Abstract
The current invention relates to gene therapy approaches for the treatment of SCA3, in particular RNAi based gene therapy approaches utilizing a total knockdown approach. The inventors provide for selected target regions and/or target sequences for which highly efficient knockdown of the ATXN3 gene expression can be advantegeously obtained in human neuronal cells and in mouse models relevant for SCA3.
Claims
1. An expression cassette encoding a double stranded RNA comprising a first RNA sequence and a second RNA sequence, wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementary to a target RNA sequence comprised in an RNA encoded by a human ATXN3 gene.
2. The expression cassette according to claim 1, wherein the target RNA sequence is comprised in the region 5′ to the RNA sequence encoded by the sequence corresponding with nucleotides 942-1060 of SEQ ID NO. 2 of the human ATXN3 gene.
3. The expression cassette according to claim 2, wherein the target RNA sequence is comprised in the RNA sequence encoded by the region 390-456 of SEQ ID NO.2 and sequences 3′ therefrom.
4. The expression cassette according to claim 2, wherein the target RNA sequence is selected from the group consisting of consisting of SEQ ID NOs. 9-13.
5. The expression cassette according to claim 1, wherein the first and second RNA sequences are comprised in a pre-miRNA scaffold, a pri-miRNA scaffold or a shRNA.
6. The expression cassette according to claim 5, wherein the pre-miRNA scaffold or pri-miRNA scaffold is from miR451.
7. The expression cassette according to claim 3, wherein the first RNA sequence is selected from the group consisting of SEQ ID NOs. 14-17.
8. The expression cassette according to claim 7, wherein the first RNA sequence and second RNA sequence are selected from the group consisting of the combinations of SEQ ID NOs. 14 and 18; SEQ ID NOs. 15 and 19; SEQ ID NOs. 16 and 20; SEQ ID NOs. 17 and 21.
9. The expression cassette according to claim 8, wherein the encoded RNA comprises an RNA sequence selected from the group consisting of SEQ ID NOs. 22-29.
10. The expression cassette according to claim 1, wherein the expression cassette comprises a PGK promoter, a CMV promoter, a neuron-specific promoter, a astrocyte-specific promoter or a CBA promoter operably linked to the nucleic acid sequence encoding the first RNA sequence and the second RNA sequence.
11. A gene therapy vector comprising the expression cassette according to claim 1.
12. The gene therapy vector according to claim 11, wherein the vector is an AAV vector.
13. A method of treatment, comprising administering to a subject in need thereof a gene therapy vector according to claim 11.
14. The method according to claim 13 for the medical treatment of SCA3/MJD.
15. The method according to claim 13, wherein the administration results in total knockdown of ATXN3 gene expression.
16. The method according to claim 14, wherein the knockdown of ATXN3 gene expression is in the brain stem and/or the cerebellum.
Description
FIGURES
[0136] FIG. 1. A schematic of part of the ATXN3 cDNA sequence comprising the CAG repeat region (comprised in exon 10), and with selected target RNA sequences indicated (SEQ ID NOs. 3-13). The sequence listed is part of NCBI Reference Sequence: NM_004993.5, the sequence depicted is referred to as SEQ ID NO.2 herein, and corresponds to nucleotides 1-1329 thereof, and represents DNA sequence (cDNA) of (part of) a spliced ATXN3 transcript. Hence, the corresponding RNA, has the same sequence except having instead of a T a U as depicted in FIG. 1 and SEQ ID NO.2, Nucleotides 1-93 represent exon 1, nucleotides 94-258 represent exon 2, nucleotides 259-303 represent exon 3, and nucleotides 304-389 represent exon 4. Nucleotides 390-941 encompasses exons 5, 6, 7, 8 and 9. Exons 5, 6, 7, 8, and 9 are represented respectively by 390-456, 457-544, 545-677, 678-844 and 845-941. The exon 10 sequence corresponds with 942-1060 of SEQ ID NO.2 and comprises a repeat region of 14 codons comprising 12 CAGs. The selected target RNA sequences (as listed in table 1) i.e. the DNA sequence corresponding thereto, are depicted in FIG. 1 as well. SEQ ID NO.3 corresponds with nucleotides 46-67, in exon 1; SEQ ID NO.4 corresponds with nucleotides 63-84, in exon 1; SEQ ID NO.5 corresponds with nucleotides 254-275, in exon 2-3; SEQ ID NO.6 corresponds with nucleotides 263-284, in exon 3; SEQ ID NO.7 corresponds with nucleotides 323-244, in exon 4; SEQ ID NO.8 corresponds with nucleotides 338-359, in exon 4; SEQ ID NO.9, corresponds with nucleotides 422-443, in exon 5; SEQ ID NO.10 corresponds with nucleotides 443-464, in exon 5-6; SEQ ID NO.11, corresponds with nucleotides 834-855, in exon 8-9; SEQ ID NO.12 corresponds with nucleotides 897-918, in exon 9; SEQ ID NO.13 corresponds with nucleotides 918-939, in exon 9.
