DI- AMINO ACID REPEAT-CONTAINING PROTEINS ASSOCIATED WITH ALS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Application No. 61/786,258, filed March 14, 2013, and the benefit 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 by reference herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under PO1NS058901 and

RO1NS040389 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Expansion of a GGGGCC hexanucleotide sequence within the intron of the human C90RF72 gene is associated with both amyotrophic lateral sclerosis and frontotemporal dementia in humans. Amyotrophic lateral sclerosis (ALS) is a debilitating disease with varied etiology characterized by rapidly progressing weakness, muscle atrophy, muscle spasticity, difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and difficulty breathing (dyspnea). Although the order and rate of symptoms varies from person to person, eventually most subjects are not able to walk, get out of bed on their own, or use their hands and arms. Most subjects with ALS will eventually die from respiratory failure, usually within three to five years from the onset of symptoms. Riluzole (Rilutek) is the only currently available treatment for ALS and only slows progression and increases survival to a modest extent. Frontotemporal dementia (FTD) is also a devestating group of disorders resulting from atrophy or shrinkage of the frontal and temporal lobes of the brain. This shrinkage or atrophy results in severe behavioral changes. There is currently no cure for FTD and limited medications for managing the symptoms of FTD. New methods for diagnosing and treating ALS and/or FTD would greatly benefit ALS and FTD subjects.

SUMMARY OF THE INVENTION

Expansion of a GGGGCC hexanucleotide sequence within the intron of the human 5 C90RF72 gene is associated with both amyotrophic lateral sclerosis and frontotemporal dementia in humans. As described herein, an expanded GGGGCC hexanucleotide repeat sequence within the intron of the C90RF72 gene was found to be transcribed such that RNA transcripts containing the hexanucleotide repeat in both the sense and anti-sense direction were produced. These sense and anti- sense transcripts were found to be translated to produce

10 di-amino acid repeat-containing proteins. The sense transcript (containing 5'-GGGGCC-3' hexanucleotide repeats) was found to be translated through repeat-associated non-ATG (RAN) translation such that poly- (Gly- Ala), poly-(Gly-Pro), and poly-(Gly-Arg) proteins were produced. The anti-sense transcript (containing 5'-GGCCCC-3' hexanucleotide repeats) was found to be translated through repeat-associated non-ATG (RAN) translation

15 such that poly-(Pro-Ala), poly- (Pro- Arg), poly- (Gly- Pro) proteins were produced.

Additionally, the anti-sense transcript was found to be translated through ATG-initiated translation to produce Met...poly-(Pro-Arg) and Met...poly-(Gly-Pro) proteins.

These di-amino acid repeat-containing proteins were found to be present in ALS subject blood samples. Accordingly, aspects of the disclosure relate to a method of detection

20 of di-amino acid-repeat containing protein levels in sample (e.g., blood) obtained from a subject, the method comprising measuring di-amino acid-repeat-containing protein levels in the sample of the subject. In some aspects, detection of di-amino acid-repeat containing protein levels may identify (or diagnose) or aid in identification (or aid in diagnosis) of a subject having ALS or FTD or likely to develop ALS or FTD. Alternatively or additionally,

25 detection of di-amino acid-repeat containing protein levels, e.g., in a blood sample of the subject, may identify (or diagnose) or aid in identification (or aid in diagnosis) of a subject as having a risk factor of ALS or FTD, such as an elevated level of a di-amino acid-repeat containing protein or proteins in the cerebrospinal fluid of the subject. Aspects of the disclosure also relate to treatment of a subject having ALS or FTD by decreasing or

^ stabili zing di-amino acid-repeat-containing protein levels in the blood of the subject. Additionally, expression of the anti-sense transcript (containing 5'-GGCCCC-3' hexanucleotide repeats) was found to be highly elevated in subjects having the expanded GGGGCC hexanucleotide repeat compared to controls. Foci of sense and anti-sense transcripts were also detectable using fluorescent in situ hybridization (FISH) in brain and blood cells of patients having the expanded GGGGCC hexanucleotide repeat sequence within the intron of the C90RF72 gene. Thus, other aspects of the disclosure relate to a method of detection of a hexanucleotide repeat-containing transcript, the method comprising measuring a level a hexanucleotide repeat-containing transcript and/or measuring the presence or absence of a hexanucleotide repeat-containing transcript focus. In some aspects, detection of a hexanucleotide repeat-containing transcript may identify (or diagnose) or aid in

identification (or aid in diagnosis) of a subject as having ALS or FTD or likely to develop ALS or FTD. Alternatively or additionally, detection of a hexanucleotide repeat-containing transcript, e.g., in a blood sample of the subject, may identify (or diagnose) or aid in identification (or aid in diagnosis) of a subject as having a risk factor of ALS or FTD, such as an elevated level of a di-amino acid-repeat containing protein or proteins in the cerebrospinal fluid of the subject.

In some aspects, the disclosure relates to a method for identifying a subject as having ALS or FTD or likely to develop ALS or FTD, the method comprising determining, in a blood sample obtained from a subject, a level of one or more di-amino acid repeat-containing proteins selected from a poly-(Gly-Ala), poly- (Gly- Pro), poly-(Gly-Arg), poly-(Pro-Ala), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly-Pro) protein, wherein 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 ALS or FTD or is likely to develop ALS or FTD. In some embodiments, the level of the one or more di-amino acid repeat-containing proteins is determined by performing an assay. In some embodiments, the assay comprises an immuno- based assay. In some embodiments, the immuno-based assay comprises an isolated antibody specific for an antigen comprising a sequence as set for in Tables 1, 2, or 3. In some embodiments, the immuno-based assay comprises an isolated antibody specific for the C- terminus of the one or more di-amino acid repeat-containing protein. In some embodiments, the method further comprises identifying the subject as having ALS or FTD or likely to develop ALS or FTD if the level of the di-amino acid repeat- containing protein is elevated compared to a control level. In some embodiments, the method further comprises treating the subject having ALS or FTD or likely to develop ALS or FTD. 5 In some embodiments, treating comprises administering to the subject an effective amount of one or more of riluzole, baclofen, diazepam, phenytoin, trihexyphenidyl or amitriptyline. In some embodiments, treating comprises performing a procedure selected from plasmapheresis or a bone marrow transplant.

In some embodiments, the one or more di-amino acid repeat-containing proteins is o selected from the poly-(Pro-Ala), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly-

Pro) protein. In some embodiments, the one or more di-amino acid repeat-containing proteins is two or more di-amino acid repeat-containing proteins.

Other aspects of the disclosure relate to a method for treating a subject with ALS or FTD, the method comprising decreasing or preventing an increase in a level of one or more5 di-amino acid repeat-containing proteins selected from a poly- (Gly- Ala), poly-(Gly-Pro), poly- (Gly- Arg), poly- (Pro- Ala), poly-(Pro-Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly- Pro) protein in the blood of the subject. In some embodiments, decreasing or preventing an increase of the level of one or more di-amino acid repeat-containing proteins comprises removing the one or more di-amino acid repeat-containing proteins from the blood of the o subject. In some embodiments, the one or more di-amino acid repeat-containing proteins from the blood of the subject is removed using a procedure selected from plasmapheresis or a bone marrow transplantation. In some embodiments, the bone marrow transplantation is an allogeneic bone marrow transplantation.

In yet another aspect, the disclosure relates to an isolated antibody specific for one or 5 more di-amino acid repeat proteins selected from a poly-(Gly-Ala), poly-(Gly-Pro), poly- (Gly-Arg), poly-(Pro-Ala), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly-Pro) protein. In some embodiments, the di-amino acid repeat protein is selected from a poly- (Pro-Ala), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly-Pro) protein. In some embodiments, the isolated antibody is specific for an antigen comprising a sequence or fragment of a sequence as set for in Tables 1, 2, or 3. Other aspects of the disclosure relate to a method for identifying a subject as having ALS or FTD or likely to develop ALS or FTD, the method comprising determining, in a sample obtained from a subject, a level of a 5'-GGGGCC-3' hexanucleotide repeat- containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat-containing RNA, wherein a level of the 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat-containing RNA that is elevated 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 is determined by performing an assay. In some embodiments, the assay comprises a nucleic acid-based assay, 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 comprises identifying the subject as having ALS or FTD or likely to develop ALS or FTD if the level of a 5'-GGGGCC-3' hexanucleotide repeat- containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat-containing RNA is elevated compared to a control level. In some embodiments, the method further comprises treating the subject having ALS or FTD or likely to develop ALS or FTD. In some embodiments, treating comprises administering to the subject an effective amount of one or more of riluzole, baclofen, diazepam, phenytoin, trihexyphenidyl or amitriptyline. In some embodiments, treating comprises performing a procedure selected from plasmapheresis or a bone marrow transplant. In some embodiments, the level of a 5'-GGGGCC-3'

hexanucleotide repeat-containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat- containing RNA is a level of a 5'-GGCCCC-3' hexanucleotide repeat-containing RNA.

Yet other aspects of the disclosure relate to a method for identifying a subject as having ALS or FTD or likely to develop ALS or FTD, the method comprising determining, in a sample obtained from a subject, the presence or absence of foci containing 5'-GGGGCC-3' hexanucleotide repeat-expansion-containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat-expansion-containing RNA, wherein the presence of the foci of the 5'-GGGGCC-3' hexanucleotide repeat-expansion-containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat-expansion-containing RNA indicates that the subject has ALS or FTD or is likely to develon ALS or FTD. In some embodiments, presence or absence of foci or elevated C90RF72 sense or antisense RNA levels is determined by performing an assay. In some embodiments, the assay comprises a nucleic acid-based assay, such as strand specific RT- PCR or in-situ hybridization (e.g., FISH).

Yet other aspects of the disclosure relate to transgenic mice. In some embodiments, the transgenic mouse comprises a human C90RF72 gene and optionally human flanking sequences. In some embodiments, the transgenic mouse comprises SEQ ID NO: 63.

These and other aspects are described in more detail herein and illustrated by the non- limiting figures and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described.

FIG. 1 is a drawing showing that transcripts are produced in the sense and anti-sense direction on the C90RF72 gene, and that repeat-associated non-ATG (RAN) translation proteins are translated in all three reading-frames from both the sense and anti- sense

C90RF72 transcripts. The drawing also shows that Met...poly-(Pro-Arg) and Met...poly- (Gly-Pro) proteins are translated through ATG-initiated translation on the anti-sense transcript. CT = predicted to and/or shown to contain a c-terminal domain. * = end of protein (due to stop codon). M = Methionine.

FIG. 2A is a diagram of an expression vector for expressing RAN translation proteins in cells. CMV = cytomegalovirus promoter. 6xStop = 6 stop codons, two in each frame. (GGGGCC)exp = a GGGGCC repeat sequence that extends for 4, 30, 60, or 120 repeats. (GR)HA-(GP)Flag-(GA)Myc = a HA, flag or myc tag that corresponds to the poly-(Gly-Arg), poly-(Gly-Pro), and poly- (Gly- Ala) repeat proteins, respectively. SV40 poly(a) =

transcription terminator and poly A signal.

FIG. 2B is a photograph of a western blot depicting that GR and GP RAN translation proteins are expressed in cells transfected with 30, 60 or 120 GGGGCC repeat sequences.

FIG. 3 is a photograph of an immunofluorescence staining of cells expressing GP, GR, or GA RAN proteins in cells transfected with 30, 60 or 120 GGGGCC repeat sequences.

FIG. 4 is a diagram of the poly-(GR) and GR-c-terminus antigens and a series of nhotopraphs of immunofluorescence staining showing that the poly-(GR) and (GR)-c- terminal antibodies detect poly-(GR) RAN proteins.

FIG. 5 is a series of photographs of tissue from C90RF72 ALS patients or control patients showing that poly-(GR), poly-(GP), and poly-(GA) di-amino acid-repeat-containing proteins are expressed by C90RF72 ALS patients.

5 FIG. 6 is a series of photographs of tissue from C90RF72 ALS patients or control patients showing that poly- (PA) and poly- (PR) di-amino acid-repeat-containing proteins are expressed by C90RF72 ALS patients.

FIG. 7A is a series of photographs of immunofluorescence staining showing antibodies generated to recognize the GP repeat motif (GP) or the unique C-terminal region 10 of the same GP-RAN proteins (GP-C) colocalize in 20% of patient cells. Cells that stain for and GP-C and GP express GP-RAN protein in the sense direction and that cells showing only GP staining express RAN-GP or Met...GP from the anti-sense strand.

FIG. 7B is a graph depicting the percentage of GP and GP + GP-C in patient cells.

FIG. 8 is a picture of a dot blot showing that di-amino acid repeat-containing proteins 15 are found in the blood (PBL) and the brain (FCX, frontal cortex) of subjects with ALS, but not controls.

FIG. 9 is a photograph of a western blot showing that GP-repeat proteins are present in the brain (FCX) of subjects with ALS but not controls and that PA-repeat proteins are present in the plasma and serum of subjects with ALS but not controls.

20 FIG. 10 is a schematic of the RAN translation mouse model construct containing 6X stops, a CAG repeat region, tags for detecting each CAG repeat frame, and a terminator sequence.

FIG. 11 depicts two photographs showing that poly-Gin proteins accumulated in the brain of RAN translation (RANT) mice containing the construct in FIG. 10, but not in control 25 mice.

FIG. 12 is a series of schematics, graphs and images showing that G2C4 antisense transcripts are elevated by strand specific RT-PCR and accumulate as RNA foci in C90RF72 patient tissues. (A) Schematic diagram of C90RF72 gene and antisense transcripts and relative location of primers for strand- specific RT-PCR and RACE primers. (B) Strand- ^ snecific RT-PCR of sense (S) and antisense (AS) transcripts (across intron lb and exon 1) from frontal cortex of C9(+) and C9(-) ALS patients. (C) strand- specific qRT-PCR showing elevated antisense mRNA in C9(+) compared to C9(-) ALS patients. (D) In situ hybridization with G4C2-Cy3 probe showing G2C4 antisense RNA foci (arrowheads) in C9(+) frontal cortex and peripheral blood leukocytes (PBLs) which are absent in C9(-) cases. Nuclear foci in FCX are indicated by arrow heads. FCX=frontal cortex. PBL=peripheral blood leukocytes.

FIG. 13 is a series of schematics, graphs and images showing in vitro evidence for RAN translation of antisense G ₂ C ₄ expansion and dual immunological detection strategy. (A- C) Immunoblots (B) and IF staining (C) of HEK293T cells 48 hours post-transfection with the (G ₂ C ₄ ) _EXP -3T construct (A). (B) PR and GP expansion proteins detected by western and (C) PA, PR and GP detected by IF in transfected cells. (D) Diagram of putative proteins translated from sense and antisense transcripts. CT=C-terminal, f 1-3: reading frame 1-3. (E) Abbreviated example of validation of a-PA rabbit polyclonal antibody. IF staining of HEK293T cells transfected with constructs with 5' Flag epitope tagged PA protein and corresponding immunoblots. See FIGs. 22 and 23 for additional controls and validation of eight additional antibodies generated against repeat motifs and CT regions.

FIGs. 14A and 14B are a series of images and graphs showing in vivo evidence for RAN-translation of the G ₂ C ₄ AS repeat and toxicity studies. (A) Dot blot of C9(+) and C9(-) frontal cortex lysates probed with a-PA, a-PA-CT, a-PR, a-PR-CT antibodies. (B)

Immunoblots of C9(+) and C9(-) ALS frontal cortex lysates. (C) IHC detection of PA, PR and GP protein aggregates in hippocampal neurons from C9(+) ALS patients detected with a- PA, a-PA-CT, a-PR, a-PRCT and a-GP antibodies. (D) IF staining with mouse a-GP

(arrowhead) and rabbit a-GP-CT (arrow) of C9(+)hippocampal tissue with sense inclusions positive for both antibodies (upper panel) and antisense inclusions positive for only GP repeat antibody (lower panel). (E) IF staining of larger region showing sense (S) and antisense (AS) staining. (F) Quantitation of double (sense) and single (antisense) labeled aggregates. (G-J) RAN and PR toxicity studies (G) G ₂ C ₄ expansion constructs (+/-ATG-PR-3T) +/ - ATG initiation codon in PR frame and 3 'epitope tags. (H) Protein blots showing levels of PR and GP in cells transfected with constructs in (G). (I) LDH and (J) MTT assays of transfected HEK293T cells.

FIGs. 15A and 15B are a series of images showing in vivo evidence for RAN translation in both antisense and sense directions of C90RF72. Cytoplasmic inclusions detected by IHC using antibodies against sense (a-GR, a-GR-CT, a-GA, a-GP-CT) and antisense (a-PA, a-PA-CT, a-PR, a-PR-CT) and a-GP which recognizes GP proteins made in both the sense and antisense directions. Aggregates were found in neurons of cornu ammonis (CA) and dentate gyrus (DG) regions of the hippocampus and the motor cortex (MC) of C9(+) ALS autopsy tissue.

FIGs. 16A and 16B are a series of images of clustered RAN protein aggregates and RAN aggregates in motor neurons. IHC showing cytoplasmic a-GP aggregates in: (A) in layer III of motor cortex. (B) upper motor neuron in layer V of the motor cortex; (C) lower motor neurons in the spinal cord (L-S.C). (D) in cornu ammonis, CA, (E) and dentatus gyrus, DG regions of the hippocampus. (F and G) IHC showing abundant PA and PR cytoplasmic inclusions in the pre-subiculum (PrSub) from one patient.

FIG. 17 is a series of images of clustered staining of RAN proteins. (A) Low power image of IHC staining with a-PA-CT shows variations in staining intensity (dark spots are positive) in regions I- IV with insets showing higher-power images. (B) Examples of aggregates from region I show immunoreactivity against all nine antibodies with similar staining for antibodies against repeat and unique C-terminal epitopes.

FIG. 18 is a table summarizing histopathological findings in C90RF72 positive ALS/FTD cases and controls.

FIGs. 19A-19F are a series of images and datasets. (A) shows strand- specific RT-PCR detection of sense (S) and antisense (AS) transcripts (across intron 1) of PBLs of C9(+) patient and normal controls. (B) is a summary of 5' RACE products. (C) shows FISH staining of frontal cortex from a C9(+) case showing an example of cytoplasmic RNA foci. (D) shows FISH staining of peripheral blood leukocytes showing the accumulation of antisense (AS) G ₂ C ₄ and sense (S) G ₄ C ₂ RNA foci in C9(+) but not C9(-) cells. (E) shows antisense foci specificity assay showing excess unlabeled (G ₄ C ₂ ) ₄ oligo blocks labeling of G4C2-Cy3 antisense (AS) but not G ₂ C ₄ -Cy3 labeled sense foci. (F) shows additional controls for antisense RNA foci showing expected DNase I resistance and RNase I sensitivity.

FIG. 20 is a series of images of in vitro evidence for RAN translation of the sense GGGGCC repeat expansion. (A) shows constructs containing varying GGGGCC repeat lengths with upstream 6X Stop cassette and 3' tags in each reading frame. Immunoblots (B) and/or immunofluorescence staining (C) showing RAN translation occurs in all three frames (GP, GR, GA) in cells transfected with constructs containing 30, 60 and 120 repeats.

FIG. 21 is a schematic of putative protein products in sense and antisense directions for all reading frames SEQ ID NOs: 57-62, from top to bottom. Underlined sequences were used to generate polyclonal antibodies. *= Stop codon.

FIGs. 22A-22E are a series of images showing validation of dual antibodies to detect putative polyPA, polyPR, polyGP proteins by immunofluorescence and protein blot (A-D Top): Schematic diagrams of constructs expressing ATG-initiated N-terminal epitope-tagged (V5 or Flag) repeat proteins with or without endogenous C-terminal sequences. (A-D Bottom panels), co-localization of a- Flag or a-V5 staining in transfected HEK293T cells with staining using the following newly developed antibodies: (A) a-PA or a-PA-CT(antisense); (B) a-PR or a-PR-CT (C) rabbit a-GP or a-GP-CT (sense); (D) mouse a-GP;. Similar staining was not seen in preimmune or pcDNA3.1 empty vector controls; (E) Corresponding immunoblots showing six of the seven antibodies tested also detect recombinant proteins by Western.

FIGs. 23A-23C are a series of images showing validation of additional sense repeat and C-terminal polyclonal antibodies. (A, B Top): Schematic diagrams of constructs expressing ATG-initiated N-terminal V5-epitope tagged GR or GA repeat proteins with endogenous C-terminal sequences. (A-B Bottom panels), co-localization of a-V5 staining in transfected HEK293T cells with a-GR, a-GR-CT and a-GP-CT respectively. Similar staining was not seen in preimmune or pcDNA3.1 empty vector controls. (C) a-GR detection of recombinant protein in Flag-GR transfected cells by protein blot.

FIG. 24 is a series of images of immunoblots of 2% soluble lysates from C9(+) and C9(-) ALS frontal cortices with a-GP-CT, a-GR, a-GR-CT and a-GA antibodies.

FIG. 25 is a series of images showing negative IHC staining of C9(-) ALS/FTD hippocampal sections with antibodies against sense and antisense proteins.

FIGs. 26A-26D are a graph and a series showing images RAN translation and PR protein expression affect cell viability. (A) qRT-PCR shows expression of expansion transcrints are similar in HEK293T cells transfected with (-)ATG-PR-3T and (+)ATG-PR-3T constructs. (B-D) Bright-field microscopy images showing changes in cell morphology in cells expressing RNA and RAN proteins from (-)ATG-PR-3T constructs compared to empty vector control (pcDNA3.1) and worsening effects in (+)ATG-PR-3T cells expressing increased levels of PR protein.

5 FIG. 27 is a table describing primers used for RT-PCR and RACE (SEQ ID NOs: 17 of them (in order. SEQ ID NOs: 36, 37, 39, 38, 45-47, 40, 48-56).

FIG. 28 is a table describing novel sense and antisense antibodies, (in order SEQ ID NOs: 20, 23, 19, 25, 21, 21, 22, 18).

FIG. 29 is a schematic of the BAC insert used to make transgenic mice, l o FIG. 30 is a series of photographs showing sense RNA foci in transgenic mice

expressing a human C90RF72 gene containing GGGGCC repeats. Exemplary foci are indicated by arrowheads.

FIG. 31 is a series of photographs showing anti-sense (AS) RNA foci in transgenic mice expressing a human C90RF72 gene containing GGGGCC repeats. Exemplary foci are 15 indicated by arrowheads.

DETAILED DESCRIPTION OF THE INVENTION

Well-established rules of translational initiation have been used as a cornerstone in molecular biology to understand gene expression and to predict the consequences of disease causing mutations. In general, micro satellite expansion mutations (e.g., CAG, CTG) located in predicted coding- and non-coding regions have been thought to cause disease by protein gain-, or loss-, of-function or RNA gain-of-function mechanisms. It has been previously reported that the canonical rules of translation do not apply for CTG*CAG repeat expansions and that CAG and CUG expansion transcripts express homopolymeric expansion proteins in all three frames without an AUG start codon (see, e.g., T. Zu et al., Non-ATG-initiated translation directed by micro satellite expansions. PNAS 108, 260 (2011)). This translation independent of an AUG start codon is termed repeat-associated non-ATG (RAN) translation. RAN translation is hairpin dependent and occurs without frameshifting or RNA editing. AN translation has been observed from trinucleotide, tetranucleotide, and pentanucleotide repeats associated with myotonic dystrophy 1, myotonic dystrophy 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 8 and Huntington disease (see PCT publication

WO/2010/115033, which is incorporated herein by reference).

Expansion of a GGGGCC hexanucleotide repeat within the intron of the C90RF72 gene has been previously associated with both amyotrophic lateral sclerosis and

frontotemporal dementia. As described herein, it has been found that this expanded hexanucleotide repeat is contained within RNA transcripts expressed in both the sense and anti-sense direction from the C90RF72 locus. These hexanucleotide repeat-containing transcripts were found to undergo RAN translation such that poly- (Gly- Ala), poly-(Gly-Pro), poly- (Gly- Arg), poly- (Pro- Ala), or poly- (Pro- Arg) proteins were produced, depending on the frame of the hexanucleotide repeat being read from the RNA (5'-GGGGCC-3\ 5'- GGGCCG-3', and 5'-GGCCGG-3' on the sense transcript, 5'-GGCCCC-3', 5'-GCCCCG-3', and 5'-CCCCGG-3' on the anti-sense transcript, see FIG. 1). In addition, the anti-sense transcript was found to be translated through ATG-initiated translation to produce

Met...poly-(Pro-Arg) and Met...poly-(Gly-Pro) proteins. These RAN and ATG-initiated proteins are referred to as di-amino acid-repeat-containing proteins herein. The sense and anti-sense 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 (anti-sense).

As further described herein, these di-amino acid-repeat-containing proteins unexpectedly were found to be present in blood samples from subjects with ALS.

Additionally, expression of the anti-sense 5'-GGCCCC-3' hexanucleotide repeat-containing RNA transcript was found to be highly elevated in subjects having a C90RF72 gene containing the expanded GGGGCC hexanucleotide repeat sequence. Further, foci of both the sense and anti-sense hexanucleotide repeat-expansion-containing RNA transcripts were found to be present in subjects having a C90RF72 gene containing the expanded GGGGCC hexanucleotide repeat sequence. Without wishing to be bound by theory or mechanism, it is believed that di-amino acid-repeat-containing proteins in the blood of subjects with ALS accumulate within the brain parenchyma over time, leading to neuroinflammatory changes, C!NS dvsfunction, and neuronal death. Accordingly, aspects of the disclosure relate to identification of a subject as having ALS or likely to develop ALS by providing novel assays for determining di-amino acid-repeat-containing protein levels in the blood of the subject and/or hexanucleotide repeat-containing RNA levels in a sample from the subject. Aspects of the disclosure also relate to treatment of a subject having ALS or FTD by decreasing or stabilizing di-amino acid-repeat-containing protein levels in the blood of the subject.

Identification of a subject having ALS or FTD or likely to develop ALS or FTD

Aspects of the disclosure relate to identification of a subject having ALS or FTD or likely to develop ALS or FTD based on a level of one or more di-amino acid-repeat- containing proteins in a blood sample from a subject. In some embodiments, a method comprises, determining, in a blood sample obtained from a subject, a level of one or more di- amino acid-repeat-containing proteins selected from a poly- (Gly- Ala), poly-(Gly-Pro), poly- (Gly-Arg), poly-(Pro-Ala), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly-Pro) protein, wherein 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 ALS or FTD or is likely to develop ALS or FTD. In some embodiments, a level of one or more di-amino acid-repeat- containing proteins is determined by performing an assay. Non-limiting assays are described herein.

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

Yet other aspects of the disclosure relate to identification of a subject having ALS or FTD or likely to develop ALS or FTD based on the presence or absence of RNA foci containing a 5'- GGGGCC-3' hexanucleotide repeat-expansion-containing RNA and/or a 5'- GGCCCC-3' hexanucleotide repeat-expansion-containing RNA in a sample from a subject, wherein the presence of the focus of the 5' -GGGGCC-3' hexanucleotide repeat-expansion- containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat-expansion-containing RNA indicates that the subject has ALS or FTD or is likely to develop ALS or FTD. As used herein, a focus of a 5'- GGGGCC-3' hexanucleotide repeat-expansion-containing RNA and/or a 5'-GGCCCC-3' hexanucleotide repeat-expansion-containing RNA refers to an area of accumulation of the 5'- GGGGCC-3' hexanucleotide repeat-expansion-containing RNA and/or the 5'-GGCCCC-3' hexanucleotide repeat-expansion-containing RNA, which may be detectable using a nucleic acid-based assay, such as FISH. In some embodiments, the focus may be, e.g., 0.1 to 2 micrometers in diameter, 0.1 to 1.5 micrometers in diameter, or 0.1 to 1 micrometers in diameter. In some embodiments, the focus may be at least 0.1 micrometers in diameter. It is to be appreciated that a sample may contain more than one focus and that each focus may be a different size. For example, one focus may be 0.2 micrometers in diameter, while second focus may be 1 micrometer in diameter. Non-limiting examples of foci and methods detecting such foci are provided in Example 3.

It is to be understood that a subject may be identified based on a level of one or more di-amino acid-repeat-containing proteins, a level of a hexanucleotide repeat-expansion containing RNA, the presence or absence of a hexanucleotide repeat-expansion containing RNA, or any combination thereof. In some embodiments, the method further comprises identifying the subject as having ALS or FTD or likely to develop ALS or FTD if the level of the di-amino acid-repeat-containing protein or hexanucleotide repeat-containing RNA is elevated compared to a control level. In some embodiments, the method further comprises identifying the subject as having ALS or FTD or likely to develop ALS or FTD if the focus or foci of the hexanucleotide repeat-expansion-containing RNA are present in the sample. In some embodiments, the method further comprises identifying the subject as not having ALS or FTD or unlikely to develop ALS or FTD if the level of the di-amino acid-repeat-containing protein or hexanucleotide repeat-containing RNA is decreased or the same compared to a control level. In some embodiments, the method further comprises identifying the subject as not having ALS or FTD or unlikely to develop ALS or FTD if the focus or foci of the hexanucleotide repeat-expansion-containing RNA are absent in the sample.

In some embodiments, a level of one or more di-amino acid-repeat-containing proteins or the identity of a subject may be recorded. In some embodiments, recordation comprises inputting a level or identity of subject into a computer, such as a medical record database.

Other aspects of the disclosure relate to treatment of a subject identified as having ALS or FTD or likely to develop ALS or FTD. As used herein, "treat" or "treatment" refers to (a) preventing or delaying the onset of ALS or FTD; (b) reducing the severity of ALS or FTD; (c) reducing or preventing development of symptoms characteristic of ALS or FTD; (d) preventing worsening of symptoms characteristic of ALS or FTD; and/or (e) reducing or preventing recurrence of ALS or FTD symptoms in subjects that were previously

symptomatic for ALS or FTD.

In some embodiments, treatment comprises 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, treatment comprises 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, treatment comprises administering an effective amount of a therapeutic agent, such as baclofen, diazepam, phenytoin, trihexyphenidyl and/or amitriptyline, which reduces one or more symptoms of ALS or FTD in a subject identified as having ALS or FTD. In some embodiments, treatment comprises one or more of physical therapy, occupational therapy, or speech therapy. In some embodiments, treatment comprises a method as described herein for decreasing or stabilizing di-amino acid-repeat-containing protein levels in the blood of the subject, such as bone marrow transplantation or plasmapheresis. In some embodiments, treatment comprises any combination of the above-mentioned treatments or anv other treatments described herein. An effective amount is a dosage of a therapeutic agent sufficient to provide a medically desirable result, such as treatment of ALS or FTD. The effective amount will vary with the age and physical condition of the subject being treated, the severity of ALS or FTD in the subject, the duration of the treatment, the nature of any concurrent therapy, the specific route of administration and the like factors within the knowledge and expertise of the health practitioner.

Administration of a treatment may be accomplished by any method known in the art (see, e.g., Harrison's Principle of Internal Medicine, McGraw Hill Inc.). Administration may be local or systemic. Administration may be parenteral (e.g., intravenous, subcutaneous, or intradermal) or oral. Compositions for different routes of administration are well known in the art (see, e.g., Remington's Pharmaceutical Sciences by E. W. Martin). Dosage will depend on the subject and the route of administration. Dosage can be determined by the skilled artisan.

Other aspects of the disclosure relate to methods for monitoring responsiveness to a treatment in a subject having ALS or FTD or suspected of having ALS or FTD. In some embodiments, the method comprises: determining, in a blood sample obtained from the subject at a first time point, a first level of one or more di-amino acid-repeat-containing proteins selected from a poly-(Gly-Ala), poly- (Gly- Pro), poly-(Gly-Arg), poly-(Pro-Ala), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly-Pro) protein; and determining, in a blood sample obtained from the subject at a second time point, a second level of one or more di-amino acid-repeat-containing proteins selected from a poly- (Gly- Ala), poly-(Gly-Pro), poly- (Gly- Arg), poly- (Pro- Ala), poly-(Pro-Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly- Pro) protein, wherein a second level that is elevated or the same compared to a first level indicates that the subject is unresponsive or likely unresponsive to treatment and wherein a second level that is decreased compared to a first level indicates that the subject is responsive or likely responsive to treatment. 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. This method may also be performed by determining a level of a hexanucleotide repeat-containing RNA or the presence or absence of a focus or foci of a hexanucleotide repeat-expansion-containing RNA in addition to or in place of the level of di- amino acid protein.

As used herein, "elevated" means that the level of one or more di-amino acid-repeat- containing proteins or a hexanucleotide repeat-containing RNA is above a control level, such as a pre-determined threshold or a level of one or more di-amino acid-repeat-containing proteins or a hexanucleotide repeat-containing RNA in a control sample. Controls and control levels are described in detail herein. An elevated level includes a level that is, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more above a control level. An elevated level also includes increasing a phenomenon from a zero state (e.g., no or undetectable di-amino acid-repeat- containing protein expression or hexanucleotide repeat-containing RNA expression) to a nonzero state (e.g., some or detectable di-amino acid-repeat-containing protein expression or hexanucleotide repeat-containing RNA).

As used herein, "decreased" means that the level of one or more di-amino acid-repeat- containing proteins or a hexanucleotide repeat-containing RNA is below a control level, such as a pre-determined threshold or a level of one or more di-amino acid-repeat-containing proteins or a hexanucleotide repeat-containing RNA in a control sample. Controls and control levels are described in detail herein. A decreased level includes a level that is, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more below a control level. A decreased level also includes decreasing a phenomenon from a non-zero state (e.g., some or detectable di-amino acid- repeat-containing protein expression or hexanucleotide repeat-containing RNA) to a zero state (e.g., no or undetectable di-amino acid-repeat-containing protein expression or hexanucleotide repeat-containing RNA expression).

Hexanucleotide Repeat- Containing RNAs and Di- Amino Acid Repeat- Containing Proteins

As described herein, an expanded GGGGCC hexanucleotide repeat sequence within the intron of the C90RF72 gene was found to be transcribed such that RNA transcripts containing the hexanucleotide repeat in both the sense and anti-sense direction were produced. The GenBank Gene ID for the human C90RF72 gene is 203228. Both the sense and anti- sense hexanucleotide repeat-containing transcripts were found to undergo translation independent of an AUG start codon (repeat-associated non-ATG (RAN) translation) such that poly- (Gly- Ala), poly-(Gly-Pro), poly-(Gly-Arg), poly-(Pro-Ala), or poly- (Pro- Arg) di-amino 5 acid repeat-containing proteins were produced, depending on the frame of the hexanucleotide repeat being read (5' -GGGGCC-3', 5'-GGGCCG-3', and 5'-GGCCGG-3' on the sense transcript, 5' -GGCCCC-3', 5'-GCCCCG-3', and 5'-CCCCGG-3' on the anti-sense transcript, see FIG. 1). In addition, the anti-sense hexanucleotide repeat-containing transcript was found to be translated through ATG-initiated translation to produce Met...poly-(Pro-Arg) and o Met...poly-(Gly-Pro) proteins.

Accordingly, aspects of the invention relate to the sense and anti- sense RNAs containing an expanded hexanucleotide repeat and uses thereof. The sense RNA is a 5'- GGGGCC-3' hexanucleotide repeat-containing RNA and the anti-sense RNA is a 5'- GGCCCC-3' hexanucleotide repeat-containing RNA.

5 The 5 ' -GGGGCC -3 ' and 5 ' GGCCCC-3 ' hexanucleotide repeat-containing RNAs comprise a repeat nucleic acid sequence of the formula (GGGGCC) _X or (GGCCCC) _X , respectively, where X may be at least 10, at least 20, at least 25, or at least 30, or in 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- o containing RNA may further comprise additional N- and/or C-terminal nucleic acids. In

some embodiments, an N-terminal nucleic sequence comprises a nucleic acid sequence upstream of the 5 '-GGGGCC-3' hexanucleotide repeat within the intron of the C90RF72 for the sense transcript or a nucleotide sequence upstream of the 5 '-GGCCCC-3' hexanucleotide repeat within the intron of the C90RF72 for the anti-sense transcript. In some embodiments, 5 a C-terminal nucleic acid sequence comprises a nucleotide sequence downstream of the 5'- GGGGCC-3' hexanucleotide repeat within the intron of the C90RF72 for the sense transcript or a nucleotide sequence downstream of the 5 '-GGCCCC-3' hexanucleotide repeat within the intron of the C90RF72 for the anti-sense transcript.

Other aspects of the invention relate to one or more di-amino acid repeat-containing n nrotein and uses thereof. The one or more di-amino 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.

The sense 5'-GGGGCC-3' hexanucleotide repeat-containing RNA and the anti-sense 5'-GGCCCC-3' hexanucleotide repeat-containing RNA both encode poly-(Gly-Pro) proteins. 5 Accordingly a poly- (Gly- Pro) protein may include a protein translated from the sense strand, the anti-sense strand, or both. It is predicted that the C-terminus of the sense and anti-sense translated poly-(Gly-Pro) proteins may differ (see Table 1). Accordingly, a sense poly-(Gly- Pro) protein may comprise the poly- (Gly- Pro) a C-terminal sequence as described in Table 1, while an anti-sense poly- (Gly- Pro) protein may comprise the repeat region with no additional o C-terminal sequence. Methods described herein may comprise use of a poly-(Gly-Pro)

protein translated from the sense strand, the anti-sense strand, or both. Antibodies described herein may be specific for a poly-(Gly-Pro) protein translated from the sense strand, the anti- sense strand, or both.

Each di-amino acid repeat-containing protein comprises a repeat amino acid5 sequence, which contains a di-amino acid repeat unit of the formula (YZ) _X , where X can be from 2-10,000, 5-10,000, 2-5,000, 5-5,000, 2-1000, 5-1000, 5-500, 5-300, 5-200, 10-500, 10- 300, or 10-200. The di-amino acid repeat unit for each di-amino acid repeat-containing protein is provided in Table 1. o Table 1. Di- Amino Acid-Repeat-Containing Proteins

RWRVGE (SEQ ID NO: 2, sense),

PGRGRGGPGGGPGAGLRLRCLRPRRRRRR RWRVGE (SEQ ID NO: 28, sense) or none (anti- sense)

poly-(Gly-Arg) (GR) _X or (RG) _X GVVGAGPGAGPGRGCGCGACARGGGGA

GGGEWVSEEAASWRVAVWGSAAGKRRG

(SEQ ID NO: 3) or

RGVVGAGPGAGPGRGCGCGACARGGGG AGGGEWVSEEAASWRVAVWGSAAGKRR

G (SEQ ID NO: 29)

poly-( Pro- (AP) _X or (PA) _X PSARLLSSRACYRLRLFPSLFSSG (SEQ ID Ala) NO: 4) OR

APSARLLSSRACYRLRLFPSLFSSG (SEQ ID NO: 30)

poly- (Pro- Arg) (PR) _X or (RP) _X PLARDS (SEQ ID NO: 5) or RPLARDS (SEQ

ID NO: 31)

Met...poly- (PR)x PLARDS (SEQ ID NO: 5)

(Pro-Arg)

Met...poly- (GP) _X None

(Gly-Pro)

X=number of repeats of the sequence in the parentheses

Each di-amino acid repeat-containing protein may further comprise an N- and/or C- terminal amino acid sequence that comprises a non-di-amino acid repeat sequence. In some embodiments, a N-terminal amino acid sequence comprises an amino acid sequence translated from a nucleotide sequence of a C90RF72 RNA transcript, such as a nucleotide sequence upstream of the 5'-GGGGCC-3' hexanucleotide repeat within the intron of the C90RF72 for the sense transcript or a nucleotide sequence upstream of the 5' -GGCCCC-3' hexanucleotide repeat within the intron of the C90RF72 for the anti-sense transcript. In some embodiments, a C-terminal amino acid sequence comprises an amino acid sequence translated from a nucleotide sequence of a C90RF72 RNA transcript, such as a nucleotide sequence downstream of the 5'-GGGGCC-3' hexanucleotide repeat within the intron of the C90RF72 for the sense transcript or a nucleotide sequence downstream of the 5'-GGCCCC- 5 3' hexanucleotide repeat within the intron of the C90RF72 for the anti-sense transcript. Such a nucleotide sequence downstream of the 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat may be translated until a stop codon or multiple stop codons are reached.

A portion of a C90RF72 gene sequence (sense and anti-sense) is shown below. The 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat is underlined and in bold. The

10 nucleotide sequence upstream of the 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat precedes the underlined and bolded sequence. The nucleotide sequence downstream of the 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat follows the underlined and bolded sequence. It is to be understood that this 5'-GGGGCC-3' or 5'-GGCCCC-3' hexanucleotide repeat can be repeated more than the number of times present in these

15 sequences.

