ALS-related diamino acid repeat-containing protein

Abstract

To provide methods and compositions for identifying and / or treating subjects having or likely to have amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).SOLUTION: A method comprises determining a level of one or more di-amino acid repeat-containing proteins contained in a blood sample from a subject, where a level of the one or more di-amino acid repeat-containing proteins that is elevated compared to a control level indicates that the subject has or is likely to develop ALS or FTD. Antibodies specific for one or more di-amino acid repeat-containing proteins that can be used in this assay are also provided herein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits as of the filing date of U.S. Provisional Application No. 61 / 786,258, filed March 14, 2013, and the benefits as of the filing date of U.S. Provisional Application No. 61 / 883,219, filed September 27, 2013. The entire contents of both of these referenced applications are incorporated herein by reference.

[0002] Federally funded research or development This invention was made with government assistance under PO1NS058901 and RO1NS040389, granted by the National Institutes of Health. The United States Government has certain rights to this invention. [Background technology]

[0003] Elongation of the GGGGCC hexanucleotide sequence within the intron of the human C9ORF72 gene is associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in humans. Amyotrophic lateral sclerosis (ALS) is a debilitating disease with various etiologies characterized by rapidly progressing weakness, muscle atrophy, muscle spasticity, dysarthria (difficulty speaking), dysphagia (difficulty swallowing), and respiratory distress (difficulty breathing). Although the order and rate of symptoms vary from person to person, eventually most individuals are unable to walk, get out of bed on their own, or use their hands and arms. Most individuals with ALS eventually die from respiratory failure, usually within 3 to 5 years of symptom onset. Riluzole (Rilutek) is the only currently available treatment for ALS and only slows progression and increases survival to a limited extent. Frontotemporal dementia (FTD) is also a destructive group of diseases resulting from atrophy or shrinkage of the frontal and temporal lobes of the brain. This shrinking or atrophy leads to serious behavioral changes. Currently, there is no treatment for FTD, and only limited medications exist to manage its symptoms. Novel methods for diagnosing and treating ALS and / or FTD are expected to bring significant benefits to those with ALS and FTD. [Overview of the Initiative]

[0004] Extension of the GGGGCC hexanucleotide sequence within the intron of the human C9ORF72 gene is associated with both amyotrophic lateral sclerosis and frontotemporal dementia in humans. As described herein, the extension of the GGGGCC hexanucleotide repeat sequence within the intron of the C9ORF72 gene was found to be transcribed in such a way that it yields RNA transcripts containing the hexanucleotide repeat in both sense and antisense directions. These sense and antisense transcripts were found to be translated into diamino acid repeat-containing proteins. The sense transcript (containing the 5'-GGGGCC-3' hexanucleotide repeat) was found to be translated via repeat-associated non-ATG (RAN) translation, resulting in poly(Gly-Ala), poly(Gly-Pro), and poly(Gly-Arg) proteins. Antisense transcripts (containing the 5'-GGCCCC-3' hexanucleotide repeat) were found to be translated via repeat-associated non-ATG (RAN) translation, resulting in poly(Pro-Ala), poly(Pro-Arg), and poly(Gly-Pro) proteins. In addition, antisense transcripts were found to be translated via ATG-initiated translation, resulting in Met...poly(Pro-Arg) and Met...poly(Gly-Pro) proteins.

[0005] These diamino acid repeat-containing proteins have been found to be present in blood samples from subjects with ALS. Accordingly, aspects of the present disclosure relate to a method for detecting levels of diamino acid repeat-containing proteins in a sample (e.g., blood) obtained from a subject, the method comprising measuring the level of diamino acid repeat-containing proteins in the sample of the subject. In some embodiments, detection of diamino acid repeat-containing protein levels may identify (or diagnose) or assist in the identification (or assistance in the diagnosis) of subjects who have ALS or FTD or are likely to develop ALS or FTD. Alternatively or in addition, detection of diamino acid repeat-containing protein levels in, for example, a blood sample of a subject may identify (or diagnose) or assist in the identification (or assistance in the diagnosis) of a subject as having a risk factor for ALS or FTD, such as elevated levels of diamino acid repeat-containing proteins in the subject or proteins in cerebrospinal fluid. Aspects of the present disclosure also relate to the treatment of subjects with ALS or FTD by reducing or stabilizing levels of diamino acid repeat-containing proteins in the subject's blood.

[0006] In addition, the expression of antisense transcripts (containing the 5'-GGCCCC-3' hexanucleotide repeat) was found to be significantly elevated in subjects with the extended GGGGCC hexanucleotide repeat compared to controls. Aggregation of sense and antisense transcripts was also detectable using fluorescence in-situ hybridization (FISH) in brain and blood cells of patients with the extended GGGGCC hexanucleotide repeat sequence in the intron of the C9ORF72 gene. Thus, other aspects of the present disclosure relate to a method for detecting hexanucleotide repeat-containing transcripts, the method comprising measuring the level of hexanucleotide repeat-containing transcripts and / or measuring the presence or absence of hexanucleotide repeat-containing transcript aggregation. In some embodiments, the detection of hexanucleotide repeat-containing transcripts may identify (or diagnose) a subject as having ALS or FTD or being highly likely to develop ALS or FTD, or assist in such identification (or assist in diagnosis). Alternatively, or in addition, for example, the detection of hexanucleotide repeat-containing transcripts in a blood sample of the subject may identify (or diagnose) or assist in the identification (or diagnosis) of the subject as having a risk factor for ALS or FTD, such as elevated levels of the subject's diamino acid repeat-containing protein or protein in cerebrospinal fluid.

[0007] In some embodiments, the disclosure relates to a method for identifying a subject as having ALS or FTD or being highly likely to develop ALS or FTD, the method comprising determining the level of one or more diamino acid repeat-containing proteins selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins in a blood sample obtained from the subject, the elevated level of one or more diamino acid repeat-containing proteins compared to a control level indicates that the subject has ALS or FTD or is highly likely to develop ALS or FTD. In some embodiments, the level of one or more diamino acid repeat-containing proteins is determined by performing an assay. In some embodiments, the assay comprises an immunobased assay. In some embodiments, the immunobased assay comprises an isolated antibody specific to an antigen containing a sequence described in Tables 1, 2, or 3. In some embodiments, the immunobased assay comprises an isolated antibody specific to the C-terminus of one or more diamino acid repeat-containing proteins.

[0008] In some embodiments, the method further includes identifying a subject as having ALS or FTD or being at high risk of developing ALS or FTD if the level of diamino acid repeat-containing protein is elevated compared to a control level. In some embodiments, the method further includes treating a subject who has ALS or FTD or is at high risk of developing ALS or FTD. In some embodiments, the treatment includes administering to the subject an effective dose of one or more of riluzole, baclofen, diazepam, phenytoin, trihexyphenidyl, or amitriptyline. In some embodiments, the treatment includes performing a treatment selected from plasmapheresis or bone marrow transplantation.

[0009] In some embodiments, one or more diamino acid repeat-containing proteins are selected from poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins. In some embodiments, one or more diamino acid repeat-containing proteins are two or more diamino acid repeat-containing proteins.

[0010] Other aspects of the present disclosure relate to methods for treating subjects having ALS or FTD, the methods comprising reducing or preventing an increase in the levels of one or more diamino acid repeat-containing proteins selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins in the subject's blood. In some embodiments, reducing or preventing an increase in the levels of one or more diamino acid repeat-containing proteins comprises removing one or more diamino acid repeat-containing proteins from the subject's blood. In some embodiments, one or more diamino acid repeat-containing proteins from the subject's blood are removed using a procedure selected from plasmapheresis or bone marrow transplantation. In some embodiments, bone marrow transplantation is allogeneic bone marrow transplantation.

[0011] In another embodiment, the disclosure relates to an isolated antibody specific to one or more diamino acid repeat proteins selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins. In some embodiments, the diamino acid repeat protein is selected from poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins. In some embodiments, the isolated antibody is specific to an antigen containing a sequence or fragment of the sequence listed in Tables 1, 2, or 3.

[0012] Other aspects of the present disclosure relate to a method for identifying a subject as having ALS or FTD or being highly likely to develop ALS or FTD, the method comprising determining the level of 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat-containing RNA in a blood sample obtained from the subject, wherein elevated levels of 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat-containing RNA compared to a control level indicate that the subject has ALS or FTD or is highly likely to develop ALS or FTD. In some embodiments, this level is determined by performing an assay. In some embodiments, the assay includes nucleic acid-based assays, such as in-situ hybridization (e.g., FISH) or RT-PCR (e.g., quantitative RT-PCR or strand-specific quantitative RT-PCR). In some embodiments, the method further includes identifying a subject as having ALS or FTD or being at high risk of developing ALS or FTD if the level of 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat-containing RNA is elevated compared to a control level. In some embodiments, the method further includes treating subjects who have ALS or FTD or are at high risk of developing ALS or FTD. In some embodiments, the treatment includes administering an effective dose to the subject of one or more of riluzole, baclofen, diazepam, phenytoin, trihexyphenidyl, or amitriptyline. In some embodiments, the treatment includes performing a treatment selected from plasmapheresis or bone marrow transplantation. In some embodiments, the level of 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat-containing RNA is the level of 5'-GGCCCC-3' hexanucleotide repeat-containing RNA.

[0013] Another aspect of the present disclosure relates to a method for identifying a subject as having or being likely to develop ALS or FTD. The method includes determining the presence or absence of aggregates containing RNA with a 5'-GGGGCC-3' hexanucleotide repeat expansion and / or RNA with a 5'-GGCCCC-3' hexanucleotide repeat expansion in a sample obtained from the subject. The presence of aggregates of RNA with a 5'-GGGGCC-3' hexanucleotide repeat expansion and / or RNA with a 5'-GGCCCC-3' hexanucleotide repeat expansion indicates that the subject has or is likely to develop ALS or FTD. In some embodiments, the presence or absence of aggregates or elevated C9ORF72 sense or antisense RNA levels is determined by performing an assay. In some embodiments, the assay includes a nucleic acid-based assay, such as strand-specific RT-PCR or in situ hybridization (e.g., FISH).

[0014] Another aspect of the present disclosure relates to transgenic mice. In some embodiments, the transgenic mice include the human C9ORF72 gene and optionally human flanking sequences. In some embodiments, the transgenic mice include SEQ ID NO: 63.

[0015] These and other aspects are described in more detail herein and illustrated by non-limiting drawings and examples.