[0137] FIG. 2A Schematic of miR451 scaffold RNA structure indicating the first RNA sequence as it is designed. FIG. 2B. Schematic of expression cassette of a miRNA scaffold. FIG. 2c, schematic showing Renilla/Firefly construct, with Renilla construct comprising an inserted target sequence (black box).
[0138] FIG. 3. Graph showing silencing of ATXN3 reporters by targeting SEQ ID NOs. 3-13. HEK293T cells were co-transfected in a 1:0.1 to 100 ratio with the luciferase reporter constructs and the different scaffolds targeting SEQ ID NOs. 3-1. Renilla and firefly were measured 2 days post-transfection and renilla was normalized to firefly expression. Scrambled miRNA (CTRL) served as a negative control and was set at 100% (y-axis). Targeting SEQ ID NOs 9-13 resulted in most strong knockdown, achieving about 75% or more knockdown at the highest level, with SEQ ID NO.11 targeting showing the most prominent knockdown (>90%).
[0139] FIG. 4A. Western Blot and FIG. 4B quantification thereof showing lowering of endogenous ataxin-3 protein in 293T cells.
[0140] FIG. 5a. Graph showing dose response of AAV-miRNA transduction in iPSC-derived neurons. Increased dosages ranging from 10exp 10, 10exp 11 and 10exp 12 were used to transduce neurons. Mature miRNA was determined in the neurons and a dose response curve was shown, with the higher dose showing the highest level of expression. FIG. 5b. This dose response curve resulted in a dose response curve having the reverse image when determining ATXN3 mRNA levels, the higher the dose of AAV, the lower the amount of ATXN3 mRNA levels detected. The lowest amount of ATXN3 mRNA was detected when targeting SEQ ID NO.11.
[0141] FIG. 6a. In vivo administration of AAV targeting SEQ ID NO. 9, 11 and 13. AAV was injected in the mouse. The amount of gc detected per genomic DNA was determined for each administration and each area. The gc detected per area were similar per injection site, with variation between injection sites. FIG. 6b. The knockdown of ATXN3 mRNA was determined in the medulla. All three target regions showed similar reduction in mRNA.
