Optogenetic induction of neurodegenerative disease pathology

文档序号：1471337 发布日期：2020-02-21 浏览：28次中文

阅读说明：本技术 神经退行性疾病病理的光遗传学诱导 (Optogenetic induction of neurodegenerative disease pathology ) 是由 C·J·唐纳利 J·R·曼恩于 2018-03-07 设计创作，主要内容包括：本公开涉及用于诱导神经退行性疾病病理的化合物、组合物和方法。在一个方面，本文公开了编码嵌合多肽的核苷酸序列，所述核苷酸序列包含：编码光诱导的寡聚化结构域的第一核苷酸序列和编码神经退行性疾病靶蛋白的第二核苷酸序列。本文公开了一种在细胞中诱导神经退行性疾病病理的方法，所述方法包括以下步骤：将编码嵌合多肽的表达载体导入细胞，所述表达载体包含：编码光诱导的寡聚化结构域的第一核苷酸序列和编码来自神经退行性疾病靶蛋白的低复杂性结构域的第二核苷酸序列，其中，第一核苷酸序列与启动子可操作地连接；表达嵌合多肽；和通过用蓝光刺激诱导嵌合多肽的寡聚化。(The present disclosure relates to compounds, compositions and methods for inducing a neurodegenerative disease pathology. In one aspect, disclosed herein is a nucleotide sequence encoding a chimeric polypeptide, the nucleotide sequence comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a neurodegenerative disease target protein. Disclosed herein is a method of inducing a neurodegenerative disease pathology in a cell, the method comprising the steps of: introducing into a cell an expression vector encoding a chimeric polypeptide, said expression vector comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter; expressing the chimeric polypeptide; and inducing oligomerization of the chimeric polypeptide by stimulation with blue light.)

1. A nucleotide sequence encoding a chimeric polypeptide, said nucleotide sequence comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein.

2. The nucleotide sequence of claim 1, wherein the light-induced oligomerization domain is selected from the group consisting of: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, VFAU1, YtvA, EL222, RsLOV, and AsLOV2.

3. The nucleotide sequence according to claim 2, wherein the light-induced oligomerization domain is NcVVD.

4. The nucleotide sequence of claim 2, wherein the light-induced oligomerization domain is CRY2 OLIG.

5. The nucleotide sequence according to claim 2, wherein the light-induced oligomerization domain is CRYPHR.

6. The nucleotide sequence of claim 1, wherein the light-induced oligomerization domain comprises a LOV domain.

7. The nucleotide sequence of claim 1, wherein the light-induced oligomerization domain comprises a PHR domain.

8. The nucleotide sequence of any one of claims 1 to 7, wherein the low complexity domain from a neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA2B1, EWS RNA binding protein 1 and TATA box binding protein factor 15.

9. The nucleotide sequence of claim 8, wherein the low complexity domain from a neurodegenerative disease target protein is TDP-43.

10. The nucleotide sequence of claim 8, wherein the low complexity domain from a neurodegenerative disease target protein is an alpha synuclein.

11. The nucleotide sequence of claim 8, wherein the low complexity domain from a neurodegenerative disease target protein is Tau.

12. An expression vector encoding a chimeric polypeptide, said expression vector comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter.

13. A cell comprising a nucleotide sequence encoding a chimeric polypeptide, the cell comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a neurodegenerative disease target protein.

14. A chimeric polypeptide comprising:

a light-induced oligomerization domain; and

low complexity domains from neurodegenerative disease target proteins.

15. A method of inducing a neurodegenerative disease pathology in a cell, the method comprising the steps of:

introducing into a cell an expression vector encoding a chimeric polypeptide, said expression vector comprising:

a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter;

expressing said chimeric polypeptide; and

inducing oligomerization of the chimeric polypeptide by stimulation with blue light.

16. The method of claim 15, wherein the light-induced oligomerization domain is selected from the group consisting of: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, VFAU1, YtvA, EL222, RsLOV, and AsLOV2.

17. The method of claim 16, wherein the light-induced oligomerization domain is NcVVD.

18. The method of claim 16, wherein the light-induced oligomerization domain is CRY2 OLIG.

19. The method of claim 16, wherein the light-induced oligomerization domain is crypthr.

20. The method of claim 15, wherein the light-induced oligomerization domain comprises an LOV domain.

21. The method of claim 15, wherein the light-induced oligomerization domain comprises a PHR domain.

22. The method of any one of claims 15 to 21, wherein the low complexity domain from a neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA2B1, EWS RNA binding protein 1 and TATA box binding protein factor 15.

23. The method of claim 22, wherein the low complexity domain from a neurodegenerative disease target protein is TDP-43.

24. The method of claim 22, wherein the low complexity domain from a neurodegenerative disease target protein is an a synuclein.

25. The method of claim 22, wherein the low complexity domain from a neurodegenerative disease target protein is Tau.

26. The method of any one of claims 15 to 25, wherein the cell is a mammalian cell.

27. The method of claim 26, wherein the cell is a human cell.

28. The method of any one of claims 15-27, wherein the blue light has a wavelength between 405nm and 499 nm.

29. A method of screening for an agent that modulates protein aggregation, the method comprising the steps of:

introducing into a cell an expression vector encoding a chimeric polypeptide, said expression vector comprising:

expressing said chimeric polypeptide;

introducing the agent into a medium comprising the cell;

inducing oligomerization of the chimeric polypeptide by stimulation with blue light; and

determining the modulation of protein aggregation by the agent.

30. The method of claim 29, wherein the light-induced oligomerization domain is selected from the group consisting of: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, VFAU1, YtvA, EL222, RsLOV, and AsLOV2.

31. The method of claim 30, wherein the light-induced oligomerization domain is NcVVD.

32. The method of claim 30, wherein the light-induced oligomerization domain is CRY2 OLIG.

33. The method of claim 30, wherein the light-induced oligomerization domain is crypthr.

34. The method of claim 29, wherein the light-induced oligomerization domain comprises an LOV domain.

35. The method of claim 29, wherein the light-induced oligomerization domain comprises a PHR domain.

36. The method of any one of claims 29 to 35, wherein the low complexity domain from a neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA2B1, EWS RNA binding protein 1 and TATA box binding protein factor 15.

37. The method of claim 36, wherein the low complexity domain from a neurodegenerative disease target protein is TDP-43.

38. The method of claim 36, wherein the low complexity domain from a neurodegenerative disease target protein is an a synuclein.

39. The method of claim 36, wherein the low complexity domain from a neurodegenerative disease target protein is Tau.

40. The method of any one of claims 29 to 39, wherein the cell is a mammalian cell.

41. The method of claim 40, wherein the cell is a human cell.

42. The method of any one of claims 29-41, wherein the blue light has a wavelength between 405nm and 499 nm.

Technical Field

The present disclosure relates to compounds, compositions and methods for inducing a neurodegenerative disease pathology.