[0016] The drawings will first be described. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] [Figure 1]Figure 1 shows that transcripts are produced in both sense and antisense directions on the C9ORF72 gene, and that repeat-associated non-ATG (RAN) translation proteins are translated from both sense and antisense C9ORF72 transcripts in all three reading frames. This figure also shows that Met... poly(Pro-Arg) and Met... poly(Gly-Pro) proteins are translated through ATG-initiated translation on the antisense transcript. CT = predicted and / or shown to contain the c-terminal domain. *= end of the protein (by stop codon). M = methionine. [Figure 2] Figure 2A is a diagram of an expression vector for expressing RAN translation proteins intracellularly. CMV = cytomegalovirus promoter. 6×Stop = six stop codons, two in each frame. (GGGGCC)exp = GGGGCC repeat sequences that extend with 4, 30, 60, or 120 repeats. (GR)HA-(GP)Flag-(GA)Myc = HA, flag or myc tags corresponding to poly(Gly-Arg), poly(Gly-Pro), and poly(Gly-Ala) repeat proteins, respectively. SV40 poly(a) = transcription terminator and polyA signal. Figure 2B is a photograph of a western blot showing that GR and GP RAN translation proteins are expressed in cells transfected with 30, 60 or 120 GGGGCC repeat sequences. [Figure 3] Figure 3 is a photograph of immunofluorescent staining of cells expressing GP, GR, or GA RAN proteins in cells transfected with 30, 60 or 120 GGGGCC repeat sequences. [Figure 4] Figure 4 is a series of photographs of immunofluorescent staining showing that poly(GR) and GR-c-terminal antigen and poly(GR) and (GR)-c-terminal antibody detect poly(GR) RAN protein. [Figure 5]Figure 5 shows a series of photographs of tissue from C9ORF72 ALS patients or control patients, demonstrating that C9ORF72 ALS patients express poly(GR), poly(GP), and poly(GA) diamino acid repeat-containing proteins. [Figure 6] Figure 6 shows a series of photographs of tissue from C9ORF72 ALS patients or control patients, demonstrating that C9ORF72 ALS patients express poly(PA) and poly(PR) diamino acid repeat-containing proteins. [Figure 7] Figure 7A is a series of immunofluorescence staining images showing that antibodies produced to recognize the GP repeat motif (GP) or the specific C-terminal region of the same GP-RAN protein (GP-C) co-localize in 20% of patient cells. Cells stained for GP-C and GP express the GP-RAN protein in the sense direction, while cells showing only GP staining express RAN-GP or Met...GP from the antisense strand. Figure 7B is a graph showing the percentages of GP and GP+GP-C in patient cells. [Figure 8] Figure 8 is a dot blot image showing that diamino acid repeat-containing proteins are found in the blood (PBL) and brain (FCX, frontal cortex) of subjects with ALS, but not in controls. [Figure 9] Figure 9 is a Western blot image showing that GP repeat protein is present in the brain (FCX) of subjects with ALS but not in controls, and that PA repeat protein is present in the plasma and serum of subjects with ALS but not in controls. [Figure 10] Figure 10 is a schematic diagram of a RAN translation mouse model construct containing 6X stops, CAG repeat regions, tags for detecting each CAG repeat frame, and a terminator sequence. [Figure 11] Figure 11 shows two photographs demonstrating that polyGln protein accumulated in the brains of RAN-translating (RANT) mice containing the construct shown in Figure 10, but not in control mice. [Figure 12]Figure 12 shows a series of schematic diagrams, graphs, and images illustrating the elevation of G2C4 antisense transcripts by strand-specific RT-PCR and their accumulation as RNA aggregates in C9ORF72 patient tissue. (A) Schematic diagram of the relative positions of the C9ORF72 gene, antisense transcripts, and primers and RACE primers for strand-specific RT-PCR. (B) Strand-specific RT-PCR of sense (S) and antisense (AS) transcripts (spanning intron 1b and exon 1) from the frontal cortex of C9(+) and C9(-) ALS patients. (C) Strand-specific qRT-PCR showing elevated antisense mRNA in C9(+) patients compared to C9(-) ALS patients. (D) In-situ hybridization with a G4C2-Cy3 probe showing G2C4 antisense RNA aggregates (arrowheads) in the frontal cortex and peripheral blood leukocytes (PBL) of C9(+) patients, which were not observed in the C9(-) patient. Nucleic acid aggregation in the FCX is indicated by arrowheads. FCX = Frontal Cortex. PBL = Peripheral Blood Leukocytes. [Figure 13] Figure 13 is a series of schematic diagrams, graphs, and images illustrating in vitro evidence and a dual immunodetection strategy for RAN translation of antisense G2C4 elongation. (A-C) Immunoblot (B) and IF staining (C) of HEK293T cells 48 hours after transfection of (A) (G2C4)EXP-3T construct. (B) PR and GP elongation proteins detected by Western blotting in transfected cells, and (C) PA, PR, and GP detected by IF. (D) Figure of putative proteins translated from sense and antisense transcripts. CT = C-terminus, f1-3: reading frames 1-3. (E) Summary of an example of validation of α-PA rabbit polyclonal antibody. IF staining and corresponding immunoblot of HEK293T cells transfected with a construct having 5' Flag epitope-tagged PA protein. See Figures 22 and 23 for additional controls and validation of eight additional antibodies generated against repeat motifs and CT regions. [Figure 14-1]Figures 14A and 14B are a series of images and graphs showing in vivo evidence for RAN translation and toxicity studies of the G2C4AS repeat. (A) Dot blots of C9(+) and C9(-) prefrontal cortex lysates probed with α-PA, α-PA-CT, α-PR, and α-PR-CT antibodies. (B) Immunoblots of C9(+) and C9(-) ALS prefrontal cortex lysates. (C) IHC detection of PA, PR, and GP protein aggregates in hippocampal neurons from C9(+) ALS patients, detected with α-PA, α-PA-CT, α-PR, α-PRCT, and α-GP antibodies. (D) IF staining of C9(+) hippocampal tissue using mouse α-GP (arrowheads) and rabbit α-GP-CT (arrowheads), where sense inclusions are positive for both antibodies (upper panel), and antisense inclusions are positive for GP repeat antibody only (lower panel). (E) IF staining of larger areas showing sense (S) and antisense (AS) staining. (F) Quantification of double (sense) and single (antisense) labeled aggregates. (G-J) RAN and PR toxicity studies. (G) G2C4 elongation construct (+ / -ATG-PR-3T) + / -ATG start codon and 3' epitope tag within the PR frame. (H) Protein blot showing PR and GP levels in cells transfected with the construct of (G). (I) LDH and (J) MTT assays of transfected HEK293T cells. [Figure 14-2]Figures 14A and 14B are a series of images and graphs showing in vivo evidence for RAN translation and toxicity studies of the G2C4AS repeat. (A) Dot blots of C9(+) and C9(-) prefrontal cortex lysates probed with α-PA, α-PA-CT, α-PR, and α-PR-CT antibodies. (B) Immunoblots of C9(+) and C9(-) ALS prefrontal cortex lysates. (C) IHC detection of PA, PR, and GP protein aggregates in hippocampal neurons from C9(+) ALS patients, detected with α-PA, α-PA-CT, α-PR, α-PRCT, and α-GP antibodies. (D) IF staining of C9(+) hippocampal tissue using mouse α-GP (arrowheads) and rabbit α-GP-CT (arrowheads), where sense inclusions are positive for both antibodies (upper panel), and antisense inclusions are positive for GP repeat antibody only (lower panel). (E) IF staining of larger areas showing sense (S) and antisense (AS) staining. (F) Quantification of double (sense) and single (antisense) labeled aggregates. (G-J) RAN and PR toxicity studies. (G) G2C4 elongation construct (+ / -ATG-PR-3T) + / -ATG start codon and 3' epitope tag within the PR frame. (H) Protein blot showing PR and GP levels in cells transfected with the construct of (G). (I) LDH and (J) MTT assays of transfected HEK293T cells. [Figure 15A] Figures 15A and 15B are a series of images showing in vivo evidence for RAN translation of C9ORF72 in both antisense and sense directions. Cytoplasmic inclusions detected by IHC using antibodies against sense (α-GR, α-GR-CT, α-GA, α-GP-CT) and antisense (α-PA, α-PA-CT, α-PR, α-PR-CT) proteins, as well as α-GP that recognizes GP proteins produced in both sense and antisense directions. Aggregates were found in neurons of the Ammon's horn (CA) and dentate gyrus (DG) regions of the hippocampus, as well as the motor cortex (MC), in C9(+) ALS autopsy tissue. [Figure 15B]Figures 15A and 15B are a series of images showing in vivo evidence for RAN translation of C9ORF72 in both antisense and sense directions. Cytoplasmic inclusions detected by IHC using antibodies against sense (α-GR, α-GR-CT, α-GA, α-GP-CT) and antisense (α-PA, α-PA-CT, α-PR, α-PR-CT) proteins, as well as α-GP that recognizes GP proteins produced in both sense and antisense directions. Aggregates were found in neurons of the Ammon's horn (CA) and dentate gyrus (DG) regions of the hippocampus, as well as the motor cortex (MC), in C9(+) ALS autopsy tissue. [Figure 16-1] Figures 16A and 16B are a series of images of clustered RAN protein aggregates and RAN aggregates within motor neurons. IHCs showing cytoplasmic α-GP aggregates in the following locations: (A) within layer III of the motor cortex; (B) upper motor neurons within layer V of the motor cortex; (C) lower motor neurons in the spinal cord (LS.C); (D) Ammon's horn, CA, (E) and dentate gyrus, DG region of the hippocampus; (F and G) IHCs showing abundant PA and PR cytoplasmic inclusions within the prehippocampal subcolum (PrSub) from one patient. [Figure 16-2] Figures 16A and 16B are a series of images of clustered RAN protein aggregates and RAN aggregates within motor neurons. IHCs showing cytoplasmic α-GP aggregates in the following locations: (A) within layer III of the motor cortex; (B) upper motor neurons within layer V of the motor cortex; (C) lower motor neurons in the spinal cord (LS.C); (D) Ammon's horn, CA, (E) and dentate gyrus, DG region of the hippocampus; (F and G) IHCs showing abundant PA and PR cytoplasmic inclusions within the prehippocampal subcolum (PrSub) from one patient. [Figure 17] Figure 17 shows a series of images of clustered staining of RAN protein. (A) Low-magnification images of IHC staining using α-PA-CT show variations in staining intensity in regions I-IV (gray dots indicate positive staining), and the inset shows high-magnification images. (B) An example of aggregates from region I shows immunoactivity against all nine antibodies with similar staining for antibodies against repeats and specific C-terminal epitopes. [Figure 18]Figure 18 is a table summarizing the histopathological findings in C9ORF72-positive ALS / FTD cases and controls. [Figure 19-1] Figures 19A–19F show a series of images and datasets. (A) shows strand-specific RT-PCR detection of sense (S) and antisense (AS) transcripts (across intron 1) of PBL from C9(+) patients and normal controls. (B) is a summary of 5'RACE products. (C) shows FISH staining of the frontal cortex from a C9(+) case, illustrating an example of cytoplasmic RNA aggregation. (D) shows FISH staining of peripheral blood leukocytes, showing accumulation of antisense (AS) G2C4 and sense (S) G4C2 RNA aggregation in C9(+) cells, but not in C9(-) cells. (E) shows an antisense aggregation specificity assay, demonstrating that excessive amounts of unlabeled (G4C2)4 oligo inhibit the labeling of G4C2-Cy3 antisense (AS) but not G2C4-Cy3 labeled sense aggregation. (F) shows an additional control for antisense RNA aggregation exhibiting the expected DNase I resistance and RNase I sensitivity. [Figure 19-2] Figures 19A–19F show a series of images and datasets. (A) shows strand-specific RT-PCR detection of sense (S) and antisense (AS) transcripts (across intron 1) of PBL from C9(+) patients and normal controls. (B) is a summary of 5'RACE products. (C) shows FISH staining of the frontal cortex from a C9(+) case, illustrating an example of cytoplasmic RNA aggregation. (D) shows FISH staining of peripheral blood leukocytes, showing accumulation of antisense (AS) G2C4 and sense (S) G4C2 RNA aggregation in C9(+) cells, but not in C9(-) cells. (E) shows an antisense aggregation specificity assay, demonstrating that excessive amounts of unlabeled (G4C2)4 oligo inhibit the labeling of G4C2-Cy3 antisense (AS) but not G2C4-Cy3 labeled sense aggregation. (F) shows an additional control for antisense RNA aggregation exhibiting the expected DNase I resistance and RNase I sensitivity. [Figure 19-3]Figures 19A–19F show a series of images and datasets. (A) shows strand-specific RT-PCR detection of sense (S) and antisense (AS) transcripts (across intron 1) of PBL from C9(+) patients and normal controls. (B) is a summary of 5'RACE products. (C) shows FISH staining of the frontal cortex from a C9(+) case, illustrating an example of cytoplasmic RNA aggregation. (D) shows FISH staining of peripheral blood leukocytes, showing accumulation of antisense (AS) G2C4 and sense (S) G4C2 RNA aggregation in C9(+) cells, but not in C9(-) cells. (E) shows an antisense aggregation specificity assay, demonstrating that excessive amounts of unlabeled (G4C2)4 oligo inhibit the labeling of G4C2-Cy3 antisense (AS) but not G2C4-Cy3 labeled sense aggregation. (F) shows an additional control for antisense RNA aggregation exhibiting the expected DNase I resistance and RNase I sensitivity. [Figure 20] Figure 20 shows a series of images of in vitro evidence for RAN translation of sense GGGGCC repeat extension. (A) shows a construct containing varying GGGGCC repeat lengths with an upstream 6X Stop cassette and a 3' tag in each reading frame. Immunoblot (B) and / or immunofluorescence staining (C) show that RAN translation occurs in all three frames (GP, GR, GA) in cells transfected with constructs containing 30, 60, and 120 repeats. [Figure 21] Figure 21 is a schematic diagram of the sense and antisense direction putative protein products for all reading frame sequence numbers 57–62, from top to bottom. Underlined sequences were used to generate polyclonal antibodies. * = stop codon. [Figure 22-1]Figures 22A–22E are a series of images showing the validation of dual antibodies for detecting putative polyPA, polyPR, and polyGP proteins by immunofluorescence and protein blotting. (A–D top): Schematic diagrams of constructs expressing ATG-start N-terminal epitope-tagged (V5 or Flag) repeat proteins, with or without endogenous C-terminal sequences. (A–D bottom panel), co-localization of α-Flag or α-V5 staining in transfected HEK293T cells by staining with the following novel antibodies: (A) α-PA or α-PA-CT (antisense), (B) α-PR or α-PR-CT, (C) rabbit α-GP or α-GP-CT (sense), (D) mouse α-GP. Similar staining was not observed in pre-immuno or pcDNA3.1 blank vector controls. (E) Corresponding immunoblots showing that 6 of the 7 antibodies tested also detect recombinant proteins by Western blotting. [Figure 22-2] Figures 22A–22E are a series of images showing the validation of dual antibodies for detecting putative polyPA, polyPR, and polyGP proteins by immunofluorescence and protein blotting. (A–D top): Schematic diagrams of constructs expressing ATG-start N-terminal epitope-tagged (V5 or Flag) repeat proteins, with or without endogenous C-terminal sequences. (A–D bottom panel), co-localization of α-Flag or α-V5 staining in transfected HEK293T cells by staining with the following novel antibodies: (A) α-PA or α-PA-CT (antisense), (B) α-PR or α-PR-CT, (C) rabbit α-GP or α-GP-CT (sense), (D) mouse α-GP. Similar staining was not observed in pre-immuno or pcDNA3.1 blank vector controls. (E) Corresponding immunoblots showing that 6 of the 7 antibodies tested also detect recombinant proteins by Western blotting. [Figure 23-1]Figures 23A–23C are a series of images showing validation of additional sense repeats and C-terminal polyclonal antibodies. (A, B top): Schematic diagrams of constructs expressing ATG-start N-terminal V5 epitope-tagged GR or GA repeat proteins with endogenous C-terminal sequences. (A, B bottom panels), co-localization of α-V5 staining in transfected HEK293T cells using α-GR, α-GR-CT, and α-GP-CT, respectively. Similar staining was not observed in pre-immuno or pcDNA3.1 empty vector controls. (C) Detection of recombinant protein in Flag-GR transfected cells by protein blotting. [Figure 23-2] Figures 23A–23C are a series of images showing validation of additional sense repeats and C-terminal polyclonal antibodies. (A, B top): Schematic diagrams of constructs expressing ATG-start N-terminal V5 epitope-tagged GR or GA repeat proteins with endogenous C-terminal sequences. (A, B bottom panels), co-localization of α-V5 staining in transfected HEK293T cells using α-GR, α-GR-CT, and α-GP-CT, respectively. Similar staining was not observed in pre-immuno or pcDNA3.1 empty vector controls. (C) Detection of recombinant protein in Flag-GR transfected cells by protein blotting. [Figure 24] Figure 24 shows a series of immunoblot images of 2% soluble lysates from the frontal cortex of C9(+) and C9(-) ALS, using α-GP-CT, α-GR, α-GR-CT, and α-GA antibodies. [Figure 25] Figure 25 shows a series of images illustrating negative IHC staining of a C9(-)ALS / FTD hippocampal section using antibodies against sense and antisense proteins. [Figure 26-1]Figures 26A–26D are graphs and a series of images illustrating the effects of RAN translation and PR protein expression on cell viability. (A) qRT-PCR shows similar expression of elongated transcripts in HEK293T cells transfected with (-)ATG-PR-3T and (+)ATG-PR-3T constructs. (B–D) Bright-field microscopy images showing changes in cell morphology of cells expressing RNA and RAN protein from the (-)ATG-PR-3T construct compared to an empty vector control (pcDNA3.1), as well as adverse effects in (+)ATG-PR-3T cells expressing increased levels of PR protein. [Figure 26-2] Figures 26A–26D are graphs and a series of images illustrating the effects of RAN translation and PR protein expression on cell viability. (A) qRT-PCR shows similar expression of elongated transcripts in HEK293T cells transfected with (-)ATG-PR-3T and (+)ATG-PR-3T constructs. (B–D) Bright-field microscopy images showing changes in cell morphology of cells expressing RNA and RAN protein from the (-)ATG-PR-3T construct compared to an empty vector control (pcDNA3.1), as well as adverse effects in (+)ATG-PR-3T cells expressing increased levels of PR protein. [Figure 26-3] Figures 26A–26D are graphs and a series of images illustrating the effects of RAN translation and PR protein expression on cell viability. (A) qRT-PCR shows similar expression of elongated transcripts in HEK293T cells transfected with (-)ATG-PR-3T and (+)ATG-PR-3T constructs. (B–D) Bright-field microscopy images showing changes in cell morphology of cells expressing RNA and RAN protein from the (-)ATG-PR-3T construct compared to an empty vector control (pcDNA3.1), as well as adverse effects in (+)ATG-PR-3T cells expressing increased levels of PR protein. [Figure 27]Figure 27 is a table listing the primers used for RT-PCR and RACE (the 17 sequence numbers are sequence numbers 36, 37, 39, 38, 45-47, 40, 48-56 in order). [Figure 28] Figure 28 is a table listing the novel sense and antisense antibodies (in order, SEQ ID NOs: 20, 23, 19, 25, 21, 21, 22, 18). [Figure 29] Figure 29 is a schematic diagram of the BAC insert used to produce transgenic mice. [Figure 30] Figure 30 is a series of photographs showing sense RNA aggregation in transgenic mice expressing the human C9ORF72 gene containing the GGGGCC repeat. Exemplary aggregations are indicated by arrowheads. [Figure 31] Figure 31 is a series of photographs showing antisense (AS)RNA aggregation in transgenic mice expressing the human C9ORF72 gene containing the GGGGCC repeat. Exemplary aggregations are indicated by arrowheads. [Modes for carrying out the invention]

[0018] Well-established translation initiation rules have been used as a foundation in molecular biology to understand gene expression and predict the consequences of disease-causing mutations. Generally, microsatellite elongation mutations (e.g., CAG, CTG) located in predicted coding and non-coding regions have been thought to cause disease through protein gain-or-loss or RNA gain-of-function mechanisms. Standard translation rules do not apply to CTG-CAG repeat elongation, and it has been previously reported that CAG and CUG elongation transcripts express homopolymer elongation proteins within all three frames without an AUG start codon (see, e.g., T. Zu et al., Non-ATG-initiated translation directed by microsatellite expansions. PNAS 108, 260 (2011)). This translation, independent of the AUG start codon, is called repeat-associated non-ATG (RAN) translation. RAN translation is hairpin-dependent and occurs without frameshift or RNA editing. RAN translation has been observed from trinucleotides, tetranucleotides, and pentanucleotide repeats associated with myotonic dystrophy type 1, myotonic dystrophy type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 8, and Huntington's disease (see PCT Publication WO / 2010 / 115033, which is incorporated herein by reference).

[0019] Elongation of the GGGGCC hexanucleotide repeat within the intron of the C9ORF72 gene has long been associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. As described herein, this elongated hexanucleotide repeat has been found to be contained within RNA transcripts expressed from the C9ORF72 locus in both sense and antisense directions. These hexanucleotide repeat-containing transcripts underwent RAN translation, resulting in poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), or poly(Pro-Arg) proteins, depending on the frame of the hexanucleotide repeat read from the RNA (5'-GGGGCC-3', 5'-GGGCCG-3', and 5'-GGCCGG-3' on sense transcripts, and 5'-GGCCCC-3', 5'-GCCCCG-3', and 5'-CCCCGG-3' on antisense transcripts; see Figure 1). In addition, antisense transcripts were found to be translated via ATG-initiated translation, yielding Met...poly(Pro-Arg) and Met...poly(Gly-Pro) proteins. These RAN and ATG-initiated proteins are referred to herein as diamino acid repeat-containing proteins. Sense and antisense hexanucleotide repeat-containing transcripts are referred to herein as 5'-GGGGCC-3' hexanucleotide repeat-containing RNA (sense) and 5'-GGCCCC-3' hexanucleotide repeat-containing RNA (antisense).