[0142] FIG. 7. DNA sequence of an expression construct (SEQ ID NO. 49) encoding a miR451 scaffold comprising a first RNA sequence of 22 nucleotides targeting SEQ ID NO.11. The expression cassette comprises a CAG promotor shown in bold (position 43-1712), the sequence encoding the first RNA sequence shown in bold and underlined (position 2031-2052, encoding SEQ ID NO. 16), the sequence encoding the second RNA sequence is shown underlined (position 2053-2070, encoding SEQ ID NO. 20), the hGH poly A signal shown in bold and italics (2318-2414). The pri-miRNA sequence comprises a pre-miRNA sequence. The pri-miRNA encoding sequence is shown between [ brackets ] (position 2015-2086, encoding SEQ ID NO. 28). The pre-miRNA sequence comprises the first RNA sequence and the second RNA sequence and the sequence encoding it is shown underlined, either normal or bold, (position 2031-2070) (encoding SEQ ID NO. 24). The pre-miRNA or pri-miRNA encoding sequence may be replaced e.g. by a sequence encoding a pre-miRNA or pri-miRNA as listed in tables 4 and 5, respectively and as depicted in FIG. 8. The first RNA sequence of the pre-miRNA or pri-miRNA can be any sequence of 22 nucleotides selected to bind and target a sequence in the ATXN3 gene, preferably a target nucleotide sequence 5′ from the CAG repeat region of the ATXN3 gene and such as listed e.g. in table 1. The second RNA sequence is selected and adapted to be complementary to the first RNA sequence. The secondary structure is checked on mfold by folding the RNA sequence using standard settings utilizing the RNA folding form, with folding temperature fixed at 37 degrees Celcius (as available online <URL:http://unafold.rna.albany.edu/?q=mfold>; Zuker et al., Nucleic Acids Res. 31 (13), 3406-15, (2003)) for folding, and adapted if necessary, into a miR-451 pri-miRNA structure as depicted in FIGS. 2a and 8.
[0143] FIG. 8. Predicted RNA structures of selected pre-miRNA (FIGS. 8A-8D) and pri-miRNA (E-H) sequences in an miR451 scaffold. FIG. 8A and FIG. 8E, FIG. 8B and FIG. 8F, FIG. 8C and FIG. 8G, FIG. 8D and FIG. 8H are predicted pre-miRNA and pri-miRNA structures targeting the respective target sequences SEQ ID NO. 9, 10, 11 and 13. Sequences of the secondary RNA sequences depicted are listed in Tables 4 and 5. Structures were made using M-fold using standard settings, utilizing the RNA folding form, (as available online <URL:http://unafold.rna.albany.edu/?q=mfold>; Zuker et al., Nucleic Acids Res. 31 (13), 3406-15, (2003). Standard settings used for m-fold version 3.5 were as follows: RNA sequence is linear, folding temperature is fixed at 37° , ionic conditions: 1M NaCl, no divalent ions, percent suboptimality number is 5, interior/bulge loop size is 30, maximum asymmetry of an interior/bulge loop is 30, and no maximum distance between paired bases.
[0144] FIG. 9. Vector copy distribution of AAV5 in F512 SCA3 mice.
[0145] FIG. 9A) Schematic representation of the routes of administration. Three months old mice (N-3) were injected ICV, or in the cisterna magna, or DCN with AAV5-miATXN3_9, AAV5-miATXN3_11 or AAV5-miATXN3_13. 10 μl of AAV5 were injected either ICV or in the cisterna magna and 2μl were injected bilaterally in the DCN. The injection sites are depicted in dark grey, indicated with arrow. All mice were sacrificed 6 weeks after surgeries. FIGS. 9B-9D) Vector copy distribution in cortex, cerebellum and brain stem. DNA was isolated from the cortex, cerebellum and brain stem tissues and qPCR was performed to determine the vector copy distribution. The genomic copies per μg DNA was calculated for each brain region using a standard curve.
[0146] FIG. 10. Silencing of mutant ataxin-3 in F512 mice.
