the modified cyclin F polypeptides described herein or nucleic acid molecules encoding the modified cyclin F polypeptides can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and sterols such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the bloodstream. Examples of suitable lipids for liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Additional examples of lipids include, but are not limited to, polylysine, protamine, sulfate, and 3.beta.-[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol. Various methods are known for preparing liposomes, as described, for example, in Szoka et al., Annual Rev. Biophysics & Bioengineering, 9:467-08 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, which are incorporated herein by reference.

[0154] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (MLVs). MLVs generally have diameters between 25 nm and 4 m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200-500 angstroms, which contain an aqueous solution in their core.

[0155] Liposomes for use with modified cyclin F polypeptides or nucleic acid molecules encoding modified cyclin F polypeptides may also be modified to avoid clearance by the mononuclear macrophage system ("MMS") and reticuloendothelial system ("RES"). Such modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure.

[0156] Opsonization-inhibiting moieties for use in preparing liposomes described herein are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization-inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by intercalation of a lipid-soluble anchor into the membrane itself or by direct binding to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly reduces uptake of liposomes by MMS and RES, as described, e.g., in U.S. Pat. No. 4,920,016, which is incorporated by reference.

[0157] In some embodiments, the opsonization-inhibiting moiety for modifying liposomes is a water-soluble polymer having a number average molecular weight of about 500 to about 40,000 daltons, or about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives, such as methoxy PEG or PPG, and synthetic polymers such as PEG or PPG stearates, polyacrylamides or poly N-vinylpyrrolidones, linear, branched, or dendrimer polyamidoamines, polyacrylic acids, polyalcohols to which carboxy or amino groups are chemically linked, such as polyvinyl alcohol and polyxylitol, and gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization-inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, a polysaccharide, a polyamidoamine, a polyethyleneamine, or a polynucleotide. Opsonization inhibiting polymers can also be, for example, naturally occurring polysaccharides that contain amino acids or carboxylic acids, such as galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan, aminated polysaccharides or oligosaccharides (linear or branched), or carboxylic acid polysaccharides or oligosaccharides that have been reacted with a derivative of carbonic acid resulting in the linkage of a carboxy group. In some embodiments, the opsonization inhibiting moiety is PEG, PPG, or a derivative thereof. Liposomes modified with PEG or PEG derivatives are sometimes referred to as "PEGylated liposomes."

[0158] 5. Delivery of Modified Cyclin F Polypeptides and Polynucleotides Neurons (e.g., motor neurons) can be contacted with a modified cyclin F polypeptide or encoding nucleic acid, e.g., in cell culture, in vitro or ex vivo, or administered to a subject, e.g., in vivo. In some embodiments, a modified cyclin F polypeptide or encoding nucleic acid described herein (optionally in a delivery vehicle as described herein) can be administered to a subject to treat or inhibit the development of neurodegenerative conditions, including those associated with neuronal TDP-43 proteinopathies, such as ALS, FTD, and AD.

[0159] For in vitro methods, neurons may be obtained from different sources. For example, neurons may be obtained from a subject. In some embodiments, neurons are whole cells. In some embodiments, the subject is afflicted with a neurodegenerative condition (e.g., a neurodegenerative condition associated with a neuronal TDP-43 proteinopathy). In some embodiments, the subject is at risk of developing a neurodegenerative condition (e.g., a neurodegenerative condition associated with a neuronal TDP-43 proteinopathy). In some embodiments, the subject is suspected of having a neurodegenerative condition (e.g., a neurodegenerative condition associated with a neuronal TDP-43 proteinopathy). In some embodiments, the subject is at risk of developing a condition characterized by neuronal cell death. In some embodiments, the subject is suspected of suffering from a condition characterized by neuronal cell death. In some embodiments, the subject is suffering from neuronal cell death. In some embodiments, the subject is afflicted with ALS. In some embodiments, the subject is afflicted with FTD. In some embodiments, the subject is afflicted with AD. In some embodiments, the subject is a carrier, e.g., an asymptomatic carrier. In some embodiments, the motor neuron cells are derived from embryonic stem cells (ESCs) of the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a mouse. In some embodiments, the mouse is a transgenic mouse. Methods for inducing motor neuron differentiation from embryonic stem cells are known in the art and are described, for example, in Di Giorgio et al., Nature Neuroscience (2007), published online 15 April 2007; doi:10.1038 / nn1885 and Wichterle et al., Cell (2002) 110:385-397. In some cases, induced pluripotent stem cells can be generated from the subject and then differentiated into motor neurons. One exemplary method of deriving motor neurons from a subject is described in Dimos, JT, et al. Science (2008) 321, 1218-122 (Epub Jul. 31, 2008).

[0160] For in vivo methods, an effective amount of a modified cyclin F polypeptide or encoding nucleic acid described herein can be administered to a subject. Methods of administering agents to a subject are known in the art and readily available to one of skill in the art.

[0161] Those skilled in the art will also understand that the modified cyclin F polypeptides or encoding nucleic acids described herein may be used to inhibit neuronal degeneration or enhance neuronal survival, which may lead to the treatment, inhibition of the onset, or amelioration of several conditions characterized by degeneration of neurons (e.g., motor neurons).

[0162] In certain embodiments, neuronal degeneration includes motor neuron degeneration. Motor neuron diseases (MNDs) are a group of neurodegenerative conditions that selectively affect motor neurons, the nerve cells that control voluntary muscle activity, including speech, walking, breathing, swallowing, and general body movements. Skeletal muscles are innervated by a group of neurons (lower motor neurons) located in the anterior horn of the spinal cord, which project anterior roots to muscle cells. These nerve cells are themselves innervated by the corticospinal tract or upper motor neurons that project from the motor cortex of the brain. In gross pathology, there is degeneration of the anterior horn of the spinal cord, and atrophy of the anterior roots. In the brain, there may be atrophy in the frontal and temporal lobes. On microscopic examination, neurons may show spongiosis, the presence of activated astrocytes and microglia, and several inclusions, including characteristic "skeletal-like" inclusions, Bunina bodies, and vacuolization. Motor neuron diseases are variable and destructive in their effects. They generally have specific differences in their origin and causes, but have a similar outcome for the patient: severe muscle weakness. Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), pseudobulbar palsy, progressive bulbar palsy, spinal muscular atrophy (SMA), and post-polio syndrome are all examples of MND. The primary site of motor neuron degeneration classifies the neurodegenerative conditions.

[0163] ALS, which affects both upper and lower motor neurons, is the most common form of MND. Progressive bulbar palsy affects the lower motor neurons of the brainstem, causing slurred speech and difficulty chewing and swallowing. Individuals with these conditions almost always have abnormal symptoms in the arms and legs. Primary lateral sclerosis is a disease of the upper motor neurons, while progressive muscular atrophy only affects the lower motor neurons of the spinal cord. Means for diagnosing MND are well known to those skilled in the art. Non-limiting examples of symptoms are described below.

[0164] 5.1 Amyotrophic lateral sclerosis (ALS) Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease or classical motor neuron disease, is a progressive and ultimately fatal disorder that eventually destroys signals to all voluntary muscles. In the United States, doctors use the terms motor neuron disease and ALS interchangeably. Both upper and lower motor neurons are affected. About 75% of patients with classical ALS will also develop weakness and wasting of the bulbar muscles (the muscles that control speech, swallowing, and chewing). Symptoms are usually seen first in the arms and hands, legs, or swallowing muscles. Muscle weakness and atrophy occur disproportionately on both sides of the body. Affected individuals lose strength and the ability to move their arms, legs, and body. Other symptoms include spasticity, tonic reflexes, muscle spasms, fasciculations, and increased problems with swallowing and speech formation. Speech may become slurred or nasal. When the diaphragm and chest wall muscles do not function properly, individuals lose the ability to breathe without mechanical support. The disease does not usually impair a person's mind or personality, but some recent studies suggest that some people with ALS may have changes in cognitive function, such as problems with decision-making and memory. ALS most commonly affects people between the ages of 40 and 60, but younger and adult people can also develop the disease. Men are affected more frequently than women. Most cases of ALS occur sporadically, and family members of those individuals are not thought to be at higher risk for developing the disease. However, familial forms of ALS exist in adults, which often result from mutations in genes involved in RNA metabolism (e.g., TDP-43 and FUS) and protein degradation (e.g., UBQLN2, TBK1, and CCNF). In addition, rare early-onset forms of ALS are genetic. Most individuals with ALS die of respiratory failure, usually within 3 to 5 years of the onset of symptoms. However, approximately 10% of affected individuals survive beyond 10 years.