Background

The world is aging. By 2050, the proportion of people over the age of 60 increased from 6.05 to 20 billion in 2000. Unfortunately, aging is the greatest risk factor for developing fatal neurodegenerative diseases. In turn, the number of patients suffering from dementia, such as Alzheimer's Disease (AD), dementia with Lewy Bodies (LBD), frontotemporal dementia (FTD), and movement disorders, such as Parkinson's Disease (PD) and Amyotrophic Lateral Sclerosis (ALS), will increase significantly. Currently, nearly 650 million people in the united states suffer from one of these diseases, and the associated costs are not sustainable.

The economic burden of AD, PD and ALS is currently estimated to be $ 2410 million per year in the united states. AD and ALS/FTD patients can incur individual medical costs of up to $ 100,000 to $ 250,000 per year. For AD, 1380 thousands of people will be diagnosed in the united states by 2050 and 470 thousands by 2010, while the number of global ALS cases will increase by about 31% by 2040, and there is no effective treatment for these diseases at present. Although there are many gene mutations that cause these neurodegenerative diseases, there is no known single cause. However, despite this diversity, almost all patients in each disease exhibit a common neuropathological hallmark in the form of intracellular protein aggregates. Animal models that reproduce these neuropathologies currently require the use of genetically mutated or severely overexpressed proteins, which often do not mimic the pathology of the patient. What is needed are new and improved methods for inducing neurodegenerative disease pathology in cell lines and animal models.

The compounds, compositions, and methods disclosed herein address these and other needs.

Disclosure of Invention

Disclosed herein are compounds, compositions and methods for inducing a neurodegenerative disease pathology in a cell or animal. The present inventors have developed a novel method for inducing neurodegenerative disease pathology in cells and animals without the need for gene mutations or severely overexpressed neurodegenerative disease proteins.

In one aspect, disclosed herein is a nucleotide sequence encoding a chimeric polypeptide, the nucleotide sequence comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein.

In one aspect, disclosed herein is an expression vector encoding a chimeric polypeptide, the expression vector peptide comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter.

In one aspect, disclosed herein is a cell comprising a nucleotide sequence encoding a chimeric polypeptide, the cell comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a neurodegenerative disease target protein.

In one aspect, disclosed herein is a chimeric polypeptide comprising: a light-induced oligomerization domain; and low complexity domains from neurodegenerative disease target proteins.

In one aspect, disclosed herein is a method of inducing a neurodegenerative disease pathology in a cell, the method comprising the steps of:

introducing into a cell an expression vector encoding a chimeric polypeptide, said expression vector comprising:

expressing the chimeric polypeptide; and

oligomerization of the chimeric polypeptide was induced by stimulation with blue light.

In another aspect, disclosed herein is a method of screening for an agent that modulates protein aggregation, the method comprising the steps of:

introducing into a cell an expression vector encoding a chimeric polypeptide, said expression vector comprising:

expressing the chimeric polypeptide;

introducing the agent into a medium comprising cells;

inducing oligomerization of the chimeric polypeptide by stimulation with blue light; and

determining the modulation of protein aggregation by the agent.

In one embodiment, the light-induced oligomerization domain is selected from the group consisting of: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, VFAU1, YtvA, EL222, RsLOV, and AsLOV2. In one embodiment, the light-induced oligomerization domain is selected from the list of domains in table 2. In one embodiment, the light-induced oligomerization domain is NcVVDY 50W. In one embodiment, the light-induced oligomerization domain is CRY2 OLIG. In one embodiment, the light-induced oligomerization domain is crypt.

In one embodiment, the light-induced oligomerization domain is selected from the group consisting of: a CRY2PHR domain (e.g., CRY2PHR, CRY2OLIG) or a photo-oxygen voltage sensing (LOV) domain (e.g., NcVVD, NcVVDY50W, VfAU1, YtvA, EL222, RsLOV, AsLOV 2).

In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntingtin (Huntingtin), Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1, and TATA box binding protein factor 15. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is selected from table 3. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is TDP-43. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is a synuclein. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is Tau.

In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell.

In one embodiment, the blue light has a wavelength between 405nm and 499 nm. In one embodiment, the blue light has a wavelength of about 465 nm.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

Figure 1 shows examples of genetic causes and common neuropathologies of ALS. The left panel shows a graph of the number of ALS-causing genes on the Y-axis, and the X-axis indicates the year each mutation was found. In fig. 1, panel a shows that ALS exhibits extreme genetic heterogeneity, particularly since these mutations are only found in about 10% of ALS cases. As shown in table 1, despite these genetic causes of ALS, almost all patients exhibit the same neuropathology in the motor cortex and spinal cord. The right panel shows an example of ALS neuropathology by H and E staining of paraffin tissue sections from the motor cortex of ALS patients. Cytoplasmic aggregates of TDP-43 are shown. TDP-43 is predominantly nuclear in normal cells, as indicated by x. In ALS, TDP-43 is absent from the nucleus and accumulates in the cytoplasm as indicated by the arrows. Although ALS is exemplified, cytoplasmatic accumulation neuropathology is a common feature of many neurodegenerative diseases, see table 1.

FIGS. 2A-2B depict a method of producing neuropathological aggrecan containing LCD/IDR/prion-like domains using TDP-43 and CRY2OLIG as examples of light. Various protein alignment and blue light exposure paradigms were developed to induce protein aggregation of proteins and protein fragments containing LCD/IDR/prion-like domain domains, promote the mislocalization of nuclear proteins and reproduce the neuropathology of neurodegenerative diseases. TDP-43 is primarily a nucleoprotein containing the DR/IDD/prion-like domain and, as an example, mis-localized and aggregates in ALS, FTD and some AD patients. The DNA sequence was designed to generate an amino acid sequence encoding a CRY2OLIG protein that clusters when exposed to blue light, producing a fusion protein, either the entire TDP-43 protein (CRY2-TDP43-mCH) or only the low complexity domain (LCD/IDD/prion-like domain), which makes TDP-43 susceptible to aggregation (Cry 2-274-mCH). As a control, only CRY2OLIG was used. All constructs were fused to a fluorescent protein called mcherry (mch) to visualize the protein in living cells. CRY2OLIG-mCH was stimulated with blue light to reversibly aggregate, but with the specific blue light stimulation paradigm, CRY2OLIG-TDP-43-mCH or truncated CRY2OLIG-274-mCH forms irreversible aggregates. FIG. 2A shows an example of the CRY2OLIG-TDP-43 arrangement and a truncated CRY2OLIG-274 arrangement used in this work. Fig. 2B shows a model of the described technique.

Fig. 3 shows a schematic depicting blue-light induced oligomerization and aggregation of proteins using NcVVD, NcVVDY50W or NcLOV photoreceptors. The NcVVD or LOV domains show homodimerization only with blue light stimulation. A light exposure paradigm that induces oligomerization and aggregation was developed. The upper panel shows a schematic representation of how a single acute stimulus with blue light (405nm-499nm) induces homodimerization of LOV proteins when fused to a protein of interest. The lower panel shows that chronic stimulation with blue light promotes homologous oligomerization of prion-like domain/LCD/IDD containing NcVVD or LOV fusion proteins.