[0020] As further described herein, these diamino acid repeat-containing proteins were unexpectedly found to be present in blood samples from subjects with ALS. In addition, the expression of antisense 5'-GGCCCC-3' hexanucleotide repeat-containing RNA transcripts was found to be significantly increased in subjects with the C9ORF72 gene containing the elongated GGGGCC hexanucleotide repeat sequence. Furthermore, aggregation of both sense and antisense hexanucleotide repeat elongation-containing RNA transcripts was found in subjects with the C9ORF72 gene containing the elongated GGGGCC hexanucleotide repeat sequence. While we do not wish to be bound by theory or mechanism, it is thought that diamino acid repeat-containing proteins in the blood of subjects with ALS accumulate in the brain parenchyma over time, leading to neuroinflammatory changes, CNS dysfunction, and neuronal cell death. Accordingly, aspects of the present disclosure relate to the identification of subjects having or being likely to develop ALS by providing novel assays for determining the level of diamino acid repeat-containing protein in the subject's blood and / or the level of hexanucleotide repeat-containing RNA in a sample from the subject. Aspects of the present disclosure also relate to the treatment of subjects having ALS or FTD by reducing or stabilizing the level of diamino acid repeat-containing protein in the subject's blood.

[0021] Identification of individuals who have ALS or FTD, or who are highly likely to develop ALS or FTD. Aspects of this disclosure relate to the identification of subjects who have ALS or FTD or are likely to develop ALS or FTD, based on the level of one or more diamino acid repeat-containing proteins in a blood sample from a subject. In some embodiments, the method involves determining the level of one or more diamino acid repeat-containing proteins selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins in a blood sample obtained from a subject, wherein an elevated level of one or more diamino acid repeat-containing proteins compared to a control level indicates that the subject has ALS or FTD or is likely to develop ALS or FTD. In some embodiments, the level of one or more diamino acid repeat-containing proteins is determined by performing an assay. Non-limiting assays are described herein.

[0022] Other aspects of this disclosure relate to the identification of subjects who have or are likely to develop ALS or FTD based on levels of 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat-containing RNA in a sample from a subject. In some embodiments, the identification of subjects who have or are likely to develop ALS or FTD is based on levels of 5'-GGCCCC-3' hexanucleotide repeat-containing RNA in a sample from a subject. The sample may be, for example, a fluid or tissue sample obtained from the subject. In some embodiments, the method comprises determining levels of 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat-containing RNA in a blood sample obtained from a subject, wherein elevated levels of hexanucleotide repeat-containing RNA compared to a control level indicate that the subject has or is likely to develop ALS or FTD. In some embodiments, the level of hexanucleotide repeat-containing RNA is determined by performing an assay. Non-limiting assays are described herein.

[0023] Another aspect of this disclosure relates to the identification of subjects who have or are likely to develop ALS or FTD based on the presence or absence of RNA aggregates containing 5'-GGGGCC-3' hexanucleotide repeat elongation RNA and / or 5'-GGCCCC-3' hexanucleotide repeat elongation RNA in a sample obtained from a subject, wherein the presence of aggregates of 5'-GGGGCC-3' hexanucleotide repeat elongation RNA and / or 5'-GGCCCC-3' hexanucleotide repeat elongation RNA indicates that the subject has or is likely to develop ALS or FTD. As used herein, aggregation of 5'-GGGGCC-3' hexanucleotide repeat extension-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat extension-containing RNA refers to the area of ​​accumulation of 5'-GGGGCC-3' hexanucleotide repeat extension-containing RNA and / or 5'-GGCCCC-3' hexanucleotide repeat extension-containing RNA, which may be detectable using nucleic acid-based assays, such as FISH. In some embodiments, the aggregation may be, for example, 0.1 to 2 micrometers in diameter, 0.1 to 1.5 micrometers in diameter, or 0.1 to 1 micrometer in diameter. In some embodiments, the aggregation may be at least 0.1 micrometers in diameter. It should be acknowledged that a sample may contain two or more aggregations, and that each aggregation may be of a different size. For example, one aggregation may be 0.2 micrometers in diameter, while a second aggregation may be 1 micrometer in diameter. Non-limiting examples of aggregations and methods for detecting such aggregations are provided in Example 3.

[0024] It should be understood that subjects may be identified based on the level of one or more diamino acid repeat-containing proteins, the level of hexanucleotide repeat-extension-containing RNA, the presence or absence of hexanucleotide repeat-extension-containing RNA, or any combination thereof. In some embodiments, the method further includes identifying a subject as having ALS or FTD or being likely to develop ALS or FTD if the level of diamino acid repeat-containing protein or hexanucleotide repeat-containing RNA is elevated compared to control levels. In some embodiments, the method further includes identifying a subject as having ALS or FTD or being likely to develop ALS or FTD if aggregation (focus or foci) of hexanucleotide repeat-extension-containing RNA is present in the sample. In some embodiments, the method further includes identifying a subject as not having ALS or FTD or being unlikely to develop ALS or FTD if the level of diamino acid repeat-containing protein or hexanucleotide repeat-containing RNA is decreased or the same compared to control levels. In some embodiments, the method further includes identifying a subject as not having ALS or FTD, or being unlikely to develop ALS or FTD, if aggregation of hexanucleotide repeat elongation-containing RNA is not present in the sample.

[0025] In some embodiments, the level or identity of one or more diamino acid repeat-containing proteins of interest may be recorded. In some embodiments, the recording includes entering the level or identity of interest into a computer, for example, a medical record database.

[0026] Other aspects of this disclosure relate to treatments for subjects identified as having ALS or FTD, or as being highly likely to develop ALS or FTD. As used herein, “treatment” or “treatment” means (a) preventing or delaying the onset of ALS or FTD; (b) reducing the severity of ALS or FTD; (c) reducing or preventing the onset of symptoms characteristic of ALS or FTD; (d) preventing the exacerbation of ALS or FTD symptoms; and / or (e) reducing or preventing the recurrence of ALS or FTD symptoms in subjects who have previously shown symptoms of ALS or FTD.

[0027] In some embodiments, the treatment includes administering an effective amount of a known ALS therapeutic agent, such as riluzole (Rilutek, Sanofi-Aventis), to a subject identified as having ALS. In some embodiments, the treatment includes administering an effective amount of a known FTD therapeutic agent, such as trazodone (Desyrel, Oleptro), or a selective serotonin reuptake inhibitor (SSRI), to a subject identified as having FTD. In some embodiments, the treatment includes administering an effective amount of a therapeutic agent that reduces one or more symptoms of ALS or FTD in a subject identified as having ALS or FTD, such as baclofen, diazepam, phenytoin, trihexyphenidyl, and / or amitriptyline. In some embodiments, the treatment includes one or more of physiotherapy, occupational therapy, or speech therapy. In some embodiments, the treatment includes the methods described herein for reducing or stabilizing diamino acid repeat-containing protein levels in the subject's blood, such as bone marrow transplantation or plasmapheresis. In some embodiments, the treatment includes any combination of the treatments described above or any other treatment described herein.

[0028] The effective dose is the amount of therapeutic agent sufficient to deliver the medically desired outcome, such as treatment for ALS or FTD. The effective dose will vary with factors within the scope of the healthcare professional's knowledge and expertise, including the age and health status of the patient being treated, the severity of ALS or FTD in the patient, the duration of treatment, the nature of any concomitant therapies, and the specific route of administration.

[0029] The administration of the treatment may be achieved by any method known in the art (see, for example, Harrison's Principle of Internal Medicine, McGraw Hill Inc.). Administration may be topical or systemic. Administration may be parenteral (e.g., intravenous, subcutaneous, or intradermal) or oral. Compositions for various routes of administration are well known in the art (see, for example, Remington's Pharmaceutical Sciences by EW Martin). The dose will depend on the subject and route of administration. The dose may be determined by those skilled in the art.

[0030] Other aspects of this disclosure relate to methods for monitoring response to treatment in subjects having or suspected of having ALS or FTD. In some embodiments, the method involves determining a first level of one or more diamino acid repeat-containing proteins selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins in a blood sample obtained from the subject at a first time point, and determining the level of poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg) in a blood sample obtained from the subject at a second time point. g) The method comprises determining a second level of one or more diamino acid repeat-containing proteins selected from poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins, wherein an elevated or equal second level compared to a first level indicates that the subject is unresponsive to the treatment or is likely to be unresponsive, and a decreased second level compared to a first level indicates that the subject is responsive to the treatment or is likely to be responsive. In some embodiments, the first blood sample is obtained before treatment of the subject, and the second blood sample is obtained during or after treatment of the subject. The method may also be carried out by determining, in addition to or instead of, the level of hexanucleotide repeat-containing RNA or the presence or absence of aggregation of hexanucleotide repeat-extension-containing RNA.

[0031] As used herein, “elevated” means that the level of one or more diamino acid repeat-containing proteins or hexanucleotide repeat-containing RNAs exceeds a control level, for example, a predetermined threshold or level of one or more diamino acid repeat-containing proteins or hexanucleotide repeat-containing RNAs in a control sample. The control and control level are described in detail herein. Elevated levels include, for example, levels exceeding the control level by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more. The elevated level also includes increasing the phenomenon from a zero state (e.g., no or undetectable diamino acid repeat-containing protein expression or hexanucleotide repeat-containing RNA expression) to a non-zero state (e.g., some diamino acid repeat-containing protein expression or hexanucleotide repeat-containing RNA is present or detectable).

[0032] As used herein, “reduced” means that the level of one or more diamino acid repeat-containing proteins or hexanucleotide repeat-containing RNAs is below a control level, for example, a predetermined threshold or level of one or more diamino acid repeat-containing proteins or hexanucleotide repeat-containing RNAs in a control sample. The control and control level are described in detail herein. Reduced levels include, for example, levels that are 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more below the control level. The reduced level also includes reducing the phenomenon from a non-zero state (e.g., some level of diamino acid repeat-containing protein expression or hexanucleotide repeat-containing RNA is present or detectable) to a zero state (e.g., diamino acid repeat-containing protein expression or hexanucleotide repeat-containing RNA expression is absent or undetectable).

[0033] Hexanucleotide repeat-containing RNA and diamino acid repeat-containing proteins As described herein, it was found that the extended GGGGCC hexanucleotide repeat sequence within the intron of the C9ORF72 gene is transcribed in such a way that it yields RNA transcripts containing hexanucleotide repeats in both sense and antisense directions. The GenBank Gene ID for the human C9ORF72 gene is 203228. Both sense and antisense hexanucleotide repeat-containing transcripts underwent translation independent of the AUG start codon [repeat-associated non-ATG (RAN) translation], resulting in poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), or poly(Pro-Arg) diamino acid repeat-containing proteins, depending on the frame of the hexanucleotide repeat read (5'-GGGGCC-3', 5'-GGGCCG-3', and 5'-GGCCGG-3' on sense transcripts, and 5'-GGCCCC-3', 5'-GCCCCG-3', and 5'-CCCCGG-3' on antisense transcripts; see Figure 1). In addition, antisense hexanucleotide repeat-containing transcripts were found to be translated via ATG-initiated translation, yielding Met...poly(Pro-Arg) and Met...poly(Gly-Pro) proteins.

[0034] Accordingly, aspects of the present invention relate to sense and antisense RNAs containing extended hexanucleotide repeats and their uses. The sense RNA is RNA containing a 5'-GGGGCC-3' hexanucleotide repeat, and the antisense RNA is RNA containing a 5'-GGCCCC-3' hexanucleotide repeat.

[0035] RNAs containing 5'-GGGGCC-3' and 5'GGCCCC-3' hexanucleotide repeats are given the formula (GGGGCC), respectively. x or (GGCCCC) xThe hexanucleotide repeat RNA contains a repeat nucleic acid sequence, where X is at least 10, at least 20, at least 25, or at least 30, or a range selected from 10-100,000, 10-50,000, 10-5,000, 20-1,000, 20-100,000, 20-50,000, 20-5,000, 20-1,000, 25-100,000, 25-50,000, 25-5,000, or 25-1,000. The hexanucleotide repeat-containing RNA may further contain additional N and / or C-terminal nucleic acids. In some embodiments, the N-terminal nucleic acid sequence includes a nucleic acid sequence upstream of the 5'-GGGGCC-3' hexanucleotide repeat in the intron of C9ORF72 for the sense transcript, or a nucleotide sequence upstream of the 5'-GGCCCC-3' hexanucleotide repeat in the intron of C9ORF72 for the antisense transcript. In some embodiments, the C-terminal nucleic acid sequence includes a nucleotide sequence downstream of the 5'-GGGGCC-3' hexanucleotide repeat in the intron of C9ORF72 for the sense transcript, or a nucleotide sequence downstream of the 5'-GGCCCC-3' hexanucleotide repeat in the intron of C9ORF72 for the antisense transcript.

[0036] Another aspect of the present invention relates to one or more diamino acid repeat-containing proteins and their uses. The one or more diamino acid repeat-containing proteins are selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins.

[0037] Both sense 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and antisense 5'-GGCCCC-3' hexanucleotide repeat-containing RNA encode poly(Gly-Pro) proteins. Therefore, poly(Gly-Pro) proteins may include proteins translated from the sense strand, the antisense strand, or both. The C-terminus of sense and antisense translated poly(Gly-Pro) proteins is expected to be diverse (see Table 1). Therefore, sense poly(Gly-Pro) proteins may include the poly(Gly-Pro) C-terminal sequences listed in Table 1, while antisense poly(Gly-Pro) proteins may include repeat regions without additional C-terminal sequences. The methods described herein may include the use of poly(Gly-Pro) proteins translated from the sense strand, the antisense strand, or both. The antibodies described herein may be specific to poly(Gly-Pro) proteins translated from the sense strand, the antisense strand, or both.

[0038] Each diamino acid repeat-containing protein contains a repeat amino acid sequence, which is given by formula (YZ) x The formula contains diamino acid repeat units, where X can be 2-10,000, 5-10,000, 2-5,000, 5-5,000, 2-1,000, 5-1,000, 5-500, 5-300, 5-200, 10-500, 10-300, or 10-200. The diamino acid repeat units for each diamino acid repeat-containing protein are provided in Table 1.

[0039] [Table 1] X = Number of repetitions of the array in parentheses

[0040] Each diamino acid repeat-containing protein may further include an N and / or C-terminal amino acid sequence containing a non-diamino acid repeat sequence. In some embodiments, the N-terminal amino acid sequence includes an amino acid sequence translated from the nucleotide sequence of the C9ORF72 RNA transcript, for example, the nucleotide sequence upstream of the 5'-GGGGCC-3' hexanucleotide repeat in the intron of C9ORF72 for the sense transcript, or the nucleotide sequence upstream of the 5'-GGCCCC-3' hexanucleotide repeat in the intron of C9ORF72 for the antisense transcript. In some embodiments, the C-terminal amino acid sequence includes an amino acid sequence translated from the nucleotide sequence of the C9ORF72 RNA transcript, for example, the nucleotide sequence downstream of the 5'-GGGGCC-3' hexanucleotide repeat in the intron of C9ORF72 for the sense transcript, or the nucleotide sequence downstream of the 5'-GGCCCC-3' hexanucleotide repeat in the intron of C9ORF72 for the antisense transcript. Such nucleotide sequences downstream of a 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat may be translated until one or more stop codons are reached.

[0041] A portion of the C9ORF72 gene sequence (sense and antisense) is shown below. The 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat is underlined and in bold. The nucleotide sequence upstream of the 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat is before the underlined and in bold sequence. The nucleotide sequence downstream of the 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat is after the underlined and in bold sequence. It should be understood that this 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat can be repeated more times than the sequence shown.

[0042] C9ORF72 (partial array, sense) [ka]

[0043] C9ORF72 (partial array, antisense) [ka]

[0044] In some embodiments, the Met...poly(Pro-Arg) or Met...poly(Gly-Pro) protein comprises an N-terminal amino acid sequence containing an N-terminal methionine. In some embodiments, the Met...poly(Pro-Arg) protein comprises an N-terminal amino acid sequence or fragment thereof containing MQAIPPVARGESPTPSFGQRNERESKNASSSEESPRFYPRLFPAAEPQTATRQDAASSLTHSPPPAPPPPRAQAPQPQPRPGPAPGPAPTT (SEQ ID NO: 41), which is located on the N-terminal side of a poly(Pro-Arg) repeat amino acid sequence. In some embodiments, the Met...Poly(Gly-Pro) protein comprises an N-terminal amino acid sequence or fragment thereof, including MRGKVKMRRALRRAPASTRASSRQPNPKQPPARMPPPHSPTRHRLRLRRRGRRHRNRSPAPGPPPGPPRPRP (SEQ ID NO: 42), MRRALRRAPASTRASSRQPNPKQPPARMPPPHSPTRHRLRLRRRGRRHRNRSPAPGPPPGPPRPRP (SEQ ID NO: 43), and MPPPHSPTRHRLRLRRRGRRHRNRSPAPGPPPGPPRPRPRP (SEQ ID NO: 44), which is located at the N-terminus of a poly(Gly-Pro) repeat amino acid sequence.