[0165] 5.2 Frontotemporal dementia (FTD) Frontotemporal dementia (FTD) is a clinical manifestation of frontotemporal lobe degeneration, which is characterized by progressive neuronal loss primarily involving the frontal and / or temporal lobes, and a typical loss of over 70% of spindle neurons, while other neuronal types remain intact in parts of FTD, the frontal and temporal lobes atrophy or shrink. The frontal and temporal lobes of the brain are commonly associated with personality, behavior, and language. Common signs and symptoms vary depending on the part of the brain that is affected. Some patients with FTD have a dramatic change in their personality, becoming socially inept, obsessive, or emotionally indiscriminate, while others lose the ability to use language. Signs and symptoms include changes in social and personal behavior, lethargy, emotional blunting, and significant changes in deficits in both expressive and receptive language. Currently, there is no cure for FTD, but there are treatments that help alleviate symptoms.

[0166] 5.3 Spinal muscular atrophy (SMA) Spinal muscular atrophy (SMA) refers to several different disorders, all with a common genetic cause and symptoms of weakness resulting from loss of motor neurons in the spinal cord and brain stem. Skeletal muscle weakness and wasting is caused by progressive degeneration of the anterior horn cells of the spinal cord. This weakness is often more severe in the legs than the arms. SMA has a variety of forms, with different ages of onset, inheritance patterns, and severity and progression of symptoms. Some of the more common SMAs are described below.

[0167] Defects in the SMN gene product are considered the primary cause of SMA, and SMN protein levels correlate with survival in subjects affected by SMA. The most common form of SMA is caused by mutations in the SMN gene. The region of chromosome 5 that contains the SMN (survival motor neuron) gene has a large duplication. A large sequence containing several genes occurs twice in adjacent segments. Thus, there are two copies of the gene, SMN1 and SMN2. The SMN2 gene has an additional mutation that makes it less efficient at making the protein, albeit at a lower level. SMA is caused by the loss of the SMN1 gene from both chromosomes. The severity of SMA, ranging from SMA1 to SMA3, is partially related to the extent to which the remaining SMN2 gene can compensate for the loss of SMN1.

[0168] SMA type I, also called Werdnig-Hoffmann disease, becomes evident by the time a child is 6 months of age. Symptoms may include hypotonia (severely reduced muscle tone), decreased limb movement, loss of tendon reflexes, fasciculations, tremors, swallowing and feeding difficulties, and breathing problems. Some children also develop scoliosis (curvature of the spine) or other skeletal abnormalities. Affected children cannot sit or stand, and most die of respiratory failure, usually before the age of 2.

[0169] Symptoms of SMA type II usually begin after a child reaches 6 months of age. Features may include inability to stand or walk, breathing problems, hypotonia, reduced or absent tendon reflexes, and fasciculations. These children learn to sit but may not stand. Life expectancy varies, with some individuals surviving into adolescence or beyond.

[0170] Symptoms of SMA type III (Kugelberg-Welander disease) appear between the ages of 2 and 17 and include abnormal walking, difficulty running, climbing stairs, or rising from a chair, and fine tremors in the fingers. The lower limbs are most often affected. Complications include scoliosis and contractures, which are chronic shortening of muscles or tendons around joints caused by abnormal muscle tone and weakness, preventing the joints from moving freely.

[0171] Other forms of SMA include, for example, hereditary bulbospinal SMA Kennedy disease (X-linked, androgen receptor), SMA with respiratory distress (SMARD1) (chromosome 11, IGHMBP2 gene), distal upper limb predominant SMA (chromosome 7, glycyl-tRNA synthetase), and X-linked infantile SMA (gene UBE1).

[0172] Current treatment for SMA consists of preventing and managing the secondary effects of chronic motor unit loss. Several drugs in clinical trials for the treatment of SMA include butyrate, valproic acid, hydroxyurea, and riluzole.

[0173] Symptoms of Fazio-Rondo disease appear between 1 and 12 years of age and may include facial weakness, dysphagia (difficulty swallowing), stridor (a high-pitched breath sound often associated with acute obstruction of the larynx), difficulty speaking (dysarthria), and eye muscle paralysis. Most individuals with SMA type III die from respiratory complications.

[0174] Kennedy disease, also known as progressive spinal-bulbar muscular atrophy, is an X-linked recessive disorder. Daughters of patients with Kennedy disease are carriers and have a 50% chance of having affected sons. Onset occurs between the ages of 15 and 60. Symptoms include facial and tongue muscle weakness, hand tremors, muscle spasms, dysphagia, dysarthria, and overdeveloped male breasts and mammary glands. Weakness usually begins in the pelvis before spreading to the limbs. Some individuals develop non-insulin-dependent diabetes mellitus.

[0175] The course of the disorder is variable but generally progresses slowly. Individuals tend to remain ambulatory until the late stages of the disease. Life expectancy for patients with Kennedy disease is usually normal.

[0176] Congenital SMA with arthrogryposis (persistent contractures of the joints with fixed abnormal postures of the limbs) is a rare disorder. Symptoms include severe contractures, scoliosis, chest deformities, breathing problems, an abnormally small chin, and drooping upper eyelids.

[0177] Progressive bulbar palsy, also called progressive bulbar atrophy, involves the bulbar brainstem, an area that controls the lower motor neurons necessary for swallowing, speaking, chewing, and other functions. Symptoms include pharyngeal muscle weakness (involved in swallowing), jaw and facial muscle weakness, progressive loss of speech, and muscle atrophy of the tongue. Weakness of the limbs, with both lower and upper motor neuron signs, is almost always evident but less pronounced. Affected individuals laugh, cry, and act violently (called emotional lability). Individuals eventually become unable to eat or speak, and are at increased risk of choking and aspiration pneumonia, caused by liquids and food passing through the vocal cords and entering the lower airways and lungs. Stroke and myasthenia gravis each have certain symptoms similar to those of progressive bulbar palsy and must be ruled out before diagnosing this disorder. In approximately 25% of ALS cases, the initial symptoms begin with bulbar damage. Approximately 75% of individuals with classical ALS eventually show some bulbar damage. Many clinicians consider progressive bulbar palsy itself, without evidence of abnormalities in the arms or legs, to be quite rare.

[0178] Pseudobulbar palsy, which shares many symptoms of progressive bulbar palsy, is characterized by upper motor neuron degeneration and progressive loss of ability to speak, chew, and swallow. Progressive weakness of the facial muscles leads to a blank face. Individuals may develop a hoarse voice and an increased gag reflex. The tongue may become immobile and unable to protrude from the mouth. Individuals may also experience emotional lability.