C90RF72 (partial sequence, sense)

CCCCATTTCGCTAGCCTCGTGAGAAAACGTCATCGCACATAGAAAACAGACAGA CGTAACCTACGGTGTCCCGCTAGGAAAGAGAGGTGCGTCAAACAGCGACAAGTT

2 o CCGCCCACGTAAAAGATGACGCTTGGTGTGTCAGCCGTCCCTGCTGCCCGGTTGC TTCTCTTTTGGGGGCGGGGTCTAGCAAGAGCAGGTGTGGGTTTAGGAGGTGTGTG TTTTTGTTTTTCCCACCCTCTCTCCCCACTACTTGCTCTCACAGTACTCGCTGAGG GTGAACAAGAAAAGACCTGATAAAGATTAACCAGAAGAAAACAAGGAGGGAAA CAACCGCAGCCTGTAGCAAGCTCTGGAACTCAGGAGTCGCGCGCTAGGGGCCG

25 GGGCCGGGGCCGGGGCGTGGTCGGGGCGGGCCCGGGGGCGGGCCCGGGGCGG GGCTGCGGTTGCGGTGCCTGCGCCCGCGGCGGCGGAGGCGCAGGCGGTGGCGAG TGGGTGAGTGAGGAGGCGGCATCCTGGCGGGTGGCTGTTTGGGGTTCGGCTGCC GGGAAGAGGCGCGGGTAGAAGCGGGGGCTCTCCTCAGAGCTCGACGCATTTTTA CTTTCCCTCTCATTTCTCTGACCGAAGCTGGGTGTCGGGCTTTCGCCTCTAGCGAC

^■ \ n TGGTGGAATTGCCTGCATCCGGGCCCCGGGCTTCCCGGCGGCGGCGGCGGCGGC GGCGGCGCAGGGACAAGGGATGGGGATCTGGCCTCTTCCTTGCTTTCCCGCCCTC AGTACCCGAGCTGTCTCCTTC (SEQ ID NO: 6)

C90RF72 (partial sequence, anti- sense)

5 GAAGGAGACAGCTCGGGTACTGAGGGCGGGAAAGCAAGGAAGAGGCCAGATCC CCATCCCTTGTCCCTGCGCCGCCGCCGCCGCCGCCGCCGCCGGGAAGCCCGGGGC CCGGATGCAGGCAATTCCACCAGTCGCTAGAGGCGAAAGCCCGACACCCAGCTT CGGTCAGAGAAATGAGAGGGAAAGTAAAAATGCGTCGAGCTCTGAGGAGAGCC CCCGCTTCTACCCGCGCCTCTTCCCGGCAGCCGAACCCCAAACAGCCACCCGCCA

1 o GGATGCCGCCTCCTCACTCACCCACTCGCCACCGCCTGCGCCTCCGCCGCCGCGG

GCGCAGGCACCGCAACCGCAGCCCCGCCCCGGGCCCGCCCCCGGGCCCGCCCCG ACCACGCCCCGGCCCCGGCCCCGGCCCCTAGCGCGCGACTCCTGAGTTCCAGA GCTTGCTACAGGCTGCGGTTGTTTCCCTCCTTGTTTTCTTCTGGTTAATCTTTATCA GGTCTTTTCTTGTTCACCCTCAGCGAGTACTGTGAGAGCAAGTAGTGGGGAGAGA

15 GGGTGGGAAAAACAAAAACACACACCTCCTAAACCCACACCTGCTCTTGCTAGA CCCCGCCCCCAAAAGAGAAGCAACCGGGCAGCAGGGACGGCTGACACACCAAG CGTCATCTTTTACGTGGGCGGAACTTGTCGCTGTTTGACGCACCTCTCTTTCCTAG CGGGACACCGTAGGTTACGTCTGTCTGTTTTCTATGTGCGATGACGTTTTCTCACG AGGCTAGCGAAATGGGG (SEQ ID NO: 7)

20 In some embodiments, a Met...poly-(Pro-Arg) or Met...poly-(Gly-Pro) protein

comprises an N-terminal amino acid sequence comprising an N-terminal methionine. In some embodiments, a Met...poly-(Pro-Arg) protein comprises an N-terminal amino acid sequence comprising

MQAIPPVARGESPTPSFGQRNERESKNASSSEESPRFYPRLFPAAEPQTATRQDAASSL

25 THSPPPAPPPPRAQAPQPQPRPGPAPGPAPTT (SEQ ID NO: 41 ) or a fragment thereof, wherein the sequence is N-terminal to a poly-(Pro-Arg) repeat amino acid sequence. In some embodiments, a Met...poly-(Gly-Pro) protein comprises an N-terminal amino acid sequence comprising

MRGKVKMRRALRRAPASTRASSRQPNPKQPPARMPPPHSPTRHRLRLRRRGRRHRN

■ n PAPGPPPGPPRPRP (SEQ ID NO: 42), MRRALRRAPASTRASSRQPNPKQPPARMPPPHSPTRHRLRLRRRGRRHRNRSPAPGP PPGPPRPRP (SEQ ID NO: 43),

MPPPHSPTRHRLRLRRRGRRHRNRSPAPGPPPGPPRPRP (SEQ ID NO: 44), or a fragment thereof, wherein the sequence is N-terminal to a poly-(Gly-Pro) repeat amino acid 5 sequence.

In some embodiments, a C-terminal amino acid sequence comprises a C-terminus amino acid sequence shown in Table 1 or a fragment of a C-terminus amino acid sequence shown in Table 1. It is to be understood that C-terminal amino acid sequences other than those in Table 1 are also contemplated,

l o Exemplary di-amino acid repeat-containing proteins may comprise a sequence

provided in Table 2.

Table 2.

(GA) _x WSGRARGRARGGAAVAVPAPAAAEAQAVASG (SEQ ID NO: 8)

(AG) _x AWSGRARGRARGGAAVAVPAPAAAEAQAVASG (SEQ ID NO: 9)

(GP) _x GRGRGGPGGGPGAGLRLRCLRPRRRRRRRWRVGE (SEQ ID NO: 10)

(PG) _x PGRGRGGPGGGPGAGLRLRCLRPRRRRRRRWRVGE (SEQ ID NO: 11)

(GR) _x GVVGAGPGAGPGRGCGCGACARGGGGAGGGEWVSEEAASWRVAVWG SAAGKRRG (SEQ ID NO: 12)

(RG) _x RGVVGAGPGAGPGRGCGCGACARGGGGAGGGEWVSEEAASWRVAVW GSAAGKRRG (SEQ ID NO: 13)

(AP) _x APSARLLSSRACYRLRLFPSLFSSG (SEQ ID NO: 14)

(PA) _x PSARLLSSRACYRLRLFPSLFSSG (SEQ ID NO: 15)

(PR) _x PLARDS (SEQ ID NO: 16)

(RP) _x RPLARDS (SEQ ID NO: 17)

MQAIPPVARGESPTPSFGQRNERESKNASSSEESPRFYPRLFPAAEPQTATRQDA ASSLTHSPPPAPPPPRAQAPQPQPRPGPAPGPAPTT(PR)xPLARDS (SEQ ID NO: 32)

MRGKVKMRRALRRAPASTRASSRQPNPKQPPARMPPPHSPTRHRLRLRRRGR RHRNRSPAPGPPPGPPRPRP(GP)x (SEQ ID NO: 33)

MRRALRRAPASTRASSRQPNPKQPPARMPPPHSPTRHRLRLRRRGRRHRNRSP APGPPPGPPRPRP(GP)x (SEQ ID NO: 34)

MPPPHSPTRHRLRLRRRGRRHRNRSPAPGPPPGPPRPRP(GP)x (SEQ ID NO: 35)

X = a number between 2-10,000, 5-10,000, 2-5,000, 5-5,000, 2-1000, 5-1000, 5-500, 5-300,

5-200, 10-500, 10-300, or 10-200.

In some embodiments, the one or more di-amino acid repeat-containing proteins are 5 selected from the poly-(Pro-Ala), poly-(Gly-Pro), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly-(Gly-Pro) protein. In some embodiments, the one or more di-amino acid repeat- containing proteins are selected from the poly-(Pro-Ala), poly-(Pro-Arg) protein, Met...poly- (Pro-Arg) or Met...poly-(Gly-Pro) protein.

In some embodiments, the one or more di-amino acid repeat-containing proteins is two or o more, three or more, four or more, or five or more, or six or more, seven or more, or eight di- amino acid repeat-containing proteins.

Subjects

Aspects of the disclosure relate to identification and treatment of a subject, such as a5 human, with ALS or FTD or likely to develop ALS or FTD. In some embodiments, a subject may have ALS. In some embodiments, a subject may have one or more symptoms of ALS, such as difficulty breathing, difficulty swallowing, muscle cramps, muscle contractions, muscle weakness, paralysis, speech problems, or weight loss. In some embodiments, a subject may not have any symptoms of ALS. In some embodiments, a subject may have a o family history of ALS .

In some embodiments, a subject may have frontotemporal dementia (FTD). In some embodiments, a subject may have one or more symptoms of FTD, such as lethargy, aspontaneity, disinhibition, loss of empathy and other interpersonal skills, apathy, progressive nonfluent aphasia, semantic dementia, binge eating, compulsive behavior, tremor, rigidity, muscle spasums, poor coordination, difficulty swallowing, and muscle weakness. In some embodiments, a subject may not have any symptoms of FTD. In some embodiments, a subject may have a family history of FTD.

In some embodiments, a subject may have GGGGCC hexanucleotide repeats within one or both alleles of a C90RF72 gene (NCBI Entrez Gene ID: 203228). In some embodiments, GGGGCC hexanucleotide repeats are within a promoter and/or intron of the C90RF72 gene. In some embodiments, the number of GGGGCC hexanucleotide repeats is greater than 25, 50, 100, 150, 200, 250, 300, 500, 5,000, 10,000 or more. The number of repeats may be detected using any assay known in the art, e.g., using as a nucleic acid-based assay such as a southern blot (see, e.g., Dejesus-Hernandez et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C90RF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245 (2011); Renton et al. A hexanucleotide repeat expansion in C90RF72 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)).

Controls and Control levels

Aspects of the disclosure relate to comparison of a level of one or more di-amino acid repeat-containing proteins and/or hexanucleotide repeat-containing RNAs to a control level. In some embodiments, the control level is a level of one or more di-amino acid repeat- containing proteins and/or hexanucleotide repeat-containing RNAs in sample, such as a fluid sample or tissue sample, obtained from a healthy subject or population of healthy subjects. In some embodiments, the sample is a blood sample. As used herein, a healthy subject is a subject that is apparently free of disease and has no history of disease, such as ALS or FTD. In some embodiments, a healthy subject is a subject that has 25 or fewer GGGGCC hexanucleotide repeats within a C90RF72 gene.

In some embodiments, a control level is a level of one or more di-amino acid repeat- containing proteins and/or hexanucleotide repeat-containing RNAs that is undetectable or below a background/noise level obtained using standard methods of detection (e.g., Western blot, qPCR, northern blot, or immunohistochemistry). Such a level could be obtained, for example, by measuring a level of one or more di-amino acid repeat-containing proteins and/or hexanucleotide repeat-containing RNAs in a sample that is known to be free of the di- 5 amino acid repeat-containing proteins and/or hexanucleotide repeat-containing RNAs.

The disclosure also involves comparing the level of one or more di-amino acid repeat- containing proteins and/or hexanucleotide repeat-containing RNAs with a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. It can be single cut-off value, such as a median or o mean. It can be established based upon comparative groups, such as where one defined

group is known not to have ALS or FTD and another defined group is known to have ALS or FTD. It can be a range, for example, where the tested population is divided equally (or unequally) into groups, such as a subject that has 25 or fewer GGGGCC hexanucleotide repeats, a subject that has 25-50 GGGGCC hexanucleotide repeats, and a subject that has 505 or more GGGGCC hexanucleotide repeats.

Samples

Aspects of the disclosure relate to determining a level of one or more di-amino acid repeat-containing proteins in a blood sample (e.g., whole blood, plasma, or serum) obtained o from a subject. The blood sample may be obtained by any method known in the art, e.g., using a needle or fingerprick device. The blood may be processed before use in the methods described herein. Such processing includes, for example, addition of an anti-coagulant, removal of blood cells, and/or freezing of the blood. However, it should be appreciated that other samples may be used, such as a tissue sample (e.g., brain tissue) or other fluid samples 5 such as saliva, or urine.

Other aspects of the disclosure relate to determining a level of hexanucleotide repeat- containing RNA in sample obtained from a subject. The sample may be a fluid or 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). Assays

Aspects of the disclosure relate to performing an assay to determine a level or presence/absence of one or more di-amino acid repeat-containing proteins and/or

hexanucleotide repeat-containing RNAs. Assays known in the art for detecting proteins and RNAs (see, e.g., 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, F.M. 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, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York) can be used alone or in combination with techniques and compositions described herein for measuring a di-amino acid repeat- containing protein level.

Assays for detecting protein levels include, but are not limited to, immunoassays (also referred to herein as immune-based or immuno-based assays, e.g., Western blot,

immunohistochemistry and ELISA assays), Mass spectrometry, and multiplex bead-based assays. Such assays for protein level detection are well-known in the art. Other examples of protein detection and quantitation methods include multiplexed immunoassays as described for example in U.S. Patent Nos. 6939720 and 8148171, and published US Patent Application No. 2008/0255766, and protein microarrays as described for example in published US Patent Application No. 2009/0088329, all of which are incorporated herein by reference in their entirety.

Any suitable binding partner for a di-amino acid repeat-containing protein is contemplated for detection of a di-amino acid repeat-containing protein level. In some embodiments, the binding partner is any molecule that binds specifically to a di-amino acid repeat-containing protein as described herein. As described herein, "binds specifically to a di-amino acid repeat-containing protein" means that the molecule is more likely to bind to a portion of or the entirety of a di-amino acid repeat-containing protein than to a portion of or the entirety of a non-di-amino acid repeat-containing protein. In some embodiments, the binding partner is an antibody or antigen-binding fragment thereof, such as Fab, F(ab)2, Fv, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, scFv, or dAb fragments. Methods for producing antibodies and antigen-binding fragments thereof are well known in the art (see, e.g., 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). Binding partners also include other peptide molecules and aptamers that bind specifically to a di-amino acid repeat-containing protein. Methods for producing peptide molecules and aptamers are well known in the art (see, e.g., published US Patent Application No. 2009/0075834, US Patent Nos. 7435542, 7807351, and 7239742). The binding partner may comprise a label including, but not limited to, a fluorescent, enzymatic, affinity or isotopic label.

In some embodiments, an assay comprises an immuno-based assay. In some embodiments, the immuno-based assay comprises an isolated antibody specific for one or more di-amino acid repeat-containing proteins. In some embodiments, the isolated antibody specific for one or more di-amino acid repeat-containing proteins is an isolated antibody as described herein in further detail. In some embodiments, the isolated antibody specific for one or more di-amino acid repeat-containing proteins is an isolated antibody specific for an antigen or sequence, or a fragment of an antigen or sequence described in Table 1, Table 2 or Table 3.

Accordingly, a di-amino acid repeat-containing binding partner (e.g., a di-amino acid repeat- containing -specific antibody) can be labeled with a detectable moiety.

Assays for detecting RNA include, but are not limited to, hybridization-based assays such as Northern blot analysis, RT-PCR, sequencing technology, RNA in situ hybridization (using e.g., DNA or RNA probes to hybridize to 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 microarray (e.g., by hybridization of polynucleotide sequences derived from a sample to oligonucleotides attached to a solid surface (e.g., a glass wafer) with addressable locations, such as an Affymetrix microarray (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 partners bind to a part of or an entire nucleic acid sequence of a hexanucleotide repeat-containing RNA provided herein.

5

Treatment

As described herein, it was found that di-amino acid repeat-containing proteins were present in samples of blood from patients with ALS. Without wishing to be bound by theory or mechanism, it is believed that di-amino acid repeat-containing proteins in the blood of o subjects with ALS accumulate within the brain parenchyma over time, leading to

neuroinflammatory changes, CNS dysfunction, and neuronal death. Accordingly, aspects of the disclosure relate to treatment of a subject having ALS or FTD by decreasing or stabilizing di-amino acid repeat-containing protein levels in the blood of the subject.

In some embodiments, decreasing or preventing an increase of the level of one or5 more di-amino acid repeat-containing proteins comprises removing the one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) di-amino acid repeat-containing proteins from the blood of the subject. In some embodiments, the one or more di-amino acid repeat-containing proteins from the blood of the subject is removed using a procedure selected from plasmapheresis or a bone marrow transplantation. In some embodiments, it may be advantageous to decrease or prevent an o increase of the level of all di-amino acid repeat-containing proteins expressed by a subject.

Accordingly, in some embodiments, a method comprises decreasing or preventing an increase of the level of all forms of di-amino acid repeat-containing proteins expressed by a subject.

In some embodiments, the one or more di-amino acid repeat-containing from the 5 blood of the subject is removed using a hematopoietic stem cell (HSC) transplantation. HSC transplantation is the transplantation of hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood, into a subject. The source of

hematopoietic stem cells may be allogeneic (e.g., from a donor such as a healthy subject). Methods of HSC transplantation are well known in the art (see, e.g., Bishop MR, Pavletic SZ. n Hematonoietic 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).

In order to prepare a subject for HSC transplantation, the HSCs present in the subject may be removed or depleted so that the transplanted cells can become the dominant HSC population in the subject. HSCs in the subject may be depleted, for example, by treating the subject with a chemotherapy, radiation, or both in order to cause the HSC cells of the subject to undergo apoptosis or cell cycle arrest.

In allogeneic HSC transplantation, the HSCs are obtained from a donor. The donor is preferably a healthy subject, such as a subject that is apparently free of disease and has no history of disease, such as ALS or FTD. It is preferable that the donor is HLA-compatible with the subject receiving the transplant in order to reduce the risk of graft versus host disease. HLA-compatibility can be determined, e.g., using HLA typing. HLA typing generally involves examination of at least 8 HLA markers: two A, two B, two C, and two DRBl markers, and optionally also two DQ markers. HLA typing can be accomplished, e.g., through a blood test. HLA allele identities can be determined using serology or a nucleic acid-based assay. Generally, a match of at least 4-6 markers between host and donor is preferred. In some embodiments, the donor is a subject that has 25 or fewer GGGGCC hexanucleotide repeats within a C90RF72 gene.

HSCs can be obtained from a donor using any method known in the art. Exemplary methods include bone marrow harvest and leukapheresis (see, e.g., 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 a bone marrow harvest, the bone marrow is typically removed from the back of one or both hip bones of the donor.

Leukapheresis involves separation of HSCs from blood obtained from the donor using, e.g., continuous flow centrifugation or filtering. The growth factor G-CSF may be administered to the donor to stimulate the growth of new HSCs so that more HSCs are present in the blood. Once obtained, the allogeneic HSCs are then administered to the subject receiving the transplant. Any suitable method of administration known in the art is contemplated, e.g., by central venous catheter.

In some embodiments, during or after HSC transplantation, the subject receiving the HSC transplant may receive additional treatments and/or therapies, such as antibiotics,

5 antifungals, antivirals, blood transfusions and/or immunosuppressive therapies. Such

treatments and/or therapies may help to prevent infection and/or graft versus host disease during a HSC transplant recovery period.

In some embodiments, the HSC transplantation is bone marrow transplantation. In some embodiments, the bone marrow transplantation is an allogeneic bone marrow

o transplantation.

Plasmapheresis is a medical procedure that occurs outside the body (an

"extracorporeal therapy") and refers to the removal, treatment, and return of (components of) blood plasma from blood circulation. Plasmapheresis is well-known in the art and has been used to treat several diseases including Goodpasture's syndrome, myasthenia gravis,

5 Guillain-Barre syndrome, lupus, and thrombotic thrombocytopenic purpura (see, e.g.,

Madore, Plasmapheresis Technical aspects and indications, Crit Care Clin 18: 375-392. 2002). During plasmapheresis, blood is initially taken out of the body, e.g., through a needle or previously implanted catheter. Plasma is then separated from the blood cells, e.g., by using a cell separator. After plasma separation, the blood cells are combined with a

o replacement fluid and readministered to the subject. The replacement fluid may be either the separated plasma treated to remove disease-associated components or a replacement plasma (also called plasma exchange).

Exemplary procedures used to separate the plasma from the blood cells include:

1) Discontinuous flow centrifugation: One venous catheter line is used. Typically, one 5 or more batches of blood are removed at a time and centrifuged to separate plasma from

blood cells. The blood cells are then combined with the replacement fluid and returned to the subject.

2) Continuous flow centrifugation: Two venous lines are used. Plasma is continuously spun out of the blood and the separated blood cells are fed through a line that combines with n a replacement fluid before return to the subject. 3) Plasma filtration: Two venous lines are used. The plasma is filtered using standard hemodialysis equipment, e.g., a parallel-plate or hollow-fiber filter. The separated blood cells are fed through a line that combines with a replacement fluid before return to the subject. The filters usually have pores of 0.2-0.6 μιη diameter, sufficient to allow passage of plasma, 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).

If the separated plasma is to be used as the replacement fluid, the separated plasma is first treated to decrease the levels of di-amino acid repeat-containing proteins present in the separated plasma. In some embodiments, decreasing the levels of di-amino acid repeat- containing proteins present in the separated plasma comprises contacting the separated plasma with one or more isolated antibodies specific for a di-amino acid repeat-containing protein as described herein, whereby the di-amino acid repeat-containing proteins present in the separated plasma bind to the one or more isolated antibodies. In some embodiments, a binding partner for the one or more isolated antibodies is contacted with the separated plasma. A binding partner for the one or more isolated antibodies may be, for example, a capture moiety such as biotin or streptavidin, protein A, or a secondary antibody specific for the one or more isolated antibodies. Such binding partners allow for the one or more isolated antibodies to be removed from the separated plasma.

In some embodiments, the one or more isolated antibodies are attached to a filter, column, and/or solid support. In such embodiments, the separated plasma is contacted with the filter, column, and/or solid support, whereby the di-amino acid repeat-containing proteins bind to the isolated antibodies attached to the filter, column and/or solid support.

Without wishing to be bound by theory, it is believed that the di-amino acid repeat-containing proteins may form aggregates in the blood. Accordingly, the di-amino acid repeat-containing proteins may be removed from the separated plasma using a filter, such that the aggregates are isolated from the separated plasma.

Tn some embodiments, a subject expressing one or more di-amino acid repeat- containing proteins may develop autoantibodies. In some embodiments, autoantibodies to one or more di-amino acid repeat-containing proteins may be removed from the separated plasma. Autoantibodies may be removed using any method known in the art, e.g., using a binding partner (e.g., bound to a solid support or attached to a tag) that recognizes the autoantibodies. In some embodiments, the binding partner may be one or more di-amino acid repeat-containing proteins as described herein.

If plasma exchange is to be used, the subject receives replacement plasma.

Replacement plasma may be, e.g., donor plasma or a solution of albumin (e.g., 5-70% albumin in saline). An exemplary replacement plasma is 5% albumin combined with 0.9% saline in a 50%:50% (vohvol) solution. Medication to keep the blood from clotting (e.g., an anticoagulant such as citrate, acid-citrate dextrose or heparin) may be given to the subject or contacted with the blood of the subject during the procedure.

In some embodiments, decreasing or preventing an increase of the level of one or more di-amino acid repeat-containing proteins comprises decreasing a level of a

hexanucleotide repeat-containing RNA. Decreasing a level of a hexanucleotide repeat- containing RNA may comprise administration of an effective amount of an inhibitory nucleic acid molecule such as an shRNA, an siRNA, miRNA, or an antisense nucleic acid molecule that targets the hexanucleotide repeat-containing RNA.

Methods for producing shRNAs, siRNAs, miRNAs, and antisense nucleic acid molecules are well known in the art (see e.g., Sambrook, Fritsch and Maniatis,

MOLECULAR CLONING: A LABORATORY MANUAL, (Current Edition); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (Current Edition)); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition). In some embodiments, a nucleic acid inhibitor comprises or corresponds to at least a portion of sequence of a target hexanucleotide repeat-containing RNA sequence or comprises at least a portion of a sequence complementary to a target hexanucleotide repeat-containing RNA sequence.

In some embodiments, treatment may comprise decreasing or stabilizing a level of an autoantibody to one or more di-amino acid repeat-containing proteins in a subject. A level of autoantibody may be decreased or stabilized using any method known in the art. In some embodiments, decreasing or stabilizing a level of an autoantibody comprises administration of an effective amount of atacicept, belimumab, blisibimod, BR3-Fc, rituximab, ocrelizumab, atumumab, epratuzumab, corticosteroid (e.g., prednisone), mycophenolic acid, methotrexate, cyclophosphamide, azathioprine, and/or cyclosporin. In some embodiments, decreasing or stabilizing a level of an autoantibody comprises plasmapheresis.

Antibodies

Aspects of the disclosure relate to isolated antibodies specific for a di-amino acid repeat-containing protein (e.g., a RAN protein) selected from a poly- (Gly- Ala), poly-(Gly- Pro), poly- (Gly- Arg), poly-(Pro-Ala), poly- (Pro- Arg), Met...poly-(Pro-Arg) or Met...poly- (Gly-Pro) protein. The isolated antibody may recognize a region or regions of the di-amino acid repeat-containing protein (such as a repeat sequence or the C-terminus) or may recognize the entire di-amino acid repeat-containing protein.

An antibody that "specifically binds" to a target or an epitope is a term understood in the art, and methods to determine such specific binding are also known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically binds to a poly- (Gly- Ala) protein or an epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood by reading this definition 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 can include) exclusive binding. Generally, but not necessarily, reference to binding means specific binding. In some embodiments, antibodies described herein have a suitable binding affinity to a di-amino acid repeat-containing protein (e.g., a RAN protein). As used herein, "binding affinity" refers to the apparent association constant or K _A . The K _A is the reciprocal of the dissociation constant (K _D ). The antibody 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 lower. An increased binding affinity corresponds to a decreased K _D . Higher affinity binding of an antibody to a first target relative to a second target can be indicated by a higher K _A (or a smaller numerical value K _D ) for 5 binding the first target than the K _A (or numerical value K _D ) for binding the second target. In such cases, the antibody has specificity for the first target (e.g., a protein in a first

conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, i o 70, 80, 91, 100, 500, 1000, 10,000 or 10 ⁵ fold.

Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in, e.g., TRIS-buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaC12 at pH7.5).

15 These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein

([Bound]) is related to the concentration of free target protein ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation:

20 [Bound] = [N][Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K _A , though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K _A , and thus can be used for

25 comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

In some embodiments, the isolated antibody is specific for a di-amino acid repeat- containing protein selected from a poly- (Pro- Ala) poly-(Pro-Arg), Met...poly-(Pro-Arg) or

30 Met... poly- (Gly- Pro) protein. In some embodiments, the isolated antibody is specific for an antigen comprising a di- amino acid repeat and/or C-terminus sequence or fragment thereof as defined in Table 1. In some embodiments, the isolated antibody is specific for an antigen comprising a sequence or fragment of a sequence defined in Table 2.

In some embodiments, the isolated antibody is specific for an antigen in Table 3 or in

FIG. 28. In some embodiments, an antigen in Table 3 does not contain an N- and/or C- terminal modification.

Table 3. Di- Amino Acid Repeat- Containing Protein Antigens

Poly-( Pro-Arg) GGGGCC- Ac-CRPRPLARDS-OH (SEQ ID C-terminus

AS F2 CT NO: 25)

Poly-(Gly-Ala) GGGGCC F2 Ac-CSGRARGRARGGA-amide C-terminus

CT (SEQ ID NO: 26)

Fl = reading frame 1, F2 = reading frame 2, F3 = reading frame 3, AS F = anti-sense reading frame 1, AS F2 = anti-sense reading frame 2, AS F3 = anti-sense reading frame 3.

An isolated antibody may be a monoclonal or polyclonal antibody, or an antigen- binding fragment thereof. An antigen-binding fragment thereof includes, for example, an Fab, F(ab)2, F(ab')2, Fv, single chain antibody, Fab fragment, sFab fragment, Fd fragment, scFv, or dAb fragment. Methods for producing polyclonal and monoclonal antibodies and antigen -binding fragments thereof are well known in the art (see, e.g., 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 encompassed are antibodies made by recombinant means such as chimeric antibodies (variable region and constant region derived from different species) and CDR-grafted antibodies (complementary determining region derived from a different species) as described in U.S. Patent Nos. 4, 816, 567 and 5, 225, 539, which are incorporated herein by reference in their entirety. Also encompassed are humanized antibodies, typically produced by recombinant methods, wherein the human sequences comprise part or all of the antibody. Also included are fully human antibodies, such as those produced in genetically- altered mice (see PCT Application No. 93/12227, which is incorporated herein by reference in its entirety).

In some embodiments, an isolated antibody specific for a di-amino acid repeat-containing protein is a rabbit polyclonal antibody as listed in Table 4.

Table 4. Di- Amino Acid Repeat-Containing Protein Rabbit Polyclonal Antibodies GGGGCC Fl repeat H3148 1,956,500

GGGGCC-AS F2 repeat H3149 2,399,600

GGGGCC-AS F2 repeat H3150 3,225,000

GGGGCC-AS Fl repeat H3151 660,200

GGGGCC-AS Fl repeat H3152 2,082,600

GGGGCC F3 repeat H3154 752,300

GGGGCC F3 repeat H3155 590,500

GGGGCC F3 CT H3156 231,300

GGGGCC F3 CT H3157 616,700

GGGGCC-AS Fl CT H3158 6,300

GGGGCC-AS Fl CT H3159 32,800

GGGGCC Fl CT H3160 573,900

GGGGCC Fl CT H3161 363,000

GGGGCC-AS F2 CT H3162 2,261,700

GGGGCC-AS F2 CT H3163 176,300

GGGGCC F2 CT H3164 1,549,500

GGGGCC F2 CT H3165 115,700

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

Isolated antibodies of the disclosure may also have a detectable label attached thereto. The label may be, for example, a fluorescent, enzymatic, affinity or isotopic label. Examples inrlnHe fluorescein isothiocyanate (FITC) for detection by fluorescence, horseradish peroxidase which allows detection by cleavage of a chromogenic substrate, radioisotopes such as I 125 for detection by autoradiography and avidin/biotin for antibody detection and affinity purification of antigens and antigen-bearing cells.

Also encompassed by the disclosure are hybridoma cell lines producing a monoclonal antibody specific for a di-amino acid repeat-containing protein selected from a poly-(Gly- Ala), poly-(Gly-Pro), poly-(Gly-Arg), poly-(Pro-Ala), poly- (Pro- Arg) protein, Met...poly- (Pro-Arg), Met...poly-(Gly-Pro), a C-terminal peptide of a di-amino acid repeat-containing protein as described herein, and/or a combination of two or more thereof.

In some embodiments, an isolated antibody is an isolated auto-antibody obtained from a subject having ALS, wherein the isolated auto-antibody is specific for one or more di- amino acid repeat-containing proteins as described herein.

In some embodiments, an isolated antibody described herein is contained within a buffered solution. In some embodiments, an isolated antibody described herein is attached to a solid support (e.g., the surface of a plate or a bead).

Transgenic mouse

In another aspect, the disclosure relates to a transgenic mouse comprising a human C90RF72 gene comprising a GGGGCC hexanucleotide repeat sequence. In some embodiments, the mouse comprises a human C90RF72 gene comprising a GGGGCC hexanucleotide repeat sequence and flanking human sequences on the 5' and 3' end of the human C90RF72 gene. In some embodiments, the flanking human sequences on the 5' and 3' end are each independently at least Ikilobases (kB), at least 5kB, at least lOkB, 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' end each independently comprise a promoter capable of driving transcription of the human C90RF72 gene in the sense and anti-sense direction, respectively. Accordingly, in some embodiments, the transgenic mouse expresses both sense and anti-sense transcripts (e.g., 5'-GGGGCC -3' and 5'GGCCCC-3'

hexanucleotide repeat-containing RNAs described herein). In some embodiments, the human C90RF72 gene and flanking sequences comprise the sequence below, wherein (GGGGCC) _n indicates the location of the GGGGCC hexanucleotide repeat sequence: Chr9:27,527,137-27,625,470 (reverse complement)

AAGCTTGATAATATTATCAAATATTAGATAAATGTAATATTAGAAGAAAACTTTTTTGAA AAGATATATAAAAAT AATTTCATTCAAAATTTTTATATTTAATTTAAATTTTTAATGAAAATATATCTAAGTTTT GTACGCTTTAAATGT AATTATGTTTGATAATTTAATCATTTACTATTCGTTCTCTATTGCTGCCCTAACAAATTA CCATAGTTCAGTGGC TTACAAAACACAAATTTATTATCTTACCATTCTGTGAGTCAAAATTCCAAAATAGGTGTC ACTAGGCTAAAATGA AGGACTGCATTTCTTCCTGCAGGCTCCAGGAGAGATCTATGTCTTACTCTTTTCGGCTTC TAAAGGCTGCCCACA TTCCTCGACTAGTGGCGTCCCTCCTTCGTCTCTAAACCCAGCAACAACAGGTTGAGTCCT CATGTCACATCTTTC TTACCTTTCTGTCATCTCATCTCGCTGACTGCTGCTGGGAAAAATTCTCCACTTTTAAGG GCTATCATGATTAGA CTATGCCCACTAGATAATACAAGATCTCAGATCCTTAACTTCCATCACATCTGCAAAGTC GCTTTTGCCTCATAA AAGAGTCTGAGGTTTAGACGGGAGATCTTAAGGGGGCTATTAATATGCCTACCATAATCA CTGAGAATAAGTACA AGTTAAGATTATAATAGCAATAGAATATACAAACGTGAAGCTCCAAAAGAACAACAACAA CAAAAAAGGTGAACA GGAAAAAGAAACTGAAAATCTTTAAAAAGGCAGTCTGTTTAAATCTATAAAAACTGGAAA AAAATGAGAGTGGAC AAATATCTGGTAAGCATGATGGACTTAAAATTTGTGACTAGGGCATTACATTTTTTATAT TAATATAATGAAGAT TGAATTACTGATCAAAACAATTAAAAAGCAAGAGAACTATTCTCATCAAATCTGCAACAC GAAAAGTTCAGACAA AATTCCAACAACTTCACATTCTGAACTAAATGAGGACTAATTACCAGTTCGAGCAATGAG AATATATGAGGTCCT CCGTTTGCACTTTGCCAGGGATCTGAAAACGTTGGGAGTAGGTCGGCTTCACCCTGAAGC CAGACCATCGACAGC CAGTTTTCCCTCCCTTCTCCACCCACAGGTCTTAGGCCCTCATCCTTCCCAGCCTCAGAA CTAGTCTCCAAAGAA GAGGAAAGTTAGAGGAGAGAGTAAATCGTTGAATAGGATGAAGGAGATGTGGGAAAAAGA AAAAGAGAGGCTGCA AGAGAGAGGGTCCCAGGGATAACTCTGCTCTTGGAAGGGTGGCCACAGTCATGTGGTCCC AAGAGGCAACAACAA GCTTAGGAAGCCAGAGAAACCAGTTACAATCACTGCTACTCTTTTCGATTCTGTGTTGTT TAAGAAATATCACCC GCCAGGAGTTCTCCAGAAACATTTTCCCTGATTCCATGTAAGTGCTCAACCAGTGAATGG TAATCCCATTTTGGT TTAGTCTGTACCATCCCCTATTCCAAAATAAAGGGAAAAATGGTGGGTTTATATCTTAAA TTTTCTACTTTACTA AACTCAAGGGAAATAGCCAAGCAAAAACGAAAGCTGAGACTCTTGCTAATTATCCTTTCC ATAGAATGTTTGCTA AAATTCCTTGTCAAGGAAGGAATAACAAAGCTAGTCCACGCTCTGTATAGGGTGTTTCCA ATTAGTTATACTTTA

AAGTATAAGTATTTAACAAAATCTATAAATTTTGTTAATTATTTACTTGTAGTGAAA AATGAGCCATTCTCAAGC AAATCACTTTTTATTACACATTCCAGAGAATAACCATAAAAGGACATTTATTATAGCAAA AATAACCACATCTGG ATGGAACTTCAATCACCAGTATTTACTAAATAAATGCCCAGAAAAAAAATAGTTCATCTT TAATTTCAGTCATCA TTAATAAAAGCTGAAGTACCTCTTCAGATCTTTTGATCATTTTCTGTTGGATTGTTTTCT TTTTACTGAGTTGCA AATGCTCTTTATATATTTTGGATACAAAGCTTTATCACATAGGCATTTTGCAAGTATTTT TTCCAAGTTTTTTTA

TCTTTTCATTTATTTAATAATATCTTTCAAAGAACGGGAATTTTATAATTTTTATGA AGTCCATTTATAATTTTT TCTTTTATGGGTTGGTGGGGGTTGGGGGTTGTGTTGTCCTAAGAAATCTTGGCTCAACAC AAAAAGATTAGTTTC TATATTTTCTTCTAGAAGTTTTATAGTACGATCTCAGATCCATTTCAGATGATGAATAAG CACATAAAAAAAGGA TACTCATCGTTAGTCATTAGAGAAATGCATATTAAAACCATAAGGAAATACTACTATATA CATATATTAGATAGG ATGAAGAGCAACTGGAATCTCATACAGTGCTGATTGAAATGCAAAATGGCAAAACAACTT TAGAAACCAATTTGG AAGCAGCTGTACTGACATGGAATTTTGAGCTGGAAGAATCTTAGAAAAAGAATACTTTAC CACCTCCCCCATTCT CTTCACCCTGGGGAACTGTTAAATGAGGAAATTGTGGTTCAAGGAGGAACTTGTCTATAT GCTTTCTCAGCTTTC CCGTGGTAATTACCATCTTGATAATATAACGTAATGTATGTATATGTTATCAAATAATAT AATATCTTCATCATA TATTTATCATCTTCATAATGTTAGCTGTCTAGTGGTAACTTTTTTTTGCTCTTTATTGCC TCCCTCTTTTTTCCC TCTTTGTTGTTTTTTGTCATACAATTATGATATATGTGTATATATTCTCACTGTAAAGAT GTAAACAACACAAAG

ATTATTGAACAAATCACGAAAGTAACCCTTCCTTCATTCTTACCCTATCCAACCCTC ATCTCCTCAGAAGAATAC ACCATTTTAGTTGTAAATGTTTTTCTAGCTCTTTTTCAATGTTTCTACCTATATGCATGT ATGTATAATGTATAT ACATACATATATACATACATATTGATATATACATATATAGAGGTATGGTTTTTTAACTTA AATGGAATTGCATTG TGGATATTGTCCTATGACTTGCTTTCAACCAAATTATATGTCTTGGAAATACATACATAT ATTTAAAAAATATGT TATGTATATGTAACATACTATATGTGCATAATATATATTACATAGATATAATAAGGCCTA GGAAGAAATTGTGTG CAACCTCTAGTACATCTTCCTCTATATCTACTGTACATACATACAACCCATTCTTTTTTT AATTTTTTTATTTTT TTAGACAGAATCTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCACAATCTCGGCTCACT GCAAGCTCCACCTCC TGGGTTCACGCCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGAATACAGGCACCTGCC ATCAGGCCCAGCTAA TTTTTTTTTGTATTTTTAGTACAGATGGGGTTTCACCGTGTTAGCCAGGATGGTCTCCAT CTCCTGACCTCGTGA TCCGCCCACCTCATCCTCCCAAAGTGCTGGGATTTACAGGCGTGAGCCACCGCGCCCAGC CACAACTCATTGCAG