[0045] In some embodiments, the C-terminal amino acid sequence includes the C-terminal amino acid sequences shown in Table 1 or fragments of the C-terminal amino acid sequences shown in Table 1. It should be understood that C-terminal amino acid sequences other than those in Table 1 are also considered.

[0046] Exemplary diamino acid repeat-containing proteins may include sequences provided in Table 2.

[0047] [Table 2] X = 2 to 10,000, 5 to 10,000, 2 to 5,000, 5 to 5,000, 2 to 1,000, 5 to 1,000, 5 to 500, 5 to 300, 5 to 200, 10 to 500, 10 to 300, or any number between 10 and 200.

[0048] In some embodiments, one or more diamino acid repeat-containing proteins are selected from poly(Pro-Ala), poly(Gly-Pro), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins.

[0049] In some embodiments, the one or more diamino acid repeat-containing proteins are proteins containing two or more, three or more, four or more, five or more, six or more, seven or more, or eight diamino acid repeats.

[0050] subject Aspects of this disclosure relate to the identification and treatment of subjects, such as humans, who have or are highly likely to develop ALS or FTD. In some embodiments, subjects may have ALS. In some embodiments, subjects may have one or more symptoms of ALS, such as dyspnea, dysphagia, muscle spasms, muscle contractions, muscle weakness, paralysis, speech impairment, or weight loss. In some embodiments, subjects may not have any symptoms of ALS. In some embodiments, subjects may have a family history of ALS.

[0051] In some embodiments, the subject may have frontotemporal dementia (FTD). In some embodiments, the subject may have one or more symptoms of FTD, such as lethargy, loss of spontaneity, disinhibition, lack of empathy and other social skills, apathy, progressive non-fluent aphasia, semantic dementia, bulimia, obsessive-compulsive behavior, tremor, rigidity, muscle spasms, coordination disorder, dysphagia, and muscle weakness. In some embodiments, the subject may not have any symptoms of FTD. In some embodiments, the subject may have a family history of FTD.

[0052] In some embodiments, the subject may have a GGGGCC hexanucleotide repeat in one or both alleles of the C9ORF72 gene (NCBI Entrez Gene ID: 203228). In some embodiments, the GGGGCC hexanucleotide repeat is located within the promoter and / or intron of the C9ORF72 gene. In some embodiments, the number of GGGGCC hexanucleotide repeats is 25, 50, 100, 150, 200, 250, 300, 500, 5,000, or 10,000 or more. The number of repeats can be detected using any assay known in the art, for example, nucleic acid-based assays, such as Southern blotting [see, for example, Dejesus-Hernandez et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245 (2011), Renton et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257 (2011), and Gijselink et al. A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: A gene identification study. Lancet Neurol. 11, 54 (2011)].

[0053] Control and control level Aspects of this disclosure relate to the comparison of levels of one or more diamino acid repeat-containing proteins and / or hexanucleotide repeat-containing RNAs with a control level. In some embodiments, the control level is the level of one or more diamino acid repeat-containing proteins and / or hexanucleotide repeat-containing RNAs in a healthy subject or a sample obtained from a population of healthy subjects, such as a fluid sample or a tissue sample. In some embodiments, the sample is a blood sample. A healthy subject as used herein is a subject that appears disease-free and has no history of diseases such as ALS or FTD. In some embodiments, a healthy subject is a subject having 25 or fewer GGGGCC hexanucleotide repeats in the C9ORF72 gene.

[0054] In some embodiments, the control level is the level of one or more diamino acid repeat-containing proteins and / or hexanucleotide repeat-containing RNAs that are undetectable or below the resulting background / noise level using a standard detection method (e.g., Western blotting, qPCR, Northern blotting, or immunohistochemistry). Such a level can be obtained, for example, by measuring the level of one or more diamino acid repeat-containing proteins and / or hexanucleotide repeat-containing RNAs in a sample known to be free of diamino acid repeat-containing proteins and / or hexanucleotide repeat-containing RNAs.

[0055] This disclosure also involves comparing the levels of one or more diamino acid repeat-containing proteins and / or hexanucleotide repeat-containing RNAs to a predetermined level or value, without requiring the measurement of a control level each time. The predetermined level or value can take various forms. It may be a single cutoff value, e.g., median or mean. It may be established based on a comparison group, for example, when one defined group is known to be free of ALS or FTD and another defined group is known to be free of ALS or FTD. It may be a range, for example, when the test population is divided equally (or unequally) into multiple groups, e.g., subjects with 25 or fewer GGGGCC hexanucleotide repeats, subjects with 25 to 50 GGGGCC hexanucleotide repeats, and subjects with 50 or more GGGGCC hexanucleotide repeats.

[0056] sample Aspects of this disclosure relate to determining the levels of one or more diamino acid repeat-containing proteins in a blood sample (e.g., whole blood, plasma, or serum) obtained from a subject. The blood sample is obtained by any method known in the art, for example, using a needle or finger lancet. The blood may be treated before use in the methods described herein. Such treatments include the addition of anticoagulants, the removal of blood cells, and / or the freezing of the blood. However, it should be acknowledged that other samples, such as tissue samples (e.g., brain tissue) or other fluid samples, such as saliva or urine, may be used.

[0057] Other aspects of this disclosure relate to determining the level of hexanucleotide repeat-containing RNA in a sample obtained from a subject. The sample may be a fluid or a tissue sample. In some embodiments, the tissue sample is brain tissue. In some embodiments, the fluid sample is blood (e.g., whole blood, plasma, or serum), saliva, or urine. In some embodiments, the fluid sample is a blood sample (e.g., whole blood, plasma, or serum).

[0058] Assay Aspects of this disclosure relate to performing assays to determine the level or presence / absence of one or more diamino acid repeat-containing proteins and / or hexanucleotide repeat-containing RNAs. Assays known in the art for detecting proteins and RNAs (see, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001, Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York.; microarray technology is described in Microarray Methods and Protocols, R. Matson, CRC Press, 2009, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York) can be used alone or in combination with the methods and compositions for measuring diamino acid repeat-containing protein levels described herein.

[0059] Assays for detecting protein levels include, but are not limited to, immunoassays [also known herein as immune-based or immuno-based assays, e.g., Western blotting, immunohistochemistry, and ELISA assays], mass spectrometry, and multiplex bead-based assays. Such assays for detecting protein levels are well known in the art. Other examples of protein detection and quantification methods include, for example, the multiplex immunoassay described in U.S. Patent Nos. 6,939720 and 8,148171, and published U.S. Patent Application No. 2008 / 0255766, as well as the protein microarray described in, for example, published U.S. Patent Application No. 2009 / 0088329, all of which are incorporated herein by reference.

[0060] Any suitable binding partner for diamino acid repeat-containing proteins is considered for the detection of diamino acid repeat-containing protein levels. In some embodiments, the binding partner is any molecule that specifically binds to the diamino acid repeat-containing proteins described herein. “Specifically binds to diamino acid repeat-containing proteins” as used herein means that the molecule is more likely to bind to some or all of the diamino acid repeat-containing proteins than to some or all of the non-diamino acid repeat-containing proteins.

[0061] In some embodiments, the binding partner is an antibody or its antigen-binding fragment, e.g., Fab, F(ab)2, Fv, single-chain antibody, Fab and sFab fragments, F(ab')2, Fd fragment, scFv, or dAb fragment. Methods for producing antibodies and their antigen-binding fragments are well known in the art [see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Cold Spring Harbor Laboratory Press (1989), Lewin, "Genes IV", Oxford University Press, New York, (1990), and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), WO2006 / 040153, WO2006 / 122786, and WO2003 / 002609]. The binding partner also includes other peptide molecules and aptamers that specifically bind to diamino acid repeat-containing proteins. Methods for producing peptide molecules and aptamers are well known in the art (see, for example, U.S. Patent Application No. 2009 / 0075834, U.S. Patents No. 7435542, No. 7807351, and No. 7239742). Binding partners may include, but are not limited to, fluorescent, enzymatic, affinity, or isotopic labels.

[0062] In some embodiments, the assay comprises an immunobased assay. In some embodiments, the immunobased assay comprises an isolated antibody specific to one or more diamino acid repeat-containing proteins. In some embodiments, the isolated antibody specific to one or more diamino acid repeat-containing proteins is an isolated antibody described in further detail herein. In some embodiments, the isolated antibody specific to one or more diamino acid repeat-containing proteins is an isolated antibody specific to an antigen or sequence, or a fragment of an antigen or sequence, as listed in Table 1, Table 2, or Table 3.

[0063] Therefore, a diamino acid repeat-containing binding partner (for example, a diamino acid repeat-containing specific antibody) can be labeled with a detectable component.

[0064] Assays for detecting RNA include, but are not limited to, hybridization-based assays, such as Northern blot analysis, RT-PCR, sequencing techniques, RNA in situ hybridization (e.g., using DNA or RNA probes that hybridize with RNA molecules present in the sample, as in FISH), in situ RT-PCR (e.g., as described in Nuovo GJ, et al. Am J Surg Pathol. 1993, 17: 683-90; Komminoth P, et al. Pathol Res Pract. 1994, 190: 1017-25), and oligonucleotide microarrays [e.g., hybridization with oligonucleotides bound to a solid surface (e.g., a glass wafer) having addressable locations of polynucleotide sequences derived from a sample, such as Affymetrix microarrays [Affymetrix®, Santa Clara, CA]]. Methods for designing nucleic acid binding partners, such as probes, are well known in the art. In some embodiments, the nucleic acid binding partner binds to part or all of the nucleic acid sequence of the hexanucleotide repeat-containing RNA provided herein.

[0065] treatment As described herein, diamino acid repeat-containing proteins have been found to be present in blood samples from patients with ALS. While we do not wish to be bound by theory or mechanism, it is thought that diamino acid repeat-containing proteins in the blood of subjects with ALS accumulate in the brain parenchyma over time, leading to neuroinflammatory changes, CNS dysfunction, and neuronal cell death. Accordingly, aspects of this disclosure relate to the treatment of subjects with ALS or FTD by reducing or stabilizing the levels of diamino acid repeat-containing proteins in the blood of the subjects.

[0066] In some embodiments, reducing or preventing an increase in the level of one or more diamino acid repeat-containing proteins involves removing one or more (e.g., one, two, three, four, five, six, seven, or eight) diamino acid repeat-containing proteins from the subject's blood. In some embodiments, one or more diamino acid repeat-containing proteins from the subject's blood are removed using a procedure selected from plasmapheresis or bone marrow transplantation. In some embodiments, reducing or preventing an increase in the level of all diamino acid repeat-containing proteins expressed by the subject may be advantageous. Therefore, in some embodiments, the method involves reducing or preventing an increase in the level of all forms of diamino acid repeat-containing proteins expressed by the subject.

[0067] In some embodiments, the presence of one or more diamino acid repeats from the subject's blood is removed using hematopoietic stem cell (HSC) transplantation. HSC transplantation is the transplantation of hematopoietic stem cells, typically derived from bone marrow, peripheral blood, or umbilical cord blood, into the subject. The source of the hematopoietic stem cells may be allogeneic (for example, from a donor such as a healthy subject). The method of HSC transplantation is well known in this field (see, for example, Bishop MR, Pavletic SZ. Hematopoietic stem cell transplantation. In: Abeloff MD, Armitage JO, Niederhuber JE, Kastan MB, McKena WG, eds. Clinical Oncology. 4th ed. Philadelphia, Pa: Elsevier Churchill Livingstone; 2008: chap 32, and Vose JM, Pavletic SZ. Hematopoietic stem cell transplantation. In: Goldman L, Schafer AI. Cecil Medicine. 24th ed. Philadelphia, Pa: Saunders Elsevier; 2011: chap 181).

[0068] To prepare for transplantation of target HSCs, HSCs present in the target may be removed or depleted, resulting in transplanted cells that may constitute a dominant HSC population in the target. To induce apoptosis or cell cycle arrest in the target HSC cells, the HSCs in the target may be depleted by treating the target, for example, with chemotherapy, irradiation, or both.

[0069] In allogeneic HSC transplantation, HSCs are obtained from a donor. The donor is preferably a healthy subject, for example, a subject that appears disease-free and has no history of diseases such as ALS or FTD. The donor is preferably HLA compatible with the recipient subject to reduce the risk of graft-versus-host disease. HLA compatibility can be determined, for example, using HLA typing. HLA typing generally involves testing for at least eight HLA markers, namely two A, two B, two C, and two DRB1 markers, as well as optionally two DQ markers. HLA typing can be achieved, for example, through a blood test. HLA allele identity can be determined using serology or nucleic acid-based assays. Generally, matching of four to six markers between the host and donor is preferred. In some embodiments, the donor is a subject having 25 or fewer GGGGCC hexanucleotide repeats in the C9ORF72 gene.

[0070] HSCs are obtained from a donor using any method known in the art. Exemplary methods include bone marrow harvesting and leukapheresis [see, for example, Transfusion. 2003 Feb;43(2):259-64. Leukapheresis after high-dose chemotherapy and autologous peripheral blood progenitor cell transplantation: a novel approach to harvest a second autograft. Schwella N, Braun A, Ahrens N, Rick O, Salama A]. In bone marrow harvesting, bone marrow is typically taken from the posterior aspect of one or both hip bones of the donor. Leukapheresis involves the separation of HSCs from the blood obtained from the donor, for example, using continuous flow centrifugation or filtration. Growth factor G-CSF may be administered to the donor to stimulate the proliferation of new HSCs, resulting in a greater number of HSCs in the blood. Once the HSCs are obtained, the allogeneic HSCs are then administered to the recipient. Any preferred method of administration known in the art, such as by central venous catheter, is considered.

[0071] In some embodiments, during or after HSC transplantation, subjects receiving HSC transplantation may receive additional treatments and / or therapies, such as antibiotics, antifungals, antivirals, blood transfusions, and / or immunosuppressive therapy. Such treatments and / or therapies may help prevent infection and / or graft-versus-host disease during the HSC transplantation recovery period.

[0072] In some embodiments, the HSC transplant is a bone marrow transplant. In some embodiments, the bone marrow transplant is an allogeneic bone marrow transplant.

[0073] Plasmapheresis is an extracorporeal medical procedure ("extracorporeal procedure") that involves the removal, treatment, and return of plasma (or its components) from the blood circulation. Plasmapheresis is well known in the art and has been used to treat several diseases, including Goodpasture syndrome, myasthenia gravis, Guillain-Barré syndrome, lupus, and thrombotic thrombocytopenic purpura (see, for example, Madore, Plasmapheresis Technical aspects and indications, Crit Care Clin 18: 375-392. 2002). During plasmapheresis, blood is first removed from the body, for example, through a needle or a previously implanted catheter. The plasma is then separated from the blood cells, for example, by using a blood component separator. After plasma separation, the blood cells are combined with replacement fluid and re-administered to the subject. The replacement fluid may be either separated plasma or replacement plasma (also called plasma exchange) that has been treated to remove disease-related components.

[0074] Exemplary procedures used to separate plasma from blood cells include: 1) Discontinuous flow centrifugation: A single intravenous catheter line is used. Typically, one or more batches of blood are removed at a time and centrifuged to separate the plasma from the blood cells. The blood cells are then combined with replacement fluid and returned to the subject. 2) Continuous flow centrifugation: Two intravenous lines are used. Plasma is continuously removed from the blood, and the separated blood cells are supplied with fluids through a connected line before being returned to the subject. 3) Plasma filtration: Two intravenous lines are used. Plasma is filtered using standard hemodialysis equipment, such as parallel plate or hollow fiber filters. The separated blood cells are supplied through a line coupled with replacement fluid before being returned to the subject. The filters typically have pores with a diameter of 0.2–0.6 μm, sufficient to allow plasma to pass through while retaining cells. Several membrane plasma separators are commercially available (e.g., Plasmaflo from Asahi Medical Co., Ltd., Tokyo, Japan; Plasmax from Toray Industries, Tokyo, Japan; CPS-10 from Baxter, Deerfield, IL, USA; Plasmaflux from Fresenius Medical Care AG, Bad Homburg, Germany; Prisma TPE 2000 from Hospal, Lyon, France).