[0179] Primary lateral sclerosis (PLS) affects only upper motor neurons and is nearly twice as common in men as in women. Onset generally occurs after age 50. The cause of PLS ​​is unknown. It occurs when certain nerve cells in the cerebral cortex (a thin layer of cells covering the brain involved in the highest levels of mental function) that control voluntary movement gradually degenerate, weakening the muscles under their control. The syndrome, which scientists believe to be rarely inherited, progresses gradually over years or decades, leading to stiffness and clumsiness in the affected muscles. The disorder usually affects the legs first, then the trunk, arms, and hands, and finally the bulbar muscles. Symptoms may include balance difficulties, weakness and stiffness in the legs, clumsiness, spasticity in the legs that causes slowness and stiffness, shuffling of the feet (leading to an inability to walk), and facial lesions resulting in dysarthria (slurred speech). The main differences between ALS and PLS (considered a variant of ALS) are the motor neurons involved and the rate of disease progression. PLS can be mistaken for spastic paraplegia, a genetic disorder of the upper motor neurons that causes spasticity in the legs and usually begins in adolescence. Most neurologists follow the clinical course of affected individuals for at least three years before making a diagnosis of PLS. The disorder is not fatal but can affect quality of life. PLS often progresses to ALS.

[0180] Progressive muscular atrophy (PMA) is characterized by a slow but progressive degeneration of only the lower motor neurons. It affects men more prevalently and develops earlier than other MNDs. Weakness is typically first seen in the hands, then spreads to the lower body and can be severe. Other symptoms may include muscle fatigue, clumsy hand movements, fasciculations, and muscle cramps. Trunk muscles and breathing may become affected. Exposure to cold temperatures may worsen symptoms. The disease progresses to ALS in many cases.

[0181] Post-polio syndrome (PPS) is a condition that polio survivors can develop decades after recovery from poliomyelitis. PPS is thought to occur when injury, disease (such as degenerative joint disease), weight gain, or the aging process damage or kill spinal motor neurons that remained functional after the initial polio attack. Many scientists believe that PPS is an underlying weakening of muscles previously affected by polio, and not a new MND. Symptoms include fatigue, slowly progressive muscle weakness, muscle atrophy, fasciculations, cold intolerance, and muscle and joint pain. These symptoms are most often manifested in muscle groups affected by the initial disease. Other symptoms include skeletal deformities such as scoliosis, and difficulty breathing, swallowing, or sleeping. Symptoms are more frequent among older people and individuals most severely affected by the initial disease. Some individuals experience only minor symptoms, while others develop SMA, a rare but apparently not a form of ALS. PPS is not usually life-threatening. Doctors estimate the incidence of PPS at about 25-50% of parasitic polio survivors.

[0182] Neuronal TDP-43 proteinopathies contemplated herein may also be associated with diseases other than ALS, such as frontotemporal dementia (FTD), AD, Perry syndrome, chronic traumatic encephalopathy, Guam ALS / Parkinson-Dementia Complex, hippocampal sclerosis, and multisystem proteinopathies. A non-exclusive list of relevant TDP-43 proteinopathies includes, for example, Chornenkyy et al. (Laboratory Neuropathy and Neuropathology, 2002). As presented in Investigation 99:993-1007 (2019), the following diseases are considered to be afflicted by Alzheimer's disease (AD), frontotemporal lobar degeneration, corticobasal degeneration, progressive supranuclear palsy, Gerstmann-Sträussler-Scheinker, neurodegeneration with cerebral iron deposition, microglobular glial tauopathy, primary age-related tauopathy, age-related tau astrogliosis, postencephalitic Parkinson's disease, subacute sclerosing panencephalitis, pantothenate kinase-associated neurodegeneration, chronic traumatic encephalopathy, Down's syndrome, early-onset AD, myotonic dystrophy, lipofuscinosis, Niemann-Pick disease, type C, Alexander disease, Perry syndrome, Cockayne syndrome, ganglioglioma / ganglioma, pilocytic astrocytoma, lead encephalopathy, traumatic brain injury (acute), and inclusion body myositis.

[0183] 5.4 Alzheimer's disease The main features of AD are: (1) the progressive accumulation of beta-amyloid (Aβ peptides in so-called neuronal plaques) outside neurons, which interferes with interneuronal communication at synapses and may contribute to cell death; (2) Aβ peptides also accumulate as so-called vascular amyloid around the blood vessels of the brain, thereby disrupting the uptake of essential nutrients from the blood into the brain; and (3) the abnormal deposition of the protein tau (neurofibrillary tangles) inside neurons, which blocks the transport of cargo inside the neurons. This is the main driver of neuronal dysfunction and cell death. Eventually, both amyloid deposits and tangles cause irreversible damage to the brain, leading to brain atrophy and loss of cognitive function. The most common early symptom of AD is difficulty remembering recent events, and as the disease progresses, symptoms may include language problems, disorientation (including being easily lost), mood swings, loss of motivation, lack of management of self-care, and behavioral problems. As a person's condition worsens, they often withdraw from their family and society. Gradually, the disease causes loss of bodily functions and ultimately death. The rate at which the disease progresses can vary, but typical life expectancy after diagnosis is 3 to 9 years.

[0184] In some embodiments, the methods described herein further comprise selecting a subject diagnosed with a neurodegenerative condition, preferably a subject associated with neuronal TDP-43 proteinopathy. A subject suffering from a neurodegenerative condition may be selected based on the symptoms presented. For example, a subject suffering from ALS may show symptoms of fasciculations, spasms, stiff and tense muscles (spasticity), twitching of the arms, shoulders, or tongue, muscle weakness affecting the hands, arms, or legs, slurred or nasal speech, or difficulty chewing or swallowing.

[0185] In some embodiments, the methods described herein further include selecting a subject at risk of developing a neurodegenerative condition, preferably a subject associated with a neuronal TDP-43 proteinopathy. Subjects at risk of developing a neurodegenerative condition can be selected based on a genetic diagnostic test (e.g., for a mutation in a gene associated with the neurodegenerative condition, or based on the symptoms presented).

[0186] 6. Treatment Methods Certain aspects of the disclosure relate to methods for treating neurodegenerative conditions, particularly neurodegenerative conditions associated with neuronal TDP-43 proteinopathy and / or conditions characterized by neurodegeneration. Accordingly, aspects of the disclosure relate to a method of treating or inhibiting the onset of a neurodegenerative condition preferably associated with neuronal TDP-43 proteinopathy in a subject in need thereof, comprising administering to the subject an effective amount of a modified cyclin F polypeptide or a nucleic acid molecule encoding a modified cyclin F polypeptide. In another aspect, the disclosure relates to a method of treating or inhibiting the onset of a condition characterized by neuronal degeneration and TDP-43 proteinopathy in a subject in need thereof, comprising administering to the subject an effective amount of a modified cyclin F polypeptide or a nucleic acid molecule encoding a modified cyclin F polypeptide.

[0187] Suitably, the modified cyclin F polypeptide or a nucleic acid molecule encoding the modified cyclin F polypeptide (referred to as an "agent") enhances or increases the level or activity of cyclin F and enhances neuronal survival (e.g., motor neuron survival) and / or inhibits neuronal degeneration (e.g., motor neuron degeneration) in a subject. In some embodiments, the agent ameliorates at least one symptom associated with a neurodegenerative condition in a subject. In some embodiments, the agent treats a neurodegenerative condition in a subject. In some embodiments, the agent prevents a subject from developing a neurodegenerative condition. In some embodiments, the agent prevents a neurodegenerative condition in a subject from progressing.

[0188] In some embodiments, the agent reduces the amount of cytoplasmic TDP-43 and enhances neuronal survival (e.g., motor neuron survival) and / or inhibits neuronal degeneration (e.g., motor neuron degeneration) in the subject. In some embodiments, the agent reduces the amount of cytoplasmic TDP-43 and ameliorates at least one symptom associated with a neurodegenerative condition in the subject. In some embodiments, the agent reduces the amount of cytoplasmic TDP-43 and treats a neurodegenerative condition in the subject. In some embodiments, the agent reduces the amount of cytoplasmic TDP-43 and prevents the subject from developing a neurodegenerative condition. In some embodiments, the agent reduces the amount of cytoplasmic TDP-43 and prevents a neurodegenerative condition in the subject from progressing.

[0189] In some embodiments, the subject is a human.