FIGS. 4A-4E show light-induced aggregation of low complexity domain proteins. Using light stimulation, the optogenetic TDP-43 fragment containing the Low Complexity Domain (LCD) undergoes progressive aggregation. (FIGS. 4A-4B) the C-terminal fragment of TDP-43 (optoRRM2+ LCD/optoLCD) oligomerized rapidly after a brief pulse of blue light (8 sec, 10% laser power, 488nm) as monitored by live confocal microscopy. These oligomers last longer than Cry2 photoreceptors alone (about 10 minutes of disassembly). (FIGS. 4C-4D) the optogenetic LCD fragments also continued to aggregate after a brief pulse of light, increasing in size over time. (FIG. 4E) sustained blue light stimulation of TDP-43LCD forms intracellular aggregates in cells. Fluorescence Recovery After Photobleaching (FRAP) experiments showed that persistent light LCD contents could not be recovered from FRAP, indicating that these were insoluble.

Figures 5A-5K show that chronic blue light stimulation induces mislocalization and aggregation of optoTDP43, which summarizes the pathological features seen in patient CNS tissues. HEK293 cells expressing optoTDP43-mCH were exposed to 488nm LED stimulation or dark for up to 36 hours. (FIGS. 5A-5C) representative images show that optoTDP43 (FIG. 5B) first undergoes gradual cytoplasmic mislocalization, which is confirmed by the nuclear/cytoplasmic fraction (FIG. 5C). (FIG. 5D) there was mis-localization after optoTDP43 concentration as measured by simultaneous chronic blue light exposure and high throughput auto-confocal microscopy, which tended to increase with increasing exposure. (fig. 5E) Fluorescence Recovery After Photobleaching (FRAP) imaging was performed to assess the kinetics of the optoTDP43 structure. The lack of fluorescence recovery indicates that the photo-induced aggregates of optoTDP43 are non-dynamic, fixed particles, reminiscent of aggregate structure. (FIG. 5F) detergent solubility of the structure of optoTDP43 was assessed by subcellular fractionation to confirm the aggregation state of light-induced optoTDP43 particles. (left lane) non-optogenetic TDP-43(TDP43-mCh) showed no change in solubility with and without light treatment, while optoTDP43 (right lane) showed a sharp change to the insoluble fraction under chronic blue light stimulation. In addition to increasing the insolubility of exogenous full-length optoTDP43 (top band), chronic blue light exposure also resulted in the recruitment of aberrant optoTDP43 cleavage products (middle band) as well as endogenous full-length TDP-43 and disease-related cleavage products (bottom band), detergent insoluble, urea soluble fractions of cellular cuttings as observed in patient tissues. (FIG. 5G) to confirm the direct recruitment of non-photoreheritable TDP-43 material into the exogenous photoinduced optoTDP43 content, EGFP-tagged TDP-43 was co-expressed with optoTDP43 or just Cry2 photoreceptor control. In cells exposed to chronic blue light stimulation, strong co-localization was observed between the optoTDP43 content and EGFP-TDP43, confirming the ability of the optoTDP43 content to directly recruit other TDP-43 species. This recruitment appears to be dependent on TDP 43: TDP43 interaction, as co-localization of Cry2-mCh spots was not observed after blue light exposure. (FIGS. 5H-5J) immunofluorescence analysis of light-induced optoTDP43 aggregates was performed to confirm pathological markers seen in patient tissues. The optoTDP43 content appeared (fig. 5H) ubiquitinated, (fig. 5I) hyperphosphorylated and (fig. 5J) p62 positive, all of which had been observed with TDP-43 content in the patient CNS. (FIG. 5K) an automated high throughput confocal microscope was performed to assess neurotoxicity of the contents of photo-induced optoTDP 43. Human ReN neurons expressing TDP43-mCh or optoTDP43 were exposed to chronic blue light stimulation and their viability was simultaneously monitored by longitudinal imaging. Neurons expressing non-optogenetic TDP43-mCh showed no significant decrease in survival with or without exposure to blue light. However, optoTDP 43-expressing neurons exposed to blue light stimulation showed significantly reduced viability over time, indicating that the optoTDP43 inclusion was neurotoxic. P 05, p 01

FIGS. 6A-6E show chronic stimulus-induced clustering and aggregation of CRY2OLIG α -synuclein or α -synuclein LOV chronic stimulus of CRY2-asyn-mCH was tested to induce α -synuclein aggregation FIG. 6A shows a chronic stimulus paradigm, FIG. 6B shows representative images of α -synuclein clustering with light over time and quantification of clustering in FIG. 6℃ these data indicate that this optogenetic system can be used to induce clustering and aggregation of a variety of neurodegenerative disease proteins prone to aggregation (i.e., proteins containing prion-like domains/LCD/IDD). FIG. 6D shows that α -synuclein fused to LOV (dimerized photoreceptor) forms α intracellular clusters of synuclein. FIG. 6E shows that light-induced aggregates α -synuclein LOV exhibit synucleinopathic features, including phosphorylation of serine 129 positive and p 62.

FIGS. 7A-7D show Cry2-Tau fusion proteins showing pathological markers of various tauopathies. (FIGS. 7A-7C) HEK293 cells expressing individual Cry2 photoreceptors or Cry2-Tau fusion proteins were exposed to chronic blue light stimulation (16 hours, 488nm, 10 mW). Cry 2-Tau-expressing cells showed fibrillar-like aggregates co-stained with phosphorylated Tau antibodies AT8 (fig. 7A) and PHF1 (fig. 7B) and with pathological conformation-dependent Tau antibody MC1 (fig. 7C). (FIG. 7D) outlines various optogenetic Tau constructs co-localized with pathological Tau antibodies after light-induced neurofibrillary tangle formation.

FIGS. 8A-8E show that the LOV-Tau fusion protein also shows pathological markers for various tauopathies and is insoluble. (FIGS. 8A-8B) HEK293 cells expressing VVD-Tau or VFACU-Tau fusion proteins were exposed to chronic blue light stimulation (16 h, 488nm, 10 mW). Both fusion proteins were co-stained with the phosphorylated Tau antibodies AT8 and PHF1 and the pathological conformation-dependent Tau antibody MC 1. (FIG. 8C) differentiated MAP2+ human ReN neurons expressing VVD-Tau also showed neurofibrillary tangle formation following chronic light treatment co-localized with AT 8. (FIG. 8D) fluorescence recovery after photobleaching analysis of light-induced entanglement was performed and showed a lack of fluorescence recovery, indicating a non-dynamically fixed structure. (FIG. 8E) Urea extraction was next performed to confirm insoluble Tau species with light treatment in HEK293 cells expressing VVD-Tau. Cells exposed to chronic light show increased levels of soluble and insoluble 150kDa tau material present in tissues of patients with Alzheimer's disease and frontotemporal dementia.

FIGS. 9A-9B show the aggregation potential of CRY2 or VVD fusion proteins based on a combination of protein alignment and light stimulation paradigm. Studies have shown that the ability of Cry2 or VVD photoreceptors to stimulate clustering and activation depends on protein arrangement and photostimulation paradigms. FIG. 9A shows that the TDP-43-CRY2OLIG-mCH protein is mislocalized, but does not exhibit the ability to aggregate as does the CRY2OLIG-TDP-43-mCH arrangement. Similarly, FIG. 9B shows that mCH-aSyn-NvVVDY50W has no aggregation capability. This data indicates that the protein alignment and light stimulation paradigm are important for inducing neuropathological protein aggregation, as observed in CNS tissues of patients with neurodegenerative diseases.