[0147] FIG. 10A) Expression of mature miATXN3 guide strands in the cerebellum after DCN administration. Total RNA was isolated from the cerebellum for small RNA TaqMan. MicroRNA input levels were normalized to U6 small nuclear RNA and set relative to AAV-GFP treated mice. FIG. 10B) Lowering of total ATXN3 mRNA in cerebellum of DCN injected mice. Total RNA was isolated from cerebellum and RT-qPCR was performed to detect the mouse wildtype ATXN3 mRNA. RNA input levels were normalized to GAPDH and set relative to AAV-GFP treated mice. FIG. 10C) Expression of mature miATXN3 guide strands in the brain stem after cisterna magna administration. Performed as described for FIG. 10A. FIG. 10D) Lowering of total ATXN3 mRNA in cerebellum of cisterna magna injected mice. Performed as described for FIG. 10B. FIG. 10E) Expression of mature miATXN3 guide strands in the brain stem after cisterna magna administration. Performed as described for FIG. 10A. FIG. 10F) Lowering of total ATXN3 mRNA in brain stem of cisterna magna injected mice. Performed as described for FIG. 10B. FIG. 10G) Reduction of mutant ataxin-3 protein in the brain stem after cisterna magna delivery. TR-FRET immunoassay was performed on tissue lysates to specifically detect the mutant ataxin-3 (no detection of wildtype mouse ataxin-3). The protein expression is shown in percentage relative to the control (untreated) mice. Strong lowering of mutant ataxin-3 protein in the brainstem up to 64.5% was observed. FIG. 10H) Reduction of mutant ataxin-3 protein in the cerebellum after cisterna magna delivery. A robust ataxin-3 protein lowering of 53.1% in the cerebellum was observed.
[0148] FIGS. 11A-11D. miATXN3 mediated ataxin-3 knockdown in SCA3 mouse brain. Mice were stereotaxically injected at 2 months of age with a mixture of a mutant ataxin-3 lentiviral expression cassette and AAV5-miATXN3_11 in both striata. The lentiviral construct results in expression of mutant ataxin-3 throughout the striatum during the study period.
[0149] FIG. 11A) Bodyweight of mice was not negatively affected by any of the tested doses of miATXN3.
[0150] FIG. 11B) qPCR analysis revealed a strong dose dependent knockdown of mutant ATXN3 expression in the striatum 7 weeks after AAV5-miATXN3 treatment. FIG. 11C) Soluble ataxin-3 protein levels were reduced up to 90% in the striatum after high dose of miATXN3 as quantified through western blot analysis. FIG. 11D) The insoluble and aggregated ataxin-3 protein fraction in striatum was almost completely abolished by mid and high dose treatment of miATXN3. LD=low dose (2×10.sup.9 gc), MD=mid dose (1×10.sup.1° gc) HD=high dose (2×10.sup.10 gc) PBS n=8, AAV5 HD n=8; AAV5 MD n=8; AAV5 LD n=8. one-way ANOVA (*p<0.05, **p<0.01 ***p<0.001 and ****p<0.0001).
[0151] FIGS. 12A-12D: Reduction in ataxin-3 inclusions and darpp32 lesion size in SCA3 mice. Striatum from right hemisphere of miATXN3 treated SCA3 mice were stained for ataxin-3 and darpp-32 (dopamine- and cAMP-regulated neuronal phosphoprotein).
[0152] FIG. 12A) Ataxin-3 stained (1H9) striatum of mice sacrificed 7 weeks after miATXN3 treatment shows presence of nuclear inclusions in PBS treated SCA3 mice. FIG. 12B) Right hemisphere of mice was stained with the midbrain dopaminergic neuron marker darpp-32. A darpp-32 depleted lesion representing the early neuronal dysfunction can be seen in the PBS treated animals close to the injection site (top left of striatum). FIG. 12C) Quantification of nuclear ataxin-3 inclusions in striatum. Low dose miATXN3 treatment significantly reduced the number of ataxin-3 inclusions by about 50%. Presence of nuclear ataxin-3 inclusions was almost completely abolished in mid and high dose miATXN3 treated animals. FIG. 12D) Quantification of darpp-32 depleted volume. Total lesion size was calculated for the whole striatum based on interspaced sections. Lesion size was significantly reduced in a dose dependent matter following miATXN3 treatment compared to PBS treated animals, indicating a reduction in neuronal dysfunction. PBS n=8, AAV5 HD n=8; AAV5 MD n=7;
[0153] AAV5 LD n=8. one-way ANOVA (*p<0.05, **p<0.01 ***p<0.001 and ****p<0.0001)
[0154] FIGS. 13A-13B: effect of miATXN3 treatment on endogenous mouse ataxin-3 protein levels. Based on qPCR and western blot data from FIG. 11. Quantification of murine ataxin-3 RNA (FIG. 13A) and protein (FIG. 13B) shows only minor downregulation of endogenous ataxin-3 at the high dose of AAV5-miATXN3 (2x10.sup.9) in the mouse striatum. Endogenous mouse ataxin-3 carries a one nucleotide mismatch in the target sequence. ** p<0.01, one-way ANOVA.