[0190] In some embodiments, the subject selected for treatment has a neurodegenerative condition or a condition characterized by motor neuron degeneration. In some embodiments, the subject is at risk of developing a neurodegenerative condition, particularly a condition associated with neuronal TDP-43 proteinopathy or a condition characterized by motor neuron degeneration. In some embodiments, the subject is suspected of having a neurodegenerative condition, particularly a condition associated with neuronal TDP-43 proteinopathy or a condition characterized by motor neuron degeneration. In some embodiments, the subject is afflicted with a neurodegenerative condition, particularly a condition associated with neuronal TDP-43 proteinopathy. The neurodegenerative condition can be any neurodegenerative condition described herein. In some embodiments, the neurodegenerative condition is characterized by motor neuron degeneration. In some embodiments, the neurodegenerative condition is motor neuron degeneration. In some embodiments, the neurodegenerative condition is ALS. In some embodiments, the neurodegenerative condition is FTD. In some embodiments, the neurodegenerative condition includes neuronal degeneration other than motor neuron degeneration. In some embodiments, the neurodegenerative condition is AD.

[0191] In some embodiments, another therapeutic agent is also administered to the subject. Such another therapeutic agent or "auxiliary" agent is typically administered simultaneously with the modified cyclin F polypeptide or the nucleic acid molecule encoding the modified cyclin F polypeptide. For example, the therapeutic agent can be administered in the same formulation or in a separate formulation Ex, for example, butyrate, valproic acid, hydroxyurea, or riluzole. In some embodiments, the agents described herein are used in combination with another therapeutic agent suitable for use in treating one or more symptoms of ALS, including, but not limited to, (i) a hydrogenated pyrido[4,3-b]indole or a pharma- ceutically acceptable salt thereof, and (ii) an agent that promotes or increases the supply of energy to muscle cells, a COX-2 inhibitor, a poly(ADP-ribose) polymerase-1 (PARP-I) inhibitor, a 3OS ribosomal protein inhibitor, an NMDA antagonist, an NMDA receptor antagonist, a sodium channel blocker, a glutamate release inhibitor, a K(V)4.3 channel blocker, an anti-inflammatory agent, a 5-HT1A receptor agonist, a neurotrophic factor enhancer, an agent that promotes motor neuron phenotype survival and / or neurogenesis, an agent that protects against blood-brain barrier breakdown, an inhibitor of the production or activity of one or more pro-inflammatory cytokines, an immunomodulatory agent, a neuroprotective agent, a modulator of astrocyte function, or an antioxidant. (such as small molecule catalytic antioxidants), free radical scavengers, agents that reduce the amount of one or more reactive oxygen species, agents that inhibit the reduction of non-protein thiol content, stimulators of normal cellular protein repair pathways (such as agents that activate the molecule caperon), neurotrophic agents, inhibitors of neuronal cell death, stimulators of neurite outgrowth, agents that prevent neuronal cell death and / or promote the regeneration of damaged brain tissue, cytokine modulators, agents that reduce the level of microglial cell activation, cannabinoid CB1 receptor ligands, nonsteroidal anti-inflammatory drugs, cannabinoid CB2 receptor ligands, creatine, creatine derivatives, stereoisomers of dopamine receptor agonists such as pramipexole hydrochloride, ciliary neurotrophic factor, agents encoding ciliary neurotrophic factor, glial derived neurotrophic factor, agents encoding glial derived neurotrophic factor, neurotrophin 3, agents encoding neurotrophin 3, or any combination thereof.

[0192] In some embodiments, a modified cyclin F polypeptide or a nucleic acid molecule encoding a modified cyclin F polypeptide described herein is used in combination with another therapeutic agent suitable for use in treating one or more symptoms of ALS or FTD, including, but not limited to, an antibiotic (e.g., an aminoglycoside, a cephalosporin, a chloramphenicol, a clindamycin, an erythromycin, a fluoroquinolone, a macrolide azolide, a metronidazole, a penicillin, a tetracycline, a trimethoprim-sulfamethoxazole, a cyclosporine ... azole, vancomycin), steroids (e.g., Andranes (e.g., testosterone), cholestanes (e.g., cholesterol), cholic acids (e.g., cholic acid), corticosteroids (e.g., dexamethasone), estradiols (e.g., estradiol), pregnanes (e.g., progesterone), narcotic and non-narcotic analgesics (e.g., morphine, codeine, heroin, hydromorphone, levorphanol, meperidine, methadone, oxydone, propoxyphene, fentanyl, methadone, naloxone, buprenorphine, butorphine, anol, nalbuphine, pentazocine), anti-inflammatory drugs (e.g., alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anilolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelain, broperamol, budesonide, carprofen, cycloprofen, synthazone, cliprofen, clobetasol propionate , clobetasone butyrate, clopirac, cloticasone propionate, cormetasone acetate, cortodoxone, decanoate, deflazacort, delatestril, depotestosterone, desonide, desoximetasone, dexamethasone propionate, diclofenac potassium, diclofenac sodium, diflorasone acetate, diflumidone sodium, diflunisal, difluprednate, diphthalone, dimethylsulfoxide, drocinonide, endrisone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate, felbinac,Fenamol, Fenbufen, Fenclofenac, Fenclorac, Fendosal, Fenpiparone, Fentiazac, Furazarone, Furazacort, Flufenamic acid, Flumizole, Flunisolide acetate, Flunixin, Flunixin meglumine, Fluocortin butyl, Fluorometholone acetate, Fluquazone, Flurbiprofen, Fluretofen, Fluticasone propionate, Furaprofen, Flobufen, Halcinonide, Halobetasol propionate, Halopredone acetate, Ibufenac, Ibuprofen, Ibuprofen aluminum, Ibuprofen Lofenpiconol, Ilonidap, Indomethacin, Indomethacin sodium, Indoprofen, Indoxol, Intrazol, Isoflupredone acetate, Isoxepac, Isoxicam, Ketoprofen, Lofimizole hydrochloride, Lomoxicam, Loteprednol etabonate, Meclofenamate sodium, Meclofenamic acid, Meclorisone dibutyrate, Mefenamic acid, Mesalamine, Meseclazone, Mesterolone, Methandostenolone, Methenolone, Methenolone acetate, Methylprednisolone suleptanate, Morniflumate, Nabumetone, Nandro Lon, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxandrolan, oxaprozin, oxyphenbutazone, oxymetholone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate, preferon, prodolie acid, proquazone, proxazole, proxazole citrate, rimexolone, romazalite, sarcolex, salnacedin, salsalate, sanguinarium chloride, seclazone, selmetacin, stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, testosterone, testosterone blend, tetridamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, zidometacin, zomepirac sodium), or an antihistamine (e.g.,Ethanolamines (such as diphenhydramine carbinoxamine), ethylenediamines (such as triphenylamine pyrilamine), alkylamines (such as chlorpheniramine, dexchlorpheniramine, bromidepheniramine, triprolidine), astemizole, loratadine, fexofenadine, bromidepheniramine, clemastine, acetaminophen, pseudoephedrine, triprolidine, and other antihistamines.

[0193] In some embodiments, the modified cyclin F polypeptides or nucleic acid molecules encoding the modified cyclin F polypeptides described herein are used in combination with another therapeutic agent suitable for use in treating one or more symptoms of AD, including, but not limited to, cognitive enhancers such as donepezil, rivastigmine, memantine, and galantamine.

[0194] 7. Formulation and Administration For administration to a subject, the agents described herein can be administered orally, parenterally, for example, by subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal instillation, or by application to mucous membranes, such as the mucous membranes of the nose, throat, and bronchial tubes. One method for targeting the nervous system, such as spinal cord glia, is by intrathecal delivery. The targeted agent is released into the surrounding CSF and / or tissue, and the released compound can penetrate into the spinal cord parenchyma immediately after acute intrathecal injection. For a comprehensive review of drug delivery strategies, including CNS delivery, see Ho et al., Curr. Opin. Mol. Ther. (1999), 1:336-3443; Groothuis et al., J. Neuro Virol. (1997), 3:387-400; and Jan, Drug Delivery Systmes: Technologies and Commercial Opportunities, Decision Resources, 1998, the entire contents of which are incorporated herein by reference.