FIGS. 10A-10B show that blue light induced aggregates of TDP-43 or truncated LCD/IDR/prion-like domain are cytotoxic to cells. Figure 10A shows that acute stimulation of HEK cells expressing CRY2OLIG-TDP274-mCh can induce pathological aggregate formation within 1 hour. Over time, these aggregates grow in size and become toxic. Figure 10B is a schematic of a screening line using induction of neurodegenerative disease pathology to identify therapeutic compounds that can rescue toxicity and prevent or eliminate the formation of induced neurodegenerative disease pathology.

Detailed Description

Disclosed herein are compounds, compositions and methods for inducing a neurodegenerative disease pathology in a cell or animal. The present inventors have developed a novel method for inducing neurodegenerative disease pathology in cells and animals without the need for gene mutations or severe overexpression of neurodegenerative disease proteins.

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings and examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, 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. As used herein, the term "comprising" and variants thereof are used synonymously with the term "comprising" and variants thereof, and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of … …" and "consisting of … …" may be used in place of "comprising" and "including" to provide more specific embodiments, and are also disclosed.

The following definitions are provided for a complete understanding of the terms used in this specification.

Term(s) for

As used herein, the articles "a", "an" and "the" mean "at least one" unless the context in which the article is used clearly indicates otherwise.

As used herein, the term "about" when referring to a measurable value such as an amount, percentage, or the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, or ± 1% from the measurable value.

As used herein, the terms "may", "optionally" and "may optionally" are used interchangeably and are intended to include situations in which a condition occurs as well as situations in which a condition does not occur.

The term "nucleic acid" as used herein refers to a polymer composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides).

The terms "ribonucleic acid" and "RNA" as used herein refer to a polymer composed of ribonucleotides.

The terms "deoxyribonucleic acid" and "DNA" as used herein refer to a polymer composed of deoxyribonucleotides.

The term "oligonucleotide" means a single-or double-stranded polymer of nucleotides of about 2 up to about 100 nucleotides in length. Suitable oligonucleotides can be prepared by the phosphoramidite method described by Beaucage and Carruther, Tetrahedron Lett., 22:1859-1862(1981), or by the Matteucci et al, J.Am.chem.Soc.,103:3185(1981) triester method, both by reference or by use of a commercially automated oligonucleotide synthesizer or VLSIPS^TMOther chemical methods of the technology are incorporated herein. When an oligonucleotide is referred to as "double stranded," one of skill in the art will understand that a pair of oligonucleotides is present in a hydrogen bonded helical array commonly associated with, for example, DNA. The term is used herein with the exception of 100% complementary forms of double-stranded oligonucleotides"double-stranded" also means those forms that include such structural features (e.g., bulges and loops) as are more fully described in biochemical texts such as Stryer, Biochemistry, third edition, (1988), which are incorporated herein by reference for all purposes.

The terms "polynucleotide", "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein and refer to a single-or double-stranded polymer composed of nucleotide monomers.

The term "polypeptide" refers to a compound consisting of a single chain of D-or L-amino acids or a mixture of D-and L-amino acids linked by peptide bonds.

The term "complementary" refers to the topological compatibility or matching together of the interacting surfaces of the probe molecule and its target. Thus, the target and its probe can be described as complementary, and in addition, the contact surface properties are complementary to each other.

The term "hybridization" refers to the process of establishing a non-covalent sequence-specific interaction between two or more complementary nucleic acid strands as a single hybrid, referred to as a duplex in the case of two strands.

The term "annealing" refers to a process in which a single-stranded nucleic acid sequence is paired with a complementary sequence by hydrogen bonding to form a double-stranded nucleic acid sequence, which includes the reformation (renaturation) of the complementary strand, which is separated by heat (heat denaturation).

The term "melting" refers to the denaturation of a double-stranded nucleic acid sequence due to high temperature, thereby separating the double strand into two single strands by breaking the hydrogen bonds between the strands.

The term "promoter" or "regulatory element" refers to a region or sequence determinant located upstream or downstream of the initiation of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription, the promoter need not be of bacterial origin, e.g., promoters derived from viruses or other organisms may be used in the compositions, systems or Methods described herein the term "regulatory element" is intended to include promoters, enhancers, Internal Ribosome Entry Sites (IRES) and other Expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences), regulatory elements such as described in, for example, Goeddel, Gene Expression Technology: Methods in enzymology 185, academy Press, Sanp Diego, Calif (1990) regulatory elements including those that which are constitutively expressed in many types of host cells and those which are directed to Expression of nucleotide sequences only in certain types of host cells (e.g., tissue-specific regulatory sequences) may be primarily in the desired tissue-specific promoters of the promoter, tissue-specific promoters of promoters such as promoters of CMV promoter, promoters of genes, such as promoters, promoters of proteins of the promoter, promoters of the promoter, promoter.

The term "recombinant" refers to a nucleic acid (e.g., a polynucleotide) that is or is a copy or complement of a nucleic acid (e.g., a polynucleotide) that is artificially manipulated, or a protein encoded by a recombinant nucleic acid (e.g., a polynucleotide) if a protein (i.e., "recombinant protein") is involved. In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) can comprise a promoter that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation (e.g., by the methods described below, Sambrook et al, Molecular cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Vol.1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette can comprise nucleic acids (e.g., polynucleotides) combined in such a way that the nucleic acids (e.g., polynucleotides) are highly unlikely to be found in nature. For example, a manually manipulated restriction site or plasmid vector sequence can flank or separate the promoter from the second nucleic acid (e.g., polynucleotide). Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be manipulated in many ways and are not limited to the examples described above.

The term "expression cassette" refers to a nucleic acid construct which, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) can comprise a promoter that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation (e.g., by the methods described below, Sambrook et al, Molecular cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Vol.1-3, John Wiley & Sons, Inc. (1994-1998)). In certain embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g., a polynucleotide) can include a terminator that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In certain embodiments, the expression cassette comprises a promoter operably linked to the second nucleic acid (e.g., polynucleotide) and a terminator operably linked to the second nucleic acid (e.g., polynucleotide) as a result of the human manipulation. In certain embodiments, the expression cassette comprises an endogenous promoter. In certain embodiments, the expression cassette comprises an endogenous terminator. In certain embodiments, the expression cassette comprises a synthetic (or non-natural) promoter. In certain embodiments, the expression cassette comprises a synthetic (or non-natural) terminator.