[0155] FIG. 14: effect of intrathecal administration of miATXN3 on endogenous non-humane primate ataxin-3 protein levels. Quantification of macaca fascicularis ataxin-3 protein shows downregulation of endogenous ataxin-3 in the non-human primate brain. Time-resolved fluorescence energy transfer (TR-FRET) was used to quantify ataxin-3 protein and expression levels calculated relative to the mean of control microRNA (miCRTL)-treated samples. Brain punches analysed were; p26 motor cortex; p32 putamen; p69, p′70, p71 pons; p72 occipital cortex, p78 deep cerebellar nucleus; p89 and p91 cerebellar cortex. N=3 per treatment.
TABLE-US-00009 SEQ ID NO 2 GAGAGGGGCAGGGGGCGGAGCTGGAGGGGGTGGTTCGGCGTGGGGGCCG TTGGCTCCAGACAAATAAACATGGAGTCCATCTTCCACGAGAAACAAGA AGGCTCACTTTGTGCTCAACATTGCCTGAATAACTTATTGCAAGGAGAA TATTTTAGCCCTGTGGAATTATCCTCAATTGCACATCAGCTGGATGAGG AGGAGAGGATGAGAATGGCAGAAGGAGGAGTTACTAGTGAAGATTATCG CACGTTTTTACAGCAGCCTTCTGGAAATATGGATGACAGTGGTTTTTTC TCTATTCAGGTTATAAGCAATGCCTTGAAAGTTTGGGGTTTAGAACTAA TCCTGTTCAACAGTCCAGAGTATCAGAGGCTCAGGATCGATCCTATAAA TGAAAGATCATTTATATGCAATTATAAGGAACACTGGTTTACAGTTAGA AAATTAGGAAAACAGTGGTTTAACTTGAATTCTCTCTTGACGGGTCCAG AATTAATATCAGATACATATCTTGCACTTTTCTTGGCTCAATTACAACA GGAAGGTTATTCTATATTTGTCGTTAAGGGTGATCTGCCAGATTGCGAA GCTGACCAACTCCTGCAGATGATTAGGGTCCAACAGATGCATCGACCAA AACTTATTGGAGAAGAATTAGCACAACTAAAAGAGCAAAGAGTCCATAA AACAGACCTGGAACGAGTGTTAGAAGCAAATGATGGCTCAGGAATGTTA GACGAAGATGAGGAGGATTTGCAGAGGGCTCTGGCACTAAGTCGCCAAG AAATTGACATGGAAGATGAGGAAGCAGATCTCCGCAGGGCTATTCAGCT AAGTATGCAAGGTAGTTCCAGAAACATATCTCAAGATATGACACAGACA TCAGGTACAAATCTTACTTCAGAAGAGCTTCGGAAGAGACGAGAAGCCT ACTTTGAAAAACAGCAGCAAAAGCAGCAACAGCAGCAGCAGCAGCAGCA GCAGGGGGACCTATCAGGACAGAGTTCACATCCATGTGAAAGGCCAGCC ACCAGTTCAGGAGCACTTGGGAGTGATCTAGGTGATGCTATGAGTGAAG AAGACATGCTTCAGGCAGCTGTGACCATGTCTTTAGAAACTGTCAGAAA TGATTTGAAAACAGAAGGAAAAAAATAATACCTTTAAAAAATAATTTAG ATATTCATACTTTCCAACATTATCCTGTGTGATTACAGCATAGGGTCCA CTTTGGTAATGTGTCAAAGAGATGAGGAAATAAGACTTTTAGCGGTTTG