[0195] They may be administered alone or with suitable pharmaceutical carriers and may be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.

[0196] The agent may be formulated in a pharma- ceutically acceptable composition containing an effective amount of the agent, and may be formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. The agent may be specially formulated for administration in solid or liquid form, including those adapted for (1) oral administration, such as drenches (aqueous or nonaqueous solutions or suspensions), troches, dragees, capsules, tablets, tablets (e.g., buccal, sublingual, and targeted for systemic absorption), boluses, powders, granules, pastes for application to the tongue, (2) parenteral administration, such as, for example, sterile solutions or suspensions or sustained release formulations, such as subcutaneous, intramuscular, intravenous, or epidural injections, (3) topical application, such as, for example, creams, ointments, or sustained release patches or sprays applied to the skin, (4) vaginally or rectally, such as, for example, pessaries, creams, or foams, (5) sublingually, (6) ophthalmically, (7) transdermally, (8) transmucosally, or (9) nasally. In addition, the compounds and / or drugs can be implanted into the patient or injected using drug delivery systems. See, e.g., Urquhart, et al. (1984. Ann. Rev. Pharmacol. Toxicol. 24:199-236), Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981), U.S. Patent No. 3,773,919, and U.S. Patent No. 353,270,960.

[0197] Some examples of materials that can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, saffron oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol, (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG), (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, (22) bulking agents such as polypeptides and amino acids, (23) serum components such as serum albumin, HDL, and LDL, (22) C2-C ethanol, etc. 12 and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, perfusants, preservatives, and antioxidants may also be present in the formulation. The terms "excipient," "carrier," "pharmaceutical acceptable carrier," and the like are used interchangeably herein.

[0198] Pharmaceutically acceptable antioxidants include, but are not limited to, (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lectin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0199] PEG includes within its scope any ethylene glycol polymer containing from about 20 to about 2,000,000 linked monomers, typically from about 50 to 1,000 linked monomers, usually from about 100 to 300. Polyethylene glycol includes PEGs containing various numbers of linked monomers, e.g., PEG20, PEG30, PEG40, PEG60, PEG80, PEG100, PEG115, PEG200, PEG300, PEG400, PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000, PEG6000, PEG8000, PEG11000, PEG12000, PEG2000000, and any mixtures thereof.

[0200] The drug may be formulated in gelatin capsules, tablet form, dragees, syrups, suspensions, ointments, suppositories, injectable solutions, or in kits for preparation of syrups, suspensions, ointments, suppositories, or injectable solutions immediately before use.The compound and / or drug may also be included in a complex that facilitates its slow release into the bloodstream, e.g., silicone discs, polymer beads.

[0201] The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the art of pharmacy. Techniques, excipients and formulations can generally be found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1985, 17th edition, Nema et al., PDA J.Pharm.Sci.Tech.1997 51:166-171. The method of making the formulations of the present invention includes the step of associating or contacting active agents with one or more excipients or carriers. In general, the formulations are prepared by uniformly and intimately associating one or more agents with liquid excipients or finely divided solid excipients, or both, and then, if appropriate, shaping the product.

[0202] The preparation procedure may include sterilization of the pharmaceutical preparation. The drug may be mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, salts that affect osmotic pressure, and the like, which do not adversely react with the drug.

[0203] Examples of injectable forms include solutions, suspensions, and emulsions. Injectable forms also include sterile powders for extemporaneous preparation of injectable solutions, suspensions, or emulsions. The agents of the present invention may be injected in association with pharmaceutical carriers such as saline, physiological saline, bacteriostatic water, Cremophor.TM.EL (BASF, Parsippany, NJ), phosphate buffered saline (PBS), Ringer's solution, dextrose solution, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof, as well as other aqueous carriers known in the art. Suitable non-aqueous carriers may also be used, examples of which include fixed oils and ethyl oleate. In any case, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin. A suitable carrier is 5% dextrose in saline. It is often desirable to include additives in the carrier, such as buffers and preservatives or other substances to enhance isotonicity and chemical stability.

[0204] In some embodiments, the agents described herein can be administered encapsulated in liposomes.The manufacture of such liposomes and the insertion of molecules into such liposomes are well known in the art, for example, as described in U.S. Patent No. 4,522,811.Liposomal suspensions (including liposomes targeted to specific cells, for example, pituitary cells) can also be used as pharmaceutically acceptable carriers.

[0205] In one embodiment, the agent is prepared with a carrier that will protect the compound and / or agent against rapid elimination from the body, such as sustained release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials may also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc.

[0206] For oral ingestion, excipients useful for solid preparations for oral administration are excipients commonly used in the art, useful examples being excipients such as lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, microcrystalline cellulose, methylcellulose, glycerin, sodium alginate, gum arabic, etc., binders such as polyvinyl alcohol, polyvinyl ethers, polyvinylpyrrolidone, ethylcellulose, gum arabic, shellac, sucrose, water, ethanol, propanol, carboxymethylcellulose, potassium phosphate, etc., lubricants such as magnesium stearate, talc, etc., and further including additives such as commonly known coloring agents, disintegrating agents such as alginic acid and PRIMOGEL (trademark), etc.

[0207] The agents may be administered orally, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be directly incorporated into dietary foods. For oral therapeutic administration, these compounds and / or agents may be incorporated into excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of the compound and / or agent. The percentage of the agent in these compositions may, of course, vary and may conveniently be from about 2% to about 60% of the weight of the unit. The amount of the compound and / or agent in such therapeutically useful compositions is such that a suitable dosage is obtained. Preferred compositions according to the invention are prepared so that an oral dosage unit contains about 100 to 2000 mg of the compound and / or agent.

[0208] Examples of bases useful for formulating suppositories include oil-based bases such as cocoa butter, polyethylene glycol, lanolin, fatty acid triglycerides, witepsol (trademark, Dynamite Nobel Co. Ltd.), etc. Liquid preparations may be in the form of aqueous or oily suspensions, solutions, syrups, elixirs, etc., which may be prepared in a conventional manner using additives.

[0209] The compositions may be given as a bolus dose to maximize circulating levels for the greatest period of time after administration. Continuous infusion may also be used after the bolus dose.

[0210] The drug can also be administered directly to the airway in the form of aerosol.For administration by inhalation, the drug in solution or suspension can be delivered in the form of aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a hydrocarbon propellant such as propane, butane or isobutene.The drug can also be administered in non-pressurized form, such as an atomizer or nebulizer.

[0211] The drug may also be administered parenterally. The solutions or suspensions of these drugs may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oil. Exemplary oils are oils of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose, and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0212] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

[0213] Administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the drug is formulated into ointments, salves, gels, or creams generally known in the art.

[0214] The agent may be administered to the subject in combination with other pharmacologic active agents. Exemplary pharmacologic active compounds and / or agents include, but are not limited to, those found in Harrison's Principles of Internal Medicine, 13.sup.th Edition, Eds.TR Harrison et al. McGraw-Hill NY,NY; Physician's Desk Reference, 50.sup.th Edition, 1997; Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8.sup.th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990, the entire contents of which are incorporated herein by reference. In some embodiments, the pharmacologic active agent is selected from the group consisting of butyrate, valproic acid, hydroxyurea, and riluzole.

[0215] The agent and the other pharma- ceutical active agent may be administered to a subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times). For example, an Aurora kinase inhibitor and an additional agent for treating a neurodegenerative condition may be administered to a subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).

[0216] The amount of drug that can be combined with a carrier material to produce a single dosage form will generally be that amount of drug that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.1% to 99% of the compound, preferably from about 5% to about 70%, and most preferably from 10% to about 30%.