The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences or percentage of amino acid residues or nucleotides that are about 60% identical, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical over a specified region when compared and aligned for maximum correspondence over a comparison window or specified region, as compared to the same check using the BLAST or BLAST2.0 sequence comparison algorithm with default parameters described below or by manual alignment and visual inspection (e.g., see NCBI website, etc.) measure the same. Such sequences are then considered "substantially identical". This definition also refers to or can be applied to the complementarity of the test sequence. The definition also includes sequences with deletions and/or additions as well as sequences with substitutions. As described below, the preferred algorithm may take into account vacancies, etc. Preferably, the identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10 to 50 amino acids or 20 to 50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignments to determine percent sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. Suitable parameters for measuring alignment can be determined by known methods, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared.

For sequence comparison, one sequence is typically used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

One example of an algorithm suitable for determining sequence identity and percent sequence similarity is the BLAST and BLAST2.0 algorithms described in Altschul et al (1977) Nuc. acids Res.25:3389-3402, and Altschul et al (1990) J.mol.biol.215:403-410, respectively. Software for BLAST analysis is publicly available through the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al (1990) J.mol.biol.215: 403-. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits extend in both directions along each sequence until the cumulative alignment score can be increased. Cumulative scores were calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The expansion of the stop word hits in each direction will be stopped in the following cases: the cumulative ratio score is reduced by an amount X from its maximum realized value; the cumulative score becomes below zero due to accumulation of one or more negative scoring residue alignments; or to the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, desirably (E) of 10, M-5, N-4 and a comparison of the two strands. For amino acid sequences, the BLASTP program defaults to using a word length of 3 and an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) proc. natl. acad. sci. usa 89:10915) alignment of 50 for (B), an expectation (E) of 10, M-5, N-4, and a two-strand comparison.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability by which a match of two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, and more preferably less than about 0.01.

The phrase "codon optimized" refers to genes or coding regions of nucleic acid molecules used to transform various hosts, and refers to codon changes in genes or coding regions of polynucleic acid molecules to reflect typical codon usage of a selected organism without altering the DNA-encoded polypeptide. Such optimization includes the replacement of at least one or more, or one or a large number of, codons with one or more codons that are used more frequently in the genes of the selected organism.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader is operably linked to DNA for a polypeptide if the DNA is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are in close proximity to each other and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g., enhancers and coding sequences) need not be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide linkers or linkers are used according to conventional practice. In embodiments, a promoter is operably linked to a coding sequence when it is capable of affecting (e.g., modulating relative to the absence of the promoter) the expression of a protein from the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

The term "variant" or "derivative" as used herein refers to an amino acid sequence derived from the amino acid sequence of a parent protein having one or more amino acid substitutions, insertions and/or deletions.

Chimeric constructs

In one aspect, disclosed herein is an expression vector encoding a chimeric polypeptide, the expression vector comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter.

In certain embodiments, the expression vector encoding the chimeric polypeptide is contained in a plasmid or a viral or viral vector. The plasmid or viral vector may be capable of extrachromosomal replication or, alternatively, may integrate into the host genome. As used herein, the term "integrated" as applied to an expression vector (e.g., a plasmid or viral vector) refers to the introduction (physical insertion or ligation) of the expression vector or a portion thereof into the chromosomal DNA of a host cell. As used herein, "viral vector" refers to a virus-like particle containing genetic material that can be introduced into eukaryotic cells without causing substantial pathogenic effects on the eukaryotic cells. Transduction can be performed using a variety of viruses or viral vectors, but should be compatible with the cell type into which the virus or viral vector is transduced (e.g., low toxicity, ability to enter the cell). Suitable viral and viral vectors include, among others, adenoviruses, lentiviruses, retroviruses. In certain embodiments, the expression vector encoding the chimeric polypeptide is naked DNA or contained in a nanoparticle (e.g., a liposome vesicle, a porous silicon nanoparticle, a gold-DNA conjugate particle, a polyethyleneimine polymer particle, a cationic peptide, etc.).

In certain embodiments, the expression vectors of the present disclosure are capable of inducing a neurodegenerative disease pathology (e.g., inducing aggregation of a protein) in a cell without substantially altering the expression level of the protein involved in the neurodegenerative disease pathology. For example, but not limited to, an expression vector can induce aggregation of a protein having a low complexity domain from a neurodegenerative disease target protein without substantially increasing or decreasing the expression level of an endogenous target protein comprising the same low complexity domain. As such, a cell comprising a nucleotide sequence encoding a chimeric polypeptide disclosed herein can have a substantially unaltered expression level of an endogenous neurodegenerative disease target protein comprising a low complexity domain compared to a cell of the same cell type that does not comprise the nucleotide sequence encoding the chimeric polypeptide. As used herein, the term "substantially unaltered expression level" refers to a change in expression level, if any, to an extent that is unknown or not suspected to cause or be associated with a neurodegenerative disease pathology in a cell.

In certain embodiments, the expression vectors disclosed herein are capable of inducing a neurodegenerative disease pathology (e.g., inducing aggregation of a protein) in a cell using a wild-type form of a low complexity domain from a neurodegenerative disease target protein. Thus, in certain embodiments, the low complexity domain from the neurodegenerative disease target protein does not include a mutation that is different from the wild-type sequence and is known or suspected to cause or be associated with a neurodegenerative disease pathology in a cell. For example, but not limited to, the expression vector may comprise a low complexity domain from a wild-type TDP-43 protein that does not comprise a mutation known to cause or be associated with ALS, such as Q331K.

In certain embodiments, the cell is a cell that can be affected by a neurodegenerative disease. For example, the cell may be a glial cell or a neuronal cell.

In one aspect, disclosed herein is a chimeric polypeptide comprising: a light-induced oligomerization domain; and low complexity domains from neurodegenerative disease target proteins.

In one embodiment, the light-induced oligomerization domain is selected from the group consisting of: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, and NcLOV. In one embodiment, the light-induced oligomerization domain is selected from the group consisting of: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, VFAU1, YtvA, EL222, RsLOV, and AsLOV2. In one embodiment, the light-induced oligomerization domain is selected from the list of domains in table 2. In one embodiment, the light-induced oligomerization domain is selected from the following variants: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, VFAU1, YtvA, EL222, RsLOV or AsLOV2. In one embodiment, the light-induced oligomerization domain is selected from fragments of: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, VFAU1, YtvA, EL222, RsLOV or AsLOV2.

In one embodiment, the light-induced oligomerization domain is NcVVDY 50W. In one embodiment, the light-induced oligomerization domain is CRY2 OLIG. In one embodiment, the light-induced oligomerization domain is crypt. In one embodiment, the light-induced oligomerization domain comprises an LOV domain from a VVD protein. In one embodiment, the light-induced oligomerization domain comprises an LOV domain from an LOV protein. In one embodiment, the light-induced oligomerization domain comprises a PHR domain. In one embodiment, the light-induced oligomerization domain comprises a PHR domain from the CRY2 protein. In one embodiment, the light-induced oligomerization domain is VfAU 1. In one embodiment, the light-induced oligomerization domain is YtvA. In one embodiment, the light-induced oligomerization domain is EL 222. In one embodiment, the light-induced oligomerization domain is RsLOV. In one embodiment, the light-induced oligomerization domain is AsLOV2.