CAAACAAAATGATGGGAAAGTGGAACAATGCGTCGGTTGTAGGACTAAA TAATGATCTTCCAAATATTAGCCAAAGAGGCATTCAGCAATTAAAGACA TTTAAAATAGTTTTCTAAATGTTTCTTTTTCTTTTTTGAGTGTGCAATA TGTAACATGTCTAAAGTTAGGGCATTTTTCTTGGATCTTTTTGCAGACT AGCTAATTAGCTCTCGCCTCAGGCTTTTTCCATATAGTTTGTTTTCTTT TTCTGTCTTGTAGGTAAGTTGGCTCACATCATGTAATAGTGGCTTTCAT TTCTTATTAACCAAATTAACCTTTCAGGAAAGTATCTCTACTTTCCTGA TGTTGATAATAGTAATGGTTCTAGAAGGATGAACAGTTCTCCCTTCAAC TGTATACCGTGTGCTCCAGTGTTTTCTTGTGTTGTTTTCTCTGATCACA ACTTTTCTGCTACCTGGTTTTCATTATTTTCCCACAATTCTTTTGAAAG ATGGTAATCTTTTCTGAGGTTTAGCGTTTTAAGCCCTACGATGGGATCA TTATTTCATGACTGGTGCGTTCCTAAACTCTGAAATCAGCCTTGCACAA GTACTTGAGAATAAATGAGCATTTTTTAAAATGTGTGAGCATGTGCTTT CCCAGATGCTTTATGAATGTCTTTTCACTTATATCAAAACCTTACAGCT TTGTTGCAACCCCTTCTTCCTGCGCCTTATTTTTTCCTTTCTTCTCCAA TTGAGAAAACTAGGAGAAGCATAGTATGCAGGCAAGTCTCCTTCTGTTA GAAGACTAAACATACGTACCCACCATGAATGTATGATACATGAAATTTG GCCTTCAATTTTAATAGCAGTTTTATTTTATTTTTTCTCCTATGACTGG AGCTTTGTGTTCTCTTTACAGTTGAGTCATGGAATGTAGGTGTCTGCTT CACATCTTTTAGTAGGTATAGCTTGTCAAAGATGGTGATCTGGAACATG AAAATAATTTACTAATGAAAATATGTTTAAATTTATACTGTGATTTGAC ACTTGCATCATGTTTAGATAGCTTAAGAACAATGGAAGTCACAGTACTT AGTGGATCTATAAATAAGAAAGTCCATAGTTTTGATAAATATTCTCTTT AATTGAGATGTACAGAGAGTTTCTTGCTGGGTCAATAGGATAGTATCAT TTTGGTGAAAACCATGTCTCTGAAATTGATGTTTTAGTTTCAGTGTTCC CTATCCCTCATTCTCCATCTCCTTTTGAAGCTCTTTTGAATGTTGAATT GTTCATAAGCTAAAATCCAAGAAATTTCAGCTGACAACTTCGAAAATTA TAATATGGTATATTGCCCTCCTGGTGTGTGGCTGCACACATTTTATCAG GGAAAGTTTTTTGATCTAGGATTTATTGCTAACTAACTGAAAAGAGAAG AAAAAATATCTTTTATTTATGATTATAAAATAGCTTTTTCTTCGATATA ACAGATTTTTTAAGTCATTATTTTGTGCCAATCAGTTTTCTGAAGTTTC CCTTACACAAAAGGATAGCTTTATTTTAAAATCTAAAGTTTCTTTTAAT AGTTAAAAATGTTTCAGAAGAATTATAAAACTTTAAAACTGCAAGGGAT GTTGGAGTTTAGTACTACTCCCTCAAGATTTAAAAAGCTAAATATTTTA AGACTGAACATTTATGTTAATTATTACCAGTGTGTTTGTCATATTTTCC ATGGATATTTGTTCATTACCTTTTTCCATTGAAAAGTTACATTAAACTT TTCATACACTTGAATTGATGAGCTACCTAATATAAAAATGAGAAAACCA ATATGCATTTTAAAGTTTTAACTTTAGAGTTTATAAAGTTCATATATAC CCTAGTTAAAGCACTTAAGAAAATATGGCATGTTTGACTTTTAGTTCCT