[0217] Tablets, capsules and the like may also contain binders such as tragacanth, acacia, corn starch, or gelatin, excipients such as dicalcium phosphate, disintegrating agents such as corn starch, potato starch, alginic acid, lubricants such as magnesium stearate, and sweeteners such as sucrose, lactose, or saccharin. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil.

[0218] Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. A syrup may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor.

[0219] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0220] Guidance regarding efficacy and dosages that will deliver an effective amount of a compound and / or agent to treat ALS or FTD may be obtained from animal models of ALS or FTD, see, for example, those described in Hsieh-Li et al. (2000. Nature Genetics 24:66-70) and references cited therein.

[0221] Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are preferred.

[0222] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compounds and / or agents is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized.

[0223] Effective doses can be estimated initially from cell culture assays. Dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of therapeutic agent that achieves half-maximal inhibition of symptoms) determined in cell culture. Plasma levels can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay. Examples of suitable bioassays include DNA replication assays, transcription-based assays, GDF-8 binding assays, and immunological assays.

[0224] Dosage will be determined by the physician and may be adjusted, if necessary, to suit observed therapeutic effects. Generally, the compositions are administered such that the compound and / or agent is given at a dose of 1 μg / kg to 100 mg / kg, 1 μg / kg to 50 mg / kg, 1 μg / kg to 20 mg / kg, 1 μg / kg to 10 mg / kg, 1 μg / kg to 1 mg / kg, 100 μg / kg to 100 mg / kg, 100 μg / kg to 50 mg / kg, 100 μg / kg to 20 mg / kg, 100 μg / kg to 10 mg / kg, 100 μg / kg to 1 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 10 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 50 mg / kg, or 10 mg / kg to 20 mg / kg. For antibody compounds and / or drugs, one preferred dosage is 0.1 mg / kg (generally 10 mg / kg to 20 mg / kg) of body weight. If the antibody acts in the brain, a dosage of 50 mg / kg to 100 mg / kg is usually appropriate.

[0225] With regard to duration and frequency of treatment, it is typical for a person skilled in the art to monitor the subject to determine when the treatment is providing therapeutic benefit and to determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The administration schedule may vary from once a week to once a day, depending on several clinical factors, such as the subject's sensitivity to the polypeptide. The desired dose may be administered at once or may be divided into partial doses, e.g., 2-4 partial doses, and administered at appropriate intervals over a period of time, e.g., throughout the day or other suitable schedule. Such partial doses may be administered as unit dosage forms. Examples of administration schedules are administration once a week, twice a week, three times a week, once a day, twice a day, three times a day, or four or more times a day.

[0226] 8. Kit A modified cyclin F polypeptide or a nucleic acid molecule encoding a modified cyclin F polypeptide (i.e., an "agent") described herein may be provided in a kit. The kit includes (a) an agent, e.g., a composition including a modified cyclin F polypeptide or a nucleic acid molecule encoding a modified cyclin F polypeptide, and (b) informational material. The informational material may be descriptive, instructional, marketing, or other material related to the methods described herein and / or the use of the agent for the methods described herein. For example, the informational material describes methods for administering an agent to enhance survival of motor neurons and treat or inhibit the onset of a neurodegenerative condition, particularly a condition associated with a neuronal TDP-43 proteinopathy (e.g., ALS, FTD, AD, etc.), or at least one symptom of a neurodegenerative condition, or a condition associated with dysfunction or diminishing neurons (e.g., motor neurons).

[0227] In one embodiment, the informational material may include instructions for administering the agent in a suitable manner, e.g., a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material may include instructions for identifying a suitable subject, e.g., a human, e.g., an adult human. The informational material of the kit is not limited in form. In many cases, the informational material, e.g., instructions, is provided in print, e.g., printed text, drawings, and / or photographs, e.g., labels or printed sheets. However, the informational material may be provided in other formats, such as Braille, computer readable materials, video recordings, or audio recordings. In another embodiment, the informational material of the kit is a link or contact information, e.g., a physical address, an email address, a hyperlink, a website, or a phone number, from which a user of the kit can obtain substantial information regarding the modulator and / or its use in the methods described herein. Of course, the informational material may also be provided in any combination of formats.

[0228] In addition to the agent, the composition of the kit may include other components, such as a solvent or buffer, a stabilizer or preservative, and / or a second agent for treating a condition or disorder described herein. Alternatively, the other components may be included in the kit, but in different compositions or containers than the agent. In such embodiments, the kit may include instructions for mixing the agent with the other components or for using the agent together with the other components.

[0229] The agent may be provided in any form, for example, liquid, dry, or lyophilized. The agent is preferably substantially pure and / or sterile. When the agent is provided in a liquid solution, the liquid solution is preferably an aqueous solution, preferably a sterile aqueous solution. When the compound and / or agent is provided as a dry form, reconstitution is generally by the addition of a suitable solvent. The solvent, for example, sterile water or buffer, may optionally be provided in the kit.

[0230] The kit may include one or more containers for the composition containing the compound and / or agent. In some embodiments, the kit includes separate containers, dividers or compartments for the agent (e.g., in the composition) and the informational material. For example, the agent (e.g., in the composition) may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single undivided container. For example, the agent (e.g., in the composition) is contained within a bottle, vial, or syringe to which the informational material in the form of a label is attached. In some embodiments, the kit includes a plurality (e.g., in a pack) of individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the agent (e.g., in the composition). For example, the kit includes a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of the agent. The containers of the kit may be airtight and / or waterproof.

[0231] The agent (e.g., in a composition) can be administered to a subject, e.g., an adult subject, e.g., a subject in need of enhancing survival or viability of a neuron (e.g., a motor neuron) and / or inhibiting degeneration of a neuron (e.g., a motor neuron) and / or inhibiting abnormal protein accumulation in a neuron (e.g., a motor neuron). The method can include evaluating the subject, e.g., evaluating the presence of a neuronal TDP-43 proteinopathy in the subject, thereby identifying that the subject may be amenable to treatment with a modified cyclin F polypeptide or polynucleotide described herein.

[0232] So that the present invention may be readily understood and put to practical effect, certain preferred embodiments will now be described by way of the following non-limiting examples. EXAMPLES

[0233] experiment Our previous work demonstrated that wild-type TDP-43 is a direct ubiquitination substrate of wild-type cyclin F and is therefore targeted for degradation through the ubiquitin proteasome system. We also showed that overexpressing wild-type CCNF (the gene encoding cyclin F protein), such as by using an AAV-based gene therapy approach, can remove TDP-43 from neurons. As a result, we proposed cyclin F as a therapy for treating MND.

[0234] Therefore, additional studies were carried out to develop cyclin F variants that are further optimized for therapeutic use.

[0235] Cytoplasmic delivery of cyclin F variants Cyclin F is generally considered a nuclear protein, regulating the nuclear levels of various cell cycle proteins through ubiquitination and degradation through the proteasome system. This is suboptimal from a therapeutic perspective for targeting pathogenic TDP-43 in MND / ALS for several reasons. First, pathogenic TDP-43 is generally considered to be a form that is mislocalized from the nucleus to the cytoplasm. As a result, the ability of wild-type cyclin F (located in the nucleus) to therapeutically remove cytoplasmic TDP-43 may be limited. Second, healthy TDP-43 is located in the nucleus where it performs a series of essential functions, and loss of nuclear TDP-43 may affect cell viability. Thus, the use of wild-type cyclin F in long-term gene therapy may have a detrimental effect on healthy TDP-43.