In one embodiment, the light-induced oligomerization domain has at least 90% identity to crypthr. In one embodiment, the light-induced oligomerization domain has at least 90% identity to NcVVD. In one embodiment, the light-induced oligomerization domain is at least 90% identical to NcVVDY 50W. In one embodiment, the light-induced oligomerization domain is at least 90% identical to NcLOV. In one embodiment, the light-induced oligomerization domain has at least 90% identity to CRY2 OLIG. In one embodiment, the light-induced oligomerization domain has at least 90% identity to VfAU 1. In one embodiment, the light-induced oligomerization domain has at least 90% identity to YtvA. In one embodiment, the light-induced oligomerization domain is at least 90% identical to EL 222. In one embodiment, the light-induced oligomerization domain has at least 90% identity to RsLOV. In one embodiment, the light-induced oligomerization domain is at least 90% identical to AsLOV2.

In certain embodiments, the first nucleotide sequence may comprise a nucleotide sequence encoding a light-induced oligomerization domain selected from the group consisting of seq id no: CRYPHR, CRY2OLIG, NcVVD, NcVVDY50W, NcLOV, and VFACU 1 LOV.

In certain embodiments, the first nucleotide sequence may comprise a nucleotide sequence encoding an amino acid sequence having at least 70% identity to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, or SEQ ID NO 102. In certain embodiments, the first nucleotide sequence may encode an amino acid sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, or SEQ ID NO 102. In certain embodiments, the first nucleotide sequence may comprise SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, or SEQ ID NO 102. In certain embodiments, the first nucleotide sequence may comprise SEQ ID NO 94. The nucleotide sequence may be that of a wild-type nucleic acid sequence encoding an amino acid sequence disclosed herein. In certain embodiments, the nucleotide sequence is modified from the wild-type sequence, but may still encode the same amino acid sequence due to the degeneracy of the genetic code. In certain embodiments, the nucleotide sequence is a variant (or encodes a variant protein sequence) of one of the sequences disclosed herein. In certain embodiments, the nucleotide sequence is a fragment of one of the nucleic acids disclosed herein, or a fragment encoding one of the amino acids disclosed herein. In certain embodiments, the nucleotide sequence is codon optimized (e.g., to improve expression).

In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntington protein, Ataxin2 and hnRNPA2B 1. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1 and hnRNPA2B 1. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1 and TATA box binding protein factor 15. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of variants of: TDP-43, alpha synuclein, Tau, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1 or TATA box binding protein factor 15. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of fragments TDP-43, a synuclein, Tau, TIA1, SOD1, huntingtin, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1, or TATA box binding protein factor 15. In one embodiment, the fragments of TDP-43, alpha synuclein, Tau, TIA1, SOD1, huntingtin, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1, or TATA box binding protein factor 15 comprise a low complexity domain (or fragment thereof) within each neurodegenerative disease target protein.

In one embodiment, the low complexity domain from a neurodegenerative disease target protein is selected from table 3. In one embodiment, the low complexity domain is from any neurodegenerative disease target protein that aggregates in a neurodegenerative disease.

In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: an amino acid sequence having at least 90% identity to TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, huntingtin, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1, and TATA box binding protein factor 15. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is selected from an amino acid sequence having at least 90% identity to a low complexity domain from a neurodegenerative disease target protein selected from table 3. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from orthologs of the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1 and TATA box binding protein factor 15.

In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: an amino acid sequence having at least 60% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) identity to TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, huntingtin, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1, and TATA box binding protein factor 15 (or a fragment thereof).

In one embodiment, the low complexity domain from the neurodegenerative disease target protein is TDP-43. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is a synuclein. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is Tau. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is Fus. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is TIA 1. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is SOD 1. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is huntingtin. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is Ataxin 2. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is hnRNPA 1. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is hnRNPA2B 1. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is EWS RNA binding protein 1. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is TATA box binding protein factor 15.

In one embodiment, the VVD light-induced oligomerization domain is fused to the low complexity domain of TDP-43. In one embodiment, VVD light-induced oligomerization is fused to full-length TDP-43 (comprising a low complexity domain). In one embodiment, the light-induced oligomerization domain is fused to a low complexity domain from any neurodegenerative disease target protein that aggregates in neurodegenerative disease.

In one embodiment, the light-induced oligomerization domain is fused to the low complexity domain of TDP-43, wherein the low complexity domain sequence of TDP-43 comprises SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 9, or SEQ ID NO 98 (or fragments thereof).

In one embodiment, the nucleotide sequence encoding the chimeric polypeptide may further comprise a third nucleotide sequence encoding a reporter protein (such as a fluorescent protein to allow visualization of protein aggregates by fluorescence). In one embodiment, the fluorescent protein is mcherry (mch). In certain embodiments, the fluorescent protein is GFP or YFP. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising a PHR domain (e.g., crypthr) of an arabidopsis cryptochrome 2 protein. In certain embodiments, the light-induced oligomerization domain can comprise a wild-type CRYPHR amino acid sequence as disclosed in SEQ ID NO. 1, or alternatively, can comprise a mutated CRYPHR amino acid sequence as disclosed in SEQ ID NO. 2. In certain embodiments, the mutated crypthr amino acid sequence may comprise an E490G mutation that may increase the clustering efficiency upon blue light stimulation compared to the wild-type crypthr amino acid sequence. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain, wherein the light-induced oligomerization domain comprises a polypeptide sequence having at least 70% identity to SEQ ID No. 1 or SEQ ID No. 2. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain, wherein the light-induced oligomerization domain comprises a polypeptide sequence having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity to SEQ ID No. 1 or SEQ ID No. 2. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising the polypeptide sequence of SEQ ID NO 1 or SEQ ID NO 2.

In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising a photooxidative voltage sensing domain (LOV) from neurospora activin (e.g., LOV, NcVVD, NcVVDY50W, NcLOV, VfAU1, YtvA, EL222, RsLOV, and/or AsLOV 2). In certain embodiments, the light-induced oligomerization domain can comprise a wild-type LOV amino acid sequence as disclosed in SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, or SEQ ID NO 102, or alternatively can comprise a mutated LOV amino acid sequence such as disclosed in SEQ ID NO 5. In certain embodiments, a mutant LOV amino acid sequence may comprise a Y50W mutation, which may reduce the dissipation rate from the dimeric state as compared to the wild-type LOV amino acid sequence. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain, wherein the light-induced oligomerization domain comprises a polypeptide sequence having at least 70% identity to SEQ ID NO 3, 4, 5, 92, 93, 99, 100, 101 or 102. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain, wherein the light-induced oligomerization domain comprises a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 99, SEQ ID No. 100, SEQ ID No. 101, or SEQ ID No. 102. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising the polypeptide sequence of SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 or SEQ ID NO 102.