AGTAGTCCAAAATATGGATGGACTGTAGCTTAATTACTTATTCTCCCATTGATAGAC ACTTAGGACTTTTCTAAT TTTTATAATTTAAAAATATGCTGCAATTAACAAACATTCTTGTGTATCTTTTTGCTGTAT GTATGCATATTTCTT TAGTATGGGTTTTGGAAGAGGAATCACAAAGGAGGCATAGAATATAAATATTTTTATTTT GAAAAATACAGTTGT AATTTAATAACCCACCAAAAGACTCTAACAGTTTAGATTCACATCAACAGTGTAAGAACA TGTCTGTTTTACTGC ATCCTTACCCCCACTGGTTATAATACTTTTAATTAACAATCTTATGGATGAAGAATACTA TCGCAATGTTGTTTT AATGCATTTTTCCAATTACTAGTGAGATTGAACATTAATTCTTTTATTTTATGGATCACT GGCTTTTCTCCTTCT GTGAACTACCTGTTCACATCCTCTGCTTTTCAGCTCTTGAGCTGTTATCTTTTTCTTATT GATTTATATGAGCTC TTTATATATTCAAGATGTTAATCATTTGTATTTTATGTATATGGCAATGATTTTCTTCCA AACCAATGCTTGTCT TTTATTTATTTATTTATTTATTTATTTATTTGAGACCGAGTCTCGCTCTGTCGCCCAGGC TGGAGTGCAGTGGCG CGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCT CCTGAGTAGGTGGGA CTACAGGCGCCCGCTGCCACACCCGGCTAATTTTTTGTATTTTTAGTAGAGACAGGGTTT CACCGTGTTAGCCAG GATGCTCTCTATCTCCTGACCTCGTGATCCGCCCGCCTCGGCCTTCCAAAGTGGTCGGAT TACAGGCATGAGCCA CCACGCCTGGCCAATGCTTGTCTTTTTATCTCTGTTTATGGCATCTTTCATACTATGGAC ATTTTTATTTTTATT TTTTATGTTGATTTATTCTTGAATTGTATACATGTTAATTATACCTAAGTTATTGTAATA CCCTTAAAGCCAAGT TCTACACATATATTTAATTTGCTTTCCCAATAGGTCTCTGAGGGAACACATTTTTTCAAA TCACTTTGTTTCATC TTTTTTAGGTGTTGATCAATTATTAAGGAGTTTGAAATAATCATTTAAACGGAATTCTTC AGATGAAAACATAAA GACATTTATCGGGTCAGAGCATTGGTCGGTTCACATACTCAGGATCAGTGGCCTGGGTGG GCAGGCACTGGGTGA ATGGAGAGCTGCAGGTATTGGAAGAGAGCCCAGTTGGATATGTAGTTTCCAAAGATCATC AAGGCAGACAACCAA AGGGAAACCGTGGGAAACACCTGCTTTGGGCCATCTAAGATGAGATGATAAAGTAAGGAA AGAGTTGAGCCCAAC ACAGTGATAGCCAATCTGAAAGCGGGCAGAACTGACAAGACCAAACAAGTAGGTGAACTG GCTGCAGGCAGCCAG CCACCACAGGGACAGCGTGTACTCCAGGGACAAGCTCAAGGCTATAGGTAGTTAGTTCAA GGCTACTAGGGTGAG AAGAGCAGGAACTGAGTTCTATACCAGTGCTTCTCAAAACTAATGTGCATCCTAATCACC TGGAAATCTTGTAAA AATGTAGATTCTGATTCAGTGAGTCTGAAGCAGAGCTTAAGATACTACATGCTTAACAAG AGCCTAGTTGATGCT GACACTGCTGGTCCCTGGAGCTCTCTTTGAGTAGCAGGCTTCTGGAAGGCTTGTGTCACT AAGCACAGAGAAGCC TCACTTATCAAATCTGCACCAAAACAGGAAAACTAATGTGAAGAATAATGTGATGCACAC GTCAGAGCATGAGGC AGTTGCTTTGTCCCTGAGGTTGCGCTCCAGATGGCTTCCTAAGATGCGACAGGCTGATCT TGTGCGTGGGGGTCC CGGAGGCTTGGGCCACGGGAGAGACAGGACCTCAGAGGCTGGGAGACAGGCAGAGACAGA AGAGTGACATCCTGC

TGCTTTTGAATTTGCACATTCTGTAGAATAATAACAGCAGTAAACTGTTACACAATA TCTATTCTCAGCATCTTG AAGCCCTTTCACATATTGTTACTTCCATTAATGGGGCCCTTTGCTGCTATTTCTACTTTT CTCTTCAGCTATCAA CAATATGGCTTTCCACACCTCCATCAGACAGTAGCCAGATGAAATAAAATGTGCCAGAAT GAAAACTTGTTCATT TGTCTACTTTTTGCCAAGACTAGACAGGCAGGAAATTGAATGTATTTTTACAGAAAAGGT TTTCAAAACTTTTTC CCCTCTGTGGCTCATTTAGGTAAACTAAAAGGCATAAGACCCACCTAAAACATGGGTTCC CGCTTTTTATTGGAG

AAAGAACATAGTACTTTAAAAAAATACATAAAATAATAAAAAGGAAAGACAAAGATA ATGAAGGTTGTACATGGT ACCAAATTTTTGTATCCCATAATAACACATGAGTAGATCACTACTAAGTAGGTTTTAGTG ACATATAGGAAACAT TAAAATCTACAGAAATTTGCATTATTTTCTGTCAAAAAGGATCATTTCACAGCCTTTCAG GGGGAACCCATTGCC CACAGGAACTCATGCATTCCATGCTTTGAGGATCACTAGATCTAAGAAGCCTTCCTTGGA GGTTCTAGCCTCCAA CCCTTATTTTAGTAAAAGAAGCTCCAGTTTTATCTGTTTCTAAGTCAGACTACCACACAA CATTGGGCTTAAAGA AAGGTTTCCAGGGCTAAAGCAGACTTTGAGGATTACTAATTCCGAGTTAAATTTCTGTGT ATTATCTCTGGATTT GACTTATTCACACTGGACTATCACTCATAAATATACATAATACAGAGTTAACTATTTAAA TTTATAAAGAGAGTA TTTTCCTTTTTTATGAGCAAAACATGCTGCCAACTACTTGGACCACATACTGATCCATAA ATACTGACAGCTTTG TAATTGGAAATAATAAATACACACTAATGAAGCATCTCAAAAGGGAAGAGCCACAGGTAA TCTGAGTGATTAGGC ATTCATGTTAGGTTAGGCTTTGATCATTGTTTTTAATCGCAATTTCATTGCAGTGCATCT ATAAATCCATGTCCA

GAAGTATGAAGTGGTTCTATAGTAAGAATAAGATGCTACAGATAATGCGACTAAATA AGACACTATAGGTAATGA CACAGATTCAAGTCTTATTGTTGATGGGAAGAGGTCAATAATGGATGATATAATATACTA CAGCAATGAGAATTA TTGAATGTTTTCCAGACTCACTTGTATAATTGGCCATAACAGCAAACAAAAAACAGGTTC TGATAGCAAAATGAT ATACAGTACTAACAAAGGTGAATCTTGAGGTGAACCTTCTCTTTATAAGTTTAAATAGTT TACCCCCGACCTTTT CCCATAGTAGAACAGCCTAAAAAGTATCTTTCAGTAGAATGCTAGTGCTTATGAGGTTTT CTTAAGATATCATTT TTCAATTAAAATTTATTTCACAAAAGACTCACATCCTTGCCAGCCTTCAGGGTGAGTGTT GATTCAGGCTGTGTC CAACGGCAACGATGAGTGAACTTCTCACCCTCAGAATCACATGAGCATTCCTGAGATGTT TTATCAGAGTGATAC CAACTTCATTATTAGAATATTGAGTCCCTATTTCCTATATTCAATGTCCTTTCAAGCCCT AACTTTGTCCGGGTT GAAGGCAAAGATCCAAATAATCACATTTGTCTTTGATAACTGAAACTGGGAGAACTGGGA CTGTCTCAAGAGTTC TACGTGACTGTAGGTTGCAAGTACTGTGGTTGCATCTCCAAATATTAACCAATCCCAGTG ACAATTCAATGGGGT

CTCCTGAACCATGATCCTCATGTCTCCAGTGAAGGAAATGGGCAAAGGGGATTCAAA AATCCCTTTTGGAGGAAT AGGAAACTTCTGCTTTCCTTCATTTCATAACATTTGCGATGGAACAAAGGCTTTTTTAGA ATGGAGCAACCAGAT CCTTTTTTGGGGGAATCAGCTTAAATGTCCCTTCTTCTCATACTACTTTTATCTATGTGA TCCTATTCTTTTCTG TTGTGGATTGAATCATGTCCCTCAAAAAGATTGAATTTAGAGTGTGCTCTAAATTCAATG TGGAGAAATTTGGAC ACAGAGGCAGACACACAGGGAGAACCCCGTGTGACAATGGAGGAAGAGGATGCATTTATG CTGCCACAAGCCAAG GAACACCAAAGATTGTCAGCAGCCACCAGAAGCTAGGATAAAGGCATGGCACATCACTCC CTCTGAGCCCCCAAA AGGAGCCAAGACTGCTAATACTCTGATCTCGGACTTCTGGCCTGAAACAGTGAGAGAATA AGGTTCTGTTGTTTC AAGCTACCCAGCTTGCGGTATTTTGTCACAGAAGCACAAGGAATCAAGTACATTTTCTTT CTCAGCACTTGTGAT AATTTGATTTTTTCTTTACTCAGTGGTTGTTTCACACCTATGTCCCCATCAGACTGTAAG CTTAAAGAGACCTGG ATCTGGTCTGTCTTCACCACTGTTGATTCATTACCAGCACAGTGCCTGGCCCATGGTCAC TGAATAAACGTTTGT TGAGAGAATGAATGTGCTTAACCAGAAGTACTATTGACCTATTAGGCCAAGTTCAAGGTG CCTAACAGCTCAGCT GTGAAGGATACCTCTCCTTTCAGTCCTCTGTTACATATGTCCCTGATAGATGTGTTATTT GTATCTCCTCCTGGC CCTCAAGTTTGTTTGAGGGCAGGACCCTTTTTTGTATATCTGTAGAGCTTCGTAGTACCT AAATACTACTTTGCA TATATAATAAAGTTTCGATAAATATTCATTAAATAAAGAAATAAATGAAATGACTAAGTT TTCTAAGATGTTACA ACTAGATTGAAGATATTTAGCTCATTATTTAACAAGAAAACTATGGTTAATTATGGTGTC CTGTGTGAAAATGGT TATAGTTTGTTTTTTAATTAATATAAGCATGTATGTGCATTATCAGTATACACAATTTGT GGTATGAGTGTTTTG TGTCCCTGCACACAGACCACGGAAATCCTGAGAAACAAACTGCCACCCCAGAGCAGGTGC CTAACACAGAGACTT TTAATCCTTAAAGTTTTTCTATAACTAAGCAATGTTTTTTCAAATGCAATAACACTGATA TGCAGACATATTGAT TGTCCACTCACAAAGCCATTCCTCAATATCATTACAACATGCCTCTTTGAATGTCATTAA AAATAGATGTCTCAT TTTTCTAGGACAAGTTGGCTGAAGTTCTGCTTGAAAACTGGTAATAGAAAATACAATTTC TCAACCCGCTTTGGC CTTTTAATTCTGTTCTACAACCTTGCCAGTTCACTTTCAAAGTCAAGGGATGCATCTTGC AAAACCATGACATCT TTTGAGTAACTCCTTCTGTTCTTAACACATATTCCCAGGAGCTTAATAAATATTGTTTTT GCAACTTGTTTAGTG GCAAAATAATGAGTCCTTGGTGTATGCTTATCCTCTGCTTTGCTATTAGAGAAGATATAT TCAGACTGTTTTAAA CAAATTAATTCAAGGGCAGGGAACAGTCCTAAAACCTGTTAAAATTCAAATACTTGGTCA CTGTATGTGCAGCAT GTGTGTTCTAGAAAGTCCTATTATTTTAAAATATAAATTGAATCTTGTTGAGAAATTAAT GTCATATGAATATAT TAATAACTGAAATGCTGCCAAGTTTACAAAAAGCCCTCAATGAAACTGTGACCTTGTATA GACAAGGGCCTGTGG AGGGACATTTTTAAACCATCTCTTTTTTTATTTCCTCATGAGATCTACAATGTAAGTGCA TTAAAGTTGATGAAT GAATTGCAGTGCAACTTTTCCTGCCTCTTTTGCCTTTCATTTGTCTATATTTCAAGCTTC ACTGAAGTGATAGAT TTTGGGCTTTGCCACATTGTCCTCTGATTGCTTCCCTCTGCTCCTCCTTTTCCTAGTGAA TCTTTGTTTTACTGG

TGGAAAAATCTACATCTTTGTATCTTGGCATTTTACTTTCACATTATCTCATAGATT TTATTTCAAGTTGCTATA AAGTTATCAACTTTTATTTTTAACTAATATTATTTTTAACAATTAGAAAATTGTTGACCA GGTAATTCCAGCACT TTGGGAAGCTGAAGCGGGAGGATCACGTGAGCCCAGGAGCTCGAGACCAGCCTGGGCAAT GCAAGGAGACTGTCT CTACAAAATATAAAAATACATTAGCCAGGTTTGGCGGTGCATGCCTGGGGTCCAGCTATT CAGGAAGCTGAGGTG GGAGGATCACTTGAGCTGGAGAGGTTGAGGCTGCAGTGAGCAGTGATCGCACCACTGCAC TCCAGTCTGGGTGAC

AGAGGGAGACCCTATCTCGAAAAAAAGGAAAAGAAGAGGATTTTGCTGGCAAGATGG CTGAATAGGAATAGCTCC GTTCTGCAGCTCCCAGTGAGATCAATGCAGAAGGCAGGTGATTTCTGCATTTCCAACAGA GGTACCTGGTTCATC TCACTGGGACTGGTTGGACGGTGGGTGCAGCCCATGGAGGGTGAGCAGAAGTAGGGTGGG GCGTTGCCTCACTCA GGAAGTGCAAGGGGTCCCTCTTCTAGCCAAGTGAAGCCGTCAGGGACTGTGCCATAAGAA CAGTGCACTCTGGTC CAGGCTTTTCCCACAGTCTTTGCAACCCACAGACCAGGAGATAACAAGCGGTGCCTATGC CACCAGGGCCCGGGG TTTCAAGCACAAAACTGGGTGGCCATTTGGGCAGACATCAAGCTAGCTGCAGGAGTTTTT ATTTTCATACCCCAG TGGTGCCTGGAACGCCAGTGAGACAGAACCGTTCACTCCCCTGGATAAGGGGCAGAATCC AGGGAGCCAAGTGGT CTGGCTTGGCGGGTCCCACACCCACGGCGCCCAGCAAGCTAAGATCCACTGGCTTGAAAC TCTCGCTTCCAGCAC AGCAGTCTGAGGTCCACCTGAGACGCCCGGGCTTGGTGTGGGGAGGGGCATCCACCATTG CTGAGGCTTGAGTAG GCGGTTTTACCCTCACGGTGTAAACAAAGCTGCCTGGAAGGTCCAGCTGGGCACAGCCCA CCACAGCTCACCAAG

GCCGCTGTGGCCAGAGTGCCCCTCTGGATTCCTCCTCTCTGGGCAAGGCATCTCTGA AAAAAAGGCAGCAGCGCC AGTCAGAGACTTATAGATAAAACCCCCATCACCCTGGGACAGAGCACCTCAGGGAAGGAG TGGCTGTGGGTGCAG TTTCAGCAGATTTAAACGTTCCTGCCTGACAGCTCTGAGAGAGCAACAGATCTCCCAGCA CAGCGTTCAAGCTCT GTTAAAGATCAGACTGCCTCCTCAAGTGGGTCCCTGACTCCCATGTCTCCTGATTGAGAG ACACCTCCCAGTAGG GGCTGACAAACACCTCATAAAGGAGAGCTCCAGCTGGCATCTGGCAGGTGCCCCTCTGGG ACGAAGCTTCCAGAG GAAGGAACAGGCAGCAATCTTTGCTGTTCTGCAGTCTCAGCTGATGATACCCAGTCAAAC AGGTCCTGGAGTGGA CCTCCAGCAAACTCCAGCAGACCTGCAGCAGAGGGGCCTGACCGTTAGAAGGAAAATTAA CAAATAGAAAGGAAT AGTATCAACATCAACAAAAAGGACGTCCACTCAGAGACCCCATCCAAAAGTCACCAACAT CAAAGACCAAAGGTA GATAAATCCACAAAGATGGGGAGAAACCAGTGCAAAAAAGTCTGAAAATTCCAAAAACCA GAACGCCTCTTCTCC TCCAAAGAATCACCACTCCTCACTAGCAAGGTAACAAAACTGGACAGAGAATGAGTTTGA CAAATTCACAGAATT

AGTGTTCAGAAGGTGGGCAATAACAAACTCCTCCAAGCTAACGGAGCATGCAAGGAA GCTAAGAACCTTGAAAAA AGTTAGAGCAATTGCTAACTAGAATAACCAGTTTAGAGAAGAACATAAATGACCTGATGG AGCTGAAAAACACAG CACGAGAACTTTGTGAAGCATACACAAGTATCAATAGCCAAATCGATCACGTGGAAGAAA GGATATCAGAGATTA AAGATCAACTTAATGAAATAAATTGAGAAGACAAGATTAGAGAAAAAAGAATGAAAAGGA ATGAACAAAGCCTCC AAGCAATATAGGACTATGTGAAAAGACCAAATCTATGTTTGACTGGTGTACCAGAAAGTG ACGGGGAGCATGGAA CCAAGCTGGAAAACACTCTTCAGGATATTATCCAGGAGAACGTCCCCAACCTAGCAAAAC AGGCCAACATTTAAA TTCAAGAAATACAGACAACACCACAAAGATACTCCTCGAGAAGACCAACCCCAAGACACA TAATCGTCAGATTCA CCAAGGTTGAAATGAAGAAAAAAATGTTAAGGGCAGCCAGAGAGAAAGGTCAGGTTACCC ACAAAGGAAGCCCAT CAGACTAACAGCAGATCTCTCTGCAGAAACCCTACAAGCCAGAAGAGAGTGGGGGCCAAT ATTCAACATTTTTAA AGAAAAGAATTTTCAACCCAGAATTTCATGTCCAGCCAAACTAAGCTTCATAAGTGAAGG AGAAATAAAATCCTT TACAGACAACCAAATGCTGAGAGATTTTGTCAACAGCAAGCGTGCCTTACAAGAGCTCCT GAAGGAAGCACTAAA CGTGGAAAGGAACAATCGGTACCAGCCACTGCAAAAGCACACCAAATTTTAAAGTCCATT GACACTATGAAAAAA CTGCATCAACTAACAGGCAAAATAACCAGCTAGCATCATAATGACAGGATCAAATTAACC TTAATTAAGTTAGCC TTAAATGTAAACGGGCTAAATGCCCCAGTTAAAAGACACAGACTGGCCACCTGTATAAAG AGTAAAGACCCATCA GTGTGCTATATTCAGGAGACCCATCTCACATGAAAAGACACACATAGGCTCAAAATAAAG GGATGGAGGAATATT TACTAAGCAAATGGGAAGCAAAGAAAACAAAAAGCAGGGGTTGCAATCCTAGTCTCTGAT AAAACAGACTTTAAA CCAACAAAGATCAAAATAGACAAACAAGGGCATTACATAATGGTAAAGGGATCAATGCAA CAAGAACAGCTAACT ATCCTAAATATATATGCACCCAATACAGGAGCACCCAGATTCATAAAGCAAGTTCTTAGA GACCTACAAAGAGAC TTAGACTCCCACACAATAATAATGGGAGACTTTAACACTCCACTGTCAATATTAGACAGA TCAATGAGATAGGAA ATTAACAAGGATACTCAGGACTTGAACTCAGTTCTGGATCAAGTGGTCCTAATAGATACC TACAGAACTCTCCAC CCCAAATCAACAGAATTTACATTCTTCTCAGCACCACATCGCACTTATTCTAAAATTCAC CACATAGTTGGAAGT AAAACACTCCTCAGCAAATGCAAAAGAACGGAAATCATAACAGTCTCTTAGACCACAGTG CAGTCAAATTAGAAC TCAGGATTAAGAAACTCACTCAAAACCGCACAACTACATGGAAACTGAACCTGTTCCTGA ATGACTACTGGGTAA ATAATGAAATGAAGGGCAAAATAAAGAAGTTCTTTGAAACCAATGACAACAAACACACAA TGTACCAGAATCTCT GGGACACATTTAAAGCAGTGTTAAGAGGGAAATTTATAGCACTAGATGCCCAAAAAAGAA AGCAGAAAAGATCTA AAATCGACACCCTAGCATCACAATTAAAAGAACTAGAGAAGCAAGAGCAAACAAATTCAA AAGCTAGCAGAAGAC AATAAATAAGATCAGAGCAGAACTGAAGAGGAGAGAGACATGAAAAACCCTTCAAAAAAA TCAATGAATCCAGGA GCTGGTTTTTTGAAGAGATTGACAAAACAGATAGACCACTAGCCAGACAATAAAGAAGGA GAGAAGAATCAAATA GATGCAATAAAAAATGATAAAGGGGGTATCACCACTGATCCCACAGAAATACAAACTACC ATCAGAGAGAATACT

ATAAACAACTACACAAATAAACTAGAAAATCTAGAAGAAATGGATAAATTCCTGGAC ACATACACCCTCCCAAGT CTAAACCAGGAAGAAGTTGAATCCCTGAATAGACCAATAACAAGTTCTGAAATTCAGGTA GTAATTAATAGCCTA CCAACCAAAAAAAGTCCAGGACCAGACAGATTCACAGCCGAATTCTATCAGAGGTACAAA CAGGAGCTGGTACCA TTCCTTCTGAAACTATTCCAATAGAAAAAGAGGGAATCCTCCCTAACTGATTGTATGAAG CCAGCATCATCGTGA TACCAAAACCTGGCAGAGACACAACAAAAAAAAGAAATTTTCAGGCCAATATCCCTGATG AACATTGATGCGAAA

ATCCTCAATAAAATACTGGCAAGCGGAATCCAGCAGCGCATCAAAAAGCTTATCCGC CAGGATCAAGTCGGCTTC ATCTCTGGGATGCAAGGCTGGTTCAACATACGCAAATCAATAAACCATCATTCTCAGCAA ATTATCACAAGAACA GAAAACCAAACACCGCATGTTCTCACTCATAAGAGGGAGTTGAACAATGAGAACACGTGG ACCCAAGGAGGGGAA CATCACATACTGCGGCCTGTCGAGGGATTTGGGGTTGAGGGAGTGATAGCATTAGGAGAA ATACCTAATGTAGGT AACAGGTTGATGGGTGCAGCAAACCACAATGCGATGTGTATACCTACCTAACAAACCTGC ACGTTCTGCACATGC ACTCCAGAACTTAAAGTATAATAATAAAAGGCGCTGCCTCAGGATGTAAAGTGTAACAAG GGGGCTGGGGTGGGC AGCGTGGGCCTCTGAGACCTTTGGTTGCCCGTGTCCGCAGCTCGCCCCGCAGCCGGCTCC ACAATGGTCCGCTCC GTTTGCCACGTGCGGATTCGGGTTCCAGACTGAAGGCTGCGTGTTCTCTGCCGCCCACAG CCCAAGTTTATTGTG GCAACCGCCGGAGCAGCCTTCCCCGCTGTGGAGGAGCCTGGGGCTACCCCTCAGCGGTAT TTGGGGCTGGTCCTG GGGGAGCTAAGCAGGGTTGTGGCAGCACTGCCTGAAAGTGTGAGACCAGACTCTAATCCT TATGGTTTTCCATGG

GAGTTGGTGATATGTGCAGCTGTACATGGATTTTTTGCTGTTCTCTTTTTTTGTGTG GAGAAGTTTTAGATCGGT TGGGAGTCGGCTTTATGTGGGAAGAGAAAAAAAGCTTGCTGTAATGCTTTCTGGACTAAT TGAAGAAAAGCATAA ACTACTTGAAAAATTTAGCCATGTTCAAAAAGAGTATGAAGGCTATGAAGTAGAGTCATC TTTAAAGAATGCCAG CTTTGAGAAGGAGGCAACCTGTGAAAAGCTAAACAGGTCCAATTCTGAACTTGAGGATGA AATACTCTGTCTAGA AAAAGAGTTAAAATAAGAGAAATCTAAACATTCTGAACAAGGTGAATTGATGGTGGATAT TTGCAAAAGGATACA GTCTCTAGAAGATGAGTCAAAATCCCTCAAATGACAAGTAGCTGAAGCCAAAATGAACTT GACGATATTTCAAAT GAATGAAGAACGACTGAAGATAGCAATAAAAGATGCTTTGAATGAAAATTCTCAACTCCA GGAAAACGAGAGACA GCTTTTGCAAGAAGCTGAGGTATGGAAAGAACAAGTGAGTGAACTTAATAAACAGAAAAT AACATTTGAAGACTC CAAAGTACATGCAGAACAAGTTCTAAATGATAAAGAAAATCACATCAAGACTCTGAACGC TTGCTAAAAATGAAA GATCAGGCTGCTATGCTTGGAGAAGACATAACGGATGATGGTAACTTGGAATTAGAAATG AACAGTGAATCGGAA

AATGGTGCTTACTTAGATAATCCTCCGAAAGGAGCTCTGAAGAAACTGATTTATGCT GCTAAGTTAAATGCTTCT TTAAAAACCTTACAAGGAGAAAGAAACCAAATTTATAGTCAGTTATCTGAAGTTGATAAA GGAAGAGCTTACAGA GCATATTAAAAATCTTCAGACTGAACAAGCATCTTTGCAGTCAGAAAACACACATTTTGA AAGTGAGAATCAGAA GCTTCAACAAAAACTTAAAGTAATGATTGAATTTTATCAAGAAAATGAAATGAAACTCCA GAGGAAATTAACAGT AGATGAAATTACCGGTTAGAAAAGGAAGAAAAACTTTCTAAAGTACACGAAAAGATCAGC CGTGCCACTGAAGAG TTGGAGACCTATAGAAAGTGAGCCAAAGATCTTGAAGAAGAGTTGGCGAGAACTATTCAT TCTTATCAAGGATGG ATTATTTCCCACGAGAAAAAAGCACATAATAATTGGTTGGCAGCTTGGACTGCTGAAAGA AACCTCAATGGTTTA AGGAAAGAAAGTGCTCACAACAGACAAAAATTAACTGAAGCAGAGTTTAAATTTGAACTT TTAGAAAAAGATCCT TATGCACTTCATGTTCCAAATACAGCATTTGGCAGAGAGCATTCCCCATATGGTCCCTCA CCACTGGGTCGGCCT TCATCCTAAACAAGAGCTTTTCTCTGAGGGCCCACTGAGACTCTCATCTTTGCTAACAGG AGGAGGAGGAAGAGG CTCAAGAGGTCCAGGGAATCCTCTGGACCATCAGATTACCAATGAAAGAGGAGAATCAAG ATGTGACAGGTTAAC CAATCCTCACAGGGCTTCTCTGACACTGGGTCCCTGTCACCTCCATGGGAACAGGACCGT AGGATGATGTTTCTT CCACCAGGACAATCATATCCTGATTCAGCTCTTCCTCCACAAAGGCAAGACAGATTTTAT TCTAATTCTGGCACA CTGTCTGGACCAGCAGAACTCAGAAGGTTTAATATGACTTCTTTGGATAAAGTGGATGGG TCAATGCTTTCAGAA ATGGAATCCAGCAGAAATGATACCAAAGATGACCTTGGTAATTTAAATGTGCCTGATTCA TCTCTCCCTGCTGAA AATGAAGCAACTGGCCCTTACTTTTCTCCTCCACCTCTTGCTCCAATCAGAGGTCCATTG TTTCCGGGGGATACA AGGAGCCTGTTCATGAGAAGAGGACCTCCTTTCCCCCCACCTCCTCCAGGAACCATGTTT GGAGCTTCTCAAGAT TATTTTCCACCAAGGGATTTCCCAGATCCACCACATGCTCCATTTGCAATGAGAAATGTC TATCCAGCGAGGCGT TTCCTCCTTACCTTCCCCCAAAACCTGGATTTTTCCCCATAAACCCCACATTCTGAAGGT AGAAGTGAGTTCCCT GCAGGGCTGATTCTGCCTTCAAATGAGCCTGCTACTGAACATCCAGAACCACAGCAAGAA ACCTGACAATATTTT TGCTCTCTTCAAAAGTAATTTTGACTGATCTCATTTTCAGTTTAAGTAACTGCTGTTACT TAAGTGATTACACTT TTGCTCCCACTGAAGCTTAATGGAATTATAATTCTCAGGATAGTGTTTTCTAAATAAAGA TGATTTAAATATGAA TCTTATGAGTAAATTATTTCCATTTTATGTTATTCTGGATAGTATAACTATTTTAATTTG ATAAACTAATCCACG ATTATATAAACAATAATGGGAGTTTTATATATGTAATCTTGCAGGTAGGGAGGCTTTAAA TTATAAAGGTTGTGT CTTTATGCCAAGAACTGTATTAACTGTGGTTGTAGACAAATGTGAAAGTAATTTTATGCT TCATTAAATAAATTT TAGTTGATTTTTTTTTAAAAAAAGAAAATGGTTAATCTATCATTTAGGTGCATCATCAGT TGTTTAACCATTCTC TCTTACTGAACATTGGGTTGTTTAAAAAGTGTTGTTATTTTTGAATCATGGTTCAGTGAA CAATTTTGGACACAT AACTTTTTATCTGATGAGTTATTTCCTAAGGATCCAGCTCAGAAACTCAGCACATAAACC TAATAAGAAAAAAAC AATTTGAAGTGGCTAACCTCTTATCCCAATAAAAATGTTGTATTTATGTTTGGATTTAGA TGCCTTTCAGTGGTC

ATACCTTCACCTAACTTTTATGGATTCTACTTTTAACATGTAGAGTGACTGTTTAAA TCACCTAAACTCACTGAG TTTTAAGTTCCTTTTTATTCAACAAGACTGGATTGTATGTTCCAGCTCCTCAAACTTAGT TACCAACCACCATCC TAGAGAAGTGAATTCACATGAGGCCTGTCCAGAAGAACAATCTCCCTTTCAGTGTCCTCA TGCATGCAGTGACCA GAGACCAACCTTGATAAATTATGGAAAAAGTACAGCACATTCTGGAAGAGCCATGAAAGA TCCAGATCATCTGGT GCTGGATAAGAATATTAATGGACAGGCTGGGCGCGGTGGCTCACGCCTGTAATCCTAGCA CTTTGGGAGGCCGAG

GCGGGCGGAACATGAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCC CGTCTCTACTGAAAATAC AAAAAATTAGCCGGGCATGGTGGCGGGCGCCTGTAGTCCCAGCTACACGAGAGGCTGAGG CAGGAGAATGGCGTG AACCCGGGAGGCAGAGCTTGTAGTGAGCCCAGATGGCGCCATTGCACTTCAGCCTGGGCG ACAGAGTGAGACTCC GTTTCAAAAAAAAAAAAAAAAGAATATTAATGGACAAAAAGATTAATGAAAGAACATATT GAAGCATCCAATTAC CTGGTGTCTGCTCAAATGAGGAATCGGTGAGATAGGTCAGTTAGCAGTCAAGATTTATAA AAGAGACGATGGCCT TGGGAGGGGCTGCCCTACTCGACTTTTTAATGGCTAGAAGCTATTAAGGGCTAAGCCAGA ACCCTTCAGTATGGT TCAGTGAGGATCCCAATTTGGGGTCCAAAAGTAAATGACAACTCCCAGGAACCATTAAGA ATAAAAATCATGGAG CATTACTGAGAATTTATGTTATCTAAGTCTGAGGAAAATTAATGTTAAGGAAGCTTTCAA AAGTCTAATATTTAC ACCGAATTCCAGGGCACCATGCTCTAAGACAAAGCACTCTGGTCCTGCCCCTCTCCTTTC CTCATGTTTTTTGGT TCTTGGGATCCTTAAGGGTCAATGTTATTCTTAAAATACAGAGCATCCTGGAAACTAAAA AAGTGGAAGATATTC

AAATTCTAATGAATGTACTGGCAGTATTGTAGATCATGGAGTATAACATAAAGACAA GAATCCCTAGCCTCTTCC ACCATACTTTGTAATGGTAAGGAGAAAGGATAGAATTTTGAGAAGTCTGGGAAGACAATG TATGATAACATCTGG AGAAGCTCTGCATAAGTTACTTTTGTTCAGGCTTAAGAAAAATTCTAGCTTGCCCCTGCA CTGTCATCAGGTATC ATGAAAGTAAATAAAACCTTTAAAGATTCTTCAAGCCAGCAGACTTCTATCTTCTCTATA CTATCCTGTGATCCT AAACTCTTAACAGTTACTACGTATAATTTCCCTACATTTGCTACTAGTATTTTATCATAC ACAATATTACACTCA ATATTTCAAAAGTGGATGATTCATCTCCCGAAGAGACTGCAAAATTCATGAGTTAAGATT TGAGAATACTATTTT AGACAAGATTTAGTCAGATTTTAGAGAGTTAGAAACCTGTAACAATTCTCTAACAATACT GCTTCTCCTTTTGTG TATTAAGGAATTTTTGTCTATCAAAGATAGTACGAGGTAGACCAGAAGATAACTTGCCTT CAAAATGTCTGGAAT GTAAAATGGCAACAGTAGTATTTGGGGACTTCGTAGGGGATGGCCAATATACACCCATTC TTAGAGGTACTGATG ATATAATGTATAAGACAAAATCAAGTGGTCTCCATCACCATATAATGTTTAAAATGGCAA AGAGGGAGCAGAACA

AACACCCTTTGCAAATCTCTTCATAGAATCTACCGTAATAAACTTGTACTTGCTTAA AGTGTGTCTCTTCAGTGG TCTTATTACCACTACTTTGGGGAAAATGAGGCTGCTTAAAAGATTAACAGACATTACATT TTACATATCTGTGGC AGAGAAAACACTATGTATTCACCAAACCACTTCTTTTCCTTCCCAGTCACTCGGGAAGAG GTCATTTCTTTGTCC CCTTTCATCTAATTGAGGTGCCGTGACTACTTCTAGACAGGCAATGTGAGCAGAAGGTAT GCACGCCACGTATAG GCCTGGTCTTCAAAAATCCCTCAGATATGATCTTCTTCTCTCGTCTCTTTCATGGACAAA CTACAGGCCATGTAA TAAGGATGGTGGGGTTCCAAACTGAAAGAGCCTGGATTTCTGATTTACTGTTTTGAGAAG AGTTCACCAGGGAAA CAGCCTGGAAATACGCACAGGAAAATATGCACAGGACCCTGTGTGAGCAAGATATAAAGA TCTATTACATGGTGC CATTAAGGTGAGAGTATTGTGCTTATAGTATCCAGCATTAATTATCCTCACTACTACAAC TTCTTTGTATCCATC ATGTGGAAAAGTAGAGTATTTAATAAATGATTATTGAGTTTATTACCTTTTTTATATTCC AATCATTGCTAATTG TACGTTACCTCATTTCAAGGTAAAGGTGACCAAGGGCTAAAGCAGTGCTATCCAAACCAA GCCAGACATCAAAAT CACACAAAACCTTTTGAAAATACAACTTTGAAGATGCCATTCACATAGATATTTATTCAG TGGGTTTTCAAATGG AACCCTGGAATCTACAGTCTTTAACAAGGCTTCCCAAGTTATTCTGATATACAGCAGGCA AATCTGAGAACCACT GGACAAGAAGAAAATAAAGGCTATATCTTTCGACAACAAAGACAATGCCTTAAACATAGA ATGTATTCAATTAAA GCTTGTAGAAAGATAGGTTTGTGAACAGGCACAGGGACTAGCCTCGAGCAAATTAATAAG GGCAGCAATGTTTTT CACTGAAACCATTATTCCCCCTATTTTATTTCTTCTGGGGCTCTGTGTTTCCTTTCTCCT ATCAAAATCCATTCT AAGGTTGGAGGTTGGGGGTATCTCTTGCCTACTCCATACAGCAAGGAATAAAATTAGTAT TTCTCGAACTATCTG TGACAGCAGACCCATTGTAGGCCAGTACTTTTGTAAAATGCAATAAAAATTAACTTCTAG AGAATGAAATTTTAA AATCACAGACATTCAAAATACAAATTCCAATTTTTTTATTATTAACTGTAAGAAATTTAA AATTAAATCTCAATA AATAAAATTAAAGCAAACATAAGATAGAAAAAAATAAGCATTATGGATTGGCCCAGTCTG CAAACTGTATACACT TTGCCAAACATGGGCATAAATTACTAAGAAGCAAAATCTTCCATCTGTAAACATTTCCAT TTCCATTGACAATAT GTGTGAGGGAAAGGAGGGATGCTTCTGTTTTAGAATGCCAGGCGTCAGCTAACAAGTGAC AAATACGTATTGAGA CTGAGATCTCCCCAGCCTCTCAGTAGTCAGCAAGAACATGTTGAGGCCTCTGTTTTTGAC TAAAAAATTGGCCAG TGCATGGGCAACATGCATAGGTCCTGAATGAAAAAAATAGCAGCAGCAGAAATTTAAAAG AATTTTCACAGCTAG GCCACAGTAAATTCTCAAGCCCTTCATCAGAAGCCACTGTGGGGCCTCATTTATGCCTTT GTTTTTATTAAATTG GATGTGATCTTAAGATTCTTCTGTCAAAATTCCACTAGCATGTGAAGGCACCAAAAGTTT AAAATGTAAAATTAA CCCAAGTTAAGCTATTCCATTATTAAGCAATAGCAGATATATTTGTTATTATATGAGAAG AAAGTTAACAGGGAG CTAAGATTGATGTTACTGATAAGAAACAGAAACAAGACTTTAAAATTAAATAAATGAATT ATTTATTTAATAAGA ACCAATTGACAGATTCTCGATAAAGACTGTAAGATGTCTTAAAACATTAGGTGTATGGAG ATAACATTTGTAACT TTGACAATTTATATGATGAGAAAAATCAAGGAATGTTATTGTTTATTGGCAGAGTTCTAG AATTACAATTCCATC

ATTCTGTTTTGGGGAAGTTTCCCTTGAAGTAAATGATAACAGGGCTTGAAATAGTAC ACCTCAGCATTTTGTTTA TAAAACTGTGGAATAGGTAAGGTTTGTATTGTAACTGAACCCAGGTTCAGCTGCTTGCTG CTCTAAAGCTAGACA TAAGAGAGGAAGGTTGGTGGGAGGAAAAGCGATTTTAATCGGAGAAGCAGCAAACCAAGA AGATGGTGAACAATA GTCACAGAACCATCTTAAATTTTAAAATTTACCATAGAGTGTTCAAAGGAAAACTTGGTA TGGGAGGCATGCAGG AGGGGTGCAGGGGGCGGGGTCTGTGTGTCTTGTTCCAATGGCTATCTCAGATAGTCACCC ATCTGGAGGTCTAGT

TGGTATTATTTTGAATTCAGCCCAGTGGTGGTGGACTGTCAGTGACTCCTCGCTAAG CAGGAGGATTCTGCACTC AGGGCTCCATGCATGGTTTGTTTCAAGATTGGCCTCTGGAATTTCTCAAGCAAGAACATA ATTAAATAAGCAGGC ATTGCCAGAGGGGAGTGTCTGGAAAGGAAAGGAATGAAGAGATGAAAGGAAAGTGGGTGG TTAAACTATATTTTT AAAACTGAGGTTCCCAGTTATAGTATGTTTCGCACGCTCCCCCCATTTTAGCACCCCTGA CAGAATTTAGTAATC TCCTCATCTTGTCCTCTACTTCAGGTCCCCTATCTGTCCTTGTACTCTCCAGGGTTTCCT TTTCTTCTTCACGAC CTTCCTTCCCTGCAATTTTATAAGCTATTCCTATCCCAGTGATTTAGTTTCAGCTTATAA AACTGTGTCTTTGCC ATTGTAATCAAATTGAAGGGCCTCTGCTTCATGGTTGGATTCTGTGACCAGGAGACTCTT ACGAGGAGTTGGCCA GGTCTCTGTTAGGAAAGCAAAAAAGAACAATGGAGGCAATTATCCCATTGATTTCAGCTA TAAATCCTATTTTGC CTGAATTGTCTGAACGATGAGTATTCTGTGAAAATGCTGCTCTCTAGTGCAATAGAACTG CAAATAATGCACATC TATTTCTTATAATCTCATCCAACATACCCACAGAGATTCAGATCTAACAAAACAGAGGTG ATTTGGTTATTGAAT

CATAATATAAATATGGGGAAGAGGAGGGAAATTTCAAGCCTGAGGAAACTGTAGTAG GAGTAAGTATGCTGTGTT TAAGAGGTCACAGATAAAATTAATATTACCAATCCATCAATAGGCAATTACTAATAGCTT ACTACACACACAGGA ATAAAATGTGAAGACAGAGGAAGTGTAAAATGGAGCCGCCAACTCTACGGAGTTGTTTGC AATTTGGTCTGGTAG AAAGCTATGAAATAAGGAAGTACATGATTGAGAGCTAGAGAATGTGGCACAGGCTCTGAA CCCGGACCGTTCAAT GTAGTAAGCTCTAGCCACACTGGACACTTGCAATGTGGCTTGTCCAAACTGACATGTGCT TTAAGTATAAAATAT AATCCAGATTTCTAAGACTTCAAAAAAAATGGAAATATCTCATTAATAATCTTAAGTTTA TTACAGGTAGAAATG ATAGATTAAATAAACTATATTGTCAAAATTCATTTGATCTGTTTCTACAGTATAACAAAC TTACTTGTGTGGTTT GCATTTTATTTCTACTGGATAACATGGCTTTAAAAATGGTATTTTAGAGGAAGGAAAGCT TGGTAGAGAATGGAC TAATCCGGATCCCTGGAAGAAATGGACCTTGAATGGGTCTTGATGACTTGGAGAGGCAGA GAGAGAAAAAGAAAA GTCAAACATAGGGAATTGGTTGATAAAATGAAGGTGAGGGGAGAAGGAACAGAGGGAGGA GAAGATCCAGTTTGA