[0236] To overcome this, a modified cyclin F variant was generated that is targeted to the cytoplasm. This variant has a cytoplasmic targeting peptide (nuclear export signal (NES): LPPLERLTL (SEQ ID NO: 8)) fused to the C-terminus of the cyclin F polypeptide. The cyclin F polypeptide also has a deletion of two nuclear localization signals (NLS) present at amino acid positions 20-28 and 568-574 of the wild-type cyclin F polypeptide set forth in SEQ ID NO: 2. This new cytoplasm-specific cyclin F variant, designated CT-cyclin F, has the amino acid sequence set forth in SEQ ID NO: 4, encoded by the nucleic acid sequence set forth in SEQ ID NO: 3. As shown in Figure 3, CT-cyclin F possesses an F box at amino acid positions 20-66 (encoded by nucleotides 58-198 of SEQ ID NO:3), a cyclin domain at amino acid positions 282-395 (encoded by nucleotides 844-1185 of SEQ ID NO:3), a PEST domain at amino acid positions 565-749 (encoded by nucleotides 1693-2247 of SEQ ID NO:3), and an NES at amino acid positions 769-778 (encoded by nucleotides 2305-2334 of SEQ ID NO:3). As shown in Figure 5, CT-cyclin F exhibits predominant cytoplasmic localization in cells.

[0237] To demonstrate the ability of CT-cyclin F to remove cytoplasmic (pathological) TDP-43, a TDP-43 variant with an inactive NLS (dNLS-TDP-43), which mislocalizes TDP-43 in the cytoplasm, was overexpressed in HEK293 cells. dNLS-TDP-43 is accepted in the art as a suitable TDP-43 variant for preclinical testing of potential TDP-43 ablation therapies. Notably, transgenic mice overexpressing dNLS-TDP-43 develop MND-like symptoms and are considered the gold standard preclinical animal model of MND / ALS (Walker et al, 2015; Acta Neuropathol 130(5):643-60). As shown in Figure 6, CT-cyclin F significantly reduces the levels of dNLS-TDP-43 in these cells.

[0238] To investigate the ability of CT-cyclin F to remove cytoplasmic TDP-43 in vivo, dNLS-TDP-43 was specifically overexpressed in spinal motor neurons of zebrafish. Overexpression of CT-cyclin F in these zebrafish resulted in a significant reduction in dNLS-TDP-43 levels (see FIG. 7).

[0239] In summary, a modified cyclin F polypeptide with a heterologous NES and deletion of two endogenous NLSs is targeted to the cytoplasm. Moreover, this modified CT-cyclin significantly reduces the levels of cytoplasmic TDP-43 in vitro and in vivo, demonstrating the clinical utility of cytoplasm-targeted cyclin F.

[0240] Truncated cyclin F variants Cyclin F is a relatively large polypeptide of 786 amino acids encoded by a 2.4 kb polynucleotide. Active truncation of cyclin F may have advantages in the clinic (e.g., in the context of viral vector-based gene therapy, where limitations may exist on the size of the gene construct / vector genome) and in production (where smaller polynucleotides or polypeptides are typically preferred). To help determine the minimal functional components of cyclin F required for its enzymatic function and thus identify therapeutically active truncated variants, truncated variants were designed that maintained TDP-43 removal properties. Specifically, the PEST domain spanning amino acid residues 582-766 of wild-type cyclin F as set forth in SEQ ID NO:2 was deleted (see FIG. 1). This PEST region is not required for the ubiquitination activity of cyclin F. Rather, the PEST domain acts as a signal for protein degradation, and thus this sequence is likely involved in the degradation of cyclin F.

[0241] This new cyclin F variant, referred to herein as deltaPEST-cyclin F (also referred to herein as dPEST-cyclin F), has the amino acid sequence set forth in SEQ ID NO: 6, encoded by the nucleic acid sequence set forth in SEQ ID NO: 5. As shown in FIG. 4, deltaPEST-cyclin F possesses an NLS at positions 20-28 and 568-574 (encoded by nucleotides 58-84 and 1702-1722 of SEQ ID NO: 5, respectively), an F box at amino acid positions 29-76 (encoded by nucleotides 85-228 of SEQ ID NO: 5), and a cyclin domain at amino acid positions 292-405 (encoded by nucleotides 874-1215 of SEQ ID NO: 5).

[0242] Surprisingly, it was observed that deletion of the PEST domain from cyclin F alters its subcellular localization, leading to cytoplasmic localization of deltaPEST-cyclin F (see FIG. 8). Moreover, deltaPEST-cyclin F facilitates substantial removal of dNLS-TDP-43 in HEK cells (FIG. 9).

[0243] Effect of cyclin F variants on nuclear TDF-43 levels As detailed above, both CT-cyclin F and deltaPEST-cyclin F variants remove cytoplasmic (pathogenic) TDP-43. To determine whether this reduction is specific to cytoplasmic TDP-43 rather than nuclear TDP-43, the effect of CT-cyclin F or deltaPEST-cyclin F on wild-type (nuclear) TDP-43 expressed in spinal motor neurons of zebrafish was determined. As shown in FIG. 10, neither variant had any effect on wild-type TDP-43 removal. This is an important property of the variants for use in the clinic, since it is only (or mainly) the pathogenic cytoplasmic TDP-43 that is targeted and removed, and not the nuclear TDP-43 that is essential for normal functioning of neurons.

[0244] Enzymatically inactive cyclin F variants Cyclin F (as part of the SCF E3 ligase complex) directly binds to TDP-43 and facilitates ubiquitination by the SCF complex, after which the ubiquitin tag introduces TDP-43 to the proteasome for removal. We engineered an LP / AA variant into cyclin F, which contains a substitution of leucine at position 35 and proline at position 36 of wild-type cyclin F, as set forth in SEQ ID NO:2, with two alanines. This modification is within the F-box of cyclin F. The modification does not prevent the modified polypeptide from binding to TDP-43, but prevents it from ubiquitinating TDP-43. It functions as an enzymatically inactive variant of cyclin F. In transgenic zebrafish overexpressing WT-TDP-43 in spinal motor neurons, we confirm that the inactive LP / AA variant completely prevents TDP-43 removal (see FIG. 11).

[0245] Effect of cyclin F variants on WT TDF-43 and dNls TDP-43 levels The experiment was carried out by repeating the experiment described for Figures 7, 10, and 11. The results are shown in Figure 12. Confirming the data of Figure 11, wild-type cyclin F reduces wild-type TDP-43 levels, but inactivated (LP / AA) cyclin F does not (Figure 12A). Neither deltaPEST-cyclin F nor CT-cyclin F has any effect on wild-type TDP-43 (confirming the data of Figure 10) (Figure 12A). Wild-type cyclin F reduces the level of dNLS-TDP-43, but inactivated (LP / AA) cyclin F does not (Figure 12B). Both deltaPEST-cyclin F and CT-cyclin F reduce the level of dNLS-TDP-43 (Figure 12B).

[0246] Materials and Methods Plasmids All plasmids were synthesized and archived by Genscript. The plasmids included plasmids encoding wild-type cyclin F (having the nucleic acid sequence set forth in SEQ ID NO: 1, encoding the amino acid sequence set forth in SEQ ID NO: 2), CT-cyclin F (having the nucleic acid sequence set forth in SEQ ID NO: 3, encoding the amino acid sequence set forth in SEQ ID NO: 4), and deltaPEST-cyclin F (having the nucleic acid sequence set forth in SEQ ID NO: 1, encoding the amino acid sequence set forth in SEQ ID NO: 2) (Figures 1-4). Also included was a plasmid encoding the LP / AA cyclin F variant (encoding the amino acid sequence set forth in SEQ ID NO: 7). Briefly, DNA oligonucleotides encoding mCherry-flag-cyclin F were subcloned into the pCS2+ plasmid.

[0247] Transfection for imaging HEK293 cells were seeded on glass coverslips in 24-well plates. The next day, cells were approximately 80% confluent and transfected with mCherry-cyclin F (WT), mCherry-cyclin F (cytoplasmic), or mCherry-cyclin F (deltaPEST) plasmids (described above) using Lipofectamine 2000 according to the manufacturer's instructions.