In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising a PHR domain of an arabidopsis cryptochrome 2 protein (e.g., crypthr), and the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising a PHR domain of an arabidopsis cryptochrome 2 protein (e.g., crypthr), and the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein comprising TDP-43. In certain embodiments, the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein that comprises full length TDP-43 (e.g., SEQ ID NO:6 or SEQ ID NO: 7). In certain embodiments, the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein comprising truncated TDP-43 (e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO: 13). In some embodiments, the truncated TDP-43 comprises or consists of amino acids 105-414, 191-414, or 274-414 of the full-length TDP-43 amino acid sequence. Thus, in certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising crypt and the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein comprising full length or truncated TDP-43, wherein the first and second nucleotide sequences encode a chimeric polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13. In certain embodiments, the first and second nucleotide sequences encode a chimeric polypeptide sequence according to SEQ ID NO 6, 7, 8, 9, 10, 11, 12 or 13. In certain embodiments, SEQ ID NO 6 is selected. In certain embodiments, SEQ ID NO 11 is selected.

In certain embodiments, the nucleotide sequence encoding the chimeric polypeptide comprising the first nucleotide sequence and the second nucleotide sequence may further comprise a third nucleotide sequence encoding a reporter protein or fragment thereof (e.g., a fluorescent protein such as mCherry, also known as mCH). Thus, in certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain, comprising a CRYPHR, the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein, which comprises full-length or truncated TDP-43, and the third nucleotide sequence may encode a mCherry protein, wherein the first, second and third nucleotide sequences encode a nucleotide sequence identical to SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO:24 or SEQ ID NO:25, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity. In certain embodiments, the first, second and third nucleotide sequences encode a chimeric polypeptide sequence according to SEQ ID NO 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25. In certain embodiments, SEQ ID NO 14 is selected. In certain embodiments, SEQ ID NO 23 is selected.

In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising an LOV photoreceptor domain (e.g., NcVVD, NcVVDY50W, NcLOV, VfAU1, YtvA, EL222, RsLOV, and AsLOV2), and the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein. In certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising an LOV photoreceptor domain (e.g., NcVVD, NcVVDY50W, NcLOV, VfAU1, YtvA, EL222, RsLOV, and AsLOV2), and the second nucleotide sequence may encode a low complexity domain comprising TDP-43 from a neurodegenerative disease target protein. In certain embodiments, the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein that comprises full length TDP-43 (e.g., SEQ ID NO:2 or SEQ ID NO: 27). In certain embodiments, the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein comprising truncated TDP-43 (e.g., SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO: 33). In certain embodiments, the truncated TDP-43 consists of amino acids 105-414, 191-414, or 274-414 of the full-length TDP-43 amino acid sequence. Thus, in certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising NcVVDY50W and the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein comprising full length or truncated TDP-43, wherein the first and second nucleotide sequences encode a chimeric polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32 or SEQ ID No. 33. In certain embodiments, the first and second nucleotide sequences encode a chimeric polypeptide sequence according to SEQ ID NO 26, 27, 28, 29, 30, 31, 32 or 33.

In certain embodiments, the nucleotide sequence encoding the chimeric polypeptide comprising the first nucleotide sequence and the second nucleotide sequence may further comprise a third nucleotide sequence encoding a reporter protein or fragment thereof (e.g., a fluorescent protein, such as mCherry). Thus, in certain embodiments, the first nucleotide sequence may encode a light-induced oligomerization domain comprising NcVVDY50W, the second nucleotide sequence may encode a low complexity domain from a neurodegenerative disease target protein comprising full length or truncated TDP-43, and the third nucleotide sequence may encode a mCherry protein, wherein the first, second and third nucleotide sequences encode chimeric polypeptide sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40 or SEQ ID NO 41. In certain embodiments, the first, second and third nucleotide sequences encode a chimeric polypeptide sequence according to SEQ ID NO 34, 35, 36, 37, 38, 39, 40 or 41. In certain embodiments, SEQ ID NO 34 is selected. In certain embodiments, SEQ ID NO 41 is selected.

In certain embodiments, the first nucleotide sequence is upstream of the second nucleotide sequence. In certain embodiments, the first nucleotide sequence is located downstream of the second nucleotide sequence.

In certain embodiments, when a sequence disclosed herein contains a methionine at the beginning of the protein, then a protein that does not contain a methionine is also disclosed. In certain embodiments, where the sequences disclosed herein do not comprise a methionine at the beginning of the protein, then proteins having a methionine at the beginning of the protein are also disclosed.

In certain embodiments, the nucleotide sequence encoding the chimeric polypeptide comprises a sequence selected from the group consisting of: 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 61, 62, 63, 64, 65, 67, 65, 68, 69, 70, 61, 62, 63, 64, 65, 66, 67, 68, 71, 69, 71, 72, 73, 74, 75, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91 SEQ ID NO.

Method of producing a composite material

In one aspect, disclosed herein is a method of inducing a neurodegenerative disease pathology in a cell, the method comprising the steps of:

introducing into a cell an expression vector encoding a chimeric polypeptide, said expression vector comprising:

expressing the chimeric polypeptide; and

oligomerization of the chimeric polypeptide was induced by stimulation with blue light.

In another aspect, disclosed herein is a method of screening for an agent that modulates protein aggregation, the method comprising the steps of:

introducing into a cell an expression vector encoding a chimeric polypeptide, said expression vector comprising:

expressing the chimeric polypeptide;

introducing the agent into a medium comprising cells;

inducing oligomerization of the chimeric polypeptide by stimulation with blue light; and

determining the modulation of protein aggregation by the agent.

In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1 and hnRNPA2B 1. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1 and TATA box binding protein factor 15. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of variants of: TDP-43, alpha synuclein, Tau, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1 or TATA box binding protein factor 15. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the following fragments: TDP-43, alpha synuclein, Tau, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1, hnRNPA2B1, EWSRNA-binding protein 1, or TATA box-binding protein factor 15. In one embodiment, a fragment of TDP-43, alpha synuclein, Tau, TIA1, SOD1, huntingtin, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1, or TATA box binding protein factor 15 comprises a low complexity domain within each neurodegenerative disease target protein.

In one embodiment, the low complexity domain from a neurodegenerative disease target protein is selected from an amino acid sequence having at least 90% identity to TDP-43, a synuclein, Tau, Fus, TIA1, SOD1, huntingtin, Ataxin2, hnRNPA1, hnRNPA2B1, ewssrna binding protein 1, and TATA box binding protein factor 15. In one embodiment, the low complexity domain from a neurodegenerative disease target protein is selected from an amino acid sequence having at least 90% identity to a low complexity domain from a neurodegenerative disease target protein selected from table 3. In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from orthologs of the group consisting of: TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, Huntington protein, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1 and TATA box binding protein factor 15.

In one embodiment, the low complexity domain from the neurodegenerative disease target protein is selected from the group consisting of: an amino acid sequence that is at least 60% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) identical to TDP-43, alpha synuclein, Tau, Fus, TIA1, SOD1, huntingtin, Ataxin2, hnRNPA1, hnRNPA2B1, EWS RNA binding protein 1, and TATA box binding protein factor 15 (or a fragment thereof).

In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is selected from the group consisting of: yeast, insect, avian, fish, helminth, amphibian, xenopus, bacteria, algae and mammalian cells. In one embodiment, disclosed herein is a non-human transgenic organism, wherein the organism is an insect, fish, bird, worm, amphibian, xenopus or non-human mammal.