GGGATATTACAGCGAGCAGCCTGAGAAAGAAGGATAAGAAAGGAGAGAAAAAATGCA AGGGAAGTAACCCTTCAA AGCCAGTCAGAAGTTTCTGGGTTCCTCAGCAGCCAGAAAAGAAGCCGTTGAAAAGATCTG AGTAACGGAGATTCT GGACGAAAACTGAAGTTATGGAAGGGAAGTTTAGACATGGGTTATTAAACGCTTTAGCGC ATTAGAAGTTTCTTA TGTAATCACTAAATTCAGATCCTGAAATAATGCCACAAGAACTATACAGCTCAGCCACCC AATTCAATAAGAAGT TACAGCACAGTCTCACACATATCCAATTAACCTTGGCCTTTAGTCAACATCTGGGTTCTT TTTGTCATTTTCAAA TACTATCACCCAGAGGTGCTATGATTTATATTGGGGAGGGGATTAAAAGAAAATAAGTAA GTTGGTGATAAGAAA AAGCTTTCAGATGATTCCATCTGAATTAACAGCCCTCTTTAGTTGTCTAGGAAAGAGGAT GCTTTTTCTTGAAAG TGCTTTGAAATGATGATGTGCTTGTTAGTAAACATCAATTATTTTCAAATCGTAATGTTT GCAAGTTTGTCTTCC TGTAGCTCACCCTTTATGTAGGTCCAGAATATGATTGTCACAAATATCTGGGTGAGCAAG ACTATGAAATGTGGT CATAAAGTAAGTGATTATTTCTAAACTCATCTTTGTCACTCGTAGTGCTTCACAAAGCAC CTTTTCCTGGACTAC AATTCATTTTAATTGATCCCATCAGCACTATATCTGTATCCTGAGTGACTTCACAATACC CTCTATTTCAAGAGA AACCAATCAGGTTATGGGTTTGTTAGTAATAAAAATTACCAAGGAGCAGTTTGTGGATGG TAAAAGCAATGCAAA TTCTAAAGAGAAGTCATAAGAGCAATAATAAGCATCCTCCTCACTTCTTGGAAGTGAACA ATTCCAAGCTCCCTG AAGCAACACTTAACCTATCATATTAAACAGTAATGGACAAATATTAGAAATGTTGATGTC AGCTTTCAGAATCTG TGGGCATCAAAACATCACTTAAGTTCTCCGAAGTATTCTCTGTCAAGTTTCCTTCTACAG TATTCTTTTCCTACT AGGACAGAGCCTTAAGCCCTAGAAGAATAATTTTGCTTGTGTGTTAATTATTTGTTTACT GGTTCATTCCAGAGT GTGAGCTGGAAAAAGGGGGAAGTGTCATAAATAGTTTTTTATGGCCCATGGTTTTTCAAC TACGTCACTATTGGT AGCAGTTTCCACTGCAGGATCTATTTGCAAAGCCTAGGAAATTAGCATTAAGCAAGCTGC TAGGAAGACTTCAAC AGTAACTAGGCCACAGGCCTCACACATTTTTCCTCCACCCCAGCCTCCTCTGGAGAGTAC TTGCTAAACCTCTGT GACACATAATGAAGCAAAGAAAGTGATAGAACAACAGAATTACACGGGCAGATCCTTGTT TCTTCTTCTCTCTCT AAAGAATTCCTTGGACTGAAAAGCAGTTTATTTTGGAGGAGTGAGAAAGTGGTGACAGAA TTAGAAGGGCCTGGG AGGGCTTCATTTTAGGAGACAGTTTTAGGCTGAAAAGAGATTTCATGAGTGTGATTTACC TGAGGTGACTTTTGG GGGCTCTTATAAAAAGGAAGTTCATGCTGAATGGGAGGTGGCTTCTGAGATGCAGATTCT GGTGAGCTAAGAGGG CTCGGTAAAGAGGAGGCAGGAGTTAAGTAGCGTGAACTATGCAGTAGCAGCCTTCTTCCC CCCTTGCTTGGGGCA GGTCATCACAACCCTTCTCAATAAAGGGGTCCAGGAACCACTAGGAATAAATGGGCATTT GCACTTCAGGTGAAA CCCATTTGTCATAACTGCTTGGACTTTAAGCTTACAAATAAAAAGAACCACATATTTCCC TTTGCAGCTTGATTT AGTTAATGTCATTTTGAGAAAGAAAGAAGACATTGTTATCCCGTCCCTTTTTTTTTTTTT TTTTTTTTTTTATGA AGAGACTGGGACTCAGAGAAGTCAAGTGATTTTCCCAGAACCAGAAAACACAGAAGTAGC AGAGCTGAGATGACT ACTCCGGTCTTCTGATTCCAAATTCCAAATTCATTCTTCTAAGCGATTTCCCAAAACGGG AAATGGGTTTATCTT

CTATTTATGGGAAGTGATAGTGGTATTCTATTTAGAGAACTTATATAAAATCTTACT TTAAAATAAATAATATTT CAAAAAGTAAGCTTAATTTAAAGAAAATAATCAAGAAAGTCTGGTATATTTTTACAAATA TACCAAATGACCTTG CTCTAAAATACATCTACTTTCCAGCAAGCCAAAGTGAAACAATTTGAAATAAGTGGCATT TACTGACCACTCCCT AAAGTTCACACAAAAGAGGTAGTACTCTAACTTAAATATACAAGGTGAAGAAATAGCTTA CTCAGCCTGTTGGGC TTCCTCTTCTACACTCTTGGGAAATGCCCTCCGTGTTAACCAAGAATTCTCAGGCCTTGG AGGGAGTTTTCCATT

CTCAGTAAACTGAGATTGCAGTTGCGGAAATTAAGAGGTATCTGTCCAGCACTTCAT TCCCTTAAGGTCAGGATC TGTGCTTTTAATAATGACAATTAGCTAACATATACAATTAAGCCATGCAAATGAAGTAAG AGAAAGCTAGAGGAG AAATTCAGGAGCCAGTTGCCTTTTCCAGACATCTTGTACAAATAGTGTTCAAAGGACTAA TTCAAAAGATGGGAT TCTTCGCTTGAACCCAGGAGGTGGAGTTTGCAGTGAGCGGAGATCGCTCCACTGCACTCC AGCCTGGGTGACAAA GTGAGACCCCATCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATGGGATTCTTTTTTAA AAAATAAATTTTACT

GCGTATTTTTAAGGTATACAACGTGATGTTATAAGATGGATATAGATAGTGAAAAGG TAACTGTAGTGAAGCAAA TTAACATATTCATCATCTCACATAGTTATCTTTTATTTGTTTTGTTTTGATGGGATTTTT AAGATAGTAGAAAGG AATGGTAGACAATAAACATTTGAGGGAAAGTGGGGCTTTGTAGAACTCCTAAAATGACAG CACGCACAAATGTCC CCATTATGTCTAAAGGGTAACTCGTTCCTACTTCTAGGGACAGCTGAGGGACATCAATGT AAATTTCTAAATGAC TTCCTGAACTTTTTATTTTTATTTTTTGTATTTTTAGAGGAAATTATAATAACATCAAGC CACCTCTGGACCATA

TCGCTGCTGATATCATCAGCAAATGGCACTATTCCTAAATCCTAAGATGCACTTTTC CCTTCACATTTCAACATT TGTGAAACTCGATTGTACCTACACCTGATTTTATATACAATGCAGCCTTTCCTTTTCTTT TGTCATTGCATCTTA CGCCTGATTTCTCCTTGGAATTGAGTAAATATAATGCTTACATGTGTTAATAAGAATTGA GGTCACTCATAATTT TTGAAATATGCCACCAAATATAAGCCTTTCTACATATTGTTGACTTTGAAGTCATTTCTT TTTTTAACTACTAAA CAATAACACTTTTTGTTGAGAAAAATTGCATATGAACAAGAGACCAAGCAGGTAGAGAGA AAAAAACTTTTAATA

ATCAAGAGAATGTTACTGTGTCCCAAAGGCTAAAGTCACCTTACTATCAAGAGAGAA GGACAGGAACAGAGAGAA CCAGGTAAATTACGAATTGAAAATTCCATGGTTCATTTATCTTTATTTTTAATAATTCCA TTTGTGTGATTGTGT TGACCACAAGGTCATAATGTTACTCTTCATACTGACTTCTCATGTAAATTATAAATAAGT TTTTATGCTAATGAT TTATGGAGTAAGCTATTCATCTTTCCGACAGAGAGTTACCTACAAAGAAATAATTATTCT ACCTCTGAGATGAAA TATCATGAAAGGAGTGGTTTCCAGATATTTTGACTTTTAAAAGCTTAAAGAATATATGTA GTATAAAATTCTAAA

GCAGGCAAAATTAATCCTTTTAGCAATCAAGATAGCGGCTACTTTTGGTGAGAAGGA CAAGGTAGTGATAGAGAA GGGGCTCAGGGGTCTTTCCTGAAGACAGTGAGGTGGGCAATGGTATTTTCCTTGACCTGG ATGGTGATTAAACAG ATGTGTTTACTTTGTGATAATTGACTAGGCTGTGCACCTATGAACTGCATACTTTTCCAT ATATGTACTGTATTC TTATACTTAAAAAGAAGTTTAAAAATAAATGCAACAGATATAGGACTTCCTATATTACTC GTTGACCAAAAAAAT GGATTCATTTTTCTTTCAGGTAAAACGTACTAGTGGTTTTAATATTATATTGACCAGGGA GTAAATGTTTACCTT AGGAACCTTAATCTTGATGTTCTCCAAAGTCATTATCTGTTCTTTCTGATTATCAGAATA GAGTATATCTCTATA TAAATGAAAATTTCTGGTCATTCTCAAAAAATAACACTAAGCATGAAAATCAGAAATATT GATCTTGTTTTGTAA TGATGTTTCTATTGATGTGAAGTAGTTTCTAGTAGAGTTGCTGTCCTAACACACAAATGA AATTGCACTGTTTGG AAGACACAACTGTGAATGACTTGCTTCAGTAAGGAATTTCCAACATGATGGTTTAGGGAT AGAGGTGCTCGATTC CTCTGTCTCCGGTTACCCAGGTTATTGAGGACAGGGAGGTCAATAAGTAATGCCCTCCTC CCACCCATAGCACAA AACAGAGCGGGGTTCAGAGAATAGGTAAGGCTTTGGCCAGGGTGTTGAGGAGACTTACAT CCCTGGGAACCAGTC AGAATGGGGGCGCTGAAAACAATGTTTTAAATTCTAGCACCCAGCAACATATGTGTGAAG ATTAAATGTACTCGT GCTAAATTCACTTGCTCCATTACTGAATTTGGGTGGTGTCTGTTAAAGATGGGAACAAAG GCATTCAGGTCCTGG TATCTTCTACCACTCCCAGCATGAACAGACTCATGTCAGTGGGTAAGGGATGGTATTTCC CGAGAAGGCTTTGAA CTCTTGTAGTGGGTCAAATAATGGCCCCCCACTTAAAAATGTTCATGTCCAAATCCCTGG AAGCTGTGAAAAGGG GTTTTTGCACATGTAATTAAGTCAAAGATATTGAAATTAGATCATCCTGGATTACATAGG TGGGCCCTACATTTA ATGACAAGTATCCTCATAACAGAAGAGGAGAAGGTGATGTGAGATTTGGAGCAGCAGAGA TTGGAGTGATGTGGC CACCAATCAAGGAAACCAAGGACTTCCAGCAGCCACCAGAAGCTGGAAGAGGCAAGGAAG GACTCTTCCCTAAAG CCTTTAAAGGAGCACAGCCCTACTAACACCTTGCTTTTGGGCTCTGGCCCGCAAAACTGT GAAAGGATACATTGC TGTTATTTGAAGCCACAGTTCGTAGTAAATTTATTACAGCAGCCCTAGAAACTGATACAA CTCCTAAATACACCC TTAGCAACACTGCTCAACAAGAAGTAGGCAATTTCCTCCTGACTGAAAAATACTGATACT GTTATGGGATCCTTG GGGGTGTTGCTTTTCTGTCCAGAAACCTCTGTGGCGGTGGCACCTTTGCATGAGTTTTGC TCGGGTCCACTGGGC CCACTCATCCTGGCAGGCTGCGCTCAGCTGACACTACTGGCGTGGATCCCATGCCTCCAA AGAGACTGGAGCGAA GCGGTGAGGGATGTGTGAGGAAGTGAGCGTGGGGTCTGGCACACAGTCAGGCTCAATGGC TGCTACAGCGGGATG GGCAGCTTCAGGTGCTGGCACGGGTGCTGGCTCACTGCAAGGCTGTGGCTGCACCAAGCA GCGCAGCAACGGAAC GCATTGGTGCCTGGAAACTTGGAGACTCCAGGAACCTCAGGGCTCCAAAAGGCAAATCAC AGCCCTAGCTTCGGG AGCTCCCAGGTCTGGGCTGCCAAAGGGCTGCAGCTCTTCTCTCCTCTCTCTCTCTTCGCT CCTCTCCCTTTCTCT CTTCACTCCTCCCTCTTTCTCTCTTCACTCCTCCTGTCGCCTATGAACAGCGAATTCAAC CTTCCAGTTTTCAGA CTAGGAATGCTGGAGTTGTCCTTGATTACTCTGAATTGTTCACTCCGCATATGGGCACTG AGGATACGTTGATGA

ACTACACAGACAAAAAGGATAGAAATTCCTGTCAAGACTACATTCAATAGGGATGAA GCAGGCAATAATGAATAA ACATACTAAGTTGAATATGACTATTTAAATATATATAACACATATGACTTGTATAATGTT AAATATTTTAAGTTT TTTAAATTCTTCCCTTCATAGATTTTACATTATAGTAGAAGAGGCATTTTTGTTGTTGTT CTTTTTGTTTTGGAT TCAGAGGGTAAATGTGCGGGGTTGTTACATGGGTATATTGCATAATGCTGATGATGGTCC CATCACCCAGGTGGT AAACATAGTACGTAATAGGTGAATTTTTAGCCCGTGCTTCCCTCTCCCATCTAGTCGTCC TGAGTGTTTATCGTT

GCTACGTTTATGTCAATGTGTATTCAATATTTAGCTCCCACTTATAATTGAGAATAT GCAGTATTTCGTTTTTTG TTCTCGTGTTAATTTGTTTAGGATAATGGCCTACAAAGAACATGATTTCATTATTTTTAT GGACATGTAGTATTT CATGGTGTATATGTACCACGGTTTCTTTATACAATCCCACTGTTGATGGGCACCTAGGTT GATTCTATTGCTGTT GTGAATAGGGCTGCAATGAACATACAAGTGCATGTATCTTTTTGGTAACAAAAATTTTAT ATTTGGATTACCCAG TAGAATTGCTGGGTTGAATAATAGTTTTGGTTTAAGTTCTCTGAGAAATCTCCAAACTGC TTTCCACAGTAGCTG AACTAATTTACATTTCCACTAGCAGTGTATAAGCGTTCTCTTTTCTCCACAATCTTTTCA CCAGCATCTGTTATG TTTTGGCTTTTTAATAGCCTTTTGATGACTGTGAAATGGTATCTCACTGTGGTTTGGATT TCCATTTCTCTAATG ATTAGTGAATGTTGAGCATTTTTTTCATATGTTTATTGGCCGTTTGTATGTCTTCTTTTG ATAAGCGTCTGTTCA TGTCCTTTACACATTTTCAATTAAAATATTTGTTTTTTGCTTGCTGATTTAAGTTCTTTG TATATTCTGGAAATT AGATCTTTGTCAGATGCATAGTTTGCAAATATTTTCTCCCATTCTGTAGCCTGTTTACTC TGTTGGTAATTTCTT

TTGCTGTACAGAAACTCTTTAATTAGGTCCCACTTGCCTATTTTTAGTTTTGTTGCA ATTATTCTCTGGAACTTA GCCATAAATTGTTTGCCAAAGCCAACGTGGAGAAGGATATTTTCTAGGTTTTCTTCTAGG ATTTTATAGTTTAAG TTTTACATTTAAATCTTTAATCCATCTTGAGTTAATTTTTGTATATGTTGAGAAGCAGGA GTCTAATTTCATTCT TCTGCATAGGGCTAGCCATTATCTTGGCACCATTTATTGAATAGAGAGTCCTTTCCTTAT TGCTTATTTCTGTCA ATTTTGTTGAATATCAGATCGTCGTAGGTGTATGGGTCCATTTCTGGGTTTTCTATTCTG TTCTATTTGTCTCTG TGTCTGTTTTTGTACCAGAACCATGCTGCTTGGTTACTGTAGCCTTTTAGTATAGTTTGA AGTTGGGTAATGTGA TGTCTCTGGCTTCGTTCTTTTTGCTTAGGATTGCTTTGGCTATTCAGGCTCCTTTTTGGT TCCATATGAATTTTA GAATATTTTTCTGATTCTGTGAAAAATGACTTGATATTTTGCTAGGGATAGCATTGGAGT GGTAACTTGCTTTGG ACAGTGTGGCCATTTTAATGATATTGATTATTCCAATCCATGAGCATGGAGTATTTTTAT ATTTATTCAGTCATC TTGATTTCTTTCAGCAGTGTTTTGTAGTTCACCCTGTAGAACATTTCACTTCCATGGTTA GATGTATTCCTATTT

TGTGGCTATTGTAAATGGCATTGTATTTTTTTTTATTTGGCCCTAAACTAGAATGTT ATTGGTGTATAGAATTGC TACTGATTTTTGTACATTGATTTTGTATCCTTAAACTTTACTGAAGTTATTTATCAGTTC TAGGAGACTTTTGGA GAAGTCTTTAGGGTTTTCTATGTATGAAATCATATCATCAGCAAAGAGAGACAGTTTGAC TTCTTCTTCTTTTTG GATGCCATTTATTTCTTTCTCTTGCCTAGTTGCTCTGACTAGGACTTCCAGGGCAATGCT GAATAGGAGTGGTGA GAGTGGGCATCCTTGTCTTGTTCCAGTACTCAAGAGAAATGCTTCCAGCATTTACCTGTT TAGTATGATGTTGGC TGTGGTTTGTCATAGGTGGATCTTATTATTCTAAGGTATATTCCTTTGATGCCTAGCCTG TCGAGGGTTTTTAAT CATGAATGGATATTGAATTTTATTGAAGGTTTTTTCTGAAACTATTGAGATGATCATATG GTTTTTGTTTTTTCA TTCTGTTTATGTGGTGAATCACACTTATTGATTTGTTATGTTGAACCAGCCTTGCATCCC AGGAATAAAGCCTAC TTGATTGTTGTGAATTAACTTTTTGATGTGCTTCTTGATTTAGTTTGCTCATATTTTGTT GAGGATTTTCGTGTT TATGTTAATCAGAGATATTGTCCTGAAGTTTTCTTTTTTCATTGTGTCTCTGGCAGATTT TGATATCAGGATGAT GCTGGCATTGTAGAATGAGTTAGGGAGGAGCCCCTCTCCTTAATATTATGGAATAGTTTC AGTAAGATTACTATC AGTTCTTCTTTGTATGCTTGGTAGAATTCAGTTGTGAATCCATCTGGTCCAGGGCTAAAT TTGGTTGGTAGGTTT TTTATTACTGATTCAATTTTGGAACTTGTTATAGGTCTGTTCAAGTTTTCACTTCCGTCC TGGTTCAATCTTGGG AGGTTGTATGTTTCCAGGAATTTATCCATTTCCTCTAGATTTCCTACTTTGTGTGCATAG AGGTGTTCATAACGG TCTCTGAAAATCTTTGGCATTTCTGTGGGATTGGTCGTAATGTCATTTTTGTCATTTCTT GTGCTTTTTGGAACT TCTGTCTGTTTTTCCTCGTTTTTCTAGCTAGCAGTCTATTAGTCTTGTTTATTCTTATGA AAAACCAACTCTTTG TTTCACTAACATTTTATGGACTTTTGCATCTCAATTTTATTTAGTCATTATCTGATTTTA GTTATGTCTTTTCCT CTGCTAGCTGTGAGATTGAATTGTGCTCTTTTTTTCTAGTTCCTCTAGTGTTATGTTAGA TTGTTTAGTTGAGAT CTTTCTAACCTCTTGATGAAGGCATTTTAGCACTATAAACTTTCCTCTTAACACTGCTTT TGCTACATCCCAAAG ATTTTGGAAAGTTGTGTCTCTATTTTCATTAATTTCAAATAATTTTTTGATTTCTGCCTT AATTTCATTGTTCAC CCAACAGTTATTCGGGAGCATGTGGCTTAATTTCCATGCTTTTGTGTAGTTTTGAGAGAT CTTCTTGGTATTGAT TTCTATTGTTATTTCACTATGATTTGAGAGTGGCCTTTGTATGATTTTAATTTTTTTTAA TTTATTGAGACTTGC TTTATGACTGAGCATGTGGGGCAATCTTAGAATACGTTCCATGTGCATATGAGAAGAATG TGTGTTCTGTCATTG TTGGCTTGAGTATCCTAGAGAGGTCTATTAGGTCCAACTGGTCAAGTGTCAAGTTTAATT CCAGAATTCCTTCGT CAGTTTTCTGCCTCAGTGATCTGTCTAATGCTATCAGTGGAGTGATAAAGCCCCCACTAA TATTGTGCTGCCATC TACGTTTTATTGTAGGCCAATAATTTGTTTTATGAATCTGAGTGCTCCAGTGTTGGGTGC ATATATGTTTAGAAT AGTTAAGTCTTTTTGTTCAATTGAACCTTTTATCATTTTATAATGCCCTTCTTTGTCCTT CCTGATTGTTGTTGG TTTAAAGTATGTTTTAATCTGATTTAAGGGTAGCAACTCCTGCTCTTTTTTGTTTTTCAT TTGCATGGTAGATCT TTCTTCATTCTTTCACTTTGAGCCTGTGAGTGTCATTCATGTAGGATGCATCTTCTGAAA ACAGCAGACAGTTGT

GTCTTGTCTTTTTATCCAGCTTACCACTTTATGCATTTTAAAGGGAGAGTGTAGACT GTTTACATTTAGGGTTAG CATTGACATGTGAGATTTTGCTCCTGTCATTGTGTTGTTTAGCTGGTTGTTTTGTAGACT TCATTGTGTAATAAG TGTATTTTTATTGGTAGCAGGTTTCGTCTTTCATTTCCATGTTTAGCAATCACTTACGGA TTTCCTGTAAGAATC ATCTGGTGGTAATGAATCTCCTTGGTGCTTGCTTGTCTGAGAAGGATTGTATTTCTCCTT CACTTATGAAACTCA GTTTGGTGGGATATGAGTTCTTGGTTGAAATTTATTTTCTTTAATAATGCTGAAAATATA GGCCCCCCCATATCT

TCTGGCTTGTAAGGTTTCTGCTGACAGAACTGTTGCTGGCCTGATGAGGTTCTTTTT GTAGGTGACCTGACCTTT CTCACTAGCTGCCTTAACAATTTTTTCTTTTGCATTGACCTTGGTGAATCTGATGACTAT GTGACTTGGCAATGG TTGTCTTGTATAGTGTCTCACAGGAGTTCTCTGTATTTCTTGAATTTGTATGCCCACCTC TCTGGTGAGATAGGG GAAATTTTCATGGACTGCATCCTCAGATGTATGTTCTAAGTTGCTTACTCTCTTTCTCAG GAATGACTGTGAGTC ATAGACTTGGTCTCTTTACATAACCTCATAAATCTTGAAGGTTTTGTTCATGTTTTAAAT TCTTTTTTCTTTATT TTTGTCCAACCAAGTTGATTCAAATAACTGGTCTTCAAACTCTGAGATTCTTTCCTCAGC TTGGTCTGTTCTGCT GTTAATGCCTCTGACTATATTATGAAATTTTTGAAGTTGATCCCTCAATTTCTGAAGTTC AGTTTTGTTCTTTCT TAAAATAGCTATTTCATCTTTAAGCTCTTTGATCATTTTTCTGGATTCCTTGAGTTCCTT GTATTGGGTTTCAAT GATCTCCTGGATCTTGATGTACTTCCTTGCCATCCAGATTCTGAATTCTATGTATGTCAT TTGAGTCATTTTAAT CTGGTTAAAATCCTTTGCTGGAGGACTTGTGTGTTTGTCTGGAGGTAAGGAGACACCAGC TTTTTTGAATTGCTA

GAGTTCTTGAGATGACTCTTTAACATATGAGGGCTGGTGTTCCATTAACAATAGTGT ACATTGAGTATAGTCAGT TGGCTTCATTCTGAGTGCTTTCAAAGGGCCAAAGCTCTGTACAGCATCTTTATTTGTGGC TAGATTTTTGCTTTA GGTTTCACAGGTGCTGTATATTGGAAAAATGTTTTTGGTGTTGTCATTTGGGGTGCAATC CAGTAGGTGATGCTT AAGAGTGGTAGCTGGCAGATAGGCTCTTACTCAGTCCACAGCTCTTTTGTATTTTGGTGC AGTCCTCAGTAGTGC TCTGTGGTGGTAGGGAGAGATGACCCCCTCACCAGATACATTCCTGGGCCTTGGGGGAGC CCTCTCTTATTACTG GCACTGCACCTGCATTTCATTTATTAGGTGTCCTGGGCTGCAGGGTGCCCTCAGGCAGAG GCTGCGGCTGGAAAA TAGACCATACCCTTCCCTGGCTGGCCCTGCACAAGGAGGCACACCCTGTTCCTGAGCCAG TCCATGAACCCAGCT GTCTCACCCCTCTCAGTGTTCTGAGAGTAGGGGATCCCCCACTGCTTGAGCACCATGAGC CCCTCCTGGCTACAG GCAGTGGGGGTAGGTATAGTCTCTCAACCCACTGTCCAACTGATTTCCAGGGTAACAGAG AGCTGTGCCTGCCCA CAGAGTTCAGGCAGAGGCCAGGCCATTGTGCTGGAAGCTGATGCTAAGCCTTGTCTGATG ATGGGGAGTGAAGCA

ATGTAACGGCTCCCTAACTGTGGCTTCTCTCAGGGCTATGGCAGCTGGCATGAGACT GCTCCAGGTCCAAGGCCT GTGGGACTTCCTGTGGACTTGAGTTTTGCCTCTGCAAACACTCCAGCAACTCTCTATGTC AGTCTAGAGGCCCAG GGACACGGATCAGGTATTGGGATGAAGGGGTTCTCCAGTTCCCAGGATTTCACAGGTCCC TGTGGAAAGTGAGGA TCCCCCAGGGGCTCTCACTCACTCACCCTTTCTCTATGTTGGGGAGCTTCCCCTGGCTCC ATGCCCATCTTGGGT GGCCAGCTGCCCAGCTTCACTCTTCCCTGTTCTCTGTGTCCCCTCACTCCCTTAATTGTC CTGATATCGTTCCTT AGGTGATCTACTTGCAGAGGCAGTGTTTACTCGCCACTTGTTTTCTCTCTGTGAGAGTAG CACACACTAGCTGCT ACTCATCTAGCATCTTGAATTCTTCCCATCTGAAAAAGTTTCAACTGCAATCACAGTTAA AGAAATACAAAAACA ATAGCACTCTAAGTTACAACTTCTCACCTATAGAATTCAAAAACATCCAAATGATTAACT AAACATTTGTTTGGT AGATCTGTGGGAAAACATGAATTCCTTGTGAATTACTGGAGAAAATGAAAATGATGCAAC ACTTATGGAAGAAAA TTTGGGGATTTTTGGGGGGGAGGGGAACAATATATTTAAAACTATAAATGCATTTATCCT AGCAATTCTATGAAT GGGGATTTATCTTAGGGTACACCTGCACACTTAGGAAATAATGTATGCAGTCATTCATTA CAGAATTGTTTGTAA TAGCAACAACCTGAAAAGCAACTCATATATCCATCCATCACACAGGGACTGGTTTCATGA CTACGGTTCATGAAT ACTCTGCAGCCCTTAGAAAGAATGAGGAAGTGGCCGGGCACGGTGGCTCATGCCTGTAAT CCCAGCACTTTGGGA GGCCGAGGCGGGTGGATCACGAGGTCAGGAGATCAAGACCATCCTGGCTAACACGGTGAA ACCCCGTCTCTACTA AAAACAATACAAAAAAATTAGCCAGGCAGGCGCCTATAGTCCCAGCTATTCGGGAGGCTG AGGCCGGAGAATGGC ATGAACCCGGGAGGCAGAGCTTGCAGTGAGCCGAGATAACGCCACTGCACTCCATCCAGC CTGGGCGACAGAGCG AGACTCCGTCAAAAAAAAAAAAAAAGAGGAAGTTCTCTATGCGCTGACATGGAAGGAAGA CAGATGGTTGAATGA AAAAAGTACATAATTAGCCATAAAGTGTAAGACTTTTTGTCTAAAAAAGAAGGGTGATAT AATTGCATATTTATA TTTTCTTCCATTTATATTAAGAGATAATAAAGGTACACAAATTGGCTAGAATAAAGTGGT TTCCTATAAAGGGTA AGAGTAATTGAGTGGATGAAGACTAGGGTTAGGGATAGATTTCTCAGTGTATTCATTTTA ATATATGTATTCATT TTATATATGTACTAATTTTTATATATGTATTTATTTTATATTTTGATTTTCTTAACATAA ATATATTATTCCTTC ATAAAATTAAACTTGATACATTTTTGATTACTAGATATGTAGAAAGCATTATGTTCAGTA CCACAGTAATACTTT CAAACCAGCTACAATTAGTATTTATGAGCATCTATGTGCCAGACATTGTGTTCTGCTTTG GTTGGTGGGGGTAGA GGAGGAAAGGAAACCATGGCTTACATAGGAGTGGAAGTCTTGTCTTTCACTTTGCACCTC TCTCCTTCAGACCTA GCATAAATATGACCTTAGGGGAGGCAGAACACATATGATAAAGAGATAACTAGCAAGAGA CATAATAGTAGCTAA ATAAATACTGAAGGAAAAATTCAGGAAGAGGTAGGAAGGATATGCCTCATCACTTCCACC TGTTAAGAAAAACTT TAGACATTCTTGCCAATATTCCTTATTGCCTGTCTTTTGAACAAATGCCATTATCACTAG AGTGAAATGATATTT CATTGTAGTTTTGATTTGCATTTCTCTCATGATCGGTGATGTTGAGCACCTTTTTATATA CCTGTTTGCCATTTG TATGTCTTCTCTTGAAAAATGTCTATTCAGATCTTTGCCCATTTTTAAATGGCGTAATAC ATTTTTTCCTATTGA

GTTGTTTGAGTTCTTTATATATTCTGGTTATTAATCCCTTGTCAGATGAATAATTTG CAAATATTTTCTCCCATT CTGAGGATTACCAGAGGCTCAGAGGGGTAATGGTGGTGGGGGAGAATAAAAATGGTTAAT GAGTACAAAAATATA GATAGGAGTAATAAGATCTAGTATCTGATAGCACAACAGGGTAATTACAGCCAACAAAAA TTTATTGTGCATTTC AAAATAACTAAGAGTATAATTGGAATGTCTGTAACACAAAGAAGCAATAAATGCTTGAGG TGATGTGAGGGGATG GATATCTAATTTACCTTGATGTGATTATTACATATTGTATGCCTGCATCAAAATAGCTCA TGTATCTTATAAGTA

TATACACCTATTATGTACCCATTAAATTTTTTAAGAACTTTAAACAAATCAAATTTA ACAGAGTTTAATTGGGCA AAGAATGATTTGAGGATCAGGCAACCCCCAGAAACAGAAGAGGTTCAAAGCAACTCAGTG CTGTCACATGGTTGG AGAGGATTTATGGGCAGAAAAGGGAAAGAGAGATACAGAAAATGGAAGTGAGGTACACAA ACAGCTGGATTGGTT ACAGCTTGCCATTTGCGTTATTTGAACATAATCTGAACAGTTGGCTGTCTTTGCTTGACC AAAACTTGGTGTTTG GTACAAGAGCAGATTACAGTCTATTTACACATCCAGTTAGTTTACAGTTCACTATACACG AAGAAGAAACCTTTA AGCAGAACTTAAAATATGCAAAGAGGAAGCTTTAAGTTAAACTTAATTTAACACACCCAA TTATCAAAAAATGAG TAGCTCTGCAAAAGTGGATTTTCCTGGTCATCTTTGGTACTTCCTTAAAAAAGAGAAAAG TAGTACTCACGATAA AAAAAAAAAAGTCCTCAAGTCTTTATTTTATTCCTTTCCAATTTAAAATGTTACATCATC TGAGGAAGGTTTTTC CCTTTGACCGCTTTCATAGACATTTCTTCTGCATGGGTTGGCCAGAATCAGAAGAGTAAT TGTAACTTTCTGTTC TTGTCCTACAGTTACAAAGCGGTTTCACTTTGTAAATGCTCTTTGGATGGCAGGAACCAA GCAGCCATGAAAAGA

GGAGTTACACCTTTAAAGGAGTCATTCCATCATGACTCTCAGGACTGGAACATGGAA TACCTGAATGGCCTCTTT GGCACAGATAGGCCACCCTTGAAAGGTGTTCCAAGCTAGGAACTCACTACCACTGTTACA TCGATGCAACTCTGT GAGAAGTTTTTATCTGGTGATGGAAAATCTCATCTCTTCAACACACTGACTACTACCAGT CTCAGAACCCTGTAA ACAAGATTCATTCATCTCAAATTGGGTTAAAGCAGTCACCCTGCCTTACATTAGTTTGGA ATAAGGATGTGGGGA TGGTGGTAGAGGAGGGGAGTGGATGATGATTTTTTTATTGTTATTTGATTCTAAAGAAAC TTCTATACATTTTGC ATTTAAAATAATTATGTTTTTAACAATGTTTGGATTAATTCAAAATAGGATATTATATCC TATTATATTAAATAT ACTATTTAATCATCTTGTTGACCAAATGCAACTTAAACATGTAAAATGGTAAATAGCATA ATAATTGTCTTCTAA GCCTGCACTATAAAGTATTTCAGTGGCCTCATTATTAAAGGACCAAGGTGCCCAAAGAAA CAAAATTTAGTAATC ATAAACAAGAGACAAACCTACTTCTTTTCCCCCAGAGTTCTGGCCACATTGAAATAAGGT GTTTGAATGCTTAAT AAGAATTATTTTGGCCCACACAGTGGCTCATGCCTGTAATCTCAGCACTTTGGGATGCCA AGGTGAGCAGATCAC

TTGAGGCCAGGAGTTCAAGACCAGCGTGGCCAACGTGGTGAAACCCCATCTCTACTA AAAATACAAAAATTAGCC CGGTGTGGTGGTACACGCCTATAGTCCCAGCTACTCGGGAGACTGAGGTGGGAGAATCAC TTGAACCCGGGAGGC CAAGGCTGCAATATCGAGATCACACCACTGCACTCTAGCCTGGGCAACAGAGTGAGAGTG AGACTCTTTCTCGGA AAAAAAAAAAAAGAATTATTTTGAACAAAGTGCTGTCACCTAAGTTAGCAAAACTCCAAG CAAGGTTTTTGGCTC TGTAAGGAAAGAATTAGCCTACTCATTTGGAAATTTAGTGGTGTTTGTAATGCAGAAAGT GACAGTGAGACTGGA AAGGGATTGGCTTTGGGGCTTGTTCTGCTTTATAAATAATAATGAATCTTCTCCAACATG AAGTAATGTGAATTA AAAAAAAAAAATCTGTCCTTAGAGTACAAAATTACTTCATAACCCAATCTGCATTTCTCC ACTCCAAGCATATTT TCTGGGAGTTCTACTTAGAGAGTGAAAGCTGCTGTGTGTGTGATAATTAATTTTAACAAA CACTTGGCAAACTGA GCTGGACTATGTATAAGCTACCCTAGACTAAGCATGAATTTGAACTGCACTTTTTATGGT GTTTTTTCCACAATG ACATTATTTAGGCATTTAAAGTTATCTGAACTGCAATTTTTTGTTCTTTTTTTTTTAATT TGACTTTTTAAAAAA AATTATTCCTGAATAAAGAGGCAGTTTGTAAAAACTCGAGAACTGTGAGAGATAATTGGA TCTTTGTGTAGCAAA ACTAGAAGGGTGTTGGGTATCTGCTCTTTATCAAATGGACCACTTACTTTTCTTTTCTTT TTTGCCCTGTGTTCA GAAAACAAATGTGCGTGTCTCCTGATTTATAATGTATAGTTCATTAATGGAGAAAGTGCT TGAGAATTAGATCCT AATGTCATTTCCCATGCAGCATCTTCATTCTTTTCTAAAGCACTATTTGGTAAAAACAAC TGATAGTCGTCAGAG GTGATCAGCAATGTTTGAGCACTATTTCCTTTTTATATCCTGCACATGGAATATGGACAG GCAAACAAATCATTT CCAAGTAAGAAAATAAATTTTGAGGGAGTTAATACTATAATTTGAAAGTAATAACCTCCT ATTTATCCATCTAGT TTGTTGTTCTGTACTAAATTATTTGTGCATGTCTCTGTGTCTATAATTTATGTGAAACTT TGCACAATCTTAAAT AGGACAAAATAGACATTCTGTAATTTCCCAGGCAAGCTATTTAAGGTGACTATCTCTCTA CATATTTGAGATGAA AAACAATAACATGACAATCCATCCCTTCTTAGGTTTTTGTAAGCAGACTTACTACCTGTG ACTCAGTTTTGTTCT CACAGGGTACTAATTAATCCTTCACGATAATAACTTGTCAAATTCCATTACTTCTGTAAA GGCAATACTTTATAT TTGTTTGTATTCAAATTTTAAACTGATGTTAAATGCCGTGGGTGCAACTGCAGGTTAAAA ATATGTGTTTGAATC TCTTATTCTTTTTGCTTGGCAATGTATGAAATAACTGCTCTTTCTAGAAATCTTGATGAT GAAGTGGCCTGTTGT TTTGTCACCTAAAAATGCAATAATGTTCAAATTAAGCTTTTCTTTATTAACATCACTTGA TTGTGTGCCATATTT AGAGCTTAGTGAAATTTTAATCTACACATTGATTAAATACATTTTATTTATTCTTGTTTC TAATGGGAACTTTCT TTGTTTCTAATGGGAACTTTCTTAAATTAAATTACATCCAACATTTATTAAAGACCTAAA ACATAGGCAATTACT GTGCTTAGAGGAAAAGCGCAGACGAAAGTGAATCAGACAAGTTCCCTGCCCTCCGGAAGC TTTCAGTCTAGTGAT GAGAAAGACGTATACACACCTTATGTTGATTTAAAAAAAAAAAAAGCTCTTACCTGGTTG CTGGCATATGAAAGT GTTAGTTACAGATCTGCCCCAAACTAAAGGTGTCACCTCGAGTAAATCTCTTTCCCTTTC CCTTTCAATCTCTTC ATCTATAAACTAGGGGTTGGGAATACATTTATTAACAAACACAAATTGAGCGTCTACCAT GTGATAATAGTAGCT

AAACTTACTGAGCAATTACCATGGGGCAGGTATCAAGATAAACCCTTTATGATGGTA ACCTCATTTAATCCTCAA AGCAATTCCATTTTCAAGAGGAGGAAATTGAGGCTCAAAAATGTTAAGTAACTCCCCCAA GGATGCAAAGTGATT GAGCCAGAATTCAAGACTAGGTTGGTTTGACTCCAAAACTCATGCCATTAAACCCTATTG TGTCACTGCAAACAA CTCTAATAGTTTCAAATTATTAGTTCTATTAATATTATATTACCATTATTTGCCCCCAAA ATGTAAAATGTAAAT ACAAAGAGTTTGGTTTTTGTATTACTAGTGGAGGTTAAAGGTGCACAATGGAATTATTCA AACTGGGAAAATCCA

GGAAGACTTCATGGAGGAGGCAGCATATGGCTGCAGTTAATAAGGTTTGCTCACACA AAATGGAGAGGTGAGGAC ATTTCAGGCAGAGAGAATTATATGAGAGGTTACAGAGCAGTAAACAGTCATGCGTCTGCA AGATCAAAGGGAAAG GGCGGTAAGAGAGAAGCTTGAAAGTCAAGTGGAGCCAGATTGTGGAAAAACTAGAGAGTC ATGCCAAGGACCTTG ACATATAGAAAATGGGAAGCCCCTGAAAGGTGAAGAACATGAGAGTGAAATGATTAGTAA CTTTTTGGTTTAGGA CTTGTTTCTTTTGTGTTTTGGTTGCTTTCTTGTTTTGTTTTGTTTGTGGTTTTTAAATTT ACAACCAATAAGAAT ATTTAGTAAGGTTTCCAAATACATCATGAATATATAAAACTAGCCTGACTCAAGGATAAT AATTCTGGGTAGTTG GAGTGAAGTTTCAATCAGCTACGTGGCATTTGCTAATCATCTGATATGAGCTAACAATAA AGGAGTTAACAAATA AACTGTCAGCCTACAGTCCAGGGTCTCAAATAGCATGTGACATAGTTGAGAAGCAGTTTT CCATATCATACATGA AATAACTAAAGAAACTACTTACAAAGCACTATACCAGTAACTACAATAAAATACAACTAT ACATGCAAAATAATG CTGAAAGCTGCAAGTAGAGGGGTAAAGCTAGGCCAGTTGCTCAGGGAACCATTCTGAAGT GGATTTGGGAAGTAT