[0075] When isolated plasma is used as replacement fluid, the isolated plasma is first treated to reduce the level of diamino acid repeat-containing proteins present in the isolated plasma. In some embodiments, reducing the level of diamino acid repeat-containing proteins present in the isolated plasma involves contacting the isolated plasma with one or more isolated antibodies specific to diamino acid repeat-containing proteins, as described herein, so that the diamino acid repeat-containing proteins present in the isolated plasma bind to the one or more isolated antibodies. In some embodiments, a binding partner for one or more isolated antibodies is contacted with the isolated plasma. The binding partner for one or more isolated antibodies may be, for example, a capture component, such as biotin or streptavidin, protein A, or a secondary antibody specific to one or more isolated antibodies. Such a binding partner allows one or more isolated antibodies to be removed from the isolated plasma.

[0076] In some embodiments, one or more isolated antibodies are bound to a filter, column, and / or solid support. In such embodiments, the isolated plasma is brought into contact with the filter, column, and / or solid support, thereby causing diamino acid repeat-containing proteins to bind to the isolated antibodies bound to the filter, column, and / or solid support.

[0077] While we do not wish to be bound by theory, it is thought that diamino acid repeat-containing proteins can form aggregates in the blood. Therefore, diamino acid repeat-containing proteins may be removed from the separated plasma using a filter so that the aggregates are isolated from the separated plasma.

[0078] In some embodiments, subjects expressing one or more diamino acid repeat-containing proteins may form autoantibodies. In some embodiments, autoantibodies against one or more diamino acid repeat-containing proteins may be removed from isolated plasma. The autoantibodies may be removed using any method known in the art, for example, a binding partner that recognizes the autoantibodies (e.g., bound to a solid support or a tag). In some embodiments, the binding partner may be one or more diamino acid repeat-containing proteins described herein.

[0079] When plasma exchange is used, the subject receives substitute plasma. The substitute plasma may be, for example, donor plasma or albumin solution (e.g., 5-70% albumin in saline). An exemplary substitute plasma is 5% albumin combined with 0.9% saline in a 50%:50% (vol:vol) solution. Drugs that maintain blood non-clotting (e.g., anticoagulants, e.g., citrate, dextrose citrate, or heparin) may be administered to the subject or brought into contact with the subject's blood during the procedure.

[0080] In some embodiments, reducing or preventing an increase in the level of one or more diamino acid repeat-containing proteins includes reducing the level of hexanucleotide repeat-containing RNA. Reducing the level of hexanucleotide repeat-containing RNA may include administering an effective amount of inhibitory nucleic acid molecules, such as shRNA, siRNA, miRNA, or antisense nucleic acid molecules, that target the hexanucleotide repeat-containing RNA.

[0081] Methods for producing shRNA, siRNA, miRNA, and antisense nucleic acid molecules are well known in the art [see, for example, Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, (Current Edition), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al. eds., (Current Edition)), Oligonucleotide Synthesis (N. Gait, ed., Current Edition), Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition), and Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition)]. In some embodiments, the nucleic acid repressor includes or is equivalent to at least a portion of the target hexanucleotide repeat-containing RNA sequence, or includes at least a portion of a sequence complementary to the target hexanucleotide repeat-containing RNA sequence.

[0082] In some embodiments, the treatment may include reducing or stabilizing the level of autoantibodies against one or more diamino acid repeat-containing proteins in a subject. The level of autoantibodies may be reduced or stabilized using any method known in the art. In some embodiments, reducing or stabilizing the level of autoantibodies includes administering an effective amount of atacicept, belimumab, bricibimod, BR3-Fc, rituximab, ocrelizumab, atumumab, epratuzumab, corticosteroids (e.g., prednisone), mycophenolic acid, methotrexate, cyclophosphamide, azathioprine, and / or cyclosporine. In some embodiments, reducing or stabilizing the level of autoantibodies includes plasmapheresis.

[0083] antibody Aspects of this disclosure relate to isolation antibodies specific to diamino acid repeat-containing proteins (e.g., RAN proteins) selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins. The isolation antibodies may recognize a region of the diamino acid repeat-containing protein (e.g., a repeat sequence or C-terminus) or the entire diamino acid repeat-containing protein.

[0084] An antibody that "specifically binds" to a target or epitope is a term understood in the art, and methods for determining such specific binding are also known in the art. A molecule is said to exhibit "specific binding" if it reacts or binds to a particular target antigen more frequently, rapidly, for a longer time, and / or with greater affinity than to alternative targets. An antibody "specifically binds" to a target antigen if it binds to it with greater affinity, binding strength, more readily, and / or for a longer period than it binds to other substances. For example, an antibody that specifically binds to a poly(Gly-Ala) protein or an epitope thereof is an antibody that binds to this target antigen with greater affinity, binding strength, more readily, and / or for a longer period than it binds to other antigens or other epitopes of the same antigen. By reading this definition, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically bind to a second target antigen. As such, "specific binding" does not necessarily require (although it may include) exclusive binding. Although not always, generally, a reference to binding means specific binding. In some embodiments, the antibodies described herein have a suitable binding affinity for a dipeptide repeat-containing protein (e.g., a RAN protein). As used herein, "binding affinity" refers to the apparent binding constant or K A . K A is the reciprocal of the dissociation constant (K D ). The antibodies described herein may have a binding affinity (K D ) of at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or less. An increased binding affinity corresponds to a decreased K D . A higher affinity binding of an antibody to a first target compared to a second target is a K A for binding to the second target (or numerical value K D ) higher than the K A for binding to the first target (or a smaller numerical value K DThis may be indicated by the following: In such cases, the antibody has specificity for the first target (e.g., the protein or its mimicry in the first conformation) compared to the second target (e.g., the same protein or its mimicry in the second conformation, or the second protein). The difference in binding affinity (e.g., with respect to specificity or other comparisons) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 It could be double.

[0085] Binding affinity can be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays). Exemplary conditions for evaluating binding affinity are, for example, in Tris buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2 at pH 7.5). These techniques can be used to measure the concentration of bound protein as a function of the target protein concentration. The concentration of bound protein ([Bound]) is related to the concentration of free target protein ([Free]) and the concentration of binding sites on the target for the binding protein, where (N) is the number of binding sites per target molecule by the following equation: [Bound]=[N][Free] / (Kd+[Free])

[0086] However, K A It is not always necessary to determine this precisely, because K A This is because it may be sufficient to obtain quantitative measurements of affinity determined by methods such as ELISA or FACS analysis, which are proportional to the affinity and can therefore be used for comparisons such as determining whether a higher affinity is twice as high; to obtain qualitative measurements of affinity; or to obtain an estimate of affinity by functional assays, such as activity in in vitro or in vivo assays.

[0087] In some embodiments, the isolated antibody is specific to one or more diamino acid repeat-containing proteins selected from poly(Pro-Ala), poly(Pro-Arg), Met...poly(Pro-Arg), or Met...poly(Gly-Pro) proteins.

[0088] In some embodiments, the isolated antibody is specific to an antigen containing the diamino acid repeats and / or C-terminal sequences or fragments thereof as defined in Table 1. In some embodiments, the isolated antibody is specific to an antigen containing the sequences or fragments thereof as defined in Table 2.

[0089] In some embodiments, the isolated antibody is specific to the antigen in Table 3 or Figure 28. In some embodiments, the antigen in Table 3 does not contain N and / or C-terminal modifications.

[0090] [Table 3] F1 = Reading Frame 1, F2 = Reading Frame 2, F3 = Reading Frame 3, AS F1 = Antisense Reading Frame 1, AS F2 = Antisense Reading Frame 2, AS F3 = Antisense Reading Frame 3

[0091] The isolated antibodies may be monoclonal or polyclonal antibodies, or their antigen-binding fragments. These antigen-binding fragments include, for example, Fab, F(ab)2, F(ab')2, Fv, single-chain antibodies, Fab fragments, sFab fragments, Fd fragments, scFv, or dAb fragments. Methods for producing polyclonal and monoclonal antibodies and their antigen-binding fragments are well known in the art [see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Cold Spring Harbor Laboratory Press (1989), Lewin, "Genes IV", Oxford University Press, New York, (1990), and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), WO2006 / 040153, WO2006 / 122786, and WO2003 / 002609]. Also included are antibodies produced by recombinant means, such as chimeric antibodies (variable and constant regions derived from different species) and CDR-grafted antibodies (complementarity-determining regions derived from different species), which are described in U.S. Patents 4,816,567 and 5,225,539, and these documents are incorporated herein by reference in their entirety. Also included are humanized antibodies, typically produced by recombinant methods, in which the human sequence is part or all of the antibody. Also included are fully human antibodies, such as those produced in genetically modified mice (see PCT application number 93 / 12227, which is incorporated herein by reference in its entirety).

[0092] In some embodiments, the isolated antibodies specific to diamino acid repeat-containing proteins are the rabbit polyclonal antibodies listed in Table 4.

[0093] [Table 4]

[0094] Antibodies can be produced in bacterial cells, such as Escherichia coli, or eukaryotic cells, such as yeast cells or mammalian cells. In one embodiment, the antibody is produced in mammalian cells. Mammalian host cells for expressing the antibody or its antigen-binding fragment include Chinese hamster ovary (CHO cells) (e.g., including dhfr-CHO cells as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR-selectable marker as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601 621), lymphocyte cell lines, such as NS0 myeloma cells and SP2 cells, COS cells, and cells from transgenic animals, such as transgenic mammals. Antibodies can also be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal.

[0095] The isolated antibodies of this disclosure may also have a detectable label conjugated thereto. The label may be, for example, fluorescent, enzyme, affinity, or isotope labeling. Examples include fluorescein isothiocyanate (FITC) for detection by fluorescence, horseradish peroxidase enabling detection by cleavage of a chromogenic substrate, and radioisotopes, such as I for detection by autoradiography. 125 This also includes avidin / biotin for antibody detection and affinity purification of antigens and antigen-retaining cells.

[0096] Also included by this disclosure are hybridoma cell lines for producing monoclonal antibodies specific to diamino acid repeat-containing proteins selected from poly(Gly-Ala), poly(Gly-Pro), poly(Gly-Arg), poly(Pro-Ala), poly(Pro-Arg) proteins, Met...poly(Pro-Arg), Met...poly(Gly-Pro), the C-terminal peptides of the diamino acid repeat-containing proteins described herein, and / or combinations of two or more thereof.

[0097] In some embodiments, the isolated antibody is an isolated autoantibody obtained from a subject having ALS, and the isolated autoantibody is specific to one or more diamino acid repeat-containing proteins described herein.

[0098] In some embodiments, the isolated antibodies described herein are contained in a buffer. In some embodiments, the isolated antibodies described herein are bound to a solid support (e.g., the surface of a plate or beads).

[0099] transgenic mouse In another embodiment, the disclosure relates to a transgenic mouse comprising a human C9ORF72 gene comprising a GGGGCC hexanucleotide repeat sequence. In some embodiments, the mouse comprises a human C9ORF72 gene comprising the GGGGCC hexanucleotide repeat sequence and flanking human sequences on the 5' and 3' ends of the human C9ORF72 gene. In some embodiments, the flanking human sequences on the 5' and 3' ends are independently at least 1 kilobase (kB), at least 5 kB, at least 10 kB, at least 20 kB, at least 30 kB, at least 40 kB, or at least 50 kB in length. In some embodiments, the flanking human sequences on the 5' and 3' ends each independently comprise a promoter capable of driving the transcription of the human C9ORF72 gene in the sense and antisense directions, respectively. Therefore, in some embodiments, transgenic mice express both sense and antisense transcripts (e.g., 5'-GGGGCC-3' and 5'GGCCCC-3' hexanucleotide repeat-containing RNAs as described herein). In some embodiments, the human C9ORF72 gene and flanking sequence include the following sequence: (GGGGCC) n This indicates the position of the GGGGCC hexanucleotide repeat sequence.

[0100] Chr9: 27,527,137~27,625,470 (inverse complementarity) [ka] [ka] [ka] [ka] [ka] [ka]

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[0101] In some embodiments, the human C9ORF72 gene and flanking sequences include, for example, sequences that are at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequences above. As used herein, “percent sequence identity” or “percent identity” or “% identity” is the identity fraction multiplied by 100. The “identity fraction” of a sequence optimally aligned with a reference sequence is the number of nucleotide matches in the optimal alignment divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in the entire length of the reference sequence.

[0102] In some embodiments, the GGGGCC hexanucleotide repeat [for example, (GGGGCC) in SEQ ID NO: 63] n The number of ] is 300-800, 300-700, 400-600, or 500-600. In some embodiments, the GGGGCC hexanucleotide repeat [for example, (GGGGCC) in SEQ ID NO: 63] n The number of ] is 500-600. In some embodiments, the GGGGCC hexanucleotide repeat [for example, (GGGGCC) in SEQ ID NO: 63] n The number of ] is 300, 400, 500, 600, 700, or 800. In some embodiments, the GGGGCC hexanucleotide repeat [for example, (GGGGCC) in SEQ ID NO: 63] n The number of [ ] exceeds 500. In some embodiments, the transgenic mouse is an FVB, balb-C, or C57B / 6 strain mouse. In some embodiments, the transgenic mouse is an FVB strain mouse. In some embodiments, the mouse can be used to screen for treatment methods for ALS or FTD, such as the treatment methods described herein or candidate therapeutic agents.

[0103] The transgenic mice described herein can be produced using any method known in the art or described herein, for example, in Example 4 (see also, for example, PCT publication numbers WO2001010199 and WO2013022715, US publication numbers US20110113496 and 20060031954, each of which is incorporated herein by reference). For example, the transgenic mice described herein can be produced by introducing a transgene (for example, the human C9ORF72 gene, which optionally has a flanking sequence) into the germline of a mouse. Embryonic target cells at various developmental stages can be used to introduce the transgene. Different methods are used depending on the developmental stage of the embryonic target cells. Any particular strain of animal used to carry out this disclosure is generally selected for good health, good embryo production, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, haplotype is an important factor. For example, when transgenic mice are produced, strains such as C57BL / 6 or FVB strains are often used (Jackson Laboratory, Bar Harbor, Me.). The strain may be transgenic itself and / or a knockout (e.g., obtained from an animal having one or more genes that are partially or completely repressed). The transgene construct may be introduced into a single-stage embryo. A zygote is a preferred target for microinjection. The use of a zygote as a target for gene transfer has a significant advantage in that, in most cases, the injected DNA is integrated into the host gene before the first cleavage [Brinster et al. (1985) PNAS 82:4438-4442]. Typically, the fertilized embryo is incubated in a suitable medium until the pronucleus appears. By this time, the nucleotide sequence containing the transgene has been introduced into a female or male pronucleus, as described below. In some species, such as mice, a male pronucleus is preferred. It is most preferable that the exogenous genetic material is added to the male DNA complement of the zygote before it is processed by the egg nucleus or the zygote female pronucleus.The egg nucleus or female pronucleus is thought to release molecules that affect the male DNA complement, possibly by replacing protamines in the male DNA with histones, thereby promoting the binding of the female and male DNA complements and forming a diploid zygote. Therefore, exogenous genetic material should be attached to the male complement of DNA or any other complement of DNA before it is affected by the female pronucleus. For example, exogenous genetic material should be attached to the early male pronucleus as soon as possible after its formation, when the male and female pronuclei are sufficiently separated and both are located close to the cell membrane.

[0104] Alternatively, exogenous genetic material may be added to sperm nuclei after they have been induced to undergo decondensation. The sperm containing the exogenous genetic material may then be added to the egg, or the decondensed sperm may be added to the egg, and the transgene construct may then be added as soon as possible. Any method enabling the addition of exogenous genetic material to the nuclear genetic material is available, provided that it does not disrupt cells, nuclear membranes, or other existing cellular or genetic structures. The introduction of the transgene nucleotide sequence into the embryo may be achieved by any means known in the art, such as microinjection, electroporation, or lipofection. The exogenous genetic material is preferably inserted into the nuclear genetic material by microinjection. Microinjection of cells and cellular structures is known and used in the art. In mice, the male pronucleus reaches a size of approximately 20 micrometers in diameter, which allows for the reproducible injection of 1–2 pl of DNA solution. After introducing the introduced gene nucleotide sequence into the embryo, the embryo may be incubated in vitro for various durations, retransplanted into a surrogate host, or both. In vitro incubation until maturation is within the scope of this invention. One common method involves incubating the embryos in vitro for approximately 1 to 7 days, depending on the species, and then retransplanting them into a surrogate host.

[0105] Transgenic offspring of a surrogate host may be screened for the presence and / or expression of the transgene by any preferred method. Screening is often achieved by Southern blot or Northern blot analysis using probes complementary to at least a portion of the transgene. Western blot analysis using antibodies against the protein encoded by the transgene may be used as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue and analyzed for the transgene by Southern blot analysis or PCR. Alternatively, any tissue or cell type may be used for this analysis, although tissues or cells that are expected to express the transgene at the highest levels may be tested for the presence and expression of the transgene using Southern blot analysis or PCR.