[0248] 24 hours after transfection, media was aspirated from the cells and the cells were washed with phosphate-buffered saline (PBS). The washed cells were fixed with 4% PFA in PBS for 10 minutes. The cells were then washed three times with PBS before staining the nuclei of the fixed cells using DAPI. The stained cells were then mounted on glass slides and imaged using a Zeiss AxioImager.

[0249] Transfection for soluble / insoluble studies HEK293 cells were seeded in T75 flasks. The next day, cells were co-transfected with mCherry-cyclin F (WT), mCHerry-cyclin F (deltaPEST), or mCHerry-cyclin F (CT) plasmids (described above) along with TDP-43-GFP (deltaNLS). As controls, cells were co-transfected with mCherry empty vector and GFP empty vector. 48 hours after transfection, cells were scraped into ice-cold PBS, centrifuged at 2000×g for 5 min, and stored at −80° C. until further use.

[0250] Soluble / insoluble protein fractionation for cultured cells The cells were resuspended and lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40 replacement, 5 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing complete protease inhibitor cocktail and phosSTOP (Roche) by sonication 10 times using a Sonic Ruptor250 at 50% power and pulse settings set at 30%. The resulting lysate was centrifuged at 100,000×g for 30 min at 4° C. The resulting supernatant was used as the detergent-soluble fraction. The resulting pellet was washed twice in RIPA buffer (the probe was sonicated in RIPA buffer and centrifuged at 100,000×g each time). After the final wash, the proteins were resuspended in RIPA buffer and centrifuged again at 100,000×g for 30 min at 4° C. The resulting pellet was resuspended in urea buffer (7M urea, 2M thiourea, 4% CHAPS, 30mM Tris) and the supernatant removed before being sonicated 10 times using a Sonic Ruptor250 with the power at 50% and the pulse setting set at 30%. This was considered the insoluble fraction.

[0251] mRNA-mediated CCNF overexpression in TDP-43 transgenic zebrafish Cyclin F-mediated TDP-43 ablation was assessed using transgenic zebrafish expressing GFP-tagged human TDP-43 in motor neurons. WT-, CT-, or deltaPEST-cyclin F RNA (i.e., CCNF RNA) was injected (approximately 2 nl) into zebrafish embryos at the one-cell stage. Successfully injected larvae were verified using a fluorescent reporter and were raised at 28.5 °C until 3–5 days post-fertilization. On days 3–5, confocal microscopy images of GFP-positive spinal neurons were taken using the same imaging settings for all treatment groups. Maximum intensity projection was used to calculate the TDP-43 fluorescence intensity in spinal motor neurons. The mean ratio of TDP-43 levels (nuclear to total cell intensity) for the CCNF-injected groups was compared to uninjected controls.

[0252] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[0253] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the present application.

[0254] Throughout this specification, the objective has been to describe preferred embodiments of the present disclosure, without limiting the disclosure to any one embodiment or particular collection of features. Thus, those skilled in the art will appreciate that, in light of this disclosure, various modifications and changes can be made to the specific embodiments illustrated without departing from the scope of the present disclosure. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

1. A nucleic acid molecule comprising a coding sequence for a modified cyclin F polypeptide, the modified cyclin F polypeptide (i) comprising a heterologous nuclear export signal (NES) and / or (ii) comprising a deletion of all or a portion of the PEST domain relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:

2.

2. 2. The nucleic acid molecule of claim 1, wherein the NES comprises a sequence of amino acids selected from LPPLERLTL (SEQ ID NO: 8), LQLPPLERLTLD (SEQ ID NO: 9), LALKLAGLDL (SEQ ID NO: 10), PLQLPPLERLTL (SEQ ID NO: 11), ERFEMFRELNEALEL (SEQ ID NO: 12), LSSHFQELSI (SEQ ID NO: 13), ERFEMFRELNEALEL (SEQ ID NO: 14), DHAEKVAEKLEALSV (SEQ ID NO: 15), QLVEELLKIICAFQL (SEQ ID NO: 16), and TNLEALQKKLEELEL (SEQ ID NO: 17).

3. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide comprises a nuclear localization signal (NLS)-inactivating modification at one or both endogenous NLSs.

4. The nucleic acid molecule of claim 1, wherein the NLS-inactivating modification comprises a deletion of all or a portion of the endogenous NLS relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2, or comprises one or more amino acid substitutions of amino acid residues comprising the endogenous NLS.

5. the modified cyclin F polypeptide comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues from the NLS at amino acid positions 20-28, using numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2; the modified cyclin F polypeptide comprises a deletion of 1, 2, 3, 4, 5, 6, or 7 amino acid residues from the NLS at amino acid positions 568-574, using numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:2; the NLS inactivating modification comprises an amino acid substitution of one or more of K20, R21, R22, R24, R25, R26, and R28 with a non-basic amino acid, using the numbering relative to wild-type cyclin F set forth in SEQ ID NO:2; or the NLS inactivating modification comprises one or more amino acid substitutions of R568, R569, K571, R572, K574, and R574 with a non-basic amino acid, using numbering relative to wild-type cyclin F as set forth in SEQ ID NO:2; A nucleic acid molecule according to claim 4.

6. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide comprises an amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:6, or a sequence having at least or about 95% sequence identity thereto.

7. 2. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide comprises a deletion of at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids from the PEST domain at amino acid positions 582-766, using numbering relative to the wild-type cyclin F polypeptide set forth in SEQ ID NO:

2.

8. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide binds TDF-43.

9. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide retains at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the TDF-43 binding ability of the wild-type cyclin F polypeptide set forth in SEQ ID NO:

2.

10. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide comprises at least or about 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110 amino acid residues of a cyclin domain at positions 292 to 405 of a wild-type cyclin F polypeptide set forth in SEQ ID NO:

2.

11. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide retains at least or about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the ability of the wild-type cyclin F polypeptide set forth in SEQ ID NO:2 to form a Skp1-Cul1-F box (SCF) E3 ubiquitin-protein ligase complex.

12. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide comprises at least or about 15, 20, 25, 30, 35, 40, or 45 amino acid residues of an F box at positions 29-76 of a wild-type cyclin F polypeptide set forth in SEQ ID NO:

2.

13. The nucleic acid molecule of claim 1, wherein the modified cyclin F polypeptide accumulates in and / or is introduced into the cytoplasm of the neuron when expressed in or delivered to the neuron.

14. The nucleic acid molecule of claim 1 , comprising an expression construct comprising a promoter operably linked to the coding sequence for the modified cyclin F polypeptide.

15. A modified cyclin F polypeptide encoded by the nucleic acid molecule of claim 1.

16. A delivery vehicle comprising a nucleic acid molecule according to claim 1 or a modified cyclin F polypeptide according to claim 15.

17. A nucleic acid molecule as described in claim 1, a modified cyclin F polypeptide as described in claim 15, or a delivery vehicle as described in claim 16 for use in a method for enhancing neuronal survival, inhibiting neuronal degeneration, inhibiting abnormal protein accumulation in neurons, or inhibiting aggregated or insoluble TDP-43 accumulation in neurons.

18. A nucleic acid molecule as described in claim 1, a modified cyclin F polypeptide as described in claim 15, or a delivery vehicle as described in claim 16, for use in a method for treating a subject having or at risk of developing a neurodegenerative condition.

19. The nucleic acid molecule, modified cyclin F polypeptide, or delivery vehicle of claim 18, wherein the neurodegenerative condition is associated with neuronal TDP-43 proteinopathy.

20. The nucleic acid molecule, modified cyclin F polypeptide, or delivery vehicle of claim 18, wherein the neurodegenerative condition is selected from familial amyotrophic lateral sclerosis (ALS), familial frontotemporal dementia (FTD), familial Alzheimer's disease (AD), sporadic amyotrophic lateral sclerosis (ALS), sporadic frontotemporal dementia (FTD), and sporadic Alzheimer's disease (AD).

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