GTCTAGAAGGGGAGCCATTGCTGTGAGAGTGCTGAGGCTCATCTGCTACTAGTCCCC CACTACTCAGGCATATGG TAGGTCAGTAACAAAACCATCATTGTGCACTGTTCTTTCCATCTAAATTCCATCAAATTA TGACCAACCTATCAA GGTACTAGTTCAAATTCTCTCTTCCTCTATAAGCTAGTGGTCTTCTCTAAAATTTAAGAA GATCGTGCTCATCTT CCTACTTCTTGTTCTCTTTCTTCTGTGTTTTCTGAGGCTGCAATGAACTAGGAACTTCCT CTCCCCAGAACTCTG TATTCCAGGCCTTAGATCACTCAAAACTGTTGCTTATAAAGTGCAGAGAATCAACAGAGA AGGAATAGAGGTTAA TGTCTGGTCAAAGATGTGATTCTCTTGTTGAAAAGTTCATTAGCTTATTATTTATAGAAT CATAAGTCCCAGGAA AAACCAAAAGGAAATATATATTGGATCCTAATGATATTCTCTTTTTTTCTTTTTTCTTTT CCCCCACTCCATTGC CCAGGCTGGAGTGCAGTGGCATAATCTCAGCTCACTGCAACCTCCACCTCCCGGGTTCAA GGGACTCTCCTGCCT CAGCCTTCCAAGTAGATGGGATTACAGGCATGTGCCACCACATCTGGCTAATTTTTTTTT GTATTTTTAGTAGAG ATGGGGTTTCACCATGTTAGTCAGGCTGGTGTTGAACTCCTGACCTCAAATGATCCACCA GCCTCGGCCTCCCAG

TGTGCTGGGATTGCAGGCGTGAGCCACCACACCCGGCCTGATATTCTCTTGCAAGGG CATTGTTTACATTGTCTA TCATCAGAACTGTAGAGTGTTGGCTCCAGGCACAGAACCCCTAGAGTTTTGTAAACCATT TATATCACACTGGCA ACCAGAAGTAACTTTATATACTCAAGAATCAAGATTTCACCTAGAAGTACCTCAGGTAGG TGTTGGTTCATTCAC ATTCCAACCAAAAGATAATGTACCATAAAGTGCATACCGCCTAGTCCGTAATGATTAAGG CAACCACATAAAATC TCATTATTTAAAAGAAATTAAGTCCAGGCACGGTGGCTCACACCTGTAATCTCAGCACTT CGGGAGGCCAAGGAG GGCAGATCACCTGAGGTTGGGAGTTTGAGACCAGCCTGATCAACATGGAGAAATCCCATC TCTACTAAAAATACA AAATTAGCGGGGCATGGTGGTGCATGCCTATAATCCCAGCTACTCAGGAGGCTGAGGCAG GAGAATCACTTGAAC CCAGGAGGTGGAGGTTGAGATCGTGCCATTGCACTCCAGCCTGGACAACAAGAGTGAAAC TCTGTCTCAAAAAAG AAAAAAAGAAAAAGAAATTAAATGCACTATGGTTTATGGAGCGGTATTCCTCCTCCATGT CCTACATAAGATCTT TCACATGCCAGTCACAGTTAAATCTAATTTGCTGTAATCTGGATAAATGGGAGCTAATCA ACAAGCTCTCAGCTC TAGCTCTGAATCAGCAGCAGATATTGCATTTTTGAAATACACTAATAGCAAGAATGCCTT CCTGACAACAACTGG CATTTTTGACACAGCAGGAAGTTTATCTGGATTCTGATATAATAGTTATTGGAATCATAC ATAGGTACATAGTTT AAAAGGCTAATAAGTCATTTGTTATTGCTTTTATTATCTCTGCATAGTTAGTAAAATTGA GATTAGAACCACTTC TCGAATGTACTGTTCTAAATCCTTAGCTTGCTTGATCACACATGACCCTCACAATGATCC TAGGAGAAATTATTC TGCATGCCATTTTGTAGCTGGGGAAACTGAGGCACAGAGAAATACAGTACTGCCCAAAAT GTCATAACTAATCAA AGGCAAAGACAATACTCACACCAGCTCTGATTCCAGAGCCCACTCTCTTAACCATATGCT TTTCTGCTTCCCTAG TTGTAGAGTCTTTTTGTATGACTGCATTAATTATATGTGAAGAGTTCAAAAATTTCTATA TAAGGTCTTTTAAGG GTGTCATTCTGGTTGAAAATGGAGGACTAGGCTTCTCACTTGAAGACATATTTCTGTAGA AAAACCTATTTTCAT TTAGATGCTACAGTTACTTGATGTGGTTAATAAACCAGTTAACAGAGTATGAAAAGGATA AGGGTTAAAGCCCTC CCAAGCCATCTTTCATGCTGCTAATATGAATCACATTACTAGATACTTAAATATCATTTT CTCTTTGGTTCCCAG AAGACTGCATATATGCTAGAATATTTGTCCTCCTCTTTTACCCTTTCAGGCAATAAAGTA TTTTGGACCACTGTA CTATGTTATAATTATTGTTTCTCTCCTGATTTTTTTGCTCCAATCTAATGAAAGACATAC AAGCTACTATACTGC TACACAATGACTAAATACCTGTTGGATTAGGTGGGGGGAAGATACACAGTCACTGGCTAG AAAGCATCATGCATA CAGAGCCATTTTCACCATATATTTTATTTCTCATGATCATGTAGAATTTAGGCTTTGGTG TTGATTATTTCTCTC TTAGGAAACATAGTTGTTTCAGGGTTGATATCACAAAAAAACAGAAAAACCTATTCGAGA AAAGGAAAATTATTT GTCTGTAGGCCAAATTTTGAAGTAGGAAAACCTGCTTTTGGAGTTGTATTCCCCTCCCAG GCACTTAATCCAAGT TCCAGTCTTATTCTAAACTGGGGATGCTAGTATTAACCACCATAGGAGTTATCTGAGATG AGTTATCATCAACTT GGTACCAGGTTGTTGTCCTCTGGACTCAGTGAGCTCTAGAATTGCATGAAACTGGCCTAA TTTATCAAAGTATGT AGCCTTGGGTAAATAATTCAAGCTCTCAGAGGTCCAGTTATCTCCTCTGTAAAACATATC TACATCCTAGGGATG

ACAATATCTACATCCTAGAGATGTCAGGAGGATTAAGTGTAATTTTTTTTAATTGTA TGTATTTAAAATGGGCAA CATAATGTTTTGATATACACGTGTATAGTGATTACTACAGTCAAGCAAATTAACATATCC ATCATTTCATAGCTA CCTTTTATGTATGTGATAAGATTATCTAAAATCTATTCTCTTACCAAATTTCCAGTATAC AATATTGATATGGTT TGATCCATATCCCCATCCAAATCTCATGTTCAGTTGCAATCCCCAACGTTGGAGATGGAG CCTGGTTGGAGGTGA TTGGATCACAGGGGTGGCTTCTAATGGTTCAGCACCATCCTTTCTTGGTACTGTATAGTG AGTAAGTTCTCACGA

GATCTGGTTGTTTAAAAGTGTGTAACACCTCCCCCACTTTCCCTCTCTCTGTTCCTC CTGCTCCCGCTATGTGAA GTGCCAGCTCCCTCTTTGCCTTCCGCCATGATTGTAAGTTCTCTGAGGCATCCCCAGAAG CTGATGCTGCCATGC TTCCTATACAGCCTGCAGAACCATGAGTCAATTAAACCTCTTTTCTTTGTAAATTACCCA GTCTCAAGTATTTCT TTATAGCAATGCAAGAATGGACTAATACAGAAAATTGTTACTGAGAAGAAGGGCATTGCT ATAAAGATACCTGAA AATGTAGAAGTGACTTTGGAACCGGCTAACAGGCAGAAGTTGAAACATTTTAGAGGGCTC AGAAGAAGACAGAAA GATGAGAGAAAGTTTGGAACTCGCTAGGAACTTGTTGAGTGGTTGTAACCAAAATACTGA TAGTGATATAGACAG TGAAGTCCAGGCTGAGGAGGTCTCAGATGGAAATGAGAAATTTATTGGGAATGAGTAAAG GTCAGGTTTGCTATG CTTTAGCAAAGAGCTTAGCTGCATTGTTCCTCTGTTCTAGGGATCTGTGAAATCTTAGAC TTAAGAATGATGATT TAGGGTATCTGGCAGAAGAAATTTCTAAGCAGCAGAGTGTTCAAGAAGTAACCTAGCTGC TTCTAATAGCCTATG CTCATAGGCATGAGCACAGAAATGACCTGAAATTGGAACTTACACTTAAAAGGGAAGCAG AGCATAAAAGTTTGT

AAATTTTGCAGCCTGGCCATGTGGTAGTAAAGAAAAGCTCGTTCTCAGGAGAGGAAG TCAAGCAGGCTGCATAAA TTTGCATAACTAAAAGGAAGGCAAGGGCTGATAACCAAAACAATGGGGAGAAAGACTCAT AGGACTAACAGGCAT TTTATTTTATTTTATTTTTATTTTATTATTATTATACTTTAAGTTTTAGGGTACATGTGC ACAATGTGCAGGTTA GTTGCATATGTATACATGTGCCATGCTGGTGTGCTGCACCCATTAACTCGTCATTTAGCA TTAGGTATATCTCCT AATGCTATCCCTCCCCCCTCCCCCACCCCACAACAGTCCCCAGAGTGTGATGTTCCCCTT CCTGTGTCCATGTGT TCTCATTGTTCAATTCCCACCTATGAGTGAGAACATGTGGTGTTTGGTTTTTTGACCTTG CAATAGTTTACTGAG AATGACGATTTCCAATTTCATCCATGTCCCTACAAAGGACATGAACTCATCATTTTTTAT GGCTGCATAGTATTC CATGGTGTATATGTGCCACATTTTCTTAATCCAGTCTATCACTGTTGGACATTTGGGTTG GTTCCAAGTCTTTGC TATTGTGAATAGTGCCACAATAAACATAGTGTGCATGTGTCTTTATAGCAGCAGGATTTA TAGTCCTTTGGGTAT ATACCCAGTGATGGGATGGCTGGGTCAAATGGTATTTCTAGTTCTAGATCCCTGAGGAAT CGCCACACTGACTTC

CACAATGGTTGAACTAGTTTACAGTCCCACCAACAGTGTAAAAGTGTTCCTAATAGG CATTTTAGGCTTTCATGG TGGTCCCTCTCATCACAGGCCCCGAGGCCTAGGAGGACTGAATCATTTCCTGGGCCAGGC CTAGGGCCCCTGCTC CCTCTTACAGCCTTGGGACTCTGCTCCCTGAATCCCAGCTGCTCAAAGGGGCCCAGGTAC TGTTACAGTAGGTAG CTAATCAGGCATGAGTGGGGTAAGAGAGAAGTCCCCACCACCCACCAGGAATGTCAGGCA ACCATCAGATGATGG TCAGGCAGTTGTCATACTGCCTCTCTAAAATAGTAATTGGTTGCAGCCAGCACCAGGGAG AGGCAACTTCTCAAT AGATAGAAACACCTGAAATTGGTAACTGGGCGCTTCCAATAAGATCTCAGGAACTGAGAG AGTGGGCTTAACATG CACATTAAGAGGCAAAATGGTGAAGTATGACCTTTGGGGGCATTCCACCGGAAAAGGGAA GAAAGCCTCAGGTAA GCATGTATACAACTCCAGTAAACACACTGCACACGCTCACCTTCCAAGTGCAAGCAGGGC ACCATGCATGCGGCA AGCTCACCCTTAGGGAAGGACCAAGGGAAAGGGGCACAAGATGTCAGAAGTAGGCCAGTG TATAAGATCCTAGGT TCAAGGTCAAACAGGGCACTTGACCTCCAAGGTGCCCACTTGGGCCTCTTCCAAATGTAC TTTCCTTTCATTCCT GTTCTAAAGCTTTTTAATAAACTTTTACTCCTGCTCTGAAACTTGTCGCAGTCTCTTTTT CTGCCTTATGCCTCT TGGTCAAATTCTTTCTTCTGAGGAGGCAAGAATTGAGGTTGCTGCAGACCCACATGGATT TGCAGCTGGTAACTC AGATAACTTTCACCAGTAAGAATACAGTTCAGGCTGCTGCTTCACAGGGTGCCAGGCATA AGCCTTGGTGGCTTC CATAAGCTGTGAAGCCGGCGGGCGCACATAATGCAAGAGTTGAGGCTTAAGAAGCTCTGC CTAGATTTTAGAGGA TGTATGAAAAAGCCTGGATGTCCAGACAGAAGCCTGTTACTGGGGTGGAATCCTCATGGA GAACATCTACTAGGG AAGCAAGGAGAAGAAATGTGGGGTTGCAGCCCCCACAGAGAGTCCCCTGGGGCACTGCCT AGCAGAGCTATGACA AGACAGCCACCGTCCTCCAGACCCCAGAATGGTAGATCCACCAACAACTTGCACCCTGCA GCCTGGAAAAGCTGC AAGCACTCAATGCTAGCCCATGAGAGCAGCTGTGGGAGATGAACCCTGGAAAACCACAGG GGTGGTTCTGCCCAA GGTTTTGGGAGCCCACTCATTGCATCAGTGTTCCCTGGGTGTGAGTCAAAGGAGATTATT TCAGAGCTTTAACAT TTAATGACTGCCCGGCTGGCTTTCAGACTTGCAATGGGGCCCTATAGCCTCTTTCTTTTG GCAGATTTCTCCCTT TCGGAATGGCAGTATCTGCCCAATGCCTATACCCCCATTGTATCTTTGAAGCAATTACCT TGTTTTTGATTTTAC AGGTTCATAGGTAGAAGGGACTAGCTTCGTCTCAGGTGAGACTTGGGACTTTGGACTTTT GAATGAATGCTGGAT CGAGTTAAGACTTTGGGGAACTGTTGGTAAGGCACGACAGTATTTTGCAATATGAGAAGG ACATTAGATTTGGGA GGGGCCAGAGTTGGAATAACATGGTTTGGATCTCTGTCCCCACCCAAATCTCATGTTCAA CTGTAATCCCCAGTG TTGGAGGTTGGGCCTGGTGGGAGGTGAGTGGATTATGGGGTGGCTTCTAATGGTTTTGTA CAGTCCCCTCTTGGT ACTATATAGTGAGTTCTGACAAGATCTAGTTGTTTAAACGTATGTAGCACCTCCCATTTC TCTCTTCCCCCAGTT CCTGCCATGTGAAGTCTGGGGTCTCCCTATGCCTTCCATCATGATTTTAAGTTCCCTATG GCCTGCCCAGAAGCT GATCCAGCCATGCTTCTTGTACAGCCTGCAGAACTGTGAGCCATTAAACTTTTCTTTATA AATTACCCAGTTTCA GTTATTTCTTTATAGCAGTGTAAGAATGGACTAACACAATTATTAACGCTAGTCCTCATG TTGTACATTAAATCT

CTAGATGTATTAGACGTAACTGCAACTTTGTACCCTACCCTACAATTTTCTTTCCCC CCAAGCCCCCCAACCAAG GGTCTACTCTGTTTCTATAAATTCAGTTGTTTTTTAATTCCACGTATAAGTGAAGTACAA CTCAGTGTAGAAACT TGGTAAATGCTAGCTACTTGTTATAAGCTGTCAGTCAAAATAAAAATACAGAGATGAATC TCTAAATTAAGTGAT TTATTTGGGAAGAAAGAATTGCAATTAGGGCATACATGTAGATCAGATGGTCTTCGGTAT ATCCACACAACAAAG AAAAGGGGGAGGTTTTGTTAAAAAAGAGAAATGTTACATAGTGCTCTTTGAGAAAATTCA TTGGCACTATTAAGG

ATCTGAGGAGCTGGTGAGTTTCAACTGGTGAGTGATGGTGGTAGATAAAATTAGAGC TGCAGCAGGTCATTTTAG CAACTATTAGATAAAACTGGTCTCAGGTCACAACGGGCAGTTGCAGCAGCTGGACTTGGA GAGAATTACACTGTG GGAGCAGTGTCATTTGTCCTAAGTGCTTTTCTACCCCCTACCCCCACTATTTTAGTTGGG TATAAAAAGAATGAC CCAATTTGTATGATCAACTTTCACAAAGCATAGAACAGTAGGAAAAGGGTCTGTTTCTGC AGAAGGTGTAGACGT TGAGAGCCATTTTGTGTATTTATTCCTCCCTTTCTTCCTCGGTGAATGATTAAAACGTTC TGTGTGATTTTTAGT GATGAAAAAGATTAAATGCTACTCACTGTAGTAAGTGCCATCTCACACTTGCAGATCAAA AGGCACACAGTTTAA AAAACCTTTGTTTTTTTACACATCTGAGTGGTGTAAATGCTACTCATCTGTAGTAAGTGG AATCTATACACCTGC AGACCAAAAGACGCAAGGTTTCAAAAATCTTTGTGTTTTTTACACATCAAACAGAATGGT ACGTTTTTCAAAAGT TAAAAAAAAACAACTCATCCACATATTGCAACTAGCAAAAATGACATTCCCCAGTGTGAA AATCATGCTTGAGAG AATTCTTACATGTAAAGGCAAAATTGCGATGACTTTGCAGGGGACCGTGGGATTCCCGCC CGCAGTGCCGGAGCT

GTCCCCTACCAGGGTTTGCAGTGGAGTTTTGAATGCACTTAACAGTGTCTTACGGTA AAAACAAAATTTCATCCA CCAATTATGTGTTGAGCGCCCACTGCCTACCAAGCACAAACAAAACCATTCAAAACCACG AAATCGTCTTCACTT TCTCCAGATCCAGCAGCCTCCCCTATTAAGGTTCGCACACGCTATTGCGCCAACGCTCCT CCAGAGCGGGTCTTA AGATAAAAGAACAGGACAAGTTGCCCCGCCCCATTTCGCTAGCCTCGTGAGAAAACGTCA TCGCACATAGAAAAC AGACAGACGTAACCTACGGTGTCCCGCTAGGAAAGAGAGGTGCGTCAAACAGCGACAAGT TCCGCCCACGTAAAA GATGACGCTTGGTGTGTCAGCCGTCCCTGCTGCCCGGTTGCTTCTCTTTTGGGGGCGGGG TCTAGCAAGAGCAGG TGTGGGTTTAGGAGGTGTGTGTTTTTGTTTTTCCCACCCTCTCTCCCCACTACTTGCTCT CACAGTACTCGCTGA GGGTGAACAAGAAAAGACCTGATAAAGATTAACCAGAAGAAAACAAGGAGGGAAACAACC GCAGCCTGTAGCAAG CTCTGGAACTCAGGAGTCGCGCGCTAGGGGCC (GGGGCC)„

GGGGCCGGGGCGTGGTCGGGGCGGGCCCGGGGGCGGG

CCCGGGGCGGGGCTGCGGTTGCGGTGCCTGCGCCCGCGGCGGCGGAGGCGCAGGCGG TGGCGAGTGGGTGAGTGA GGAGGCGGCATCCTGGCGGGTGGCTGTTTGGGGTTCGGCTGCCGGGAAGAGGCGCGGGTA GAAGCGGGGGCTCTC CTCAGAGCTCGACGCATTTTTACTTTCCCTCTCATTTCTCTGACCGAAGCTGGGTGTCGG GCTTTCGCCTCTAGC GACTGGTGGAATTGCCTGCATCCGGGCCCCGGGCTTCCCGGCGGCGGCGGCGGCGGCGGC GGCGCAGGGACAAGG GATGGGGATCTGGCCTCTTCCTTGCTTTCCCGCCCTCAGTACCCGAGCTGTCTCCTTCCC GGGGACCCGCTGGGA GCGCTGCCGCTGCGGGCTCGAGAAAAGGGAGCCTCGGGTACTGAGAGGCCTCGCCTGGGG GAAGGCCGGAGGGTG GGCGGCGCGCGGCTTCTGCGGACCAAGTCGGGGTTCGCTAGGAACCCGAGACGGTCCCTG CCGGCGAGGAGATCA TGCGGGATGAGATGGGGGTGTGGAGACGCCTGCACAATTTCAGCCCAAGCTTCTAGAGAG TGGTGATGACTTGCA TATGAGGGCAGCAATGCAAGTCGGTGTGCTCCCCATTCTGTGGGACATGACCTGGTTGCT TCACAGCTCCGAGAT GACACAGACTTGCTTAAAGGAAGTGACTATTGTGACTTGGGCATCACTTGACTGATGGTA ATCAGTTGTCTAAAG AAGTGCACAGATTACATGTCCGTGTGCTCATTGGGTCTATCTGGCCGCGTTGAACACCAC CAGGCTTTGTATTCA GAAACAGGAGGGAGGTCCTGCACTTTCCCAGGAGGGGTGGCCCTTTCAGATGCAATCGAG ATTGTTAGGCTCTGG GAGAGTAGTTGCCTGGTTGTGGCAGTTGGTAAATTTCTATTCAAACAGTTGCCATGCACC AGTTGTTCACAACAA GGGTACGTAATCTGTCTGGCATTACTTCTACTTTTGTACAAAGGATCAAAAAAAAAAAAG ATACTGTTAAGATAT GATTTTTCTCAGACTTTGGGAAACTTTTAACATAATCTGTGAATATCACAGAAACAAGAC TATCATATAGGGGAT ATTAATAACCTGGAGTCAGAATACTTGAAATACGGTGTCATTTGACACGGGCATTGTTGT CACCACCTCTGCCAA GGCCTGCCACTTTAGGAAAACCCTGAATCAGTTGGAAACTGCTACATGCTGATAGTACAT CTGAAACAAGAACGA GAGTAATTACCACATTCCAGATTGTTCACTAAGCCAGCATTTACCTGCTCCAGGAAAAAA TTACAAGCACCTTAT GAAGTTGATAAAATATTTTGTTTGGCTATGTTGGCACTCCACAATTTGCTTTCAGAGAAA CAAAGTAAACCAAGG AGGACTTCTGTTTTTCAAGTCTGCCCTCGGGTTCTATTCTACGTTAATTAGATAGTTCCC AGGAGGACTAGGTTA GCCTACCTATTGTCTGAGAAACTTGGAACTGTGAGAAATGGCCAGATAGTGATATGAACT TCACCTTCCAGTCTT CCCTGATGTTGAAGATTGAGAAAGTGTTGTGAACTTTCTGGTACTGTAAACAGTTCACTG TCCTTGAAGTGGTCC TGGGCAGCTCCTGTTGTGGAAAGTGGACGGTTTAGGATCCTGCTTCTCTTTGGGCTGGGA GAAAATAAACAGCAT GGTTACAAGTATTGAGAGCCAGGTTGGAGAAGGTGGCTTACACCTGTAATGCCAGAGCTT TGGGAGGCGGAGGCA AGAGGATCACTTGAAGCCAGGAGTTCAAGCTCAACCTGGGCAACGTAGACCCTGTCTCTA CAAAAAATTAAAAAC TTAGCCGGGCGTGGTGATGTGCACCTGTAGTCCTAGCTACTTGGGAGGCTGAGGCAGGAG GGTCATTTGAGCCCA AGAGTTTGAAGTTACCGAGAGCTATGATCCTGCCAGTGCATTCCAGCCTGGATGACAAAA CGAGACCCTGTCTCT AAAAAACAAGAAGTGAGGGCTTTATGATTGTAGAATTTTCACTACAATAGCAGTGGACCA ACCACCTTTCTAAAT ACCAATCAGGGAAGAGATGGTTGATTTTTTAACAGACGTTTAAAGAAAAAGCAAAACCTC AAACTTAGCACTCTA

CTAACAGTTTTAGCAGATGTTAATTAATGTAATCATGTCTGCATGTATGGGATTATT TCCAGAAAGTGTATTGGG AAACCTCTCATGAACCCTGTGAGCAAGCCACCGTCTCACTCAATTTGAATCTTGGCTTCC CTCAAAAGACTGGCT AATGTTTGGTAACTCTCTGGAGTAGACAGCACTACATGTACGTAAGATAGGTACATAAAC AACTATTGGTTTTGA GCTGATTTTTTTCAGCTGCATTTGCATGTATGGATTTTTCTCACCAAAGACGATGACTTC AAGTATTAGTAAAAT AATTGTACAGCTCTCCTGATTATACTTCTCTGTGACATTTCATTTCCCAGGCTATTTCTT TTGGTAGGATTTAAA

ACTAAGCAATTCAGTATGATCTTTGTCCTTCATTTTCTTTCTTATTCTTTTTGTTTG TTTGTTTGTTTGTTTTTT TCTTGAGGCAGAGTCTCTCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCCATCTCAGCTC ATTGCAACCTCTGCC ACCTCCGGGTTCAAGAGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTGT CCACCACCACACCCG GCTAATTTTTTGTATTTTTAGTAGAGGTGGGGTTTCACCATGTTGGCCAGGCTGGTCTTG AGCTCCTGACCTCAG GTGATCCACCTGCCTCGGCCTACCAAAGAGCTGGGATAACAGGTGTGACCCACCATGCCC GGCCCATTTTTTTTT TCTTATTCTGTTAGGAGTGAGAGTGTAACTAGCAGTATAATAGTTCAATTTTCACAACGT GGTAAAAGTTTCCCT ATAATTCAATCAGATTTTGCTCCAGGGTTCAGTTCTGTTTTAGGAAATACTTTTATTTTC AGTTTAATGATGAAA TATTAGAGTTGTAATATTGCCTTTATGATTATCCACCTTTTTAACCTAAAAGAATGAAAG AAAAATATGTTTGCA ATATAATTTTATGGTTGTATGTTAACTTAATTCATTATGTTGGCCTCCAGTTTGCTGTTG TTAGTTATGACAGCA GTAGTGTCATTACCATTTCAATTCAGATTACATTCCTATATTTGATCATTGTAAACTGAC TGCTTACATTGTATT

AAAAACAGTGGATATTTTAAAGAAGCTGTACGGCTTATATCTAGTGCTGTCTCTTAA GACTATTAAATTGATACA ACATATTTAAAAGTAAATATTACCTAAATGAATTTTTGAAATTACAAATACACGTGTTAA AACTGTCGTTGTGTT CAACCATTTCTGTACATACTTAGAGTTAACTGTTTTGCCAGGCTCTGTATGCCTACTCAT AATATGATAAAAGCA CTCATCTAATGCTCTGTAAATAGAAGTCAGTGCTTTCCATCAGACTGAACTCTCTTGACA AGATGTGGATGAAAT TCTTTAAGTAAAATTGTTTACTTTGTCATACATTTACAGATCAAATGTTAGCTCCCAAAG CAATCATATGGCAAA GATAGGTATATCATAGTTTGCCTATTAGCTGCTTTGTATTGCTATTATTATAAATAGACT TCACAGTTTTAGACT TGCTTAGGTGAAATTGCAATTCTTTTTACTTTCAGTCTTAGATAACAAGTCTTCAATTAT AGTACAATCACACAT TGCTTAGGAATGCATCATTAGGCGATTTTGTCATTATGCAAACATCATAGAGTGTACTTA CACAAACCTAGATAG TATAGCCTTTATGTACCTAGGCCGTATGGTATAGTCTGTTGCTCCTAGGCCACAAACCTG TACAACTGTTACTGT ACTGAATACTATAGACAGTTGTAACACAGTGGTAAATATTTATCTAAATATATGCAAACA GAGAAAAGGTACAGT

AAAAGTATGGTATAAAAGATAATGGTATACCTGTGTAGGCCACTTACCACGAATGGA GCTTGCAGGACTAGAAGT TGCTCTGGGTGAGTCAGTGAGTGAGTGGTGAATTAATGTGAAGGCCTAGAACACTGTACA CCACTGTAGACTATA AACACAGTACGCTGAAGCTACACCAAATTTATCTTAACAGTTTTTCTTCAATAAAAAATT ATAACTTTTTAACTT TGTAAACTTTTTAATTTTTTAACTTTTAAAATACTTAGCTTGAAACACAAATACATTGTA TAGCTATACAAAAAT ATTTTTTCTTTGTATCCTTATTCTAGAAGCTTTTTTCTATTTTCTATTTTAAATTTTTTT TTTTACTTGTTAGTC GTTTTTGTTAAAAACTAAAACACACACACTTTCACCTAGGCATAGACAGGATTAGGATCA TCAGTATCACTCCCT TCCACCTCACTGCCTTCCACCTCCACATCTTGTCCCACTGGAAGGTTTTTAGGGGCAATA ACACACATGTAGCTG TCACCTATGATAACAGTGCTTTCTGTTGAATACCTCCTGAAGGACTTGCCTGAGGCTGTT TTACATTTAACTTAA AAAAAAAAAAAGTAGAAGGAGTGCACTCTAAAATAACAATAAAAGGCATAGTATAGTGAA TACATAAACCAGCAA TGTAGTAGTTTATTATCAAGTGTTGTACACTGTAATAATTGTATGTGCTATACTTTAAAT AACTTGCAAAATAGT ACTAAGACCTTATGATGGTTACAGTGTCACTAAGGCAATAGCATATTTTCAGGTCCATTG TAATCTAATGGGACT ACCATCATATATGCAGTCTACCATTGACTGAAACGTTACATGGCACATAACTGTATTTGC AAGAATGATTTGTTT TACATTAATATCACATAGGATGTACCTTTTTAGAGTGGTATGTTTATGTGGATTAAGATG TACAAGTTGAGCAAG GGGACCAAGAGCCCTGGGTTCTGTCTTGGATGTGAGCGTTTATGTTCTTCTCCTCATGTC TGTTTTCTCATTAAA TTCAAAGGCTTGAACGGGCCCTATTTAGCCCTTCTGTTTTCTACGTGTTCTAAATAACTA AAGCTTTTAAATTCT AGCCATTTAGTGTAGAACTCTCTTTGCAGTGATGAAATGCTGTATTGGTTTCTTGGCTAG CATATTAAATATTTT TATCTTTGTCTTGATACTTCAATGTCGTTTTAAACATCAGGATCGGGCTTCAGTATTCTC ATAACCAGAGAGTTC ACTGAGGATACAGGACTGTTTGCCCATTTTTTGTTATGGCTCCAGACTTGTGGTATTTCC ATGTCTTTTTTTTTT TTTTTTTTTTTGACCTTTTAGCGGCTTTAAAGTATTTCTGTTGTTAGGTGTTGTATTACT TTTCTAAGATTACTT AACAAAGCACCACAAACTGAGTGGCTTTAAACAACAGCAATTTATTCTCTCACAATTCTA GAAGCTAGAAGTCCG AAATCAAAGTGTTGACAGGGGCATGATCTTCAAGAGAGAAGACTCTTTCCTTGCCTCTTC CTGGCTTCTGGTGGT TACCAGCAATCCTGAGTGTTCCTTTCTTGCCTTGTAGTTTCAACAATCCAGTATCTGCCT TTTGTCTTCACATGG CTGTCTACCATTTGTCTCTGTGTCTCCAAATCTCTCTCCTTATAAACACAGCAGTTATTG GATTAGGCCCCACTC TAATCCAGTATGACCCCATTTTAACATGATTACACTTATTTCTAGATAAGGTCACATTCA CGTACACCAAGGGTT AGGAATTGAACATATCTTTTTGGGGGACACAATTCAACCCACAAGTGTCAGTCTCTAGCT GAGCCTTTCCCTTCC TGTTTTTCTCCTTTTTAGTTGCTATGGGTTAGGGGCCAAATCTCCAGTCATACTAGAATT GCACATGGACTGGAT ATTTGGGAATACTGCGGGTCTATTCTATGAGCTTTAGTATGTAACATTTAATATCAGTGT AAAGAAGCCCTTTTT TAAGTTATTTCTTTGAATTTCTAAATGTATGCCCTGAATATAAGTAACAAGTTACCATGT CTTGTAAAATGATCA TATCAACAAACATTTAATGTGCACCTACTGTGCTAGTTGAATGTCTTTATCCTGATAGGA GATAACAGGATTCCA

CATCTTTGACTTAAGAGGACAAACCAAATATGTCTAAATCATTTGGGGTTTTGATGG ATATCTTTAAATTGCTGA ACCTAATCATTGGTTTCATATGTCATTGTTTAGATATCTCCGGAGCATTTGGATAATGTG ACAGTTGGAATGCAG TGATGTCGACTCTTTGCCCACCGCCATCTCCAGCTGTTGCCAAGACAGAGATTGCTTTAA GTGGCAAATCACCTT TATTAGCAGCTACTTTTGCTTACTGGGACAATATTCTTGGTCCTAGAGTAAGGCACATTT GGGCTCCAAAGACAG AACAGGTACTTCTCAGTGATGGAGAAATAACTTTTCTTGCCAACCACACTCTAAATGGAG AAATCCTTCGAAATG

CAGAGAGTGGTGCTATAGATGTAAAGTTTTTTGTCTTGTCTGAAAAGGGAGTGATTA TTGTTTCATTAATCTTTG ATGGAAACTGGAATGGGGATCGCAGCACATATGGACTATCAATTATACTTCCACAGACAG AACTTAGTTTCTACC TCCCACTTCATAGAGTGTGTGTTGATAGATTAACACATATAATCCGGAAAGGAAGAATAT GGATGCATAAGGTAA GTGATTTTTCAGCTTATTAATCATGTTAACCTATCTGTTGAAAGCTTATTTTCTGGTACA TATAAATCTTATTTT TTTAATTATATGCAGTGAACATCAAACAATAAATGTTATTTATTTTGCATTTACCCTATT AGATACAAATACATC TGGTCTGATACCTGTCATCTTCATATTAACTGTGGAAGGTACGAAATGGTAGCTCCACAT TATAGATGAAAAGCT AAAGCTTAGACAAATAAAGAAACTTTTAGACCCTGGATTCTTCTTGGGAGCCTTTGACTC TAATACCTTTTGTTT CCCTTTCATTGCACAATTCTGTCTTTTGCTTACTACTATGTGTAAGTATAACAGTTCAAA GTAATAGTTTCATAA GCTGTTGGTCATGTAGCCTTTGGTCTCTTTAACCTCTTTGCCAAGTTCCCAGGTTCATAA AATGAGGAGGTTGAA TGGAATGGTTCCCAAGAGAATTCCTTTTAATCTTACAGAAATTATTGTTTTCCTAAATCC TGTAGTTGAATATAT

AATGCTATTTACATTTCAGTATAGTTTTGATGTATCTAAAGAACACATTGAATTCTC CTTCCTGTGTTCCAGTTT GATACTAACCTGAAAGTCCATTAAGCATTACCAGTTTTAAAAGGCTTTTGCCCAATAGTA AGGAAAAATAATATC TTTTAAAAGAATAATTTTTTACTATGTTTGCAGGCTTACTTCCTTTTTTCTCACATTATG AAACTCTTAAAATCA GGAGAATCTTTTAAACAACATCATAATGTTTAATTTGAAAAGTGCAAGTCATTCTTTTCC TTTTTGAAACTATGC AGATGTTACATTGACTGTTTTCTGTGAAGTTATCTTTTTTTCACTGCAGAATAAAGGTTG TTTTGATTTTATTTT GTATTGTTTATGAGAACATGCATTTGTTGGGTTAATTTCCTACCCCTGCCCCCATTTTTT CCCTAAAGTAGAAAG TATTTTTCTTGTGAACTAAATTACTACACAAGAACATGTCTATTGAAAAATAAGCAAGTA TCAAAATGTTGTGGG TTGTTTTTTTAAATAAATTTTCTCTTGCTCAGGAAAGACAAGAAAATGTCCAGAAGATTA TCTTAGAAGGCACAG AGAGAATGGAAGATCAGGTATATGCAAATTGCATACTGTCAAATGTTTTTCTCACAGCAT GTATCTGTATAAGGT TGATGGCTACATTTGTCAAGGCCTTGGAGACATACGAATAAGCCTTTAATGGAGCTTTTA TGGAGGTGTACAGAA

TAAACTGGAGGAAGATTTCCATATCTTAAACCCAAAGAGTTAAATCAGTAAACAAAG GAAAATAGTAATTGCATC TACAAATTAATATTTGCTCCCTTTTTTTTTCTGTTTGCCCAGAATAAATTTTGGATAACT TGTTCATAGTAAAAA TAAAAAAAATTGTCTCTGATATGTTCTTTAAGGTACTACTTCTCGAACCTTTCCCTAGAA GTAGCTGTAACAGAA GGAGAGCATATGTACCCCTGAGGTATCTGTCTGGGGTGTAGGCCCAGGTCCACACAATAT TTCTTCTAAGTCTTA TGTTGTATCGTTAAGACTCATGCAATTTACATTTTATTCCATAACTATTTTAGTATTAAA ATTTGTCAGTGATAT TTCTTACCCTCTCCTCTAGGAAAATGTGCCATGTTTATCCCTTGGCTTTGAATGCCCCTC AGGAACAGACACTAA GAGTTTGAGAAGCATGGTTACAAGGGTGTGGCTTCCCCTGCGGAAACTAAGTACAGACTA TTTCACTGTAAAGCA GAGAAGTTCTTTTGAAGGAGAATCTCCAGTGAAGAAAGAGTTCTTCACTTTTACTTCCAT TTCCTCTTGTGGGTG ACCCTCAATGCTCCTTGTAAAACTCCAATATTTTAAACATGGCTGTTTTGCCTTTCTTTG CTTCTTTTTAGCATG AATGAGACAGATGATACTTTAAAAAAGTAATTAAAAAAAAAAACTTGTGAAAATACATGG CCATAATACAGAACC CAATACAATGATCTCCTTTACCAAATTGTTATGTTTGTACTTTTGTAGATAGCTTTCCAA TTCAGAGACAGTTAT TCTGTGTAAAGGTCTGACTTAACAAGAAAAGATTTCCCTTTACCCAAAGAATCCCAGTCC TTATTTGCTGGTCAA TAAGCAGGGTCCCCAGGAATGGGGTAACTTTCAGCACCCTCTAACCCACTAGTTATTAGT AGACTAATTAAGTAA ACTTATCGCAAGTTGAGGAAACTTAGAACCAACTAAAATTCTGCTTTTACTGGGATTTTG TTTTTTCAAACCAGA AACCTTTACTTAAGTTGACTACTATTAATGAATTTTGGTCTCTCTTTTAAGTGCTCTTCT TAAAAATGTTATCTT ACTGCTGAGAAGTTCAAGTTTGGGAAGTACAAGGAGGAATAGAAACTTAAGAGATTTTCT TTTAGAGCCTCTTCT GTATTTAGCCCTGTAGGATTTTTTTTTTTTTTTTTTTTTTTGGTGTTGTTGAGCTTCAGT GAGGCTATTCATTCA CTTATACTGATAATGTCTGAGATACTGTGAATGAAATACTATGTATGCTTAAACCTAAGA GGAAATATTTTCCCA AAATTATTCTTCCCGAAAAGGAGGAGTTGCCTTTTGATTGAGTTCTTGCAAATCTCACAA CGACTTTATTTTGAA CAATACTGTTTGGGGATGATGCATTAGTTTGAAACAACTTCAGTTGTAGCTGTCATCTGA TAAAATTGCTTCACA GGGAAGGAAATTTAACACGGATCTAGTCATTATTCTTGTTAGATTGAATGTGTGAATTGT AATTGTAAACAGGCA TGATAATTATTACTTTAAAAACTAAAAACAGTGAATAGTTAGTTGTGGAGGTTACTAAAG GATGGTTTTTTTTTA AATAAAACTTTCAGCATTATGCAAATGGGCATATGGCTTAGGATAAAACTTCCAGAAGTA GCATCACATTTAAAT TCTCAAGCAACTTAATAATATGGGGCTCTGAAAAACTGGTTAAGGTTACTCCAAAAATGG CCCTGGGTCTGACAA AGATTCTAACTTAAAGATGCTTATGAAGACTTTGAGTAAAATCATTTCATAAAATAAGTG AGGAAAAACAACTAG TATTAAATTCATCTTAAATAATGTATGATTTAAAAAATATGTTTAGCTAAAAATGCATAG TCATTTGACAATTTC ATTTATATCTCAAAAAATTTACTTAACCAAGTTGGTCACAAAACTGATGAGACTGGTGGT GGTAGTGAATAAATG AGGGACCATCCATATTTGAGACACTTTACATTTGTGATGTGTTATACTGAATTTTCAGTT TGATTCTATAGACTA CAAATTTCAAAATTACAATTTCAAGATGTAATAAGTAGTAATATCTTGAAATAGCTCTAA AGGGAATTTTTCTGT