[0106] Alternative or additional methods for assessing the presence of transgenes include, but are not limited to, suitable biochemical assays, such as enzyme and / or immunological assays, tissue staining for specific markers or enzyme activity, and flow cytometry analysis. Blood analysis may also be useful for detecting the presence of transgene products in the blood and for assessing the effects of transgenes on the levels of various types of blood cells and other blood components.

[0107] Progeny of transgenic animals may be obtained by mating transgenic animals with suitable partners, or by in vitro fertilization of eggs and / or sperm obtained from transgenic animals. If mating with a partner is performed, the partner may be transgenic and / or knockout, and if transgenic, it may contain the same or different transgenes, or both. Alternatively, the partner may be a parental line. If in vitro fertilization is used, the fertilized embryo may be transferred into a surrogate host, incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgenes using the methods described above or other suitable methods.

[0108] Aspects of this disclosure also relate to polynucleotides including SEQ ID NO: 63, such as bacterial artificial chromosome (BAC) vectors.

[0109] Without further detail, it is expected that those skilled in the art will utilize this disclosure to the fullest extent based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and not as limiting in any way other parts of this disclosure. All publications referenced herein are incorporated by reference for the purposes or subjects referenced herein. [Examples]

[0110] Cells were transfected with a construct containing a CMV promoter, an elongation motif (GGGGCC) containing one of the 4, 30, 60, or 120 repeats of GGGGCC, and an HA, FLAG, or MYC tag (Figure 2A). Western blotting showed that poly(GR) and poly(GP) proteins were generated in cells transfected with the construct containing the 30, 60, or 120 repeats of GGGGCC (Figure 2B). Immunofluorescence of cells further demonstrated that GP-flag, GR-HA, and GA-Myc proteins were expressed in cells transfected with the construct containing the 30, 60, or 120 repeats of GGGGCC (Figure 3). These results indicate that the GGGGCC repeat region can initiate translation independently of the AUG start codon (repeat-associated non-ATG (RAN) translation), and that poly(GP), poly(GR), and poly(GA) repeat proteins are generated.

[0111] Antibodies were generated against the C-terminus of poly(GR) sequences or poly(GR) repeat proteins. Fluorescence staining using these antibodies demonstrated their ability to detect poly(GR) repeat proteins (Figure 4).

[0112] Further antibodies against the C-terminuses of poly(GP) sequences and poly(GA) repeat proteins were generated. Next, anti-poly(GR), anti-poly(GP), and anti-poly(GA) C-terminal antibodies were used to stain cross-sections of brain tissue from patients or controls with C9ORF72 ALS (Figure 5).

[0113] Next, it was hypothesized that C9ORF72 transcripts could be generated in both sense and antisense directions (see Figure 1). It was further hypothesized that these antisense transcripts could also undergo RAN translation, generating further repeat proteins from the 5'-GGCCCC-3' repeat present in the antisense transcripts. As shown in Figure 6, both poly(PA) and poly(PR) proteins were detectable in brain tissue samples from patients with C9ORF72 ALS, but not in controls. These results indicate that diamino acid repeat-containing proteins, such as RAN proteins, are generated from both sense and antisense transcripts produced from the C9ORF72 locus.

[0114] Figure 7 shows that approximately 20% of the aggregates detected by the anti-GP antibody (GP) also co-localize with the antibody against the intrinsic C-terminus of the sense GP protein (GP-C). Consistent with the increased levels of antisense transcripts observed in affected brains, these co-localization data suggest that over 80% of GP dipeptide aggregates originate from the C9ORF72 antisense transcript.

[0115] In addition, antisense transcript levels were found to be dramatically elevated in subjects with ALS compared to controls (Figure 12). Primers for qPCR assays to detect antisense transcript levels are shown in the table below.

[0116] [Table 5]

[0117] Furthermore, diamino acid repeat-containing proteins were found to be present in the blood (including serum and plasma) and brain of subjects with ALS (Figures 9 and 10), but not in control subjects. [Examples]

[0118] According to several aspects of this disclosure, the accumulation of diamino acid repeat-containing proteins (e.g., RAN proteins) in the blood and cerebrospinal fluid (CSF) substantially contributes to C9ORF72 ALS / FTD, and plasmapheresis and bone marrow transplantation would halt disease progression. According to several aspects of this disclosure, the accumulation of diamino acid repeat-containing proteins in the blood and circulating CSF infiltrates the brain parenchyma, leading to protein accumulation, neuroinflammatory changes, CNS dysfunction, and neuronal cell death. Aspects of this disclosure are based in part on the following: Firstly, blood-brain barrier (BBB) ​​dysfunction is an early characteristic of the disease in ALS patients (4, 5), and a higher rate of ALS and other neurological diseases is found in patients with traumatic brain injury (6). In some embodiments, although we do not wish to be bound by theory, ALS is caused in part by BBB disruption, which allows immune cells and other harmful substances that promote ALS / FTD to enter the CNS. Secondly, as described herein, diamino acid repeat-containing proteins were found to accumulate in blood samples from ALS patients (Figures 8 and 9).

[0119] Although plasmapheresis and bone marrow transplantation have been tested as treatment strategies for ALS in the past, it is unclear which of these cases were C9ORF72 positive or whether the treatment was effective early enough. Therefore, in some embodiments, ALS treatment (e.g., plasmapheresis or BMT) is initiated when one or more diamino acid repeat-containing proteins are detected in the subject's blood at levels exceeding normal.

[0120] The data presented herein regarding the accumulation of diamino acid repeat-containing proteins in the tissues and blood of C9ORF72 ALS patients suggest that reducing the amount of diamino acid repeat-containing proteins in the blood (and possibly also in CSF) may be helpful in treating ALS in C9ORF72 ALS patients. According to some aspects of this disclosure, the reduction may be achieved, for example, using plasmapheresis or bone marrow transplantation.

[0121] method Detailed evaluations will be conducted on gene carriers from the C9ORF72 family (CNSA-1) and outpatients, including gene-positive patients with early signs of motor neuron disease, frontotemporal cognitive impairment, or both. Diamino acid repeat-containing protein expression will correlate with repeat length in CNSA family samples and additional samples collected in the hospital. Blood diamino acid repeat-containing protein expression will be determined in long-term collected samples and will correlate with disease onset and clinical severity. These methods are expected to characterize diamino acid repeat-containing protein expression in the C9ORF72-positive expanded study population, determine whether diamino acid repeat-containing protein expression occurs throughout life or increases with age, and determine whether diamino acid repeat-containing protein levels quantitatively correlate with disease severity.

[0122] Plasmaferesis is tested to determine whether lower levels of diamino acid repeat-containing proteins in the blood and CSF stop the signs of the disease. Plasmaferesis will be performed on five C9ORF72-positive individuals with early signs of the disease. Six plasmaferesis sessions will be performed over two weeks, each involving an exchange of 2 liters of normal human albumin, followed by weekly plasmaferesis sessions for the next six months. This study may be extended if necessary. The primary outcome measure is the Appel ALS Rating Scale (AALSRS). Clinical assessments, including neurological examinations, speech assessments, neuropsychological tests, the ALS Functional Rating Scale (ALSFRS), EMG, and needle muscle biopsy for immunohistopathological evaluation of the vastus lateralis muscle, will be performed immediately before and after the treatment period to assess disease progression. To assess serum concentrations of RAN and ATG translation products and CSF levels, venous and lumbar punctures should also be performed before and after the 6-month treatment period (or after the extension period, if applicable).

[0123] Bone marrow transplantation in animal models will be tested to determine whether BMT in the blood and brain prevents the accumulation of diamino acid repeat-containing proteins. In a first cohort of animals, bone marrow will be removed from RANT-positive mice and replaced with wild-type donor bone marrow to test whether the amount of protein aggregates in the brain will decrease. In a parallel set of experiments, RANT-negative animals will be transplanted with RANT-positive bone marrow to test whether CNS protein accumulation occurs in animals that express the transgene only in hematopoietic cells. Both groups of treated animals will be compared to wild-type and untreated RANT control animals using a combination of behavioral, functional, and neuropathological assessments.

[0124] RAN translation model mice were generated. Transgenic mice were generated using a construct containing six stop codons (two in each reading frame) immediately upstream of a CAG elongation mutation, followed by three distinct epitope tags in each reading frame (Figure 10). The CAG repeat generates a polyGln RAN protein, which has previously been associated with diseases in humans, such as fragile X syndrome. The RANT model mice yielded a polyGln RAN protein, which was found to localize to high levels beneath the pia mater surface in the brain exposed to cerebrospinal fluid (Figure 11). This RANT model mouse is used in the study summarized in Example 2. Therefore, the detection of polyamino acid repeat-containing proteins (e.g., mono- or diamino acid repeat-containing proteins) may be an indicator of the risk of brain damage associated with polyamino acid repeat-containing proteins. Thus, the methods described herein can be used to detect or treat other neurological diseases. [Examples]

[0125] Introduction The most common cause of familial FTD and ALS identified to date, the chromosome 9p21-associated morphology of ALS / FTD, is caused by an elongated GGGGCC (G4C2) hexanucleotide repeat in intron 1 of chromosome 9 open reading frame 72 (C90RF72) (1, 2). C9ORF72 mutations are found in 40% of familial ALS cases and 7% of sporadic ALS cases, as well as in 21% of familial FTD patients and 5% of sporadic FTD patients (3). The discovery of C9ORF72 elongation has been quite exciting because it links ALS and FTD to a large group of diseases caused by microsatellite elongation mutations (4).

[0126] Traditionally, microsatellite elongation mutations located in predicted coding and non-coding regions were thought to cause disease through protein gain-or-loss or RNA gain-of-function mechanisms (4). Protein loss-of-function has been proposed as the basis for C90RF72-induced ALS / FTD because the elongation mutation results in a decrease in the level of mutant 1 transcript and a potential decrease in C9ORF72 protein expression (1, 2). In addition, since the C9ORF72 G4C2 elongation mutation is located in an intron, several studies have pursued the hypothesis that C9-related ALS-FTD is a result of a toxic RNA gain-of-function mechanism in which G4C2 elongation RNA sequestrates important cellular factors in nuclear RNA condensation. Several G4C2 RNA-binding proteins have been identified, but to date, none of these candidates have been demonstrated to directly bind to endogenous elongation transcripts or co-localize with RNA condensation observed in patient cells or biopsy tissues (5-8).

[0127] In this mechanism, hairpin-forming microsatellite elongation transcripts express proteins in one or more reading frames without an AUG start codon (9). In recent years, various names have been attributed to these RAN-translating proteins (e.g., homopolymeric, dipeptide, RANT), but to avoid confusion, it is proposed that all proteins expressed through microsatellite elongation mutations in the absence of an ATG start codon be called RAN proteins, because additional elongation mutations that undergo RAN translation can be identified.

[0128] Here, we demonstrate that C9ORF72 ALS / FTD antisense transcripts containing GGCCCC(G2C4) elongation accumulate in the brains of patients as nuclear, and rarely cytoplasmic, aggregations. In addition, a novel group of antibodies against both repeat motifs and specific C-terminal regions were developed, demonstrating that both sense and antisense RAN proteins accumulate in CNS biopsy tissues from C9ORF72 patients. The discovery of antisense G2C4 RNA aggregation and three novel antisense RAN proteins in the brains of C9ORF72 patients suggests that bidirectional transcription and RAN translation are fundamental pathological features of C9ORF72 ALS / FTD.

[0129] result Antisense RNA aggregation in patients with C9ORF72 elongation A series of experiments were conducted to test the hypothesis that antisense (AS) C9ORF72 elongated transcripts form AS G2C4 RNA aggregates, leading to AS protein expression via RAN translation or from short AS open reading frames (AS-ORFs). First, the expression of the C9ORF72 antisense transcript was confirmed using a linkered strand-specific RT-PCR strategy to compare the expression of sense and antisense transcripts in intron 1b, 5' of the antisense G2C4 elongation, and exon 1a. For the antisense strand in intron 1b, strand-specific RT-PCR was performed using LK-ASORF-R primers for the RT reaction and ASORF-F and LK for PCR to specifically amplify the antisense cDNA (Figure 12A). A similar strategy was used to amplify the sense transcript from the same region of intron 1b, as well as the sense and antisense transcripts in exon 1a. Intron 1b antisense transcripts were detected by RT-PCR in the frontal cortex from C9(+) ALS / FTD patients but not from C9(-) ALS / FTD or normal controls (Figure 12B). qRT-PCR showed a dramatic increase in these transcripts among six C9(+) ALS / FTD cases (Figure 12C). In contrast, intron 1b sense transcripts were not detected by RT-PCR in the frontal cortex (Figure 12B). In blood, both intron 1b sense and antisense transcripts were detectable, and no dramatic increase in C9(+) was observed for intron 1b antisense transcripts. 5'RACE showed that intron 1b AS transcription initiated at various sites 251–455 base pairs (bp) upstream of the G2C4 repeat (Figures 12A, 19B). In contrast, 3'RACE using 3'GSP1 or 3'GSP2 primers located at 40 and 90 bp3' of the G2C4 repeat did not detect the transcript. These data indicate that the 3' end of the AS transcript does not overlap with the sense exon 1a region located at 170 bp3' of the antisense G2C4 repeat.Consistent with these results, sense transcripts were detected by strand-specific linker RT-PCR using a primer overlapping exon 1a, but antisense transcripts were not detected (Figure 12B). To determine whether antisense transcripts contain G2C4 repeat elongation, RNA fluorescence in situ hybridization (FISH) was performed using a Cy3-labeled (G4C2)4 probe to detect putative antisense G2C4 RNA condensation. The results showed that G2C4 RNA condensation in the nucleus (Figure 12D), and rarely in the cytoplasm (Figure 19C), accumulates in the C9(+) ALS prefrontal cortex, but not in the C9(-) ALS prefrontal cortex. The detection of cytoplasmic condensation indicates that antisense elongation transcripts can be found in the same intracellular compartment as the protein translation mechanism, where RAN translation likely occurs. Since RNA aggregation in peripheral tissues can provide a biomarker for disease, peripheral blood leukocytes (PBLs) were examined, and both sense and antisense RNA aggregation was detected in C9(+) PBLs but not in C9(-) PBLs (Figure 12D, Figure 19D). RNA-FISH signals from the Cy3-G4C2 probe that detects AS aggregation can compete with excessive unlabeled G4C2 oligonucleotides, and these aggregations were found to be resistant to DNase I and sensitive to RNase I digestion (Figure 19E, F). In summary, this indicates that C9ORF72 antisense transcripts are elevated in the prefrontal cortex in C9(+) ALS but not in C9(-) ALS or normal controls. It was also shown for the first time that antisense transcripts containing G2C4 elongation mutations are expressed and accumulate in nuclear, and rarely cytoplasmic, RNA aggregation in the C9(+) prefrontal cortex. In addition, sense and antisense aggregates were shown to accumulate in the blood and provide potential biomarkers for C9ORF72 ALS / FTD in easily accessible tissues.

[0130] RAN translation of GGCCCC repeat extension in vitro To test whether antisense G2C4 elongation receives RAN translation, a triple-tagged G2C4 minigene lacking the ATG start codon was created by inserting a 6X STOP codon cassette (two stops per frame) upstream of three different C-terminal epitope 8 tags to monitor 40 or 70 repeat G2C4 elongation and protein expression in all reading frames [for example, a G2C4EXP transcript translated in three frames yields Gly-Pro (GP), Pro-Ala (PA), and Pro-Arg (PR) RAN proteins]. EXP-3T was generated (Figure 13A). Immunoblotting detected two epitope-tagged RAN proteins, PR-Myc and GP-Flag, but not PAHA (Figure 13B). The (PR)40- and (PR)70-3xMyc proteins migrated to predicted sizes of approximately 20 and 27 kDa, respectively. In contrast, the (GP)40- and (GP)70-3xFlag proteins migrated substantially higher than their predicted sizes (10–15 kDa), to 50 and 75 kDa, respectively (Figure 13B). A faint low molecular weight band on this blot may be due to repeat contraction or differences in translation initiation sites observed during bacterial culture. Immunofluorescence (IF) showed that the antisense RAN protein was expressed in all three reading frames (Figure 13C). The detection of PA-HA by IF, which was not detected by Western blotting, may be due to the lower frequency of cells expressing RAN PA-HA from these constructs. In addition, recombinant GP-flag and PA-HA proteins were localized in the cytoplasm, while PR-Myc protein was distributed in both the nucleus and cytoplasm. These differences in localization may result from different characteristics of the repeat motif or C-terminal flanking sequence found in this epitope-tagged construct. A series of additional experiments also showed that sense G4C2 elongation constructs containing 30, 60, and 120 repeats expressed GP-Flag, GR-HA, and GA-Myc RAN proteins (Figure 20). In summary, these data demonstrate that recombinant G2C4 and G4C2 elongation transcripts express RAN proteins in all six reading frames.

[0131] A dual immunological strategy for detecting RAN proteins Because amino acid repeats can be found in a variety of different proteins, a dual immunological strategy was used to develop antibodies that recognize the predicted repeat motifs or their corresponding unique C-terminal regions as described herein. Schematic diagrams showing eight putative C9ORF72 RAN proteins are shown in Figures 13D and 21. The predicted proteins include six putative RAN proteins and two putative proteins with an additional ATG-start N-terminus. Unique C-terminal regions are predicted in five of the six predicted leading frames. To test the accumulation of these proteins in vivo, a series of polyclonal antibodies against the predicted repeat motifs or available corresponding C-terminal regions were developed (Figures 13D and 21). Antibodies were generated to test for putative antisense proteins [rabbit α-PA, α-PA-CT, α-PR, α-PR-CT, α-GP, α-GP-CT(sense), and mouse α-GP], and their specificity was demonstrated by Western blotting and IF detection in cells transfected with constructs expressing epitope-tagged recombinant proteins (Figure 13E, Figure 22). Additional antibodies detecting repeats and C-terminal regions expressed in the sense direction are characterized in Figure 23.

[0132] Antisense G2C4RAN protein accumulates in the brain. Multiple approaches were used to determine whether novel antisense (AS) proteins are expressed in C9ORF72 elongation-positive biopsy tissue. To overcome the difficulty of isolating aggregated proteins from human brain, a sequential protein extraction protocol (23) was used on frozen C9(+) and C9(-) ALS prefrontal cortex autopsy specimens. Antisense PA and PR proteins were detected in a subset of C9(+) ALS patients, but not in C9(-) ALS patients, using 1% Triton-X100 insoluble material, 2% SDS soluble extracts, and immunodot blots as α-PA, α-PA-CT, α-PR, and α-PR-CT (Figure 14A). Additional immunodot blots showing evidence for sense RAN protein (GP, GR, GA)10 accumulation in the C9(+) ALS / FTD prefrontal cortex are shown in Figure 24. α-PA, α-PR, and α-GP antibodies also detected high molecular weight smears in the 2% SDS-insoluble fraction from C9(+)ALS prefrontal cortex samples after resuspending pellets in sample buffer containing 8% SDS (23) (Figure 3B). Differences in migration patterns were observed for recombinant proteins migrating as one or more bands (Figure 13B), and the smears observed in patient tissue extracts (Figure 14B) reflect differences in RAN proteins due to much longer repeat regions in patient samples and their extracts from highly insoluble aggregates. Immunohistochemistry (IHC) was then used to show that protein aggregates are detectable in the perinuclear bodies of hippocampal neurons from C9(+)ALS / FTD autopsy tissue, but not in C9(-)ALS patients or controls, using antibodies against the repeat motif (α-PA, α-PR, α-GP) along with antibodies against the predicted C-terminal sequences beyond the PA and PR repeat regions (α-PA-CT and α-PR-CT) (Figure 14C, Figure 25). Previous studies using antibodies against the GP repeat motif detected aggregates presumed to be expressed from the sense strand (10, 11). It is noteworthy that GP repeat-containing proteins are predicted to be expressed from both sense and antisense transcripts (Figure 13D).In the sense direction, the predicted RAN GP protein contains a unique C-terminal (CT) sequence. In contrast, the antisense GP protein has a stop codon immediately following the repeat. To distinguish sense GP RAN proteins from antisense GP proteins, dual-labeled IF experiments were performed on C9(+) human hippocampal autopsy sections, using rabbit α-GP-CT to detect the CT region of sense GP proteins and mouse α-GP to detect both sense and antisense GP elongation proteins. Dual labeling revealed two types of inclusions: a) putative sense inclusions dual-labeled with mouse α-GP and rabbit α-GP-CT sense, and b) putative antisense inclusions single-labeled with mouse α-GP (Figure 14D). Approximately 18% of the inclusions showed a sense pattern with dual labeling, and 82% of the inclusions showed an antisense pattern, positive for α-GP and negative for α-GP-CT sense (Figures 14E, F). These data highlighted the importance of characterizing protein aggregates with both repeat and C-terminal antibodies. In summary, these results indicate that the insoluble, aggregate-forming antisense RAN protein is expressed from all three antisense reading frames.

[0133] G2C4 elongation and RAN proteins are toxic to cells. In addition to the antisense GP and PR RAN proteins expressed by RAN translation, two antisense reading frames have upstream ATG start codons that can result in both ATG start GP and PR proteins (M-GPAS and M-PRAS) (Figure 13D, Figure 21). It was shown that the presence of the ATG start codon does not inhibit RAN translation from occurring in all three reading frames (9). Therefore, antisense GP and PR proteins may be expressed by both AUG start and / or RAN translation. To explore the effect of the ATG start codon on RAN protein expression with respect to G2C4 elongation, an additional minigene construct was generated by placing the ATG start codon before the G2C4 repeat (Figure 14G). The PR frame was selected for analysis because the ATG start codon occurs naturally in this reading frame. Western blotting showed that HEK293T cells transfected with (+)ATG-PR-3T expressed substantially higher levels of PR protein compared to (-)ATG-PR-3T transfected cells (Figure 14H). In contrast, qRT-PCR and Western blotting showed comparable levels of transcript (Figure 26A) and RAN-translated GP (Figure 14H). Similar to Figure 13, RAN-translated PA was undetectable by Western blotting. Next, the effects of these constructs on cell viability were tested using complementarity assays, lactate dehydrogenase (LDH) detection, and methylthiazole tetrazolium (MTT). For the LDH assay, cells transfected with (-)ATG-PR-3T or (+)ATG-PR-3T constructs showed 1.9 and 2.9-fold increases in cell death compared to vector control cells (p=0.008 and 0.001). In addition, (+)ATG-PR-3T transfected cells expressing elevated levels of PR protein showed a 1.5-fold increase in cell death compared to cells transfected with the (-)ATG-PR-3T construct (p=0.034). The MTT assay showed similar results.Cells transfected with (-)ATG-PR-3T and +ATG-PR-3T constructs showed a dramatic decrease in the number of metabolically active cells, at 33% (p<0.00001) and 43% (p<0.00001), respectively, compared to untreated cells or blank vector controls (Figure 14J). In addition, elevated PR expression in cells transfected with (+)ATG-PR-3T was associated with significantly lower levels of metabolic activity compared to (-)ATG-PR-3T cells (p<0.05). Light microscopy cell isolation revealed clear morphological changes in (-ATG-PR-3T) cells compared to control cells, and these phenotypes were exacerbated in (+)ATG-PR-3T cells expressing elevated levels of PR (Figure 26B-D). In summary, these data demonstrate the following: In other words, 1) G2C4 elongation mutations are toxic to cells, and this toxicity can be caused by the effects of DNA, G2C4RNA, and / or RAN-translated PR, GP, or PA proteins; and 2) increased PR protein expressed in cells transfected with the (+)ATG-PR-3T construct increases cytotoxicity and cell death beyond the levels caused by the effects of DNA, G2C4RNA, and RAN proteins. Therefore, it has been shown that PR proteins are inherently toxic to cells.

[0134] All six RAN proteins form aggregates in the brain. To determine whether all six RAN proteins from both sense and antisense RNA strands are expressed in C9(+) ALS patients, IHC staining was performed on paraffin-embedded brain tissue sections using nine polyclonal antibodies against repeat extensions and / or C-terminal sequences of these proteins. In C9(+) cases, abundant spherical and irregularly shaped neuronal cytoplasmic inclusions (NCIs) were present in the hippocampus, the majority of which were located in pyramidal cells of the dentate gyrus and CA region. These RAN inclusions were also detected in the C9(+) motor cortex (Figure 15). Using α-GP, GP-positive inclusions were detected in the hippocampus, as in the motor cortex, in all examined C9(+) cases but not in C9(-) cases or normal control sections. Clusters of aggregates were frequently found in the CA region of the hippocampus and in the motor cortex, with aggregates in >20% of neurons in C9(+) cases (Figure 27). Fewer aggregates were detected using α-GP-CT sense antibody, which is consistent with dual-labeling experiments showing that most GP aggregates were translated from the C9ORF72 antisense strand (Figure 14D-F). PA inclusions were detected in the hippocampus in four of the six C9(+) cases and in one of the two motor cortex samples (Figure 27). In C9(+) cases, the frequency of PA inclusions was significantly lower in the hippocampus and motor cortex compared to GP inclusions, although high-intensity regional staining with extremely large PA inclusions found in >50% of neurons was found in one patient (Figure 27). PR-positive inclusions were also found in the hippocampus in all C9(+) cases examined and in the motor cortex in one of the two C9(+) cases tested. Similar to PA staining, PR inclusions were less frequent, but high-intensity regional staining was occasionally observed. In the sense direction, GR-positive inclusions were found in the hippocampus and motor cortex in all C9(+) cases examined, but appeared to be less frequent than GP aggregates. GA inclusions were only occasionally detected as small perinuclear inclusions in the hippocampus and motor cortex by IHC (Figures 15 and 27).The apparent differences in the frequency of various aggregate types may be due to differences in protein conformation and epitope availability, or differences in the affinity of these antibodies designed for different epitopes. In summary, these data showed that all six RAN proteins form aggregates in C9(+) autopsy brains.

[0135] RAN protein inclusions in upper and lower motor neurons A central feature of ALS is the gradual degeneration and death of upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. To test for the accumulation of RAN proteins in upper and lower motor neurons, IHC was performed using all nine antibodies against the predicted proteins in both sense and antisense directions. In C9(+) cases, abundant GP-positive neuronal cytoplasmic inclusions were observed in all layers of the motor cortex, with high frequency of GP aggregates in pyramidal neurons of layer III and throughout layer V (Figure 16A). GP inclusions were found in the remaining upper motor neurons, although cell death and atrophy made identification of motor neurons in layer V difficult (Figure 16B). In addition, PA, PR, GR, and GA-positive inclusions were also found in the motor cortex (Figures 15, 27). Using a similar series of experiments performed on spinal cord sections, GP aggregates were detected in all three cases examined, and aggregates in lower motor neurons in two of the three C9(+) patients, but not in C9(-) ALS cases or normal controls (Figure 16C). This is the first report of RAN protein accumulation in motor neurons. The discovery of GP aggregates in both upper and lower motor neurons links C9 RAN protein accumulation to neurons selectively vulnerable in ALS.

[0136] High-density clustering of RAN protein aggregates Both sense and antisense proteins accumulated in neurons of the C9ORF72 autopsy brain. Generally, two types of aggregation patterns were observed: 1) isolated cytoplasmic aggregates and 2) high-density clustered cytoplasmic aggregates in which approximately 10 to over 50% of neurons were positive. Clustered aggregates were most frequently detected for GP and were found in the dentate gyrus (DG) and CA1–4 of the hippocampus (Figure 16D, E). Clustered GP aggregates in the DG were smaller and less frequent than the larger cytoplasmic aggregates in the CA region. Additional clustered GP aggregates were found as frequently in the hippocampal and prehippocampal regions of the hippocampus as in the motor cortex. Immunostaining of serial sections showed that multiple proteins were often found in the same region. For example, high-intensity clustering staining for PA, PR, GP, GA, and GR proteins was found in the same region of the prehippocampal region in serial sections from one C9(+) patient (see Figure 16F, G). Immunostaining for PA showed that some brain regions had abundant aggregates, while other regions of the same section showed relatively little change. For example, Figure 17A shows the gradient of PA inclusions across the hippocampal region of a single section from one patient (anterior hippocampus > hippocampus > CA1). In this patient, PA inclusions were numerous in the anterior hippocampus (I) (>50% of neurons), moderate in the hippocampus (II), and rare in the CA1 hippocampal region (III and IV). Consistent with the localized regional staining seen in this section, PA staining was not detected in sections from separate blocks of hippocampal tissue taken from the same patient. These data suggest that PA RAN protein expression is variable by cell, or that aggregation of PA in one cell triggers aggregation in adjacent cells, as has been proposed in mouse models of Parkinson's disease (24). Next, serial sections from this C9(+) case were used to demonstrate that antibodies against both repeat motifs (α-PA, α-PR, α-GP, α-GR) and their corresponding C-terminal regions (α-PA-CT, α-PR-CT, α-GP-CT, α-GR-CT, α-GA-CT) detected aggregates in the same intensely stained area (region I) of the anterior hippocampus (Figure 17B).These results indicated that both sense and antisense RAN protein aggregates accumulate in this region. Detection of similar aggregates using antibodies that recognize either the repeat motif or a specific C-terminal region confirms that these antibodies recognize proteins expressed across both G2C4 and G4C2 elongated transcripts, providing a novel tool for understanding the biological effects of RAN translation in C9ORF72 ALS / FTD.

[0137] essay There has been considerable excitement regarding the discovery that intron microsatellite elongation mutations in C9ORF72 cause both common familial and sporadic forms of ALS / FTD (1, 2). Three major pathological mechanisms being investigated for this disease include haploinsufficiency (1, 2), RNA gain-of-function (5-8), and RAN translation (9, 11-13). To date, efforts to understand the molecular mechanisms of this disease have focused solely on understanding the consequences of sense-directed C9ORF72 elongation mutations. The results reported herein show that C9ORF72 elongation mutations are also expressed in the antisense direction, demonstrating that antisense RNA aggregation and antisense RAN proteins contribute to C9ORF72 ALS / FTD. We are the first to demonstrate the following: Specifically, 1) antisense C9ORF72 is elevated in C9(+) autopsy tissue, but sense transcripts are not; 2) antisense G2C4 elongation transcripts form RNA aggregates that accumulate in the C9+ brain and blood; 3) RAN translation occurs throughout the antisense G2G4 elongation construct in cell culture; 4) sense and antisense RAN proteins accumulate in C9(+) autopsy brain using a dual immunological approach with both repeat and C-terminal antibodies; 5) RAN protein aggregates accumulate in upper and lower motor neurons, directly linking RAN translation to a key pathological feature of ALS. Since the initial reports (1, 2) that G4C2 RNA aggregates accumulate in C9ORF72 ALS / FTD patient tissues, the main hypothesis has been that G4C2 sense transcripts sequester and dysregulate RNA-binding proteins, similar to the sequestering of MBNL proteins in DM1, DM2, and SCA8 (4). Several groups have already reported G4C2 binding proteins and are investigating their potential roles in the disease (5-8). The finding that antisense G2C4 aggregation also accumulates in patient cells suggests that G2C4 antisense RNA and binding proteins may play a role. In addition, the discovery of sense and antisense aggregation in C9(+) peripheral blood may be useful as an easily accessible biomarker for C9ORF72 ALS / FTD.Biomarkers that monitor both sense and antisense transcripts can be particularly important because treatments that reduce the expression of one chain may reduce the expression of the other chain. Using a dual immunological approach, we have shown that the G2C4 antisense transcript induces the expression of novel antisense proteins (PA, PR, GP) via RAN translation and / or from two short ORFs (Met-AS-PR and Met-AS-GP).

[0138] material and method cDNA construct containing 6×Stop codons upstream: CCCGGGGCC(GGGGCC)2GGGGCCC(SEQ ID NO: 64) and CCCGGGGCC(GGGGCC). 28 The GGGGCCC (SEQ ID NO: 65) fragment was synthesized and cloned into pIDTSmart vectors by Integrated DNA Technologies. 6×Stops-(GGGGCC)4-3T and 6×Stops-(GGGGCC) 30 -3T constructs were generated by subcloning the NheI / XhoI fragment into a pcDNA3.1 vector containing a triple epitope. To extend the size of the GGGGCC repeat, the SmaI / XhoI fragment was used in pcDNA-6×Stops-(GGGGCC) EXP Subcloning was performed to blunt-terminated PspOMI using -3T T4 DNA polymerase / XhoI. To reverse the orientation of the GGGGCC repeat within the pcDNA-6×Stop-3T construct, the SmallI / ClaI fragment was subcloned to pBluescript SK+, and then pBluescript-(GGGGCC) EXP Generated: AfeI / XhoI fragment pBluescript-(GGGGCC) EXP This was subcloned into pcDNA-6×Stop-3T, and then pcDNA-6×Stop-(GGCCCC) EXP -3T constructs were manufactured.

[0139] RT-PCR. 1) Strand-specific RT-PCR in autopsy tissue: To detect transcripts from both strands of total RNA isolated from frontal cortical autopsy tissue and peripheral blood lymphocytes (PBL) of ALS patients and healthy controls using TRIzol (Invitrogen), cDNA was generated from 0.25 μg of total RNA using the SuperScript III system (Invitrogen) with linker strand-specific reverse primers and PCR with strand-specific forward and linker (LK) primers. The PCR reaction was performed as follows: 3 minutes at 94°C, then 35 cycles of 45 seconds at 94°C, 45 seconds at 58°C, and 1 minute at 72°C, followed by 6 minutes at 72°C. Bands were cloned and sequenced to verify the specificity of the PCR amplification. 2) RT-PCR for toxicity assay in 293T cells: Total RNA was extracted from cells using the miRNeasy Mini kit (Qiagen) according to the manufacturer's protocol. Total RNA was reverse transcribed using the Superscript III RT kit (Invitrogen) and random hexamer primers. Expression of different G4C2-3XTag constructs was analyzed by RT-PCR and qPCR using 3xTag-Fw and 3xTag-Rv primer sets. β-actin expression was used as a reference gene amplified using primer sets ACTB3 and ACTB4. The primer sequences are listed in Figure 27.

[0140] Real-time RT-PCR. Two-step quantitative PCR was performed on a MyCycler Thermal Cycler system (Bio-Rad) using SYBER Green PCR Master Mix (Bio-Rad) and ASORF strand-specific cDNA and primer sets. A control reaction was performed using human beta-actin primers ACTB3 and ATCB4, with total cDNA synthesized from oligodT as the template. The two-step PCR was performed for 40 cycles (95°C for 30 seconds, 60°C for 30 seconds) in an optical 96-well plate containing triple-stranded cDNA / primer pairs for each sample. Relative fold changes were generated by first standardizing each experimental Ct value to its beta-actin Ct value, and then to the healthy control antisense ΔΔCt. The primer sequences are listed in Figure 28.

[0141] Rapid amplification of 5' and 3' cDNA ends (5' and 3' RACE). 4 μg of total RNA from frontal cortical autopsy tissue of two C9(+) ALS patients and two C9(-) ALS patients was used for 5' and 3' RACE (5'RACE system and 3'RACE, Life Technologies). In 5' RACE, the primer ASORF R was used for gene-specific first-strand cDNA synthesis, with nested reverse primers 5'GSP1 and 5'GSP2. In 3' RACE, the nested forward primers were 3'GSP1 and 3'GSP2. 3'RACE and 5'RACE products were gel extracted, cloned using TOPO TA Cloning (Invitrogen), and sequenced. Primer sequences are listed in Figure 28.

[0142] Production of polyclonal antibodies. Polyclonal rabbit antibodies were generated using New England Peptide, and polyclonal mouse antibodies were generated at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. Antiserum was produced against the C-terminal regions of synthetic poly(GP), poly(GR) peptides and predicted GP, GR, and GA RAN proteins in the sense chain (GGGGCC) (Figure 21). Antiserum was produced against the C-terminal regions of synthetic poly(PA), poly(PR) peptides and predicted PA and PR RAN proteins in the antisense chain (GGCCCC). The peptides used to generate antibodies against both antisense and sense proteins, as well as their use for Western blotting, immunofluorescence (IF), and immunohistochemistry (IHC), are summarized in Table S3.

[0143] Cell culture and transfection. HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and incubated at 37°C in humid air containing 5% CO2. DNA transfection was performed using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's instructions.

[0144] Human samples. Frozen prefrontal cortical tissue samples for biochemical and histological analysis were used in this study, including samples from 6 C9(+)ALS patients, 5 C9(-)ALS controls, and 1 normal control. In addition, paraffin-implanted tissues from C9(+)ALS / FTD and C9(-)ALS / FTD cases and normal controls were used. Peripheral blood lymphocytes (PBLs) were isolated from the pia mater of freshly recovered whole blood after short centrifugation at 2000×g. Red blood cells (RBCs) were preferably lysed and removed using RBC Lysis Buffer (Roche), PBLs were centrifuged, washed once with PBS, and dried on slides. This study was conducted in compliance with the Declaration of Helsinki. The intra-institutional review boards of the University of Florida and Johns Hopkins University approved the samples. Written informed consent was obtained from participants or related parties at the time of enrollment.

[0145] Immunofluorescence. The intracellular distribution of high molecular weight proteins was evaluated by immunofluorescence in transfected HEK293T cells. Cells were plated on an 8-well tissue culture chamber and transfected with plasmids the following day. 48 hours after transfection, cells were fixed in 4% paraformaldehyde (PFA) in PBS for 30 minutes and permeabilized on ice in 0.5% Triton X-100 in PBS for 15 minutes. Cells were blocked in 1% normal goat serum in PBS for 30 minutes. After blocking, cells were incubated at room temperature for 1 hour in a blocking solution containing rabbit anti-Myc (Abcam), mouse anti-HA (Covance), mouse anti-Flag (Sigma), rabbit anti-GR, and rabbit anti-GR-CT primary antibodies at a dilution of 1:400. The slides were washed three times in PBS and incubated for 1 hour at room temperature in a blocking solution containing goat anti-rabbit antibody conjugated to Cy3 (Jackson ImmunoResearch, PA) and goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (Invitrogen) at a dilution of 1:200. The slides were washed three times in PBS and mounted using a mounting medium containing DAPI (Invitrogen).

[0146] RNA-FISH. Slides containing cells were fixed in 4% PFA in PBS for 10 minutes and incubated on ice for 30 minutes in pre-refrigerated 70% ethanol. After rehydration for 10 minutes in 40% formamide in 2XSSC, the slides were blocked at 55°C for 10 minutes with a hybridization solution (40% formamide, 2XSSC, 10 mg / ml BSA, 100 mg / ml dextran sulfate, and 10 mg / ml yeast tRNA), and then incubated at 55°C for 2 hours in the hybridization solution with 200 ng / ml denatured RNA probe. After hybridization, the slides were washed three times with 40% formamide in 2XSSC and then briefly washed once in PBS. The autofluorescence of lipofuscin was quenched with 0.25% Sudan Black B in 70% ethanol, and the slides were mounted in a mounting medium containing DAPI (Invitrogen). RNA aggregation specificity was determined by treating cells with RNAse (100 μg / mL in 2× SSC), DNase (1 U / μl in DNase I buffer), or protease K (120 μg / mL in 2 mM CaCl2, 20 mM Tris, pH 7.5) before FISH detection. Treated cells were incubated at 37°C for 30 minutes and washed three times with PBS, followed by three washes with 2× SSC. Subsequent FISH detection was performed as described above. Antisense aggregation specificity was determined using standard FISH detection, in which slides were first hybridized with a 10-fold overdose of unlabeled (G4C2) 4 oligonucleotide, and then hybridized with G4C2-cy3 (antisense probe) or G2C4-cy3 (sense probe). Subsequent processing and detection were performed as described above.

[0147] Western blotting. Transfected cells from each well of a 6-well tissue culture plate were rinsed with PBS and lysed on ice for 45 minutes in 300 μL of RIPA buffer with a protease inhibitor cocktail. DNA was sheared by passing a 21-gauge needle through the cells. The cell lysates were centrifuged at 16,000 × g for 15 minutes at 4°C, and the supernatant was collected. The protein concentration of the cell lysates was determined using a protein assay dye reagent (Bio-Rad). 20 micrograms of protein were separated on a 4–12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (Amersham). The membranes were blocked in 5% milk powder in PBS (PBS-T) containing 0.05% Tween-20, and probed in the blocking solution with anti-Flag (1:2000), anti-Myc (1:1000), anti-HA (1:1000), or rabbit polyclonal antibody (1:1000). After incubation of the membranes with anti-rabbit or anti-mouse HRP conjugate secondary antibody (Amersham), the bands were visualized using ECL plus Western Blotting Detection System (Amersham). Serial extraction of patient frontal cortex autopsy tissue was performed as follows: the tissues were homogenized in PBS containing 1% Triton-X100, 15 mM MgCl2, 0.2 mg / ml DNase I, and a protease inhibitor cocktail, and centrifuged at 16,000 × g for 15 minutes at 4°C. The supernatant was collected. The pellet was resuspended in 2% SDS and incubated at room temperature for 1 hour, then centrifuged at 16,000 × g for 15 minutes at 4°C. The supernatant was collected, and the 2% SDS-insoluble pellet was resuspended in 8% SDS, 62.5 mM Tris-HCl pH 6.8, 10% glycerol, and 20% 2-mercaptoethanol for protein blotting (25).

[0148] Protein slot blotting. 1% Triton-X100 soluble fractions and 2% SDS soluble fractions from serial extraction were immobilized on nitrocellulose membranes under vacuum using a Bio-Dot 96-well microfiltration system (Bio-Rad). The membranes were washed with PBS-T and blotted using the same protocol as Western blotting with each rabbit polyclonal antibody (1:2000).

[0149] Immunohistochemistry. 10-micrometer sections were deparaffinized in xylene, rehydrated with graded alcohol, incubated with 95-100% formic acid for 5 minutes, and washed with distilled water for 10 minutes. HIER was performed by steam treatment of sections in citrate buffer, pH 6.0, at 90°C for 30 minutes. Serum-free block (Biocare Medical) was applied for 30 minutes to block nonspecific immunoglobulins. Rabbit polyclonal antibodies were applied in serum-free block (Biocare Medical) at dilutions of 1:5000 to 1:15000 and incubated overnight at 4°C. Conjugation reagent (streptavidin and / or alkaline phosphatase, Covance) was applied at room temperature for 30 minutes. These sections were incubated in 3% H2O2 for 15 minutes to bleach endogenous peroxidase activity. Labeling reagent (HRP, Covance) was then applied at room temperature for 30 minutes. Peroxidase activity was developed using NovaRed substrate (Vector), and sections were counterstained with hematoxylin.

[0150] Cytotoxicity assay. All transfection experiments were performed using Lipofectamine 2000 (Invitrogen) with 60% cell confluence, according to the manufacturer's instructions. 500 ng of each vector was transfected in 35 mm wells. Cell death was determined by measuring lactate dehydrogenase (LDH) cell release using the CytoTox 96 non-radioactive cytotoxicity assay (Promega), according to the manufacturer's instructions. Absorbance was recorded at 490 nm, and total LDH release was measured by lysing cells with 1% Triton X-100. In each experiment, the determination was performed quintuplely for each experimental condition, and the mean data were calculated. Statistical significance was determined using a two-sided independent Student's t-test (p<0.05) for single comparisons and analysis of variance (ANOVA) when multiple paired conditions were compared.

[0151] Cell viability assay. HEK293T cells were transfected in 96-well plates, and cell viability was determined 42 hours after transfection using the 3-(4,5-dimethithiazol-,2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT was added to the cell culture medium at a final concentration of 0.5 mg / mL and incubated at 37°C for 45 minutes. Next, during medium removal, the cells were lysed with 100 μL of DMSO, and absorbance was measured at 595 nm. In each experiment, the determination was performed quintuple-wise. Statistical significance was determined using Student's t-test (p<0.05). [Examples]

[0152] A BAC transgenic mouse model of C9ORF72 ALS to test the hypothesis that both sense and antisense transcripts contribute to ALS / FTD. Principle: C9ORF72 ALS / FTD model mice that reproduce sense and antisense transcripts are critically important for modeling this disease. BAC clones were isolated from human patients containing approximately 800 G4C2 repeats. These BAC clones were used to generate eight established lines. These mice are useful, for example, to answer questions such as: Do both RAN protein expression and RNA gain-of-function contribute to C9ORF72 ALS / FTD? Are both sense and antisense mechanisms important in C9ORF72 pathogenicity?

[0153] Approach: A BAC clone containing the complete human C9ORF72 gene plus a flanking sequence with approximately 800 GGGGCC repeats was isolated from a human patient and inserted into the pCC1BAC® plasmid [Epicentre®]. The BAC insert selected for use in mice extended from bp27,625,470 to 27,527,137 of the human genome reference sequence on chromosome 9 (Figure 29). The coordinates above do not include extra repeats from this patient. The BAC insert DNA was found to contain approximately 800 repeats in several clonal preparations, but was highly unstable. Pronuclear injection was performed to generate eight FVB-established lines. Two independent lines showed elongation mutations. The BAC repeat size in mice is approximately 500 repeats, but it can vary between progeny, and the size can increase or decrease as mouse colonies expand and additional mouse generations are bred in the laboratory. BAC elongation mice expressed both sense and antisense versions of the C9ORF72 gene. Sense and antisense GGGGCCRNA condensation was present in mice with GGGGCC repeats but not in control mice (Figures 30-31).

[0154] At least two elongation and two control lines are selected for detailed characterization. Behavioral characterization includes rotarod analysis, grip strength, balance beam, and open-field assessments. Molecular characterization of sense and antisense transcripts, as well as RAN proteins, is performed by RT-PCR, RACE, immunoblotting, immunohistochemistry, and immunofluorescence. Immunohistochemistry, immunofluorescence, and FISH studies are performed to correlate the sites of RNA aggregation and C9-RAN protein accumulation with pathological changes. RAN protein accumulation in the CNS, CSF, muscle, blood, and other tissues is examined at various points in time during development.

[0155] Relevance: The results from these studies will lead to a better understanding of the role that RAN translation plays in C9ORF72 ALS / FTD. In addition, these studies will help determine which proteins are most frequently found in autopsy tissue and identify clear differences in the toxicity of individual RAN proteins, thereby aiding in the prioritization of individual protein targets. Information from model cells and mice will also inform future research on the effectiveness of various treatment strategies.

[0156] References [Table 6] [Table 7] [Table 8]

[0157] Without further detail, those skilled in the art will likely be able to utilize this disclosure to the fullest extent based on the above description. Accordingly, the following specific embodiments should be construed as merely illustrative and not as limiting the remainder of this disclosure in any way. All publications referenced herein are incorporated by reference for the purposes or subject matter referred to herein.

[0158] Other Embodiments All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely one example of a comprehensive set of equivalent or similar features.

[0159] From the above description, those skilled in the art will readily identify the essential features of this disclosure and can make various changes and modifications to it without departing from its spirit and scope, so as to suit various uses and conditions. Accordingly, other embodiments are also within the scope of the claims.

Claims

1. A method for producing an antibody, comprising administering a poly(Pro-Arg) diamino acid repeat-containing protein comprising the sequence described in Sequence ID No. 19 to a non-human subject, wherein the antibody specifically binds to the poly(Pro-Arg) diamino acid repeat-containing protein.

2. A method for producing an antibody for use in treating a subject having ALS or FTD, the method comprising administering a poly(Pro-Arg) diamino acid repeat-containing protein comprising the sequence described in SEQ ID NO: 19 to a non-human subject, wherein the antibody specifically binds to the poly(Pro-Arg) diamino acid repeat-containing protein.

3. A poly(Pro-Arg) diamino acid repeat-containing protein comprising the sequence described in SEQ ID NO: 19 for use in a method for producing an antibody, wherein the method comprises administering the poly(Pro-Arg) diamino acid repeat-containing protein comprising the sequence described in SEQ ID NO: 19 to a non-human subject, and the antibody specifically binds to the poly(Pro-Arg) diamino acid repeat-containing protein.

4. An isolated antibody for use in a method for reducing RAN protein translation or RAN protein aggregates in a target, wherein the antibody specifically binds to a poly(Pro-Arg) diamino acid repeat-containing protein consisting of the sequence described in SEQ ID NO:

19.

5. The isolated antibody for use according to claim 4, wherein the subject is a human subject or a mouse subject, and optionally the human subject or mouse subject is identified as having amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), is identified as being highly likely to develop ALS or FTD, and / or comprises the human C9ORF72 gene comprising the GGGGCC hexanucleotide repeat sequence.

6. A method for producing an isolated antibody for use according to claim 4 or claim 5, comprising administering a poly(Pro-Arg) diamino acid repeat-containing protein comprising the sequence described in SEQ ID NO: 19 to a non-human subject.

7. The protein for use according to claim 3, wherein the non-human subject is a mouse subject, preferably a transgenic mouse subject, or a rabbit subject.

8. The isolated antibody for use according to claim 4 or claim 5, wherein the isolated antibody is a monoclonal antibody.

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