AGAGAGTTTTTGTTTTTGTTTTTGTTTTTTTTTGAGACGGAGTCTTGCT ATGTCTCCCAGGCTGGAGGGCAGTGGCATGATCTCGGCTCACTACAACT TCCACCTCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCAGAGTAGC TGGGATTACAGGCGCCCACCACCACACCCGGCAGATTTTTGTATTTTTG GTAGAGACGCGGTTTCATCATGTTTGGCCAGGCTGGTCTCGAACTCCTG ACCTCAGGTGATCCGCCTGCCTTGGCCTCCCAAAGTGTTGGGATTACAG GCATGAGCCACTGCGCCTGGCCAGCTAGAGAGTTTTTAAAGCAGAGCTG AGCACACACTGGATGCGTTTGAATGTGTTTGTGTAGTTTGTTGTGAAAT TGTTACATTTAGCAGGCAGATCCAGAAGCACTAGTGAACTGTCATCTTG GTGGGGTTGGCTTAAATTTAATTGACTGTTTAGATTCCATTTCTTAATT GATTGGCCAGTATGAAAAGATGCCAGTGCAAGTAACCATAGTATCAAAA AAGTTAAAAATTATTCAAAGCTATAGTTTATACATCAGGTACTGCCATT TACTGTAAACCACCTGCAAGAAAGTCAGGAACAACTAAATTCACAAGAA CTGTCCTGCTAAGAAGTGTATTAAAGATTTCCATTTTGTTTTACTAATT GGGAACATCTTAATGTTTAATATTTAAACTATTGGTATCATTTTTCTAA TGTATAATTTGTATTACTGGGATCAAGTATGTACAGTGGTGATGCTAGT AGAAGTTTAAGCCTTGGAAATACCACTTTCATATTTTCAGATGTCATGG ATTTAATGAGTAATTTATGTTTTTAAAATTCAGAATAGTTAATCTCTGA TCTAAAACCATCAATCTATGTTTTTTACGGTAATCATGTAAATATTTCA GTAATATAAACTGTTTGAAAAGGCTGCTGCAGGTAAACTCTATACTAGG ATCTTGGCCAAATAATTTACAATTCACAGAATATTTTATTTAAGGTGGT GCTTTTTTTTTTTGTCCTTAAAACTTGATTTTTCTTAACTTTATTCATG ATGCCAAAGTAAATGAGGAAAAAAACTCAAAACCAGTTGAGTATCATTG CAGACAAAACTACCAGTAGTCCATATTGTTTAATATTAAGTTGAATAAA ATAAATTTTATTTCAGTCAGAGCCTAAATCACATTTTGATTGTCTGAAT TTTTGATACTATTTTTAAAATCATGCTAGTGGCGGCTGGGCGTGGTAGC TCACGCCTGTAATCCCAGCATTTTGGGAGGCCGAAGTGGGTGGATCACG AGGTCGGGAGTTCGAGACCAGCTTGGCCAAAATGGTGAAACCCCATCTG TACTAAAAACTACAAAAATTAGCTGGGCGCGGTGGCAGGTGCCTGTAAT CCCAGCTACCTGGGAGTCTGAGGCAGGAGAATTGCTTGAACCCTGGCGA CAGAGGATGCAGTGAGCCAAGATGGTGCCACTGTACTCCAGACTGGGCG ACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAAATCATGCTAGTGCC AAGAGCTACTAAATTCTTAAAACCGGCCCATTGGACCTGTACAGATAAA AAATAGATTCAGTGCATAATCAAAATATGATAATTTTAAAATCTTAAGT AGAAAAATAAATCTTGATGTTTTAAATTCTTACGAGGATTCAATAGTTA ATATTGATGATCTCCCGGCTGGGTGCAGTGGCTCACGCCTGTAATCCCA GCAGTTCTGGAGGCTGAGGTGGGCGAATCACTTCAGGCCAGGAGTTCAA GACCAGTCTGGGCAACATGGTGAAACCTCGTTTCTACTAAAAATACAAA