TTTATTGATTCTTAAAATATATGTGCTGATTTTGATTTGCATTTGGGTAGATTATAC TTTTATGAGTATGGAGGT TAGGTATTGATTCAAGTTTTCCTTACCTATTTGGTAAGGATTTCAAAGTCTTTTTGTGCT TGGTTTTCCTCATTT TTAAATATGAAATATATTGATGACCTTTAACAAATTTTTTTTATCTCAAATTTTAAAGGA GATCTTTTCTAAAAG AGGCATGATGACTTAATCATTGCATGTAACAGTAAACGATAAACCAATGATTCCATACTC TCTAAAGAATAAAAG TGAGCTTTAGGGCCGGGCATGGTCAGAAATTTGACACCAACCTGGCCAACATGGCGAAAC CCCGTCTCTACTAAA

AATACAAAAATCAGCCGGGCATGGTGGCGGCACCTATAGTCCCAGCTACTTGGGAGG ATGAGACAGGAGAGTCAC TTGAACCTGGGAGGAGAGGTTGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGAG CAATGAAAGCAAAAC TCCATCTCAAAAAAAAAAAAAGAAAAGAAAGAATAAAAGTGAGCTTTGGATTGCATATAA ATCCTTTAGACATGT AGTAGACTTGTTTGATACTGTGTTTGAACAAATTACGAAGTATTTTCATCAAAGAATGTT ATTGTTTGATGTTAT TTTTATTTTTTATTGCCCAGCTTCTCTCATATTACGTGATTTTCTTCACTTCATGTCACT TTATTGTGCAGGGTC AGAGTATTATTCCAATGCTTACTGGAGAAGTGATTCCTGTAATGGAACTGCTTTCATCTA TGAAATCACACAGTG TTCCTGAAGAAATAGATGTAAGTTTAAATGAGAGCAATTATACACTTTATGAGTTTTTTG GGGTTATAGTATTAT TATGTATATTATTAATATTCTAATTTTAATAGTAAGGACTTTGTCATACATACTATTCAC ATACAGTATTAGCCA CTTTAGCAAATAAGCACACACAAAATCCTGGATTTTATGGCAAAACAGAGGCATTTTTGA TCAGTGATGACAAAA TTAAATTCATTTTGTTTATTTCATTACTTTTATAATTCCTAAAAGTGGGAGGATCCCAGC TCTTATAGGAGCAAT

TAATATTTAATGTAGTGTCTTTTGAAACAAAACTGTGTGCCAAAGTAGTAACCATTA ATGGAAGTTTACTTGTAG TCACAAATTTAGTTTCCTTAATCATTTGTTGAGGACGTTTTGAATCACACACTATGAGTG TTAAGAGATACCTTT AGGAAACTATTCTTGTTGTTTTCTGATTTTGTCATTTAGGTTAGTCTCCTGATTCTGACA GCTCAGAAGAGGAAG TTGTTCTTGTAAAAATTGTTTAACCTGCTTGACCAGCTTTCACATTTGTTCTTCTGAAGT TTATGGTAGTGCACA GAGATTGTTTTTTGGGGAGTCTTGATTCTCGGAAATGAAGGCAGTGTGTTATATTGAATC CAGACTTCCGAAAAC TTGTATATTAAAAGTGTTATTTCAACACTATGTTACAGCCAGACTAATTTTTTTATTTTT TGATGCATTTTAGAT AGCTGATACAGTACTCAATGATGATGATATTGGTGACAGCTGTCATGAAGGCTTTCTTCT CAAGTAAGAATTTTT CTTTTCATAAAAGCTGGATGAAGCAGATACCATCTTATGCTCACCTATGACAAGATTTGG AAGAAAGAAAATAAC AGACTGTCTACTTAGATTGTTCTAGGGACATTACGTATTTGAACTGTTGCTTAAATTTGT GTTATTTTTCACTCA TTATATTTCTATATATATTTGGTGTTATTCCATTTGCTATTTAAAGAAACCGAGTTTCCA TCCCAGACAAGAAAT

CATGGCCCCTTGCTTGATTCTGGTTTCTTGTTTTACTTCTCATTAAAGCTAACAGAA TCCTTTCATATTAAGTTG TACTGTAGATGAACTTAAGTTATTTAGGCGTAGAACAAAATTATTCATATTTATACTGAT CTTTTTCCATCCAGC AGTGGAGTTTAGTACTTAAGAGTTTGTGCCCTTAAACCAGACTCCCTGGATTAATGCTGT GTACCCGTGGGCAAG GTGCCTGAATTCTCTATACACCTATTTCCTCATCTGTAAAATGGCAATAATAGTAATAGT ACCTAATGTGTAGGG TTGTTATAAGCATTGAGTAAGATAAATAATATAAAGCACTTAGAACAGTGCCTGGAACAT AAAAACACTTAATAA TAGCTCATAGCTAACATTTCCTATTTACATTTCTTCTAGAAATAGCCAGTATTTGTTGAG TGCCTACATGTTAGT TCCTTTACTAGTTGCTTTACATGTATTATCTTATATTCTGTTTTAAAGTTTCTTCACAGT TACAGATTTTCATGA AATTTTACTTTTAATAAAAGAGAAGTAAAAGTATAAAGTATTCACTTTTATGTTCACAGT CTTTTCCTTTAGGCT CATGATGGAGTATCAGAGGCATGAGTGTGTTTAACCTAAGAGCCTTAATGGCTTGAATCA GAAGCACTTTAGTCC TGTATCTGTTCAGTGTCAGCCTTTCATACATCATTTTAAATCCCATTTGACTTTAAGTAA GTCACTTAATCTCTC TACATGTCAATTTCTTCAGCTATAAAATGATGGTATTTCAATAAATAAATACATTAATTA AATGATATTATACTG ACTAATTGGGCTGTTTTAAGGCTCAATAAGAAAATTTCTGTGAAAGGTCTCTAGAAAATG TAGGTTCCTATACAA ATAAAAGATAACATTGTGCTTATAGCTTCGGTGTTTATCATATAAAGCTATTCTGAGTTA TTTGAAGAGCTCACC TACTTTTTTTTGTTTTTAGTTTGTTAAATTGTTTTATAGGCAATGTTTTTAATCTGTTTT CTTTAACTTACAGTG CCATCAGCTCACACTTGCAAACCTGTGGCTGTTCCGTTGTAGTAGGTAGCAGTGCAGAGA AAGTAAATAAGGTAG T T T AT T T T AT AAT C T AGC AAAT G AT T T G AC T C T T T AAG AC T G AT G AT AT AT C AT GG AT T G T C AT T T AAAT GG T AG GTTGCAATTAAAATGATCTAGTAGTATAAGGAGGCAATGTAATCTCATCAAATTGCTAAG ACACCTTGTGGCAAC AG T G AG T T T G AAAT AAAC T G AG T AAG AAT C AT T T AT C AG T T T AT T T T G AT AGC T C GG AAAT AC C AG T G T C AG T AG TGTATAAATGGTTTTGAGAATATATTAAAATCAGATATATAAAAAAAATTACTCTTCTAT TTCCCAATGTTATCT TTAACAAATCTGAAGATAGTCATGTACTTTTGGTAGTAGTTCCAAAGAAATGTTATTTGT TTATTCATCTTGATT TCATTGTCTTCGCTTTCCTTCTAAATCTGTCCCTTCTAGGGAGCTATTGGGATTAAGTGG TCATTGATTATTATA CTTTATTCAGTAATGTTTCTGACCCTTTCCTTCAGTGCTACTTGAGTTAATTAAGGATTA ATGAACAGTTACATT T C C AAGC AT T AGC T AAT AAAC T AAAGG AT TTTGCACTTTTCTTCACT G AC CAT TAG T T AG AAAG AG T T C AG AG AT AAGTATGTGTATCTTTCAATTTCAGCAAACCTAATTTTTTAAAAAAAGTTTTACATAGGA AATATGTTGGAAATG ATACTTTACAAAGATATTCATAATTTTTTTTTGTAATCAGCTACTTTGTATATTTACATG AGCCTTAATTTATAT TTCTCATATAACCATTTATGAGAGCTTAGTATACCTGTGTCATTATATTGCATCTACGAA CTAGTGACCTTATTC CTTCTGTTACCTCAAACAGGTGGCTTTCCATCTGTGATCTCCAAAGCCTTAGGTTGCACA GAGTGACTGCCGAGC TGCTTTATGAAGGGAGAAAGGCTCCATAGTTGGAGTGTTTTTTTTTTTTTTTTTAAACAT TTTTCCCATCCTCCA TCCTCTTGAGGGAGAATAGCTTACCTTTTATCTTGTTTTAATTTGAGAAAGAAGTTGCCA CCACTCTAGGTTGAA

AACCACTCCTTTAACATAATAACTGTGGATATGGTTTGAATTTCAAGATAGTTACAT GCCTTTTTATTTTTCCTA ATAGAGCTGTAGGTCAAATATTATTAGAATCAGATTTCTAAATCCCACCCAATGACCTGC TTATTTTAAATCAAA TTCAATAATTAATTCTCTTCTTTTTGGAGGATCTGGACATTCTTTGATATTTCTTACAAC GAATTTCATGTGTAG ACCCACTAAACAGAAGCTATAAAAGTTGCATGGTCAAATAAGTCTGAGAAAGTCTGCAGA TGATATAATTCACCT GAAGAGTCACAGTATGTAGCCAAATGTTAAAGGTTTTGAGATGCCATACAGTAAATTTAC CAAGCATTTTCTAAA

T T T AT T T G AC C AC AG AAT C C C T AT T T T AAGC AAC AAC T G T T AC AT C C C AT GG AT T C C AGG T G AC T AAAG AAT AC T TATTTCTTAGGATATGTTTTATTGATAATAACAATTAAAATTTCAGATATCTTTCATAAG CAAATCAGTGGTCTT TTTACTTCATGTTTTAATGCTAAAATATTTTCTTTTATAGATAGTCAGAACATTATGCCT TTTTCTGACTCCAGC AGAGAGAAAATGCTCCAGGTTATGTGAAGCAGAATCATCATTTAAATATGAGTCAGGGCT CTTTGTACAAGGCCT GCTAAAGGTATAGTTTCTAGTTATCACAAGTGAAACCACTTTTCTAAAATCATTTTTGAG ACTCTTTATAGACAA ATCTTAAATATTAGCATTTAATGTATCTCATATTGACATGCCCAGAGACTGACTTCCTTT ACACAGTTCTGCACA TAGACTATATGTCTTATGGATTTATAGTTAGTATCATCAGTGAAACACCATAGAATACCC TTTGTGTTCCAGGTG GGTCCCTGTTCCTACATGTCTAGCCTCAGGACTTTTTTTTTTTTAACACATGCTTAAATC AGGTTGCACATCAAA AAT AAG AT C AT T T C T T T T T AAC T AAAT AG AT T T G AAT T T T AT T G AAAAAAAAT T T T AAAC AT C T T T AAG AAGC T T ATAGGATTTAAGCAATTCCTATGTATGTGTACTAAAATATATATATTTCTATATATAATA TATATTAGAAAAAAA

TTGTATTTTTCTTTTATTTGAGTCTACTGTCAAGGAGCAAAACAGAGAAATGTAAAT TAGCAATTATTTATAATA CTTAAAGGGAAGAAAGTTGTTCACCTTGTTGAATCTATTATTGTTATTTCAATTATAGTC CCAAGACGTGAAGAA ATAGCTTTCCTAATGGTTATGTGATTGTCTCATAGTGACTACTTTCTTGAGGATGTAGCC ACGGCAAAATGAAAT AAAAAAATTTAAAAATTGTTGCAAATACAAGTTATATTAGGCTTTTGTGCATTTTCAATA ATGTGCTGCTATGAA CTCAGAATGATAGTATTTAAATATAGAAACTAGTTAAAGGAAACGTAGTTTCTATTTGAG TTATACATATCTGTA AATTAGAACTTCTCCTGTTAAAGGCATAATAAAGTGCTTAATACTTTTGTTTCCTCAGCA CCCTCTCATTTAATT ATATAATTTTAGTTCTGAAAGGGACCTATACCAGATGCCTAGAGGAAATTTCAAAACTAT GATCTAATGAAAAAA TATTTAATAGTTCTCCATGCAAATACAAATCATATAGTTTTCCAGAAAATACCTTTGACA TTATACAAAGATGAT TATCACAGCATTATAATAGTAAAAAAATGGAAATAGCCTCTTTCTTCTGTTCTGTTCATA GCACAGTGCCTCATA CGCAGTAGGTTATTATTACATGGTAACTGGCTACCCCAACTGATTAGGAAAGAAGTAAAT TTGTTTTATAAAAAT

ACATACTCATTGAGGTGCATAGAATAATTAAGAAATTAAAAGACACTTGTAATTTTG AATCCAGTGAATACCCAC TGTTAATATTTGGTATATCTCTTTCTAGTCTTTTTTTCCCTTTTGCATGTATTTTCTTTA AGACTCCCACCCCCA CTGGATCATCTCTGCATGTTCTAATCTGCTTTTTTCACAGCAGATTCTAAGCCTCTTTGA ATATCAACACAAACT TCAACAACTTCATCTATAGATGCCAAATAATAAATTCATTTTTATTTACTTAACCACTTC CTTTGGATGCTTAGG TCATTCTGATGTTTTGCTATTGAAACCAATGCTATACTGAACACTTCTGTCACTAAAACT TTGCACACACTCATG AATAGCTTCTTAGGATAAATTTTTAGAGATGGATTTGCTAAATCAGAGACCATTTTTTAA AATTAAAAAACAATT ATTCATATCGTTTGGCATGTAAGACAGTAAATTTTCCTTTTATTTTGACAGGATTCAACT GGAAGCTTTGTGCTG CCTTTCCGGCAAGTCATGTATGCTCCATATCCCACCACACACATAGATGTGGATGTCAAT ACTGTGAAGCAGATG CCACCCTGTCATGAACATATTTATAATCAGCGTAGATACATGAGATCCGAGCTGACAGCC TTCTGGAGAGCCACT TCAGAAGAAGACATGGCTCAGGATACGATCATCTACACTGACGAAAGCTTTACTCCTGAT TTGTACGTAATGCTC TGCCTGCTGGTACTGTAGTCAAGCAATATGAAATTGTGTCTTTTACGAATAAAAACAAAA CAGAAGTTGCATTTA AAAAGAAAGAAATATTACCAGCAGAATTATGCTTGAAGAAACATTTAATCAAGCATTTTT TTCTTAAATGTTCTT CTTTTTCCATACAATTGTGTTTACCCTAAAATAGGTAAGATTAACCCTTAAAGTAAATAT TTAACTATTTGTTTA ATAAATATATATTGAGCTCCTAGGCACTGTTCTAGGTACCGGGCTTAATAGTGGCCAACC AGACAGCCCCAGCCC CAGCCCCTACATTGTGTATAGTCTATTATGTAACAGTTATTGAATGGACTTATTAACAAA ACCAAAGAAGTAATT CTAAGTCTTTTTTTTCTTGACATATGAATATAAAATACAGCAAAACTGTTAAAATATATT AATGGAACATTTTTT TACTTTGCATTTTATATTGTTATTCACTTCTTATTTTTTTTTAAAAAAAAAAGCCTGAAC AGTAAATTCAAAAGG AAAAGTAATGATAATTAATTGTTGAGCATGGACCCAACTTGAAAAAAAAAATGATGATGA TAAATCTATAATCCT AAAACCCTAAGTAAACACTTAAAAGATGTTCTGAAATCAGGAAAAGAATTATAGTATACT TTTGTGTTTCTCTTT TATCAGTTGAAAAAAGGCACAGTAGCTCATGCCTGTAAGAACAGAGCTTTGGGAGTGCAA GGCAGGCGGATCACT TGAGGCCAGGAGTTCCAGACCAGCCTGGGCAACATAGTGAAACCCCATCTCTACAAAAAA TAAAAAAGAATTATT GGAATGTGTTTCTGTGTGCCTGTAATCCTAGCTATTCCGAAAGCTGAGGCAGGAGGATCT TTTGAGCCCAGGAGT TTGAGGTTACAGGGAGTTATGATGTGCCAGTGTACTCCAGCCTGGGGAACACCGAGACTC TGTCTTATTTAAAAA AAAAAAAAAAAAAATGCTTGCAATAATGCCTGGCACATAGAAGGTAACAGTAAGTGTTAA CTGTAATAACCCAGG TCTAAGTGTGTAAGGCAATAGAAAAATTGGGGCAAATAAGCCTGACCTATGTATCTACAG AATCAGTTTGAGCTT AGGTAACAGACCTGTGGAGCACCAGTAATTACACAGTAAGTGTTAACCAAAAGCATAGAA TAGGAATATCTTGTT CAAGGGACCCCCAGCCTTATACATCTCAAGGTGCAGAAAGATGACTTAATATAGGACCCA TTTTTTCCTAGTTCT CCAGAGTTTTTATTGGTTCTTGAGAAAGTAGTAGGGGAATGTTTTAGAAAATGAATTGGT CCAACTGAAATTACA TGTCAGTAAGTTTTTATATATTGGTAAATTTTAGTAGACATGTAGAAGTTTTCTAATTAA TCTGTGCCTTGAAAC

ATTTTCTTTTTTCCTAAAGTGCTTAGTATTTTTTCCGTTTTTTGATTGGTTACTTGG GAGCTTTTTTGAGGAAAT TTAGTGAACTGCAGAATGGGTTTGCAACCATTTGGTATTTTTGTTTTGTTTTTTAGAGGA TGTATGTGTATTTTA ACATTTCTTAATCATTTTTAGCCAGCTATGTTTGTTTTGCTGATTTGACAAACTACAGTT AGACAGCTATTCTCA TTTTGCTGATCATGACAAAATAATATCCTGAATTTTTAAATTTTGCATCCAGCTCTAAAT TTTCTAAACATAAAA TTGTCCAAAAAATAGTATTTTCAGCCACTAGATTGTGTGTTAAGTCTATTGTCACAGAGT CATTTTACTTTTAAG

TATATGTTTTTACATGTTAATTATGTTTGTTATTTTTAATTTTAACTTTTTAAAATA ATTCCAGTCACTGCCAAT ACATGAAAAATTGGTCACTGGAATTTTTTTTTTGACTTTTATTTTAGGTTCATGTGTACA TGTGCAGGTGTGTTA TACAGGTAAATTGCGTGTCATGAGGGTTTGGTGTACAGGTGATTTCATTACCCAGGTAAT AAGCATAGTACCCAA TAGGTAGTTTTTTGATCCTCACCCTTCTCCCACCCTCAAGTAGGCCCTGGTGTTGCTGTT TCCTTCTTTGTGTCC ATGTATACTCAGTGTTTAGCTCCCACTTAGAAGTGAGAACATGCGGTAGTTGGTTTTCTG TTCCTGGATTAGTTC ACTTAGGATAATGACCTCTAGCTCCATCTGGTTTTTATGGCTGCATAGTATTCCATGGTG TATATGTATCACATT TTCTTTATCCAGTCTACCATTGATAGGCATTTAGGTTGATTCCCTGTCTTTGTTATCATG AATAGTGCTGTGATG AACATACACATGCATGTGTCTTTATGGTAGAAAAATTTGTATTCCTTTAGGTACATATAG AATAATGGGGTTGCT AGGGTGAATGGTAGTTCTATTTTCAGTTATTTGAGAAATCTTCAAACTGCTTTTCATAAT AGCTAAACTAATTTA CAGTCCCGCCAGCAGTGTATAAGTGTTCCCTTTTCTCCACAACCTTGCCAACATCTGTGA TTTTTTGACTTTTTA

ATAATAGCCATTCCTAGAGAATTGATTTGCAATTCTCTATTAGTGATATTAAGCATT TTTTCATATGCTTTTTAG CTGTCTGTATATATTCTTCTGAAAAATTTTCATGTCCTTTGCCCAGTTTGTAGTGGGGTG GGTTGTTTTTTGCTT GTTAATTAGTTTTAAGTTCCTTCCAGATTCTGCATATCCCTTTGTTGGATACATGGTTTG CAGATATTTTTCTCC CATTGTGTAGGTTGTCTTTTACTCTGTTGATAGTTTCTTTTGCCATGCAGGAGCTCGTTA GGTCCCATTTGTGTT TGTTTTTGTTGCAGTTGCTTTTGGCGTCTTCATCATAAAATCTGTGCCAGGGCCTATGTC CAGAATGGTATTTCC TAGGTTGTCTTCCAGGGTTTTTACAATTTTAGATTTTACGTTTATGTCTTTAATCCATCT TGAGTTGATTTTTGT ATATGGCACAAGGAAGGGGTCCAGTTTCACTCCAATTCCTATGGCTAGCAATTATCCCAG CACCATTTATTGAAT ACGGAGTCCTTTCCCCATTGCTTGTTTTTTGTCAACTTTGTTGAAGATCAGATGGTTGTA AGTGTGTGGCTTTAT TTCTTGGCTCTCTATTCTCCATTGGTCTATGTGTCTGTTTTTATAACAGTACCCTGCTGT TCAGGTTCCTATAGC CTTTTAGTATAAAATCGGCTAATGTGATGCCTCCAGCTTTGTTCTTTTTGCTTAGGATTG CTTTGGCTATTTGGG

CTCCTTTTTGGGTCCATATTAATTTTAAAACAGTTTTTTCTGGTTTTGTGAAGGATA TCATTGGTAGTTTATAGG AATAGCATTGAATCTGTAGATTGCTTTGGGCAGTATGGCCATTTTAACAATATTAATTCT TCCTATCTATGAATA TGGAATGTTTTTCCATGTGTTTGTGTCATCTCTTTATACCTGATGTATAAAGAAAAGCTG GTATTATTCCTACTC AATCTGTTCCAAAAAATTGAGGAGGAGGAACTCTTCCCTAATGAGGCCAGCATCATTCTG ATACCAAAACCTGGC AGAGACACAACAGAAAAAAGAAAACTTCAGGCCAATATCCTTGATGAATATAGATGCAAA AATCCTCAACAAAAT ACTAGCAAACCAAATCCAGCAGCACATCAAAAAGCTGATCTACTTTGATCAAGTAGGCTT TATCCCTGGGATGCA AGGTTGGTTCAACATACACAAATCAATAAGTGTGATTCATCACATAAACAGAGCTAAAAA CAAAAACCACAAGAT TATCTCAATAGGTAGAGAAAAGGTTGTCAATAAAATTTAACATCCTCCATGTTAAAAACC TTCAGTAGGTCAGGT GTAGTGACTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCATATCTCTTAA GCCCAGGAGTTCAAG ACGAGCCTAGGCAGCATGGTGAAACCCCATCTCTACAAAAAAAAAAAAAAAAAAAAATTA GCTTGGTATGGTGAC ATGCACCTATAGTCCCAGCTATTCAGGAGGTTGAGGTGGGAGGATTGTTTGAGCCCGGGA GGCAGAGGTTGGCAG CGAGCTGAGATCATGCCACCGCACTCCAGCCTGGGCAACGGAGTGAGACCCTGTCTCAAA AAAGAAAAATCACAA ACAATCCTAAACAAACTAGGCATTGAAGGAACATGCCTCAAAAAAATAAGAACCATCTAT GACAGACCCATAGCC AATATCTTACCAAATGGGCAAAAGCTGGAAGTATTCTCCTTGAGAACCGTAACAAGACAA GGATGTCCACTCTCA CCACTCCTTTTCAGCATAGTTCTGGAAGTCCTAGCCAGAGCAATCAGGAAAGAGAAAGAA AGAAAGACATTCAGA TAGGAAGAGAAGAAGTCAAACTATTTCTGTTTGCAGGCAGTATAATTCTGTACCTAGAAA ATCTCATAGTCTCTG CCCAGAAACTCCTAAATCTGTTAAAAATTTCAGCAAAGTTTTGGCATTCTCTATACTCCA ACACCTTCCAAAGTG AGAGCAAAATCAAGAACACAGTCCCATTCACAATAGCCGCAAAACGAATAAAATACCTAG GAATCCAGCTAACCA GGGAGGTGAAAGATCTCTATGAGAATTACAAAACACTGCTGAAAGAAATCAGAGATGACA CAAACAAATGGAAAT GTTCTTTTTTAACACCTTGCTTTATCTAATTCACTTATGATGAAGATACTCATTCAGTGG AACAGGTATAATAAG TCCACTCGATTAAATATAAGCCTTATTCTCTTTCCAGAGCCCAAGAAGGGGCACTATCAG TGCCCAGTCAATAAT GACGAAATGCTAATATTTTTCCCCTTTACGGTTTCTTTCTTCTGTAGTGTGGTACACTCG TTTCTTAAGATAAGG AAACTTGAACTACCTTCCTGTTTGCTTCTACACATACCCATTCTCTTTTTTTGCCACTCT GGTCAGGTATAGGAT GATCCCTACCACTTTCAGTTAAAAACTCCTCCTCTTACTAAATGTTCTCTTACCCTCTGG CCTGAGTAGAACCTA GGGAAAATGGAAGAGAAAAAGATGAAAGGGAGGTGGGGCCTGGGAAGGGAATAAGTAGTC CTGTTTGTTTGTGTG TTTGCTTTAGCACCTGCTATATCCTAGGTGCTGTGTTAGGCACACATTATTTTAAGTGGC CATTATATTACTACT ACTCACTCTGGTCGTTGCCAAGGTAGGTAGTACTTTCTTGGATAGTTGGTTCATGTTACT TACAGATGGTGGGCT TGTTGAGGCAAACCCAGTGGATAATCATCGGAGTGTGTTCTCTAATCTCACTCAAATTTT TCTTCACATTTTTTG GTTTGTTTTGGTTTTTGATGGTAGTGGCTTATTTTTGTTGCTGGTTTGTTTTTTGTTTTT TTTTGAGATGGCAAG

AATTGGTAGTTTTATTTATTAATTGCCTAAGGGTCTCTACTTTTTTTAAAAGATGAG AGTAGTAAAATAGATTGA TAGATACATACATACCCTTACTGGGGACTGCTTATATTCTTTAGAGAAAAAATTACATAT TAGCCTGACAAACAC CAGTAAAATGTAAATATATCCTTGAGTAAATAAATGAATGTATATTTTGTGTCTCCAAAT ATATATATCTATATT CTTACAAATGTGTTTATATGTAATATCAATTTATAAGAACTTAAAATGTTGGCTCAAGTG AGGGATTGTGGAAGG TAGCATTATATGGCCATTTCAACATTTGAACTTTTTTCTTTTCTTCATTTTCTTCTTTTC TTCAGGAATATTTTT

CAAGATGTCTTACACAGAGACACTCTAGTGAAAGCCTTCCTGGATCAGGTAAATGTT GAACTTGAGATTGTCAGA GTGAATGATATGACATGTTTTCTTTTTTAATATATCCTACAATGCCTGTTCTATATATTT ATATTCCCCTGGATC ATGCCCCAGAGTTCTGCTCAGCAATTGCAGTTAAGTTAGTTACACTACAGTTCTCAGAAG AGTCTGTGAGGGCAT GTCAAGTGCATCATTACATTGGTTGCCTCTTGTCCTAGATTTATGCTTCGGGAATTCAGA CCTTTGTTTACAATA TAATAAATATTATTGCTATCTTTTAAAGATATAATAATAAGATATAAAGTTGACCACAAC TACTGTTTTTTGAAA CATAGAATTCCTGGTTTACATGTATCAAAGTGAAATCTGACTTAGCTTTTACAGATATAA TATATACATATATAT ATCCTGCAATGCTTGTACTATATATGTAGTACAAGTATATATATATGTTTGTGTGTGTAT ATATATATAGTACGA GCATATATACATATTACCAGCATTGTAGGATATATATATGTTTATATATTAAAAAAAAGT TATAAACTTAAAACC CTATTATGTTATGTAGAGTATATGTTATATATGATATGTAAAATATATAACATATACTCT ATGATAGAGTGTAAT ATATTTTTTATATATATTTTAACATTTATAAAATGATAGAATTAAGAATTGAGTCCTAAT CTGTTTTATTAGGTG

CTTTTTGTAGTGTCTGGTCTTTCTAAAGTGTCTAAATGATTTTTCCTTTTGACTTAT TAATGGGGAAGAGCCTGT ATATTAACAATTAAGAGTGCAGCATTCCATACGTCAAACAACAAACATTTTAATTCAAGC ATTAACCTATAACAA GTAAGTTTTTTTTTTTTTTTTGAGAAAGGGAGGTTGTTTATTTGCCTGAAATGACTCAAA AATATTTTTGAAACA TAGTGTACTTATTTAAATAACATCTTTATTGTTTCATTCTTTTAAAAAATATCTACTTAA TTACACAGTTGAAGG AAATCGTAGATTATATGGAACTTATTTCTTAATATATTACAGTTTGTTATAATAACATTC TGGGGATCAGGCCAG GAAACTGTGTCATAGATAAAGCTTTGAAATAATGAGATCCTTATGTTTACTAGAAATTTT GGATTGAGATCTATG AGGTCTGTGACATATTGCGAAGTTCAAGGAAAATTCGTAGGCCTGGAATTTCATGCTTCT CAAGCTGACATAAAA TCCCTCCCACTCTCCACCTCATCATATGCACACATTCTACTCCTACCCACCCACTCCACC CCCTGCAAAAGTACA GGTATATGAATGTCTCAAAACCATAGGCTCATCTTCTAGGAGCTTCAATGTTATTTGAAG ATTTGGGCAGAAAAA ATTAAGTAATACGAAATAACTTATGTATGAGTTTTAAAAGTGAAGTAAACATGGATGTAT TCTGAAGTAGAATGC

AAAATTTGAATGCATTTTTAAAGATAAATTAGAAAACTTCTAAAAACTGTCAGATTG TCTGGGCCTGGTGGCTTA TGCCTGTAATCCCAGCACTTTGGGAGTCCGAGGTGGGTGGATCACAAGGTCAGGAGATCG AGACCATCCTGCCAA CATGGTGAAACCCCGTCTCTACTAAGTATACAAAAATTAGCTGGGCGTGGCAGCGTGTGC CTGTAATCCCAGCTA CCTGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGTGTAGGTTGCAGTGAGTCA AGATCGCGCCACTGC ACTTTAGCCTGGTGACAGAGCTAGACTCCGTCTCAAAAAAAAAAAAAAATATCAGATTGT TCCTACACCTAGTGC TTCTATACCACACTCCTGTTAGGGGGCATCAGTGGAAATGGTTAAGGAGATGTTTAGTGT GTATTGTCTGCCAAG CACTGTCAACACTGTCATAGAAACTTCTGTACGAGTAGAATGTGAGCAAATTATGTGTTG AAATGGTTCCTCTCC CTGCAGGTCTTTCAGCTGAAACCTGGCTTATCTCTCAGAAGTACTTTCCTTGCACAGTTT CTACTTGTCCTTCAC AGAAAAGCCTTGACACTAATAAAATATATAGAAGACGATACGTGAGTAAAACTCCTACAC GGAAGAAAAACCTTT GTACATTGTTTTTTTGTTTTGTTTCCTTTGTACATTTTCTATATCATAATTTTTGCGCTT CTTTTTTTTTTTTTT TTTTTTTTTTTTCCATTATTTTTAGGCAGAAGGGAAAAAAGCCCTTTAAATCTCTTCGGA ACCTGAAGATAGACC TTGATTTAACAGCAGAGGGCGATCTTAACATAATAATGGCTCTGGCTGAGAAAATTAAAC CAGGCCTACACTCTT TTATCTTTGGAAGACCTTTCTACACTAGTGTGCAAGAACGAGATGTTCTAATGACTTTTT AAATGTGTAACTTAA TAAGCCTATTCCATCACAATCATGATCGCTGGTAAAGTAGCTCAGTGGTGTGGGGAAACG TTCCCCTGGATCATA CTCCAGAATTCTGCTCTCAGCAATTGCAGTTAAGTAAGTTACACTACAGTTCTCACAAGA GCCTGTGAGGGGATG TCAGGTGCATCATTACATTGGGTGTCTCTTTTCCTAGATTTATGCTTTTGGGATACAGAC CTATGTTTACAATAT AATAAATATTATTGCTATCTTTTAAAGATATAATAATAGGATGTAAACTTGACCACAACT ACTGTTTTTTTGAAA TACATGATTCATGGTTTACATGTGTCAAGGTGAAATCTGAGTTGGCTTTTACAGATAGTT GACTTTCTATCTTTT GGCATTCTTTGGTGTGTAGAATTACTGTAATACTTCTGCAATCAACTGAAAACTAGAGCC TTTAAATGATTTCAA TTCCACAGAAAGAAAGTGAGCTTGAACATAGGATGAGCTTTAGAAAGAAAATTGATCAAG CAGATGTTTAATTGG AATTGATTATTAGATCCTACTTTGTGGATTTAGTCCCTGGGATTCAGTCTGTAGAAATGT CTAATAGTTCTCTAT AGTCCTTGTTCCTGGTGAACCACAGTTAGGGTGTTTTGTTTATTTTATTGTTCTTGCTAT TGTTGATATTCTATG TAGTTGAGCTCTGTAAAAGGAAATTGTATTTTATGTTTTAGTAATTGTTGCCAACTTTTT AAATTAATTTTCATT ATTTTTGAGCCAAATTGAAATGTGCACCTCCTGTGCCTTTTTTCTCCTTAGAAAATCTAA TTACTTGGAACAAGT TCAGATTTCACTGGTCAGTCATTTTCATCTTGTTTTCTTCTTGCTAAGTCTTACCATGTA CCTGCTTTGGCAATC ATTGCAACTCTGAGATTATAAAATGCCTTAGAGAATATACTAACTAATAAGATCTTTTTT TCAGAAACAGAAAAT AGTTCCTTGAGTACTTCCTTCTTGCATTTCTGCCTATGTTTTTGAAGTTGTTGCTGTTTG CCTGCAATAGGCTAT AAGGAATAGCAGGAGAAATTTTACTGAAGTGCTGTTTTCCTAGGTGCTACTTTGGCAGAG CTAAGTTATCTTTTG TTTTCTTAATGCGTTTGGACCATTTTGCTGGCTATAAAATAACTGATTAATATAATTCTA ACACAATGTTGACAT

TGTAGTTACACAAACACAAATAAATATTTTATTTAAAATTCTGGAAGTAATATAAAA GGGAAAATATATTTATAA GAAAGGGATAAAGGTAATAGAGCCCTTCTGCCCCCCACCCACCAAATTTACACAACAAAA TGACATGTTCGAATG TGAAAGGTCATAATAGCTTTCCCATCATGAATCAGAAAGATGTGGACAGCTTGATGTTTT AGACAACCACTGAAC TAGATGACTGTTGTACTGTAGCTCAGTCATTTAAAAAATATATAAATACTACCTTGTAGT GTCCCATACTGTGTT TTTTACATGGTAGATTCTTATTTAAGTGCTAACTGGTTATTTTCTTTGGCTGGTTTATTG TACTGTTATACAGAA

TGTAAGTTGTACAGTGAAATAAGTTATTAAAGCATGTGTAAACATTGTTATATATCT TTTCTCCTAAATGGAGAA TTTTGAATAAAATATATTTGAAATTTTGCCTCTTTCAGTTGTTCATTCAGAAAAAAATAC TATGATATTTGAAGA CTGATCAGCTTCTGTTCAGCTGACAGTCATGCTGGATCTAAACTTTTTTTAAAATTAATT TTGTCTTTTCAAAGA AAAAATATTTAAAGAAGCTTTATAATATAATCTTATGTTAAAAAAACTTTCTGCTTAACT CTCTGGATTTCATTT TGATTTTTCAAATTATATATTAATATTTCAAATGTAAAATACTATTTAGATAAATTGTTT TTAAACATTCTTATT ATTATAATATTAATATAACCTAAACTGAAGTTATTCATCCCAGGTATCTAATACATGTAT CCAAAGTAAAAATCC AAGGAATCTGAACACTTTCATCTGCAAAGCTAGGAATAGGTTTGACATTTTCACTCCAAG AAAAAGTTTTTTTTT GAAAATAGAATAGTTGGGATGAGAGGTTTCTTTAAAAGAAGACTAACTGATCACATTACT ATGATTCTCAAAGAA GAAACCAAAACTTCATATAATACTATAAAGTAAATATAAAATAGTTCCTTCTATAGTATA TTTCTATAATGCTAC AGTTTAAACAGATCACTCTTATATAATACTATTTTGATTTTGATGTAGAATTGCACAAAT TGATATTTCTCCTAT

GATCTGCAGGGTATAGCTTAAAGTAACAAAAACAGTCAACCACCTCCATTTAACACA CAGTAACACTATGGGACT AGTTTTATTACTTCCATTTTACAAATGAGGAAACTAAAGCTTAAAGATGTGTAATACACC GCCCAAGGTCACACA GCTGGTAAAGGTGGATTTCATCCCAGACAGTTACAGTCATTGCCATGGGCACAGCTCCTA ACTTAGTAACTCCAT GTAACTGGTACTCAGTGTAGCTGAATTGAAAGGAGAGTAAGGAAGCAGGTTTTACAGGTC TACTTGCACTATTCA GAGCCCGAGTGTGAATCCCTGCTGTGCTGCTTGGAGAAGTTACTTAACCTATGCAAGGTT CATTTTGTAAATATT GGAAATGGAGTGATAATACGTACTTCACCAGAGGATTTAATGAGACCTTATACGATCCTT AGTTCAGTACCTGAC TAGTGCTTCATAAATGCTTTTTCATCCAATCTGACAATCTCCAGCTTGTAATTGGGGCAT TTAGAACATTTAATA TGATTATTGGCATGGTAGGTTAAAGCTGTCATCTTGCTGTTTTCTATTTGTTCTTTTTGT TTTCTCCTTACTTTT GGATTTTTTTATTCTACTATGTCTTTTCTATTGTCTTATTAACTATACTCTTTGATTTAT TTTAGTGGTTGTTTT AGGGTTATACCTCTTTCTAATTTACCAGTTTATAACCAGTTTATATACTACTTGACATAT AGCTTAAGAAACTTA

CTGTTGTTGTCTTTTTGCTGTTATGGTCTTAACGTTTTTATTTCTACAAACATTATA AACTCCACACTTTATTGT TTTTTAATTTTACTTATACAGTCAATTATCTTTTAAAGATATTTAAATATAAACATTCAA AACACCCCAATTAAA AGTCAGAGATTGTTAATACCACATGATCTCACTTACACACAGAATTGAAAAACTTGGAAC TCATAGAAGCAGAGA GTAAAAACATGGTTACCAGGTGCTGGGGAGAGGCGGTGGGCTGGGGAGATGTTGGTCAAA GTTAGACAGGAGGAA TAAGTTCAAGAGATCTATTGTACAACTTATTCAGTTAGATAGGAGGAATAAGCTAAAGAT CAAGAGATCTATTGT ACAATGTGACTATAACCAACAACATATATTGTACACTTGAAAATTGCTAACAGTATCTTT TAAGTGTTCTCTCTA CAAATAAATATGTGAGGTAATGTATATATTAATTAACTGTAGTCATTTCACAATGTATAC TTATTTCAAAACATC ATATTGTATGCTATAAATATATACAACTTTTATTTTTCAATTTTAGAAATGTCCTTAAAA AATCAGATTTTCAGA TCAGATAAAAAAGCAAGACCCAACTATATGCTGCCAACAGGAAACACACCTTAAAAATAA AGGACGAACAAACAG ATTAAAAGTAAAAGGATGGAGAAAAGATACATCATATTGGTAATTAGAAGAAAACTGGAG TGACAATATGAAACA AAATAGATTTCAGAGCAAAGAATATTACCAGGGGTAAAAATGATCATTTTATAATGATAA AAGAGTCAGTTCAGC AAAAGGATATAACAGTCCTAAATGTTTTTTCACCTCATAGCTGTGTCAAAATAGATGAAG CAAAAACTGATAGAA CTGTAAGAAGTAGACAAGTCCACAATTATGTTTGGAGATTTTTTTTTTTTTTTTTTTTGT CGCCCAGGCTGGAGT GCAGTGGCAGGATCTCAGCTCACTGCAAGCTCCGCCTCCCAGGTTCACGCCATTCTCCTG CTTCAGCCTCCCCAG TAGCTGGGACTACAGGCGGCCACCACCACGCCTGGCTAATTTTTTTGTATTTTTAGTAGA GACGGGGTTTCACCG TGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCTGCCTGCCTCGGCCTCCCAAA GTGCTGGGATTACAG GCATGAGCCACTGCACGCAGCCTGGAGATTTTAATATCCTTTCAATGTTTAGTAGAACAA GAATACACAAAATCA GTAAGGATATAGAAGATTAGAACAAGACTATCAAACAATTTGACTTAAATGACATTTGTA GAGCACAGCAGTCCC CAACAACAATAAATCACACATTCTTTCCAAGAGTACATGAAACATGTACCAAGATAGACC GTATTTTGAGCCATG AAACAAATCTTGATAAATTTAAAAGGATTCAAGTCATAGAAAATATGTTCTCTGACCACA ATGGAATTAAATTAT TAACCAATAACAAATATCTGGGAAAACCTCAAAAACTTGGACACCAGCGCTTTTAAAAGA CTAAATAATTTCTAA ATTATCTGTGTTGGGGGGAAAAGAGAAATGGATTAGAGAGCAAAAAGGGTATCAGAGTGC TGTGGTACGATTTTT ATGAAGAGTGGAACAGAATCTGCCTTTGGCGTTTCCCCACTACAGCCCATTCTTCACATT GATAACAGCATGATC CTTCTAAAATTAAATCTAACGATCACTTCTGCTTAATGGCTCTCCAACACTTACAGAATT AGGTCCAAAATTCTA GCACAGTTTCTGTTCATCTTTCTAACCTTTCTTCCCACAGGTCTAGCTAGTACGTATTTC TTTTATTGCATTTAT TACACTATTCCTTTGCTTATCTATCTCCCCACCTAGGCTAAAGAACAAGATTCTTGTCTT TTTCATTTTTGTGTC TCAGTGCCTAGCATGGTGCCAGGCACACAGCATGCTTCCAGTAAATGTTAGCTGGATGGA TGTAATGAGTATATT AAATATTAATTTATTTGTTTTTCCCCAAAAAGAATTATTTCCTGCAAATCAAGGAAATTG CTTTCTTTATATAAT CAAAAACTTATTTTCCCAGAAGATTCTTCATTAAAAATTAAGCCTATGCACAACCTAGCT CTAAAGTTTCAAAGA