Inducing a neurodegenerative disease pathology refers to an action that causes a neurodegenerative disease pathology or increases the phenotype, symptom, or severity of a neurodegenerative disease pathology as compared to not performing the selected action.

Examples of such pathologies include, but are not limited to, protein aggregation in the cytoplasm, mislocalization of nucleoproteins to, e.g., the cytoplasm, increased expression of ubiquitin, cellular degeneration and/or death, extracellular amyloid β (a β) aggregation, and/or intracellular and/or cytoplasmic aggregation of Tau protein.

Increased ubiquitination or increased ubiquitin expression may be a phenotypic characteristic of neurodegenerative diseases. Ubiquitin has a variety of cellular roles, including "tagging" proteins (e.g., by covalent linkage) for degradation in the proteasome. The increase in ubiquitin expression in the cell is typically compared to a control. In certain embodiments, the cell with increased ubiquitin has at least 50% increased ubiquitin expression compared to a control. In certain embodiments, a cell with increased ubiquitin has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% increased ubiquitin expression compared to a control.

Ubiquitin expression in a cell can be determined at the transcriptional level, translational level, or a combination thereof, and can be measured by a variety of methods for measuring the expression level of a gene or polypeptide. In certain embodiments, ubiquitin expression can be measured at the level of gene transcription. For example, but not limited to, the level of ubiquitin mRNA transcripts can be determined by: absorbance of radiation (e.g., uv absorption at 260nm, 280nm, or 230 nm), quantification of fluorescent dye or tag emission (e.g., ethidium bromide intercalation), quantitative polymerase chain reaction (qPCR) of cDNA generated from mRNA transcripts, southern blot analysis, gene expression microarrays, or other suitable methods. An increase in mRNA transcript levels can be used to infer or estimate an increase in polypeptide expression levels. In certain embodiments, ubiquitin expression can be measured at the post-translational level. For example, but not limited to, the level of ubiquitin polypeptide can be determined by: radiation absorption (e.g., ultraviolet), bicinchoninic acid (BCA) assay, bradford assay, biuret assay, Lowry method, coomassie brilliant blue staining, functional or enzymatic assay, immunoassay, and/or Western blot analysis or other suitable methods.

As used herein, the terms "introduce," "introduction," and grammatical variations thereof, in relation to the introduction of an expression vector into a cell, refer to any method suitable for transferring an expression vector into a cell. The term includes, but is not limited to, for example, conjugation, transformation/transfection (e.g., divalent cation exposure, heat shock, electroporation), nuclear microinjection, incubation with calcium phosphate polynucleotide precipitates, high velocity bombardment with polynucleotide-coated microparticles (e.g., by a gene gun), lipofection, cationic polymer complexation (e.g., DEAE-dextran, polyethyleneimine), dendrimer complexation, mechanical deformation of cell membranes (e.g., cell extrusion), ultrasonic perforation, optical transfection, transfection (improcection), hydrodynamic polynucleotide delivery, agrobacterium-mediated transformation, transduction (e.g., transduction with a virus or viral vector), natural or artificial competence, protoplast fusion, magnetic transfection, nuclear transfection, or combinations thereof. The introduced expression vector or polynucleotide derived therefrom may be genetically integrated or present extrachromosomally.

A range of blue wavelengths may be used in the methods of the present disclosure. In one embodiment, the blue light has a wavelength of about 400nm to about 500 nm. In one embodiment, the blue light has a wavelength of about 405nm to about 499 nm. In one embodiment, the blue light has a wavelength of about 420nm to about 490 nm. In one embodiment, the blue light has a wavelength of about 450nm to about 490 nm. In one embodiment, the blue light has a wavelength of about 460nm to about 495 nm. In one embodiment, the blue light has a wavelength of about 488 nm. In one embodiment, the blue light has a wavelength of about 475 nm. In one embodiment, the blue light has a wavelength of about 465 nm.

In one embodiment, the blue light has a wavelength of about 405nm, about 410nm, about 415nm, about 420nm, about 425nm, about 430nm, about 435nm, about 440nm, about 445nm, about 450nm, about 455nm, about 460nm, about 465nm, about 470nm, about 475nm, about 480nm, about 485nm, about 490nm, about 495nm, or about 500 nm.

The method may include various degrees of blue light stimulation. In certain embodiments, the stimulation is acute or optionally chronic. Acute stimulation refers to stimulation with a pulse of blue light for about 0.2 seconds to about 60 seconds, where the wavelength of the blue light can be any of the blue light wavelengths disclosed herein. In certain embodiments, the acute stimulus comprises a blue light pulse for about 0.5 seconds to about 30 seconds, about 1 second to about 20 seconds, or about 5 seconds. The blue light may be provided by a blue light source or a broad spectrum light source filtered for the disclosed wavelengths.

In certain embodiments, acute stimulation can result in temporary aggregation of the light-induced oligomerization domain (e.g., cytoplasmic prion-like domain/LCD/IDD protein fragment). In certain embodiments, temporary aggregation comprises aggregation of a protein that is observable by the methods disclosed herein for less than about twenty minutes, or alternatively for less than about fifteen minutes, less than about ten minutes, or for less than about five minutes. In certain embodiments, the acute stimulus does not result in aggregation of the cytoplasmic prion-like domain/LCD/IDD protein fragment for more than twenty minutes.

In certain embodiments, acute stimulation may result in aggregation of the light-induced oligomerization domain for a shorter duration than aggregation of the light-induced oligomerization domain fused to a low complexity domain from a neurodegenerative disease target protein. In certain embodiments, acute stimulation may result in aggregation of a light-induced oligomerization domain fused to a low complexity domain from a neurodegenerative disease target protein for a shorter duration than aggregation of the same protein with amino acid mutations known to cause or associated with neurodegenerative diseases (e.g., TDP-43Q 331K).

Chronic stimulation is achieved at about 0.1mW/cm²To 8mW/cm²Exposure to blue light at a wavelength of about 400nm to about 500nm for a period of time of about 1 minute or more (e.g., at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours or more) (within a wavelength of 400nm to 500 nm).

In certain embodiments, the methods disclosed herein can induce a neurodegenerative disease pathology (e.g., aggregation of a protein) in a cell without substantially altering the expression level of the protein involved in the neurodegenerative disease pathology. For example, but not limited to, the methods can induce aggregation of a chimeric polypeptide comprising TDP-43 (which can include aggregation of endogenous TDP-43) without substantially increasing or decreasing the expression level of endogenous TDP-43. In certain or other embodiments, the methods can use a wild-type form of a low complexity domain from a neurodegenerative disease target protein to induce a neurodegenerative disease pathology in a cell. Thus, in certain embodiments, the low complexity domain from the neurodegenerative disease target protein does not include a mutation that is different from the wild-type sequence and is known or suspected to cause or be associated with inducing a neurodegenerative disease pathology in a cell. For example, but not limited to, the methods may induce aggregation of a chimeric polypeptide comprising a wild-type TDP-43 protein or fragment thereof that does not comprise a mutation known to cause ALS or associated with ALS, such as Q331K.

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