AATTAGCCGGGCGTGGTTGCACACACTTGTAATCCCAGCTACTCAGGAG GCTAAGAATCGCATGAGCCTAGGAGGCAGAGGTTGCAGAGTGCCAAGGG CTCACCACTGCATTCCAGCCTGCCCAACAGAGTGAGACACTGTTTCTGA AAAAAAAAAATATATATATATATATATATATGTGTGTATATATATATGT ATATATATATGACTTCCTATTAAAAACTTTATCCCAGTCGGGGGCAGTG GCTCACGCCTGTAATCCCAACACTTTGGGAGGCTGAGGCAGGTGGATCA CCTGAAGTCCGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCCA TCTCTACTAAAAATACAAAACTTAAGCCAGGTATGGTGGCGGGCACCTG TAATCCCAGTTACTTGGGAGGCTGAGGCAGGAGAATCGTTTAAACCCAG GAGGTGGAGGTTGCAGTGAGCTGAGATCGTGCCATTGCACTCTAGCCTG GGCAACAAGAGTAAAACTCCATCTTAAAGGTTTGTTTGTTTTTTTTTAA TCCGGAAACGAAGAGGCGTTGGGCCGCTATTTTCTTTTTCTTTCTTTCT TTCTTTCTTTTTTTTTTTTTCTGAGACGGAGTCTAGCTCTGCTGCCCAG GCTGGAGTACAATGACACGATGTTGGCTCACTGCAACCTCCACCTCCTG GGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTACCTGGGATTACAG GCACCTGCCACTACACCTGGCGAATATTTGTTTTTTTTAGTAGAGACGG GCTTTTACCATGTTAGGCTGGTCTCAAACTCCTGACCTCAGGTGATCTG CCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGTGCAGGCCACCACA CCCGGCCTTGGGCCACTGTTTTCAAAGTGAATTGTTTGTTGTATCGAGT CCTTAAGTATGGATATATATGTGACCCTAATTAAGAACTACCAGATTGG ATCAACTAATCATGTCAGCAATGTAAATAACTTTATTTTTCATATTCAA AATAAAAACTTTCTTTTATTTCTGGCCCCTTTATAACCAGCATCTTTTT GCTTTAAAAAATGACCTGGCTTTGTATTTTTTTAGTCTTAAACATAATA AAAATATTTTTGTTCTAATTTGCTTTCATGAGTGAAGATTATTGACATC GTTGGTAAATTCTAGAATTTTGATTTTGTTTTTTAATTTGAAGAAAATC TTTGCTATTATTATTTTTTCCAAGTGGTCTGGCATTTTAAGAATTAGTG CTAATAACGTAACTTCTAAATTTGTCGTAATTGGCATGTTTAATAGCAT ATCAAAAAACATTTTAAGCCTGTGGATTCATAGACAAAGCAATGAGAAA CATTAGTAAAATATAAATGGATATTCCTGATGCATTTAGGAAGCTCTCA ATTGTCTCTTGCATAGTTCAAGGAATGTTTTCTGAATTTTTTTAATGCT TTTTTTTTTTTTGAAAGAGGAAAACATACATTTTTAAATGTGATTATCT AATTTTTACAACACTGGGCTATTAGGAATAACTTTTTAAAAATTACTGT TCTGTATAAATATTTGAAATTCAAGTACAGAAAATATCTGAAACAAAAA GCATTGTTGTTTGGCCATGATACAAGTGCACTGTGGCAGTGCCGCTTGC TCAGGACCCAGCCCTGCAGCCCTTCTGTGTGTGCTCCCTCGTTAAGTTC ATTTGCTGTTATTACACACACAGGCCTTCCTGTCTGGTCGTTAGAAAAG CCGGGCTTCCAAAGCACTGTTGAACACAGGATTCTGTTGTTAGTGTGGA TGTTCAATGAGTTGTATTTTAAATATCAAAGATTATTAAATAAAGATAA TGTTTGCTTTTCTA