TTTTAGGCAGCAATTTTTCAATCTTTTTGAAGTAATACATTTGAATCTTTTCAAATT TCTGTTTCTGCATTTGTG CCACACCATCTCATCTCTTGCTGAAATGTTTTTGTTAAATTAATTGCTTGATAAATTGCT AAGTACTTTTCATCA GACCAATTAGGACAATAGTAAGTATCCATCTGTGGAGCGCGGACATTCAAGAAATCTGAT CCAGTATTTAGAAAG TCATTCCTGAGCTGAGTTGGCTCAAACTGGCACCTTCTGGCATTTGCTTGTGGGTGGGGA ATGTGGAATGCTTTG AAAGCTGAATGAGTTTGTCAAGTTTTAAAATTCCCTTATGGCTAAAGGAAAACAACATTC ATTGTTTAAAAACAC

CATTGTTTGTTTTTTCTGCTTTTTTGTTCTTTGGAGCCTGAATCTGCAAAAACACTC ACACCCAGCATTTTGCTT CATGTACCACTCCTAAGATGTTTTTAGAGACTTGAATAGTGTCTCCGCACTACTTTTTAT TGTGATTGTTCAGAA TGTTCATAACAAATGGTAAAAAGTCAGTTTTAGTGCTCAAATTGAGTTTTATGGAGAAAG ACCATAATTTATGTT TGTCATTGTAAATTGATAGGAGAATTTTTGGAAGTTTGCGTCCTAGAACCAGATTTCCAA GGCTCAGATCCTTAT TTTCTCACTTCCTAGCTGTGTGACCTTAGACAAGGTATTAAACCTGTCTGTGCTGCCTCA GTGTCCTCATCTATT CTTTAAGAGTAAGAATAGAACCTACCCGATAGAGTCACTTGAAGATTAAGTGGGTTAGTA AATTCAGAATGCTTG GAACAGTAACTAGCACAGAATAAGTGTCCAATAAAATTGGGTTGCAGCTATTATCAGTAT TATTCCTGTCATAAT CATCATCACCATTAAGCAATTAAATGTAGAGTTCCAAAATTTGATTATGAAACTACAGTT ATACAGCCATGATTC CCGGTGATACCACGTCAGTAACAAGATTATTTCCTTAGCTTGAGCCAGTCACTACCTCAT TGCATGTGGCAGAGT GTGTTGCCGTAGGCAAATGTCATTGTAGGGAATGAAAAAAAAATTGCCTGTGAGCTGCTC TCCAGAGGCCTCATC

CCATTTTCCCATCGTCCACTTTACTCCATCTCCACTGCCACTATTAGGACCTTATCA TTTCTTGTCTAGATTAAT TCAACAGCTTCCTTCCTTCTAGTCTCCATGATTTCACCCACTAGCCATCCCCTCCCCTTT GCCCAATTTTCTCCA TTTATGGTAGAGTGATCTTTCTAATAGGAAACTCCTGACTTGCCTTAAAAAGCCCTCATT GAGGCCGGACGTGGT GGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCACGAGGTCAAG AGATTGAGACCATCG TGACTAACACAGTGAAACCCCATCTGTACTAAAAATACAAGAAATTAGCCAGGCGTGGTG GCGGGTGCCTGTAGT CGCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCAGAGCTTGCA GTGAGCCGAGATTGC GCCACTGCACTCCAGCCTGGGCGACAGAGTGAGACTCCGTCTCAAAAAAAAAAAGCCCTC ATTGACAACCTTCAA CCCACAATCCATGGTGAAGCACAGGAGCCTTGGGGATCTGCCCCCAGCACACCTCTCCAC CCTTGTCTCTCACTG CTCCTGCCTTCATGGAGAGCCCTGATGAACTATTTGTAGTTTCCCCTGACTCACCTTGCT GTTACTGGGCCTGTG TGCGTGTTGCTCCCACTACCTGCAATACGCTTACCCACTTCACCTGGGTGAACTTTACTT AGGATTCACCTTAGG

TGGGCATCATGTTCTTCCAGGCCCCTCCTCTAACTTTTAGTTGAGAGTATTCCAGAC TTAAGGCTCCATGGGATA GGGATCTTGTCTATGCACCAGCTTATTCCCAACTGCCTGGCACGTAATGCATTTATTAAA TATATATTGAATTGA TTACCCTACTTGGGGCTCTTGTTTGCTTCTACACTTACAGTTCTAGCATAGCACTTAACT CATTATCATGCATCA TTATTATGGGTTTGTTTTGTCTCCCATTAGACTGTGAGCTCCACAAGGCTGTGTCCTTGT CTTATACATCATTGT ATTTCCAGCTTCCAACATAGTGCTTGCCATGACACAGGAAGTCAGTAAGCTCTGAATGAA TGAATAGTATCTACA TACCATTAATCTGAGGTTTAAAGTTTCCCCAAATTCTGAAGCAAGGGGATTTACGGACTT CCCTGACAATTTTTG GATGTCATCCCAATGATACCACTAACATTTTAAGGGACAGCTTGCATATATACATTTTTC TGGATGGCAGTTTTT TTTCCCACAGGCTTCATCAGATATTTCTCCATAGCCTTCCTCAGATTCTCAAAGGGGTCT CTGATTCCCCCAAAA GATAAGAAACTGTCATAAAAAATTATTTCTAAATATCAATTGTTAAATAAAATGTTTGCA AAGCAGCCTGATGAA TCATTTCAGGCCACTTGACCCCGATGAGTTAGAGAGTTTGTGCTCTGCAATCTGACTGCT TCCAGCAGTCTCACT GCTGCTGGACTGTGGCACTTCCAATTGGCAGCAGGGCAAGTTTCTTCTGGATGAATATTC TGTCATAGGGGTCCC CCTTCCACACATACCTGTAGGAGCAGTTTGAAACTCATATGCATGGTCTTCCTGGTTCTA GGCACATGAGTCATT TAAGCTGCTGGAGCCAGGACCAGCTAGTATGCTAGCCCGGCATTCAGAAAGTTAAAATTT GGGGTCAAAACTGAG AACCTTCTTTGATCCACCTTGGCCAGACATTTTCTCTGGCTTCCATTAATAGCCTCAACA TTTTTTTTTTTTCTG GCCTAGACCCACACAGGCAAGAGACCAGAGCTTCTCTAAGGAGCTAAGGGAAAGCACATT TTAAAAATAACTTGA GCAAATGAATTCATCTGGCAAAAGCAACCCCACTACGTAAAATAAACCTTTTTAGTTTCG CAATAGCAGTTCCTG AAAATGTAAACAACCTCAGGGTCTACATGCACTGAATCATTTGCTGAACAGAAAGTCCCT GGTCCAAATTCTGCA AGAATAAACACCTTACAAAACTAGGGGTCAATGACCTTCATATGGGAACAAGGAGGGTGT GGGGGGCAGCAACCC ACCCTGAGGACAATGAGAAAGTCTTGAGACTTGATATTCAAAATGCTGGCTTTCTAAACC AAAAACTGGCATGAG TGGAGGGAGAAGGGGAGGGTGGGCACAGTCTATGCCTCAGGCTCTTGCTCAGACCCTACC AGGCCCCTGCCTTCC CTAGGGAAAGCGAGAGTCTACTCACTGTCATGAAGCCAGAGGAAGGCCCTGCAGGTTTCA CTGTGTGTTCTGTTG ACAAGATGATGGTTCCATTGAAACTGTAATAACATACTTGGCCAACTAAGCCCATACGAT CGTAGTAACTTTGTA CCCAGTCCTAGCTTTTCAAACATAATGATAATATGTTCTTTCTAATGTGGCCCATACTGT TCTAATGAACTTATG CTGAGTTTTTCTGAGTACTAGAATAATATTCGCCATAAATAATAGATATAATTATTCTCA TTTAATATTTGCGTA GCTCTTCTTTAAAGCAGAAAGTATTTTCTCATTCCTTACTAGAACCTTTCTGTGTGAGGA GCACTGAGCTAGAAC CCATATCTTAGAATGGTCAGAATTTGGAGAAATTCAGGGAAAAGGCACTGGACTCATTTT TAAAGACTAGAAAAT GCAACCTCCAGAAAAAGATTCAAGAGTTTTTTACTCCCAGAGATGTAGGAAAGATTGGAG TAAATCTTAATATTA TATTTCAGGTAAACAAAGGATCACTGTCAAAATAGCAGCATTTATTGAGTAATGGCTGTG TGCCAGGTACTTTAC AGTTTCACATTTAACCCTCATAATAACCTTGTAAAGTGGATATCCCCTCAGTACATGATG AGAACACTGAAGCTT

AGGTTAAATGATTGTCCAAATCGGACAATCATTTTCAAAATCTCCCCCTTTTTTTCT CCTTTCTTATCTGCAAGG CAGATTGCCCTTTCCCTTTCAGTGAAACTTGTGCATGACCACATGACTCTCTTTGGCCAA TGAAACATGAACAAG CAGCGTTTATCACTTTCAGATGGAAGGCTTTGCATGAGCTTTGCCTCCTTTTCACTCTGC CACAGTGGCCACTAA CATTCCAGATAGTGGCGCTCTGCAGGCTAGGTCCTATAGTGGGAGCTATGGGCAGAGCCC CCTTTCCCACCCCCA TCAAGATGTGCATGCTGCATAAGCCATGCATTAATCTTTGCAGTTTTAAGCCACTAAGTT TTGGAGTTATATTAA

TCATTAATCATGGTTCTCAAGAGAAACAGAGTGGGGGAGTGGTATTCATTATGGGAA TTGGCTTACATGATTATG GAAGCTGAGTAGTCCCCCAGTCTGCTGTTTTTGAGCTGGAGAACTAGAGGAGCCAGTGGT ATAATTCAGCCCAAG CCTGAAGGCCTGAGAAATGGGATGGGGGAATTGGGAGGGTGGGTGTGCTAGGGTAGGATA AGTCCTGAAGTTCAA AGGCCAGCCAGAAGGTGGATGTTTCAGCACCAGAAGAGAGAGCAAATTCGCTTTTCTTCT GCCTTTTTGTCCTCT CTGGGCCCTCAATGGATTGGATGATGCCCTCCCACATTGGTAAGGGTGGATCTTCTATAC TCAGTCTGCTAATTT CTTCCAGAAACATCTTCACAGACACATCCAGAAATAATGTTTTACCAGCTATCTCGGTAT CCCTTAGCCTAGTCC ATATTTAAAAATTAATGATCACAAGCAGTTGTTTGTTTCCACAGCAAAACCTGGGTGACA GACCAAGTGACCCAG ATGACTAGAATTTGACCTTCTTTTGTTGCCCACACCATACTCTGAACTAACATGCTGTGC TGCCTTCCAAGTGGA GAATGATGGCTAAGTATCTTCTACCTAATTTGAGTCACAGAAAAAAAAAAAAAAGGTTAT TAACTGCAGTGACAA GAATTGTGATTCCCCAGGGGGCAGATCAAGACTGATAGATAAGAGAAGTGAGGAACATCT GGGGAATGTCCATTG

AAAATTTACTCAGAAGAGAAGAATAATTAATATAATAATATGATATATTGAATTATA ATAAATAATATTTTGATG TATTTCCTTCCAGGCATGTTTAAGTTATAGACTTTGAGTATATTTTCTCAAAGGGGGTTC TATGTAAGAGACTAT TTCTTAATATAGTTCCTAGCTTGGAATTGCTCTTGCTGGTTTAAGCTGAGCTTATTTTAT TACAGACTTCACAAC AATAACGTTTTCCTTCACTAGTCAGTACACAAGATGGTCTTCATTTCCAGTTTGGAATCC CACACTATCAGAGCC TGAGACAAGGACTAGTATGCAGTTAGTTTGTTTGGGAGGTGATTCCAGGAAGTGGGAATG AGAGATCAGTCAGCC TGCAACACGAAGGAGGAAAAGTCAATATAAGGATGAATTTGGCAATTGGCCGTTTCATGC AACTGGGGCTAAATT TTGCTTGGCTCTCTAAGAAATGTAAAGAATGCCTCCCGTAATTGCTCACCTCAAGTATTT ATTCATTGGCTCTCA TGCTCCATTGGTTGTCCATGAGAACTTTAGCCCTCCCTCGCTGCAGCACAGACACTGTGC TTTCTCCTAGGCTGA GCAAGCTCCTGCATCTGTGGAAACCGTCCCGGGGCAGATAGTGAAATAATGACTGCTGCG TGCTTGAGATCTGGG AAAGAGGCCACATCATAAGTGCACTGAAATCAGAGATGTGTCAAGAGATGTGACACAGGG CATCTGAGGTGTCTA

CTGCACCAGCTATAACTCCCTAAACGCTAATCTCAGTTCTTACAGAGGGGATGGATG CAAGGGAACAGTCATGAT TGAGAGCACCGAAGAAGCTCTGTATGAACCTTAGGCAAGTTTCCTAATCTCCAAAATGAA GGTAATAATACCCAC CATCCAAGATCTTCGGGAGGAATAGATGAACTAATGTATGTGAAAATGTCCAGCACAGGT CCTAACCCATAGTAG GTGCTCACCAAATGTTAGTTCCCTGCCCTCCACGTTGTGTGTATCCGGAGCTGCACTAGA TGCTGAGGCAAATGG TCTCAAATGTACTTTAACACTTAATGACTGAGATTTTTTCTGAGCTGCCTACAGGTTATT GACTATATTCATTAT TAATAATAATATATATGGCCACTTCAGGCAACTGGGGCTAAATTTTGCTTGGCTCTCTAA GAAATGTAAAGAATG CCTCCTGTAATTGCTCACCTCAAGTATTTATTCATTGGCTCTCGTGCTTTATTGGTTGTC CCTGAGGACTTTAGC CCTCTCTCACTGCAGCACAGACACTGTGCTTTCTCCTAGTTTCTGTGGCAAGTGACAGGA GCCCACCTCAAACTA AAGCAAAAGGGACTTCATTGGCTCTTGTAGCTAGGAATTCCAGGGTTGGCACTGGCTTTG GGCACTACTGGATGC AGGAATTCAAACAATGTCTTCAACTCTTTCTTTTGGTGTTTCTCTCAGCTGTGCTTCTCT TGTCGTTTCTTTTTC CCATTTTACAGATAAGTTCATCCGTAACTGAGAGAGGTGAAAAGGGGATGGCTGCAGAGA ACTCTGGCTTATATC ATCCTTGCTTGCTGACCTCAAGGTCCATGTATAAATTCTCAGAGAAGAAGCCCTCTGGTT GGTGATGCTTGGAAC ATGCCCTGGAGGGTGGGCCCCTTGAAGTGGAGCTTGCTGGAACCACATGGGCTGGAGCAA GGCGCTAGGGCCAGA AGAGAGAGGTAGGCAGGGCTGCTGGCCAGGCACTCTTCACCAAGACAAGGCAAGAGGAGG GGCATGATTGAGGCA GTGATACAGAAAGCAGACAGTAGAGGTCGTGGCAAGTGTGCCGTTACTTGCTACCTGTGG TTGATGGGAGAGTCA CACCACATTTAGGAGGAGAGAATCCATTTGCCACTTCTGACAATGCCACAAGAATCACAT ATTTCATCCAGAGGT TGAATTTGGCCCATGCTGAGCTTTAAAATACAGAGCTGTCTTGGAACAATGGCTCAGTAC ATTCATTTGGTGTCC AACAAAGCCTGCCTCTGTTGCCTTCCCTCTCTCTGTGTGCCCTTCAAGATCTTCATTGTG CTTTGGGGAGAGAAA GAGAAAATGTCATATCAGGGTAGCTCACCCCATGTGTCCTGGACTCAGGAAAAGAGTATC TTATCACCTTACTCT TTTGTTATTATAAAAAATAAAGTTGAACGTCTTCAAATAAAATAAAGAAGTATAGAAAAA ATTTTAAATTAACCT GTTATGATTCTACCTAGAGAACCATTGTCAACATCTTGGTATATGTACTTCCAGATACTT TCCTATGAATATATA CATTGTAGATTTTTTAATATTAAAAGGCTATCATGCTGCTTTGTATACAGGCTTTCTTTA CTGATATGTAATATA ATACACAGACAAATATACAAATCCTAAGCCATCAACTCATTGAATTTTTATTCATTGTTT TTAATACCTGCATTG TGTTCCATTGTTAGGCTATGTCACAACATATTTAATTAAGCCCCTATTGATGAATATTAA TTTACTCTATTTGCC AGTTCATTCCAGTCCAACATTTATTGAGTGTCTACTTACGGGCCAGGCACTCTTGTATTC ATCAAGATCACCACA TTATCTGTATCAGTTATTTATTGCCACAATAAAACTGCATAACAAATCACTCCAAAATGT AGCACCTTAAAACTA CAACTACTTATTATTTCTCAAGAGTCAATGGGTCAGCTGAGCAGTTCTGCCGATAGGGGT CAAGGTCAACACATT TCAACTAGACTACTTGTAAAAAAGAATGAGTGTCTGGGTAGGTGTGTTCTTCTAAAAATA AAACAAGGAATGAGG AAATTGCAGGTAGGATAAGAGGGGTGGTTGGCAACCAAACCCCACAAAAGGCAGACAAAT TTTAAGGAAACATAA

TGCCAGACTCCTATGTCATCATCCAAGTAGATGCAGTGAAGTATAACCTGGGGCGTA GTAGGGTAGGAGTGGGGA GAGCAGAGGAGAAGGAAGGGAGATTGCTTTTCATCACTTTTGGATTCCCTAATAACAGAC ATGACTGCCAGTATT AAAATTTAACAAAGGATATCTGATCATTAATTTTCCTGTATAAGTCACTGGTGATCTTCA ACATCTCTCCCTCCC TTCCTCCCTTCCTTCCTCCCACCCTCCCTTCCTTCCTTCTTTCCTCTTTTGCTTTCAACT TCCTTTTCTCGTTTC CTTTTGCTTTCTTTCTCTTCTCCCTTTTTTCTGTCACTCTGGGCGTATGTAGTAGTGTAA AAAGGTTGACAGAGA

AATCAAATATAACAGGAGCAGGGCCCTGAGAAAAGCACCTGGCATCCTGTAGGCAAA CCATTGTTTCTAAAAGAA GGGACTGAGAGATTGAGGAGCTCAGGACATTGCCAAATGAACAAGGCAAGCACATTTATT CAGTACCAAACAAAC GGAAAACGGCCTTTCCAAATAACTGACCTATAAAACAGCCTTTTCACAAGAGTACCGTAA TTACTGGCCAACAGC AACAATGAAAAACAACTCCCAAACAAAGAAATATTTCTGGATTAAAAGCCATGAGATCTG GATTCTAACAAGCTG TGCTCCTCAAACTACAAGTACAAAATCTGGCTCTAAACTAACAAGCTATGAGCCTCAAAC TGATGACTGGCATGT TTGGGTCTCCATCTCCTTCTTGGGGGTTGGGGTCTTAGAGACCCTTTTCCACGCCCTGAT TCTCTTACTAGTGTG TATGCTTTCCTTTTGACTTCTCATGCTGACCGTCTGAGCAGGAGTGAGAAGCAATTTCAA AGGAAAACATCGTTT ATCATCTGCTGAAAGAAACCAAAAAGAACACAGGAAAACAAAAAGACAAGGAAAGGGAAT GAAAATGTAATTCAT TTTATTAAAAAGAAGAATTATTCTTCTGGGACACTGGATAGAAACCTTAATGAGTTACCT AGCTATCATAAATCC TCTAACAGAGAAGAGAAGAGAAAGAAACAAAGACGGAAGAGGGCAGGATAAAAGAAAGAA AAAAGGAAGGGAAAA

ATGAAGGAAGGAAGTTATCTATTCATTTCTACAGAGACTCTGCTGAGCAGTAGACAA GAAGACTTGGGAAAAATT TAACTGAAACTTTTCCAAAAATCTTTTCAGAGGGATTTTTTCCCTCTGAAAAGCATCATT AGAGGCTGTTCAATA CCCAAGGCAAGCCTCTTTCATATTACTTACTGTACATGAAACACTCATGCAATTGAGGCT AGCCAGAGGCCATTT AGAAATTCAATAATTATTCAACCCAAGGGGCTTTCCAAATGGTGAAGTAGCTTCTTAAGA GGAAATTAATATTGA GCAGTATAGCAAACCTAATTGGAATCTTGAGAAAATAGTTCTGTGTCGTTAGAACAGCTA GAGGCTAAAGAAGAT CAGGTTGGATGATACCTTCATTTTTGTCTCTTTCCTTAATTATGATGTAAAGGGAAAAAT CTTGTTTATTTTCTA TGCCAGGAGGGTAGAGGGTGATTTGGAGAGGTTCCAAGTTTATCAAAATCTACCTTCAGT CTGGCAGTAGAAAAG TTTACTTCCTTCATTTCTTTCCTATAGACATTCAAAGAGAGCTAAGGAGATCCAAAAACC TTTTTTTCTATATTT GCAATGCAAGGCAGTTGGGAATTAATGACTGATTTGTTGGTGAGGGCAGTGGGCATTGAT CACAAAAGCAGTAAA GCTGTGTTTCTCAAAGAGAGAAAGTCTCTTTGAGATCTTCATTATTTTACTATTTAGAAG AGAAAGGGGCGTTAT

ATCACGTTGGAAGCATCCATGAGTCACTAGTCTCTTCTCTATCTTTCTATGCCTTTC TGTATTAATTACTTTGAA AGCACAACATTCCAAACCCATTGAGCACACAGTGGTCTGATTTCTCCACTTGTGAAAGGT GCTAAAGTCTCACTG TAGGATTAATTTGGGGGTCCAGGCTATGGGCTTGTAGATATGACTACCTTAGACTTTGGT TCTCCTGGCAACTAA CCCTTTTTGGATCGTATCTAAGTTGACCTGTTTCACAGTGAGAGAACTCCTCTCCATTAC TCAGAATACTGAGGC AGATCACAAGTGTACCACACCTGGCTAATGTTAAGCCAGACAGAAACATCAGGCTCATCT CTTGAGAAGAAGGGT CGCTTATTAAGGATACAAACTATTTTTTTTTTTTTTTTTTGAGACAGGGTCTCATTGCCC AGGTTAGAGTGCAGT GGTGCAATCATAGCTCACTGCAGCCTCAACCACATGGGTATTTTTAAATAAGAAAAAAAT ACCATCTGATAGATA TGAAGGAGCATTGGGTCACTATAAACAAAACAGATTCTAAGAGCAGGAAGAAAGAGTACA GTCTCTTTTCAATAA TTTTTTTTTAAACTTGGGAAAGAACACTCACTCTATTCCTATAGACCAGAAAGCAGATAA TTGTCCATTATGATT CCACATGACACTATCTTGTTCAGCTGTCACTGAAACAACTTTGAACACTGTCATATGTTC TTCCCAGCTCCTGAA CTCTGACCTTTTTATGCCTTAGTTCCACTTTCACAAAAAGGGATTGATGTAATGTGCATT TCAGAGGAAACGACT ATAGACATTTAGTGTCATTATAAATGTTGAGAAGTATGCTGGCAGAAATTATGCCTTAAG ATCATATATGGATTC TTGTATGGTTTGAAATTGCTTAAAAGATATATATGATCTCTAAAATGTGTGTGTATATAT ATATGATGTCTTCTT ATATATCTATATGTGATATATTTATATATATATAAATCTGTGTATATCACATATATAAAT TTGCTGTTATTTGAA TTGCCATTACCTCAGTGCTTAGGGGAAGCCATGCACGTTTGTTTCTTTTCAGTACCCAGA GTTAATTAACATAAG TTATCACAGAAGCTCCCATAAGCATTGAGACAATTTCTCTATACCTGTGACTATTTAAGG TTTTGAAAACAAAAC AGAAGCAGGTAAGGAGGAAGTACGCTTTACTATTGAAGATTTATTAGGTACACATTTAGA TTTGTGAACTCACAT TGCTTAGGATGAAAGGGACTCTTGAGGATGTCTGCTGTTTGTTAGTGAACTGCCTGTAAC AATTACAATTAGCAC ACACATGAGCACAATGAACTGGGTAGTCAGACTCAGCCAAAATGAATAGAAATAGCCTCT TACCAAATTTACTTT GAGTAGCCCTTGGACTCTGAGCACTGCTGCCCAGAGCAATATGACTGTAGGTCCAAGTTT GTCAATGACTATGCA AATGTGCTTTCTTCGCTTTTACTCTATTGTCATCTGTCTATTACAATGTTGCTATGGTGA CACCTTTCCAATATC CCTGTGCTTCTTTGGTATCCTCTAAGGGGAAGCTGTAATGAAGTGGCTTGGCAAAAGAAT CCTCTTGGAATTTTT TTTTTTTCATATGCTACTGAAAACCAGCATGATTTTCCTCTTATGGGAAATGTATAAAGT ATGAGTTGGAAATGA TGGAAATTAATCTGTACTGACTTGGGCAAGGAATGTGAATGTTATTCATTCTGTTCCAAA CTACCTGAAAATATT CTCTTTCTGTTCCTACTTTCCAGGAGATAACATCTTAAGGGACACTGAAGCTTGTGCGTG TGTGAGTAGAACACG TGCTGGGGGCTCTTGAGCTCATGAGGGAGGGGCTACATGTCGGTGGGGTGATAACTGTAT GCTGGAAACAATGAT AGGTGGTGACCCTGGAGCACTTACCATGTGACAGGTGTTATGCTAAGCATGTTGTATGCA TTCCTTCATTGAATG ACAGCTACCTATATTATCCTCATTTTATAAGATGAGGTAACAGAGCTTCAGAAAGGTTAG ACTCAGCTGCTATGG GTCTGTCTGACTCTGGTGTTCTTCCTCTTAAAAACTGGGGCACTTTGGAAATGAGATTCC TCGGTGATGAACAGA

AATATTGCTTAGCGGCTGTATTTTTGTATCTGGCAGTTTTCCCATATTTGAGTCTTA TATTCACAATCGGTATCT TTACATTACACAAAAGTGACACAGAATTAGAGTCATTTAATCCAGGGTTGATATCATTAA GTCATGACTATTTAT TAAATGTTTCTTACAATATCTGAGATGATATTGCAAAAGATGTAAGTGATTTTAGAAGTT CTCACTTCGTAGTTA GTTGCAGAAACCTCTTTTGGAGGAGGGATGTTTTCTCTATATATCCTAATTTCTACTTAA TATATTTCCACACCT CTTTGAAGTGTGTAGTAAGAATGGTAAAATGCAGTACTTCGTCATTTGGTACAGTTCAAT CAATATGCATTAAGA

TGTGATCATATGGGTAATAGAAAAATGTGAAAGATCCAATTCTTTTTCTCCAGAAGG CAGGAAGCTCATATTTGA TTTCTGTTACTATAAACTATAAAAACGTTTCAAATGTAGTTTACCCGTAACCATCACCCT GCAAGGGTGATATTG CTCCCCGCCAATTTACGGAGGAGAATACTGAGGCTTTAAGGTTGTAGATAGACCAAGACC ACACAAGTAGAGAGT GGCGGGCTGTGGGTTGAGCTTTAAAATCCAGGTTCATCCATGACTCCCAGTGTGTTCTAG TAAATCCACTAGAAT CTGAGTATTTTCCAATGATTTATGCTCCGCTCTGTGTCAGGCAGTTCATGGTATTTTTCA ACAATCAGAAAATCC TGGGGAAGGCAAACTGTTTCCCCCTCTCTAGGTGCCTTGGAAGTGGCCGTTGTGGACCCA GAGATCATCCTTTCT GATCTGACACCTTCTTCACTGCCCTGGCCCAGTGTCTTTTCTGCAAGGCTGGAAGCCCCC TTAGACTGGTCATGT CCCATCTCTTTCCGGAGGGAAGATGATCCCAAAGACGACTTTTCTCTCCACGGTGCTGCC ATACCGCAGGCGGCC GCCAGGGGTCCCCGCTCGGCGTCCCCGCGAGACAGTCGAGCCCCGGCCGGCTGCGCGGCG CGCTGGGTGCATGAG GGGGCTGCTCCGGAGCGACGGCGGCTGCAGCTGGAGCCAGGCGCTCGCCCGTCCGCCGGT TGGCTCGCCGGGACC

TCGCGCACCGGCGGCAGAGTCCCTTGCGTGGATTGGCAAGCGACGCCCCACCTGCCC CGAGCTCACCATTTTCTT TCGCGCTGGCTGCAGCTGACCCGGCGAAGGGAGCCGACCGGGCCCTGGGCTGGAGGTAAA ACCCCACGGTGAGTA AGAACCCGCTCCAAGCTAGGGGAGGCGGCGCAGCCCGGTGGCTGCTCGCTCCCGATCTCG CCCGGGCGGGCGGCG AGGTTTGGGGCGCACCTGGGCGCGGGTGCAAGAAGGTGCGGGAGGCGGCGGACCGGTCTT CTGCCCGCCGGCCAC GGGCTTCCGGGGCTGGAGTCCTCTTCAGACCCCTGCCGGCGCCTGGGTTTCTGGCCGGCT CCTCGTGTGCACTTC CCGGCAGGAACAAGGGTCGCCCACTTTCCACCCCGGGATCTTGATTTGTCCTTGATTTGA AAAGATATAAATCAA TAAGATCGTCCTTCTTTCGGGGTGCAAGACTCCGAGCCCATCCCCAGCCGCGGACGCCTG CAGGGTGCGTGTTGG GCTGTGGGTGGCGGGAAGACAAACTTTTACAAAAGTGCGCCTGGGCTGGGGGACAACGCT TGGGCGTCCTGATCC TGAGGGAGGAGTCTCGGCTTGGGGCAGCGTAGGGGAAGTCCGCACCGTCAGCCAGGTCGC CCCCGGGGCTGACGA TGCCTCACGGAGGTGGGGAGCGTGTAAAGGCCGTACAAATCGCGCTTAACTTTGGGGCCA ACAACTGTCAAACAT

CTGGAATCCCAGCCCCTCCCTTTCCCTGAACTGGGGAAGAAGGTGAAAACCCTTCAA GTTTTCTTTGATTGCCCC TTCCCACCTTCAGACCCCTGCTGGGAGGGTAAAGCGCCGACCCCTGGTGCCTGGCAAGTA CCAGAGACTCTAAAT CTCTCGGGATCCCCCCCCTCGCGCTCTTTCCTGACCCTCTCCCCTAACCCTCCCCACAGA GATCTCTCTACGCAG CCGACTGAGATCGTGGCGAATGGCCTTTTGTTTCTCCGCGTTTCCCCTATTGTTTGCCTT TCCAACATCTGGCGG GGCTTGGGGAGAGAAGGAAGCCCCTCTGGTCCCCCTCCCCGGCCCCCACGCCAGCTCCGG CAGGGGATCCCAGCT GGGAAAGTGGAGGAGCCCGACCCCAGCGAGGCCGCCCCACCCCGCCCTTGTGGTTAGAGG GCGGAGGGAAAGTTG TTCCTTCCCCGCCTCCGCTGCTGCCTGTGGCCCAGGGCGCATTTCTCAGATCTCAGCCCA GGCGCGCCGCAAAGG CTCAAATCCGAGAAGGTGCTGCTTTCGAGACAGTGGAAGCGCGTTCCGCCCCAATCCAGA GCGTCCAGTGGTTGG TTCCAGAGGATTTCAATCTCTAGCCAAAGGCGTTGGGGCTGGGCCGCTGCTAGGGCAGTG GGAGGGGATCGGGGC ACCTTTGGTAGGCGGAAAGCTGAGATTCTGGGGTCCACAAGTTTCCAAGGGCGGGAGGGC AGGCTAGTCGCCAAA AAGAGAACGAAGATGCAAATAACGAGGAAGCCTTATGACGTTGCCTGGAAATAGTAGTGT GGTGGTTCACTCCGG AATGAACGTGGAGTTCTGGCTTTGAGTACCGCTCCAAGTTTAAATCCCAAGTCCCCTTTC TTCATTGTAGAAAAA GAGGACTCAGACGACGCAACACAGATACGGCTAGAGCACAGTTCCTGCTTCCACGTCCCA GAGAACAAGTGGCTT AGGATGGTCCCGAGTTCCCCTGTGGGTGCGCTTGTTGGGTTGCAGGCGGCCCTGTTTCCC TGCACAAGTCAGATG CTTACACATTGTGTTCATTCTTAGTGTGGATTATTGATTAAAGAACTGGGGCAAAAGCAA AGTAGCTACTCTGAG AAGTCAGGGTCCCCAGATGGTGCCCAGCGAGTTGTCTTGCCTCTGAGGGGAGGCTGACTG AGACTGTGCACCTGT TAGAACCTATGCTACCCCATAGCCTTGCAGTTGACTTGCTGTTGCCAGCTTTTCCTGTGG GATCCCCAATGAGTC CCTCTTCCAAGGAAGCTCAATTACACTTTTGATTCCTCCTCAACCCAGGGGAAGAAAGAG GCTTCTGTAGGAACA TTATGATCTATGTACCCACTCAGACATTGTCAGTGGATACCAGAAGCTTGGCTCTGCACA GCTCTGAGAGTTTTC CCTTTGCGAACTCAACAGAACTTTTGAGTTTCCATTTAACATAAAAGAAGTGAGACTGCT AAGCCAGGAATGCGA CACATAGAGCACTTTCTCTAGTGATTTCTGGGTATTATATCTCTTTACCTTCCCAACGGT GGAACCAGGAAAAGA AAAAAAAGCAACATCTTTGAAGTACTGCAAGGCACTTTACAAACATTTCATTATGAAAAT GATCCCCAAGGAAGG ATTCCTTTGAAATTTAGCAGCAGCAACCCAGAAGCAACAAAAAAGACCAAAGTTACTCAA GAAGTACCCAAAGGC ATCATTAACAAAATAAAAGAGCATTTCTTGTCTTGGCCTACCCCGCTAAGGAAAACAGGG TAATTATAGTGGAAG TTAAGCTTG (SEQ ID NO. 63)

In some embodiments, the human C90RF72 gene and flanking sequences comprise a sequence that is, e.g., at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequence above. As used herein, the term "percent sequence identity" or "percent identity" or "% identity" is the identity fraction times 100. The "identity fraction" for 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, e.g. the total number of nucleotides in the full length of the entire reference sequence.

In some embodiments, the number of GGGGCC hexanucleotide repeats (e.g.,

(GGGGCC) _n in SEQ ID NO: 63) is 300-800, 300-700, 400-600, or 500-600. In some embodiments, the number of GGGGCC hexanucleotide repeats (e.g., (GGGGCC) _n in SEQ ID NO: 63) is 500-600. In some embodiments, the number of GGGGCC hexanucleotide repeats (e.g., (GGGGCC) _n in SEQ ID NO: 63) is greater than 300, 400, 500, 600, 700 or 800. In some embodiments, the number of GGGGCC hexanucleotide repeats (e.g., (GGGGCC) _n in SEQ ID NO: 63) is greater than 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 therapies for the treatment of ALS or FTD, e.g., a therapy described herein or a candidate therapeutic agent. A transgenic mouse as described herein can be made using any method known in the art or described herein, e.g., Example 4 (see also, e.g., PCT Publication Number

WO2001010199 and WO2013022715; and US Publication Number US20110113496 and 20060031954, each of which are incorporated by reference herein). For example, a

5 transgenic mouse described herein may be produced by introducing transgenes (e.g., the human C90RF72 gene, optionally with flanking sequences) into the germline of the mouse. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal used to practice this disclosure are selected for general o good health, good embryo yields, good pronuclear visibility in the embryo, and good

reproductive fitness. In addition, the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, Me.). The line(s) may themselves be transgenics, and/or may be knockouts (e.g., obtained from animals which have one or more genes partially or5 completely suppressed). The transgene construct may be introduced into a single stage

embryo. The zygote is the preferred target for micro-injection. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS

82:4438-4442). Normally, fertilized embryos are incubated in suitable media until the o pronuclei appear. At about this time, the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred. It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus. It is thought that the ovum nucleus or 5 female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones, thereby facilitating the combination of the female and male DNA complements to form the diploid zygote. Thus, the exogenous genetic material should be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus. For example, the exogenous n ^g eneric material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane.

Alternatively, the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation. Sperm containing the exogenous 5 genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter. Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane, or other existing cellular or genetic structures. Introduction of the transgene nucleotide o sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible5 injection of 1 -2pl of DNA solution. Following introduction of the transgene nucleotide

sequence into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method in to incubate the embryos in vitro for about 1- 7 days, depending on the species, and then reimplant them into the surrogate host.

o Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for 5 the presence of the transgene product. Typically, DNA is prepared from tail tissue and

analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis. Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene

5 product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal. Where mating with a partner is to be performed, the partner may or may o not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a

different transgene, or both. Alternatively, the partner may be a parental line. Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.

5 Aspects of the disclosure also relate to polynucleotides, e.g., a bacterial artificial

chromosome (BAC) vector, comprising SEQ ID NO: 63.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the o remainder of the disclosure in any way whatsoever. All publications cited herein are

incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES 5 Example 1

A construct containing a CMV promoter, a (GGGGCC) expansion motif containing either 4, 30, 60, or 120 repeats of GGGGCC, and an HA, FLAG, or MYC tag were transfected into cells (FIG. 2A). It was shown by western blot that poly-(GR) and poly-(GP) proteins were produced in cells transfected with constructs containing 30, 60 or 120 repeats n of GGGGCC (FIG. 2B). It was further shown using immunofluorescence of cells that GP- flag, GR-HA, and GA-Myc proteins were expressed in cells transfected with constructs containing 30, 60 or 120 repeats of GGGGCC (FIG. 3). These results show that GGGGCC repeat regions are capable of initiating translation independent of an AUG start codon (repeat-associated non-ATG (RAN) translation), and that poly-(GP), -(GR), and (GA)-repeat proteins are produced.

Antibodies to a poly-(GR) sequence or to the C-terminus of the poly-(GR)-repeat protein were generated. Fluorescent staining using these antibodies showed that these antibodies were capable of detecting the poly-(GR) repeat protein (FIG. 4).

Antibodies were further generated to a poly-(GP) sequence and the C-terminus of the poly-(GA)-repeat protein. The anti-poly-(GR), anti-poly-(GP), and anti-poly-(GA)-C-term antibodies were then used to stain sections of brain tissue from patients with C90RF72 ALS or controls (FIG. 5).

It was then hypothesized that transcripts of C90RF72 may be produced in both a sense and anti-sense direction (see FIG. 1). It was further hypothesized that these anti-sense transcripts may also undergo RAN translation to produce further repeat proteins from the 5'- GGCCCC-3' repeats present in the anti-sense transcript. As shown in FIG. 6, both poly-(PA) and poly- (PR) proteins were detectable in brain tissue samples from patients with C90RF72 ALS but not in controls. These results indicate that di-amino acid-repeat-containing proteins, such as RAN proteins are produced from both a sense and anti-sense transcript produced from the C90RF72 locus .

FIG. 7 shows that approximately 20% of aggregates detected with the anti-GP antibody (GP) also co-localize with antibodies directed against the unique C-terminus of the sense GP protein (GP-C). Consistent with the increases levels of antisense transcripts that seen in affected brains, these co-localization data suggest the more -80 percent of the GP dipeptide aggregates are expressed from C90RF72 antisense transcripts.

Additionally, the anti-sense transcript was found to be dramatically elevated in subjects with ALS compared to controls (FIG. 12). The primers for the qPCR assay for detecting the anti-sense transcript levels are shown in the table below. ORF F2 AGTCGCTAGAGGCGAAAGC primer in c9orf72 antisense orf (SEQ ID NO: 36)

ORF R2 CGAGTGGGTGAGTGAGGAG

(SEQ ID NO: 37)

ORF F2+IK CGACTGGAGCACGAGGACACT

GAAGTCGCTAGAGGCGAAAGC

(SEQ ID NO: 38)

ORF R2+lk CGACTGGAGCACGAGGACACT for RT 1st strand

GACGAGTGGGTGAGTGAGGAG

(SEQ ID NO: 39)

Linker CGACTGGAGCACGAGGACACT for RT- per with ORF Fl and F2

GA (SEQ ID NO: 40)

Further, di-amino acid repeat-containing proteins were found to be present in the blood (including in the serum and plasma) and in the brain of subjects with ALS (FIGs. 9 and 10) but not in control subjects.

Example 2

According to some aspects of the disclosure, di-amino acid repeat-containing protein (such as RAN protein) accumulation in blood and cerebral spinal fluid (CSF) substantively contribute to C90RF72 ALS/FTD and that plasmapheresis and bone marrow transplantation will reverse progression of the disease. According to some aspects of the disclosure, di- amino acid repeat-containing protein accumulation in blood and circulating CSF infiltrates the brain parenchyma and leads to protein accumulation, neuroinflammatory changes, CNS dysfunction and neuronal death. Aspects of the disclosure are based in part on the following. First, blood brain barrier (BBB) impairment is an early feature of disease in ALS patients (4, 5) and higher rates of ALS and other neurological diseases are found in patients who have had traumatic brain injuries (6). In some embodiments, without wishing to be bound by theory, ALS is in part caused by BBB disruptions that allow for the CNS entry of immune cells and other harmful substances that accelerate ALS/FTD. Secondly, as described herein di-amino acid repeat-containing proteins were found to accumulate in ALS patient blood samples (FIGs. 8 and 9).

Although plasmapheresis and bone marrow transplants have been tested as therapeutic strategies for ALS in the past, it is not clear if any of these cases were C90RF72 positive or if treatment was early enough to have an effect. Accordingly, in some embodiments, ALS treatment (e.g., plasmapheresis or BMT) is initiated when above-normal levels of one or more di-amino acid repeat-containing proteins are detected in the blood of a subject.

The data presented herein on di-amino acid repeat-containing protein accumulation in C90RF72 ALS patient tissues and blood indicates that reduction of blood (and perhaps also CSF) di-amino acid repeat-containing -protein load may help treat ALS in C90RF72 ALS patients. According to some aspects of the disclosure, reduction may be achieved, for example, using plasmapheresis or a bone marrow transplant.

Methods

A detailed evaluation is performed on gene carriers from a C90RF72 family (CNSA- 1) and patients in the clinic including a gene-positive patient with early signs of motor neuron disease or fronto-temporal cognitive dysfunction, or both. Di-amino acid repeat-containing protein expression is correlated with repeat length in CNSA family samples and additional samples collected in clinic. Di-amino acid repeat-containing protein expression in blood is determined in longitudinally collected samples and correlated with disease onset and clinical severity. These methods are expected to characterize di-amino acid repeat-containing protein expression in C90RF72 positive expansion study subjects and to determine if di-amino acid- repeat-containing protein expression occurs throughout life or increases with age and if di- amino acid repeat-containing protein levels quantitatively correlate with disease severity.

Plasmapheresis is tested to determine if lower di-amino acid repeat-containing - protein load in the blood and CSF reverses signs of the disease. Plasmapheresis is performed on five C90RF72 positive individuals with early signs of the disease. Six plasmaphereses, each with 2-litter exchange with normal human albumin, is performed over two weeks, followed by one plasmapheresis weekly for the next six months. The study may be prolonged, if required. The primary outcome measure is the Appel ALS Rating Scale (AALSRS). Clinical evaluations including neurological examination, speech evaluation, neuropsychological testing, the ALS Functional Rating Scale (ALSFRS), EMG, and needle muscle biopsy for immunohistopathological evaluations of the vastus lateralis muscle are performed to assess disease progression immediately before and after the treatment period. Venipuncture and lumbar puncture are also performed before and after the 6-month (or if applicable, also after the prolonged) treatment period to assess the concentration of serum and CSF levels of RAN translation and ATG-translation products.

Bone marrow transplant in an animal model is tested to determine if BMT prevents di-amino acid repeat-containing -protein accumulation in blood and the brain. In a first cohort of animals, bone marrow from RANT-positive mice are ablated and replaced with wild- type donor marrow to test if protein aggregate load in the brain decreases. In a parallel set of experiments, RANT-negative animals are transplanted with RANT-positive bone marrow to test if CNS protein accumulation occurs in animals that only express the transgene in hematopoietic cells. Both groups of treated animals are compared to wild-type and untreated RANT control animals using a combination of behavioral, functional and neuropathological assessments.

A RAN translation mouse model has been generated. Transgenic mice were generated using a construct containing 6 stop codons (two in each reading frame)

immediately upstream of a CAG expansion mutation and followed by 3 separate epitope tags in each reading frame (FIG. 10). The CAG repeat generates poly-Gin RAN proteins, which have been previously associated with diseases in humans such as fragile X syndrome. The RANT mouse model produced poly-Gin RAN proteins, which were found to localize at high levels under the pia surface in the brain which is exposed to the cerebral spinal fluid (FIG. 11). This RANT mouse model is used in the studies outlined in Example 2. Accordingly, detection of poly- amino acid repeat containing proteins (e.g., mono- or di-amino acid repeat containing proteins) may be indicative of a risk for a brain disorder associated with the poly- amino acid repeat containing proteins. Accordingly, methods described herein may be used to detect or treat other neurological diseases. Example 3 INTRODUCTION

The chromosome 9p21 -linked form of ALS/FTD, the most common cause of familial FTD and ALS identified to date, is caused by an expanded GGGGCC (G ₄ C ₂ ) hexanucleotide repeat in intron 1 of chromosome 9 open reading frame 72 (C90RF72) (1, 2). The C90RF72 mutation is found in 40% of familial and 7% of sporadic ALS cases and 21% of familial and 5% of sporadic FTD patients (3). The discovery of the C90RF72 expansion has generated substantial excitement because it connects ALS and FTD to a large group of disorders caused by microsatellite expansion mutations (4).

Traditionally, microsatellite expansion mutations located in predicted coding- and noncoding regions were thought to cause disease by protein gain-, or loss-, of-function or RNA gain-of-function mechanisms (4). Protein loss-of-function has been proposed to underlie C90RF72-driven ALS/FTD because the expansion mutation leads to decreased levels of variant 1 transcripts and potential decreases in C90RF72 protein expression (1, 2). Additionally, because the C90RF72 G ₄ C ₂ expansion mutation is located in an intron, several studies have pursued the hypothesis that C9-linked ALS-FTD results from a toxic RNA gain- of-function mechanism in which G ₄ C ₂ expansion RNAs sequester important cellular factors in nuclear RNA foci. Multiple G ₄ C ₂ RNA binding proteins have been identified, but so far there is no demonstration that any of these candidates directly bind endogenous expansion transcripts or co-localize with RNA foci observed in patient cells or autopsy tissue (5-8).

In this mechanism, hairpin-forming microsatellite expansion transcripts express proteins in one or more reading frames without an AUG-initiation codon (9). While a variety of names have recently been ascribed to these RAN translated proteins (e.g. homopolymeric, dipeptide, RANT), it is proposed that all proteins expressed across microsatellite expansion mutations in the absence of an ATG-initiation codon be referred to as RAN proteins to prevent confusion as additional expansion mutations that undergo RAN translation are identified.

Here it is shown that C90RF72 ALS/FTD antisense transcripts containing the GGCr.r.C (G ₂ C ₄ ) expansion accumulated in patient brains as nuclear, and infrequent cytoplasmic, foci. Additionally, a novel panel of antibodies directed to both the repeat motifs and unique C-terminal regions was developed and both sense and antisense RAN proteins were demonstrated to accumulate in C90RF72 patient CNS autopsy tissue. The discovery of antisense G ₂ C ₄ RNA foci and three novel antisense RAN proteins in C90RF72 patient brains 5 suggests that bidirectional transcription and RAN translation are fundamental pathologic features of C90RF72 ALS/FTD.

RESULTS

Antisense RNA foci in C90R 72-expansion patients

0 A series of experiments was performed to test the hypotheses that antisense (AS)

C90RF72 expansion transcripts form AS G ₂ C ₄ RNA foci and express AS proteins by RAN translation or from short AS open-reading frames (AS-ORFs). First, it was confirmed that C90RF72 antisense transcripts are expressed using a linkered strand- specific RT-PCR strategy to compare expression of the sense and antisense transcripts in intron lb, 5' of the5 antisense G ₂ C ₄ expansion, and exon la. For the antisense strand in intron lb, strand- specific RT-PCR was performed using LK-ASORF-R primer for the RT reaction and ASORF-F and the LK for PCR to specifically amplify antisense-cDNAs (FIG. 12A). Similar strategies were used to amplify sense transcripts from the same region of intron lb and sense and antisense transcripts in exon la. Intron lb antisense transcripts were detected by RT-PCR in frontal o cortex from C9(+) ALS/FTD patients but not C9(-) ALS/FTD or normal controls (FIG. 12B) and qRT-PCR shows these transcripts are dramatically increased among six C9(+) ALS/FTD cases (FIG. 12C). In contrast, intron lb sense transcripts were not detected by RT-PCR (FIG. 12B) in frontal cortex. In blood, both intron lb sense and antisense transcripts are detectable and the dramatic C9(+) elevation of the intron lb antisense transcripts was not observed. 5' 5 RACE showed intron lb AS transcripts begin at varying sites 251-455 basepairs (bp)

upstream of the G ₂ C ₄ repeat (FIGs. l2A, 19B). In contrast, 3'RACE, using 3'GSP1 or 3'GSP2 primers located 40 and 90 bp 3' of the G ₂ C ₄ repeat, did not detect transcripts. These data showed that the 3' end of the AS transcript does not overlap the sense exon la region, located 170 bp 3' of the antisense G ₂ C ₄ repeat. Consistent with this result, sense but not antisense n transcrints are detected by strand specific linkered-RT-PCR using primers overlapping exon la (FIG. 12B). To determine if antisense transcripts include the G ₂ C ₄ repeat expansion, RNA fluorescence in situ hybridization (FISH) was performed using a Cy3-labelled (G4C2)4 probe to detect putative antisense G ₂ C ₄ RNA foci. The results showed nuclear (FIG. 12D) and rare cytoplasmic (FIG. 19C) G ₂ C ₄ RNA foci accumulate in C9(+) but not C9(-) ALS frontal 5 cortex. The detection of foci in the cytoplasm showed that antisense expansion transcripts can be found in the same cellular compartment as the protein translation machinery, presumably where RAN translation occurs. Because RNA foci in peripheral tissues may provide biomarkers of the disease, peripheral blood leukocytes (PBLs) were examined and both sense and antisense RNA foci were detected in C9(+) but not C9(-) PBLs (FIG. 12D, FIG. 19D). It o was discovered that the RNA-FISH signal from the Cy3- G4C2 probe detecting AS-foci may be competed with excess unlabeled G4C2 oligo, and these foci were resistant to DNase I and sensitive to RNase I digestion (FIG 19E, F). Taken together, this shows that C90RF72 antisense transcripts are elevated in the frontal cortex in C9(+) ALS but not C9(-) ALS or normal controls. It was also shown for the first time that antisense transcripts containing the 5 G ₂ C ₄ expansion mutation are expressed and accumulate in nuclear and rare cytoplasmic RNA foci in C9(+) frontal cortex. Additionally, it was shown that sense and antisense foci accumulate in blood, providing potential biomarkers of C90RF72 ALS/FTD in a readily accessible tissue. o RAN translation of GGCCCC repeat expansion in vitro

To test if the antisense G ₂ C ₄ expansions undergo RAN translation, a triply tagged G ₂ C4 minigene was generated, (G ₂ C ₄ ) _EXP -3T, lacking an ATG initiation codon, by inserting a 6X STOP codon cassette (two stops in each frame) upstream of G ₂ C ₄ expansions of 40 or 70 repeats and three different C-terminal epitope 8 tags to monitor protein expression in all 5 reading frames [e.g., (G ₂ C ₄ EXP transcripts translated in three frames results in Gly-Pro (GP), Pro- Ala (PA) and Pro-Arg (PR) RAN proteins] (FIG. 13A). Immunoblotting detected two epitope-tagged RAN proteins, PR-Myc and GP-Flag, but not PAHA (FIG. 13B). The (PR)40- and (PR)70-3xMyc proteins migrated at approximately their predicted sizes of 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) at 50 and 75 kDa, respectively (FIG. 13B). The faint lower molecular weight bands on this blot may result from repeat contractions seen during bacterial culture or differences in translational start site. Immunofloresence (IF) showed antisense RAN proteins are expressed in all three reading frames (FIG. 13C). The detection of PA-HA by IF but not western blotting may be caused by a lower frequency of 5 cells expressing RAN PA-HA from these constructs. Additionally, recombinant GP-Flag and PA-HA proteins had a cytoplasmic localization whereas PR-Myc proteins were distributed in both the nucleus and cytoplasm. These localization differences may result from different properties of the repeat motifs or the C-terminal flanking sequences found in this epitope tagged construct. In an additional series of experiments also it was shown that sense G4C2-0 expansion constructs containing 30, 60 and 120 repeats express GP-Flag, GR-HA and GA- Myc RAN proteins (FIG. 20). In summary, these data showed that recombinant G ₂ C ₄ and G ₄ C ₂ expansion transcripts express RAN proteins in all six reading frames.

Dual immunological strategy to detect RAN proteins

5 Since amino acid repeats can be found in a range of different proteins, a dual

immunological strategy was used and antibodies that recognize the predicted repeat motifs described herein or their corresponding unique C-terminal regions were developed. A schematic diagram showing eight putative C90RF72 RAN proteins is shown in FIGs. 13D and 21. Predicted proteins include six putative RAN proteins and two putative proteins with o additional ATG-initiated N-terminal sequence. Unique C-terminal regions are predicted in five of the six predicted reading frames. To test for 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(FIGs. 13D, 21). Antibodies to test for putative antisense proteins [rabbit a-PA, a-PA-CT, a-PR, a-PR-CT, a-GP a-GP-CT(sense), 5 and mouse a-GP] were generated and their specificities demonstrated in cells transfected with constructs expressing epitope-tagged recombinant protein by western blot and IF detection (FIGs. 13E, 22). Additional antibodies detecting repeat and C-terminal regions expressed in the sense direction are characterized in FIG. 23. n Antisense G ₂ C4 RAN proteins accumulate in brain Several approaches were used to determine if novel antisense (AS) proteins are expressed in C90RF72 expansion positive autopsy tissue. To overcome the obstacle that aggregated proteins are difficult to isolate from human brain, a sequential protein extraction protocol (23) was used on frozen C9(+) and C9(-) ALS frontal cortex autopsy samples.

Antisense PA and PR proteins were detected with a-PA, a-PA-CT, a-PR, a-PR-CT on immuno-dot blots of 1% Triton-X100 insoluble, 2% SDS soluble extracts from a subset of C9(+) but not C9(-) ALS patients (FIG. 14A). Additional immuno-dot blots showing evidence for sense-RAN protein (GP, GR, GA) 10 accumulation in C9(+) ALS/FTD frontal cortex are shown in FIG. 24. a-PA, a-PR and a-GP antibodies also detected high molecular weight smears in 2% SDS insoluble fractions from C9(+) ALS frontal cortex samples after resuspending the pellets in sample buffer containing 8 % SDS (23) (FIG. 3B). The differences in migration pattern seen for the recombinant proteins (FIG 13B), which migrate as one or more bands, and the smears observed in patient tissue extracts (FIG. 14B) reflect differences in the RAN proteins due to much longer repeat tracts in patient samples and their extraction from highly insoluble aggregates. Immunohistochemistry (IHC) was next used to show that protein aggregates were detectable in the perikaryon of hippocampal neurons from C9(+) ALS/FTD autopsy tissue but not in C9(-) ALS patients or control subjects using antibodies against the repeat motifs (a-PA, a-PR, a-GP) as well as antibodies directed to predicted C-terminal sequences beyond the PA and PR repeat tracts (a-PA-CT and a-PR-CT) (FIG. 14C, 25). Previous studies using antibodies directed against the GP repeat motif, detected aggregates, which were assumed to be expressed from the sense strand (10, 11). It is noted that GP repeat-containing proteins are predicted to be expressed from both sense and antisense transcripts (FIG. 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 after the repeat. To distinguish sense-GP RAN proteins from antisense-GP proteins, a double label IF experiments was performed on C9(+) human hippocampal autopsy sections using rabbit a-GP-CT to detect the CT region of the sense-GP protein and mouse a- GP to detect both sense and antisense GP expansion proteins. Double labeling showed two types of inclusions: a) putative sense inclusions double labeled with mouse a-GP and rabbit rc-GP-ΓΤ sense and; b) putative antisense inclusions singly labeled with mouse-a-GP (FIG. 14D). Approximately 18% of inclusions showed the sense pattern with double labeling and 82% 11 of inclusions showed the antisense pattern and were positive for a-GP and negative for a-GP-CT sense (FIG. 14E,F). These data showed the importance of characterizing protein aggregates with both repeat and C-terminal antibodies. Taken together, these results show that insoluble, aggregate-forming antisense-RAN proteins are expressed from all three antisense reading frames.

G ₂ C4 expansions and RAN proteins are toxic to cells

In addition to antisense GP and PR RAN proteins expressed by RAN translation, two of the antisense reading frames have upstream ATG initiation codons that may result in both ATG-initiated GP and PR proteins (M-GPAS and M-PRAS) (FIG. 13D and 21). It was shown that the presence of an ATG-initiation codon does not prevent RAN translation from also occurring in all three reading frames (9). Therefore antisense GP and PR proteins may be expressed by both AUG-initiated and/or RAN translation. To explore the effects that an ATG-initiation codon has on RAN protein expression for the G ₂ C ₄ expansion, an additional minigene construct was generated by placing an ATG initiation codon in front of the G ₂ C ₄ repeat (FIG. 14G). The PR frame was selected for analysis because an ATG initiation codon naturally occurs in this reading frame. Western blotting shows that HEK293T cells transfected with (+)ATG-PR-3T express substantially higher levels of PR protein compared to (-)ATG-PR-3T transfected cells (FIG. 14H). In contrast, qRT-PCR and Western blotting showed transcript levels (FIG 26 A) and levels of RAN-translated GP (FIG. 14H) were comparable. Similar to FIG. 13, RAN-translated PA was not detectable by Western blot. The effects of these constructs on cell viability was then tested using complementary assays; lactate dehydrogenase (LDH) detection and methylthiazol tetrazolium (MTT). For the LDH assay, cells transfected with the (-)ATG-PR-3T or (+)ATG-PR-3T construct showed 1.9 and 2.9 fold increases in cell death compared to vector control cells (p=0.008 and 0.001), respectively. Additionally, (+)ATG-PR- 3T transfected cells, which express elevated levels of PR protein showed a 1.5 fold increase in cell 12 death compared to cells transfected with the (-)ATG-PR-3T construct (p=0.034). The MTT assay showed similar results. Cells transfected with i-^ATG-PR-3T and +ATG-PR-3T constructs showed dramatic decreases in the number of metabolically active cells, 33% (p<0.00001) and 43% (p<0.00001), respectively compared to untreated cells or empty vector controls (FIG. 14J). Additionally, elevated PR expression in cells transfected with (+)ATG-PR-3T had significantly lower levels of metabolic activity compared to (-)ATG-PR-3T cells (p<0.05). By light microscopy cell detachment and

5 changes in cell morphology were evident in -ATG-PR- 3T compared to control cells and these phenotypes worsened in (+)ATG-PR-3T cells which express elevated levels of PR (FIG. 26B-D). Taken together, these data demonstrated that: 1) the G ₂ C ₄ expansion mutation is toxic to cells - this toxicity may be caused by effects of the DNA, G ₂ C ₄ RNA and/or RAN- translated PR, GP or PA proteins; 2) increased PR protein expressed in cells transfected with o the (+)ATG-PR- 3T construct increases cell toxicity and death above levels caused by the

DNA, G ₂ C ₄ RNA and RAN protein effects. Therefore the PR protein was shown to be intrinsically toxic to cells.

All six RAN proteins form aggregates in the brain

5 To determine if all six RAN proteins from both sense and antisense RNA strands are expressed in C9(+) ALS patients, IHC staining was performed on sections of paraffin- embedded brain tissues using nine polyclonal antibodies against repeat-expansion and/or C- terminal sequences of these proteins. In C9(+) cases there were abundant globular and irregular- shaped neuronal cytoplasmic inclusions (NCIs) in the hippocampus, the majority of o which were in the dentate gyrus and in pyramidal cells in the CA regions. These RAN

inclusions were also detected in C9(+) motor cortex (FIG. 15). GP positive inclusions were detected in all examined C9(+) cases but not in C9(-) cases or normal control sections in the hippocampus as well as in the motor cortex using a-GP. In the CA regions of the 13 hippocampus and in the motor cortex, clusters of aggregates were frequently found in C9(+) 5 cases with aggregates in >20% of neurons (FIG. 27). Fewer aggregates were detected with the a-GP-CT sense antibody, consistent with double labeling experiments (FIG. 14D-F) that showed most GP aggregates are translated from C90RF72 antisense strand. PA inclusions were detected in hippocampus in four out of six C9(+) cases tested and in one out of two motor cortex samples (FIG. 27). In C9(+) cases, the frequency of PA inclusions were n si ^p nificantly lower in the hippocampus and motor cortex compared with GP inclusions, but high-intensity regional staining with extremely large PA inclusions found in >50 of neurons were found in one patient (FIG. 27). PR positive inclusions were also seen in hippocampus in all C9(+) cases examined and in motor cortex in one out of two C9(+) cases tested. Similar to the PA staining, PR inclusions are less frequent but intense regional staining was occasionally 5 observed. In the sense direction, GR positive inclusions were found in the hippocampus and motor cortex in all C9(+) cases examined, but appeared less frequent than the GP aggregates. GA inclusions were only occasionally detected by IHC as small perinuclear inclusions in hippocampus and in motor cortex (FIG.15, 27). The apparent differences in the frequency of various types of aggregates may result from differences in protein conformation and epitope o availability or differences in the affinities of these antibodies, which were designed to

different epitopes. Taken together, this data showed that all six RAN proteins form

aggregates in the C9(+) autopsy brains.

Inclusions of RAN proteins in upper and lower motor neurons

5 A central feature of ALS is the gradual degeneration and death of upper motor

neurons in motor cortex and lower motor neurons in the brain stem and spinal cord. To test if RAN proteins accumulate in upper and lower motor neurons, IHC was performed using all nine antibodies against predicted proteins in both sense and antisense directions. In C9(+) cases, abundant GP-positive neuronal cytoplasmic inclusions were seen in all layers of motor o cortex, with frequent GP aggregates in pyramidal neurons of layer III and throughout layer V

(FIG. 16A). Although cell death and atrophy made motor-neurons in layer V difficult to identify, GP inclusions in remaining upper motor neurons were found (FIG. 16B).

Additionally, PA-, PR-, GR- and GA-positive inclusions were also found in the motor cortex (FIG. 15, 27). Using a similar series of experiments performed in spinal cord sections, GP 5 aggregates in all three cases examined and aggregates in lower motor neurons in two out of three C9(+) patients were detected, but not in C9(-) ALS cases or normal controls (FIG. 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 the neurons selectively vulnerable in ALS. High density clustering of RAN-protein aggregates

Both sense and antisense proteins accumulated in neurons of C90RF72 autopsy brains. In general, two types of aggregation patterns were observed: 1) isolated cytoplasmic aggregates and 2) high-density clustered cytoplasmic aggregates in which -10 to more than 5 50% of neurons were positive. Clustered aggregates were most frequently detected for GP and were found in the dentate gyrus (DG) and CAl -4 of the hippocampus (FIG. 16D, E). The clustered GP aggregates in DG were smaller and less frequent than the large cytoplasmic aggregates in CA regions. Additional clustered GP aggregates were frequently found in subiculum and presubiculum of the hippocampus as well as 15 the motor cortex.

o Immuno staining of serial sections showed that multiple proteins are often found in the same region. For example, intense clustered staining for PA, PR, GP, GA and GR proteins was found in the same region of the presubiculum in serial sections from one C9 (+) patient (see FIG 16F,G). Immuno staining for PA showed that some brain regions have abundant aggregates whereas other regions in the same section are relatively spared. For example, FIG.5 17A illustrates a gradient of PA inclusions (presubiculm>subiculum>CAl) across

hippocampal regions in a single section in one patient. PA inclusions in this patient were numerous (>50% of neurons) in presubiculum (I), moderate in subiculum (II), and rare in CAl hippocampal regions (III and IV). Consistent with the focal regional staining seen in this section, PA staining was not detected in sections from a separate block of hippocampal tissue o taken from the same patient. These data shows that expression of the PA RAN protein is variable from cell to cell or that aggregation of PA in one cell triggers aggregation in neighboring cells as has been proposed in a mouse model of Parkinson's disease (24). Next, serial sections from this C9(+) case were used to show that antibodies directed against both the repeat motifs (a-PA, a-PR, a-GP, a-GR) and corresponding C-terminal regions (a-PA- 5 CT, a-PR-CT, a-GP-CT, a-GR-CT a-GA-CT) detect aggregates in the same densely staining region of the presubiculum (region I) (FIG. 17B). These results showed that both sense and antisense RAN protein aggregates accumulate in this region. The detection of similar aggregates in using antibodies that recognize either the repeat motifs or specific C-terminal regions confirms that these antibodies are recognizing proteins expressed across both the G ₂ C ₄ and G ₄ C ₂ expansion transcripts and provides new tools to understand the biological impact of RAN translation in C90RF72 ALS/FTD.

DISCUSSION

There has been much excitement about the discovery that an intronic micro satellite expansion mutation in C90RF72 causes a common form of both familial and sporadic ALS/FTD (1, 2). The three major pathological mechanisms being considered 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 exclusively on understanding the consequences of the C90RF72 expansion mutation in the sense direction. The results reported here show t atC90RF72 expansion mutation is also expressed in the antisense direction and show that antisense RNA foci and antisense RAN proteins contribute to C90RF72 ALS/FTD. We show for the first time: 1) antisense

C90RF72 but not sense transcripts are elevated in C9(+) autopsy tissue; 2) antisense G ₂ C ₄ expansion transcripts form RNA foci that accumulate in C9+ brain and blood; 3) RAN translation occurs across antisense G ₂ C ₄ expansion constructs in cell culture; 4) that sense and antisense RAN proteins accumulate in C9(+) autopsy brains using a dual immunological approach with both repeat and C-terminal antibodies; 5) RAN protein aggregates accumulate in upper and lower motor neurons linking RAN translation directly to the key pathologic feature of ALS. Since the initial report that G ₄ C ₂ RNA foci accumulate in C90RF72

ALS/FTD patient tissues (1, 2), a leading hypothesis is that G ₄ C ₂ sense transcripts sequester and dysregulate RNA binding proteins similar to the sequestration of MBNL proteins in DM1, DM2 and SCA8 (4). Several groups have already reported G ₄ C ₂ binding proteins and are testing their potential role in disease (5-8). The discovery that antisense G ₂ C ₄ foci also accumulate in patient cells shows that G ₂ C ₄ antisense RNAs and binding proteins may play a role. Additionally, the discovery of sense and antisense foci in C9(+) peripheral blood may prove useful as an easily accessible biomarker of C90RF72 ALS/FTD. Biomarkers that monitor both sense and antisense transcripts may be particularly important as therapies that decrease expression of one strand may increase expression of the other strand. Using a dual immunological approach it was shown that G ₂ C ₄ antisense transcripts express novel antisense proteins (PA, PR, GP) by RAN translation and/or from two short ORFs (Met-AS-PR and Met-AS-GP).

MATERIALS AND METHODS

5

cDNA constructs. CCCGGGGCC(GGGGCC) ₂ GGGGCCC (SEQ ID NO: 64) and CCCGGGGCC(GGGGCC) ₂₈ GGGGCCC (SEQ ID NO: 65) fragments that contain upstream 6xStop codons were synthesized and cloned into pIDTSmart vector by Integrated DNA Technologies. 6xStops- (GGGGCC) ₄ -3T and 6xStops-(GGGGCC) ₃₀ -3T constructs were o generated by subcloning NheVXhoI fragment into pcDNA3.1 vector containing triple

epitopes. To expand the size of the GGGGCC repeats, SmaVXhoI fragment was subcloned into PspOMI blunted with T4 DNA polymerase/X^I of pcDNA-6xStops-(GGGGCC) _EXP -3T. To reverse the orientation of GGGGCC repeats in pcDNA-6xStop-3T construct, SmalVClal fragment was subcloned into pBluescript SK+ to generate pBluescript-(GGGGCC)Exp. The 5 AfeVXhol fragment pBluescript- (GGGGCC) _EX p was subcloned into pcDNA-6xStop-3T to make pcDNA-6xStop-(GGCCCC) _EXP -3T construct.

RT-PCR. 1) Strand-specific RT-PCR in autopsy tissues: Total RNA was isolated from Frontal cortex autopsy tissues and peripheral blood lymphocytes (PBL) of ALS patients o and healthy controls with TRIzol (Invitrogen). To detect transcripts from both strands, cDNA was generated from 0.25μg of total RNA using the Superscript III system (Invitrogen) with linkered strandspecific reverse primers and PCR with strand specific forward and linker (LK) primers. The PCR reactions were done as follows: 94 °C for 3min, then 35 cycles of 94°C for 45s, 58°C for 45s and 72 °C for 1 min followed by 6 min at 72°C. Bands were cloned and 5 sequence to verify their specificity of the PCR amplification. 2) RT-PCR for toxicity assay in 293T cells: Total RNA from cells was extracted using 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. The expression of the different G4C2- 3XTag constructs were analyzed by RT-PCR and qPCR using primer set: 3xTag-Fw and 3xTag-Rv. β-Actin expression was used as a reference gene amplified with primer set ACTB3 and ACTB4. Primer sequences are listed in FIG. 27.

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. Control reactions were performed with human beta-actin primers ACTB3 and ACTB4 using oligo dT synthesized total cDNA as template. Two stage PCR was performed for 40 cycles (95°C 30s, 60°C 30s) in an optical 96 well plate with each sample cDNA/primer pair done in triplicate. The relative fold changes were generated by first normalizing each experimental Ct value to their beta actin Ct value and then normalized to the healthy control antisense AACt. Primer sequences are listed in FIG. 28.

Rapid Ampliciation of 5' and 3' cDNA ends (5' and 3' RACE). Four μg of total RNA from 2 C9(+) ALS patients and 2 C9(-) ALS patients frontal cortex autopsy tissues were used for 5' and 3' RACE (5' RACE systems and 3' RACE; Life Technologies). In 5'RACE, Primer ASORF R was used for gene specific first strand cDNA synthesis and nested reverse primers are 5'GSP1 and 5'GSP2. In 3'RACE, nested forward primers are 3'GSP1 and 3'GSP2. The 3' RACE and 5' RACE products were gel-extracted, cloned with TOPO TA Cloning (Invitrogen) and sequenced. Primer sequences are listed in FIG. 28.

Production of polyclonal antibodies. The polyclonal rabbit antibodies were generated by New England Peptide and the polyclonal mouse antibody was generated by the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. In sense strand (GGGGCC), antisera were raised against synthetic poly(GP), poly(GR) peptides and C terminal regions of predicted GP, GR, and GA RAN proteins (FIG. 21). In antisense strand (GGCCCC), antisera were raised against synthetic poly(PA), poly(PR) peptides and the C terminal regions of predicted PA and PR RAN proteins. Peptides used to generate antibodies to both antisense and sense proteins and their use for Western blot,

immunofluorescence (IF) and immunohistochemistry (IHC) is summarized in Table S3. Cell culture and transfection. HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and incubated at 37°C in a humid atmosphere containing 5% C02. DNA transfections were performed using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's instructions.

Human Samples. Frozen frontal cortex tissue samples for biochemical and histological analysis included samples from six C9(+) ALS, five C9(-) ALS controls and one normal control were used in this research. Additionally, paraffin embedded fixed tissues from C9(+) ALS/FTD and C9(-) ALS/FTD cases as well as a normal control. Peripheral blood lymphocytes (PBL) were isolated from the buffy coat of freshly collected whole blood following brief centrifugation at 2000xg. Red blood cells (RBC) were preferentially lysed and removed using RBC Lysis Buffer (Roche), PBLs centrifuged, washed once with PBS and dried on slides. This study was conducted in compliance with the Declaration of Helsinki. Institutional review boards of the University of Florida and Johns Hopkins University approved the study. Written, informed consent was obtained from participants or relevant parties at the time of enrollment.

Immunofluorescence. The subcellular distribution of polymeric proteins was assessed in transfected HEK293T cells by immunofluorescence. Cells were plated on 8 well tissue-culture chambers and transfected with plasmids the next day. Forty-eight hours post- transfection, cells were fixed in 4% paraformaldehyde (PFA) in PBS for 30 min and permeabilized in 0.5% triton X-100 in PBS for 15 min on ice. The cells were blocked in 1% normal goat serum in PBS for 30 min. After blocking, the cells were incubated for 1 hour at RT in blocking solution containing the rabbit anti-Myc (Abeam), 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 RT in blocking solution containing Goat anti-rabbit conjugated to Cy3 (Jackson

ImmunoResearch, PA) and goat anti-mouse conjugated to Alexa Fluor 488 (Invitrogen) secondary antibodies at a dilution of 1:200. The slides were washed three times in PBS and mounted with mounting medium containing DAPI (In vitro gen).

RNA-FISH. Slides with cells were fixed in 4% PFA in PBS for 10 min and incubated in prechilled 70% ethanol for 30 min on ice. Following rehydration in 40% formamide in 2XSSC for 10 min, the slides were blocked with hybridization solution (40% formamide, 2XSSC, lOmg/ml BSA, lOOmg/ml dextran sulfate and 10 mg/ml yeast tRNA) for 10 minutes at 55 °C and then incubated with 200ng/ml denatured RNA probe in hybridization solution at 55°C for 2 hours. After hybridization the slides were washed 3 times with 40% formaminde in 2XSSC and briefly washed one time in PBS. Autofluorescence of lipofuscin was quenched by 0.25% of Sudan Black B in 70% ethanol and the slides were mounted with mounting medium containing DAPI (In vitro gen). The specificity of the RNA foci was determined by treating cells prior to FISH detection with either RNAse (100 ug/mL in 2xSSC), DNase (lU/ul in DNasel buffer) or Protease K (120 ug/mL in 2mM CaC12, 20mM Tris, pH 7.5). Treated cells were incubated at 37°C for 30 minutes, washed 3 times with PBS then 3 times with 2xSSC. Subsequent FISH detected was performed as described above. Antisense foci specificity was determined using standard FISH detection to first hybridize slides with 10- fold excess unlabeled (G4C2)4 oligo followed by hybridization with either G4C2-cy3 (antisense probe) or G ₂ C4-cy3 (sense probe). Subsequent treatment and detection were performed as described above.

Western blotting. Transfected cells in each well of a six-well tissue-culture plate were rinsed with PBS and lysed in 300 μΐ _^ RIPA buffer with protease inhibitor cocktail for 45 min on ice. DNA was sheared by passage through a 21 -gauge needle. The cell lysates were centrifuged at 16,000 x g for 15 min at 4 °C, and the supernatant was collected. The protein concentration of the cell lysate was determined using the protein assay dye reagent (Bio- Rad). Twenty micrograms of protein were separated in a 4-12% NuPAGE Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (Amersham). The membrane was blocked in 5% dry milk in PBS containing 0.05% Tween-20 (PBS-T) and probed with the anti-Fla ^p (1:2000), anti- Myc (1: 1000), anti-HA (1: 1000), or rabbit polyclonal antibodies (1: 1000) in blocking solution. After the membrane was incubated with anti-rabbit or anti- mouse HRP-conjugated secondary antibody (Amersham), bands were visualized by the ECL plus Western Blotting Detection System (Amersham). Sequential extraction of patient frontal cortex autopsy tissue was performed as follows: tissue was homogenized in PBS containing 5 1% Triton-X100, 15 mM MgC12, 0.2 mg/ml DNase I and protease inhibitor cocktail and centrifuged at 16,000 x g for 15 min 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 x g for 15 min 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- o Mercaptoethanol for protein blotting (25).

Protein slot blot. 1% Triton-XlOO soluble fraction and 2% SDS soluble fraction from the sequential extraction was immobilized onto nitrocellulose membranes with Bio-Dot 96- well microfiltration system (Bio-Rad) under vacuum. The membranes were washed in PBS-T5 and blotted with each rabbit polyclonal antibody (1:2000) using the same protocol as western blotting.

Immunohistochemistry. Ten-micrometer sections were deparaffinized in xylene and rehydrated through graded alcohol, incubated with 95-100 % formic acid for 5 min, and o washed with distilled water for 10 min. HIER was performed by steaming sections in citrate buffer, pH 6.0, at 90 °C for 30 min. To block nonspecific immunoglobulin binding, a serum- free block (Biocare Medical) was applied for 30 min. Rabbit polyclonal antibodies were applied at a dilution of from 1:5000 to 1: 15,000 in serum-free block (Biocare Medical) and incubated overnight at 4 °C. Linking reagent (streptavidin and/or alkaline phosphatase,

5 Covance) was applied for 30 min at room temperature. These sections were incubated in 3% H202 for 15 min to bleach endogenous peroxidase activity. Then labeling reagent (HRP, Covance) was applied for 30 min at room temperature. Peroxidase activity was developed with NovaRed substrate (Vector) and sections were counterstained with hematoxylin. Cell toxicity assays. All the transfection experiments were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instruction and at a 60% cell confluence. 500ng of each vector was transfected in 35mm wells.. Cell death was determined by measuring Lactate dehydrogenase (LDH) cell release, using CytoTox 96 non- 5 radioactive cytotoxicity assay (Promega) according to the manufacturer's instructions.

Absorbance was recorded at 490 nm and total LDH release was measured by lysing the cells with 1% Triton X-100. In each experiment, determinations were performed in quintuplicates for each experimental condition and average data calculated. Statistical significance was determined using the two tailed unpaired Student t test for single comparisons (p < 0.05) and o the analysis of variance (ANOVA) when multiple pairwise conditions were compared.

Cell viability assays. HeK293T cells were transfected in 96 well plates and cell viability was determined 42 hours post-transfection with the 3-(4,5- dimethythiazol-. 2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. MTT was added to cell culture media at 0.55 mg/mL final concentration and incubated for 45 minutes at 37°C. Cells were then lysed with 100 of DMSO upon medium removal and absorbance was measured at 595 nm. In each experiment, determinations were performed in quintuplicates. Statistical significance was determined using Student's t test (p < 0.05). o Example 4. BAC transgenic mouse model of C90RF72 ALS to test the hypothesis that both sense and antisense transcripts contribute to ALS/FTD.

Rationale: A mouse model of C90RF72 ALS/FTD that recapitulates the sense and antisense transcripts is critical for modeling this disease. BAC clones were isolated from a human 5 patient which contain -800 G4C2 repeats. These BAC clones were used to generate 8

founder lines. These mice are useful, for example, to answer the following questions: Does both RAN protein expression and RNA gain of function contribute to C90RF72 ALS/FTD? Are sense and antisense mechanisms both important in C90RF72 pathogenesis? n Annroach: BAC clones containing the full human C90RF72 gene plus flanking sequences were isolated from a human patient with -800 GGGGCC repeats and inserted into the pCClBAC™ plasmid (Epicentre®). The BAC insert chosen for use in the mouse extended from bp27, 625,470 to 27,527,137 of human genome reference sequence on Chromosome 9 (FIG. 29). The coordinates above do not include extra repeats from this patient. It was found 5 that the BAC insert DNA contained about 800 repeats in some clone preps but was very

unstable. Pronuclear injections were performed and 8 FVB founder lines were generated - 2 independent lines which were confirmed expansion mutations. The BAC repeat size in the mice was -500 repeats but varied between progeny and may grow or shrink in size as the mouse colony is expanded and additional generations of mice are propagated in the

o laboratory. BAC expansion mice expressed both sense and antisense versions of the

C90RF72 gene. Sense and anti-sense GGGCC RNA foci were present in mice that had the GGGGCC repeats, but not in control mice (FIGs. 30-31).

At least two expansion and two control lines are selected for detailed characterization. Behavioral characterization includes rotorod analysis, grip strength, balance beam and open5 field assessments. Molecular characterization of sense and antisense transcripts and RAN proteins are performed by RT-PCR, RACE, immunoblot, immunohistochemistry and immunofluorescence. Immunohistochemistry, immunofluorescence and FISH studies are performed to correlate sites of RNA foci and C9-RAN proteins accumulation with

pathological changes. RAN-protein accumulation in the CNS, CSF, muscle, blood and other o tissues are examined at various times during development.

Relevance: Results from these studies will lead to a better understanding of the role that RAN translation plays in C90RF72 ALS/FTD. Additionally, these studies will help to prioritize individual protein targets by determining which proteins are found most frequently 5 in autopsy tissue and identifying overt differences in the toxicities of individual RAN

proteins. Information from cellular and mouse models will also inform future studies on the effectiveness of various treatment strategies.

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Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

What is claimed is: