CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Application Ser. No. 60/140,345, the disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION The present invention relates generally to a method for the prophylaxis and/or treatment of medical disorders, and in particular proliferative and/or inflammatory skin disorders, and to genetic molecules useful for same. The present invention is particularly directed to genetic molecules capable of modulating growth factor interaction with its receptor on cells such as epidermal keratinocytes to inhibit, reduce or otherwise decrease stimulation of this layer of cells. The present invention contemplates, in a particularly preferred embodiment, a method for the prophylaxis and/or treatment of psoriasis or neovascularization conditions such as neovascularization of the retina. The present invention is further directed to the subject genetic molecules in adjunctive therapy for epidermal hyperplasia, such as in combination with UV treatment, and to facilitate apoptosis of cancer cells and in particular cancer cells comprising keratinocytes.
BACKGROUND OF THE INVENTION Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia or any other country.
Psoriasis and other similar conditions are common and often distressing proliferative and/or inflammatory skin disorders affecting or having the potential to affect a significant proportion of the population. The condition arises from over proliferation of basal keratinocytes in the epidermal layer of the skin associated with inflammation in the underlying dermis. Whilst a range of treatments have been developed, none is completely effective and free of adverse side effects. Although the underlying cause of psoriasis remains elusive, there is some consensus of opinion that the condition arises at least in part from over expression of local growth factors and their interaction with their receptors supporting keratinocyte proliferation via keratinocyte receptors which appear to be more abundant during psoriasis.
One important group of growth factors are the dermally-derived insulin-like growth factors (IGFs) which support keratinocyte proliferation. In particular, IGF-I and IGF-II are ubiquitous peptides each with potent mitogenic effects on a broad range of cells. Molecules of the IGF type are also known as “progression factors” promoting “competent” cells through DNA synthesis. The IGFs act through a common receptor known as the Type I or IGF-I receptor, which is tyrosine kinase linked. They are synthesised in mesenchymal tissues, including the dermis, and act on adjacent cells of mesodermal, endodermal or ectodermal origin. The regulation of their synthesis involves growth hormone (GH) in the liver, but is poorly defined in most tissues [1].
Particular proteins, referred to as IGF binding proteins (IGFBPs), appear to be involved in autocrine/paracrine regulation of tissue IGF availability [2]. Six IGFBPs have so far been identified. The exact effects of the IGFBPs is not clear and observed effects in vitro have been inhibitory or stimulatory depending on the experimental method employed [3]. There is some evidence, however, that certain IGFBPs are involved in targeting IGF-I to its cell surface receptor.
Skin, comprising epidermis and underlying dermis, has GH receptors on dermal fibroblasts [4]. Fibroblasts synthesize IGF-I as well as IGFBPs-3, -4, -5 and -6 [5] which may be involved in targeting IGF-I to adjacent cells as well as to the overlaying epidermis. The major epidermal cell type, the keratinocyte, does not synthesize IGF-I, but possesses IGF-I receptors and is responsive to IGF-I [6].
It is apparent, therefore, that IGF-I and other growth promoting molecules, are responsible for or at least participate in a range of skin cell activities. In accordance with the present invention, the inventors have established that aberrations in the normal functioning of these molecules or aberrations in their interaction with their receptors is an important factor in a variety of medical disorders such as proliferative and/or inflammatory skin disorders. It is proposed, therefore, to target these molecules or other molecules which facilitate their functioning or interaction with their receptors to thereby ameliorate the effects of aberrant activity during or leading to skin disease conditions and other medical conditions such as those involving neovascularization. Furthermore, these molecules may also be used to facilitate apoptosis of target cells and may be useful as adjunctive therapy for epidermal hyperplasia.
SUMMARY OF THE INVENTION Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. (<400>1), (<400>2), etc. A sequence listing is provided after the claims.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Accordingly, one aspect of the present invention contemplates a method for ameliorating the effects of a medical disorder such as a proliferative and/or inflammatory skin disorder in a mammal, said method comprising contacting the proliferating and/or inflamed skin or skin capable of proliferation and/or inflammation or a cell otherwise involved in the said medical disorder with an effective amount of a nucleic acid molecule or chemical analogue thereof capable of inhibiting or otherwise reducing a growth factor mediated cell proliferation and/or inflammation and/or other medical disorder.
According to this preferred embodiment, there is provided a method for ameliorating the effects of a medical disorder such as a proliferative and/or inflammatory skin disorder in a mammal, said method comprising contacting the proliferating and/or inflamed skin or skin capable of proliferation and/or inflammation or a cell otherwise involved with said medical disorder with an effective amount of a nucleic acid molecule or chemical analogue thereof capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation and/or inflammation and/or other medical disorder.
According to this embodiment, there is provided a method for ameliorating the effects of a proliferative and/or inflammatory skin disorder such as psoriasis said method comprising contacting the proliferating and/or inflamed skin or skin capable of proliferation and/or inflammation with effective amounts of UV treatment and a nucleic acid molecule or chemical analogue thereof capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation and/or inflammation.
According to this embodiment, there is provided in a particularly preferred aspect a ribozyme comprising a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least part of a target mRNA sequence transcribed from a genomic gene corresponding to <400>1 or <400>2 wherein said catalytic domain is capable of cleaving said target mRNA sequence to reduce or inhibit IGF-I mediated cell proliferation and/or inflammation and/or other medical disorders.
Yet another aspect of the present invention contemplates co-suppression to reduce expression or to inhibit translation of an endogenous gene encoding, for example, IGF-I, its receptor, or IGFBPs such as IGFBP-2 and/or -3. In co-suppression, a second copy of an endogenous gene or a substantially similar copy or analogue of an endogenous gene is introduced into a cell following topical administration. As with antisense molecules, nucleic acid molecules defining a ribozyme or nucleic acid molecules useful in co-suppression may first be protected such as by using a nonionic backbone.
Another aspect of the present invention contemplates a pharmaceutical composition for topical administration which comprises a nucleic acid molecule capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation such as psoriasis and one or more pharmaceutically acceptable carriers and/or diluents.
Yet another aspect of the present invention contemplates the use of a nucleic acid molecule in the manufacture of a medicament for the treatment of proliferative and/or inflammatory skin disorders or other medical disorders mediated by a growth factor.
Still a further aspect of the present invention contemplates an agent comprising a nucleic acid molecule as hereinbefore defined useful in the treatment of proliferative and/or inflammatory skin disorders, such as psoriasis or other medical disorder.
The present invention further contemplates the use of the genetic molecules and in particular the antisense molecules to inhibit the anti-apoptotic activity of IGF-I receptor.
BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a representation of the nucleotide sequence of IGFBP-2. LOCUS HSIGFBP2 1433 bp RNA PRI 31-JAN-1990 5 DEFINITION Human mRNA for insulin-like growth factor binding protein (IGFBP-2) ACCESSION X16302 KEYWORDS insulin-like growth factor binding protein. SOURCE human ORGANISM Homo sapiens Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia;
Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae. REFERENCE 1 (bases 1 to 1433) AUTHORS Binkert,C., Landwehr,J., Mary,J.L., Schwander,J. and Heinrich,G. TITLE Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2) JOURNAL EMBO J. 8, 2497-2502 (1989) STANDARD full automatic COMMENT NCBI gi: 33009
FEATURES Location/Qualifiers
source 1. .1433
/organism=“Homo sapiens”
/dev_stage=“fetal”
/tissue_type=“liver”
misc_feature 1416. .1420
/note=“pot. polyadenylation signal”
polyA site 1433
/note=“polyadenylation site”
CDS 118. .1104
/note=“precursor polypeptide;
(AA −39 to 289);
NCBI gi:33010.”
/codon start=1
/translation=“MLPRVGCPALPLPPPPLLPLLPL
LLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPP
VAPPAAVAAVAGGARNPCAELVREPGCGCCSVCARLE
GEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTC
EKRRDAEYGASPEQVADNGDDHSEGGLVENHVDSTMN
TMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQM
GKGGKNHLGLEEPKKLRPPPARTPCQQELDQVLERIS
TMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSL
NGORGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQ
QEACGVHTQRMQ”
(<400>21)
CDS 118. .234
/note=“signal peptide;
(AA −39 to −1); NCBI gi: 33011.”
/codon_start=1
/translation=“MLPRVGCPALPLPPPPLLPLLPL
LLLLLGASGGGGGARA”
(<400>22)
CDS 235. .1101
/note=“mature IGFBP-2;
(AA 1 to 289); NCBI gi: 33012.”
/codon_start=1
/translation=“EVLFRCPPCTPERLAACGPPPVA
PPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGE
ACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEK
RRDAEYGASPEQVADNGDDHSEGGLVENHVDSTMNML
GGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGI
GKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMR
LPDERGPLEHLYSLHIPNCDKHGLYNLKQCKNSLNGQ
RGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEA
CGVHTQRMQ” (<400>23)
BASE COUNT 239 a 466 c 501 g 227 t
ORIGIN
HSIGFBP2 Length: 1433 May 11, 1994 10:06 Type:
N Check: 6232 . .
FIG. 2 is a representation of the nucleotide sequence of IGFBP-3. LOCUS HUMGFIBPA 2474 bp ss-mRNA PRI 15-JUN-1990 DEFINITION Human growth hormone-dependent insulin-like growth factor-binding protein mRNA, complete cds. ACCESSION M31159 KEYWORDS insulin-like growth factor binding protein. SOURCE Human plasma, cDNA to mRNA, clone BP-53. ORGANISM Homo sapiens Eukaryota; Animalia; Chordata; Vertebrata; Mammalia; Theria;
Eutheria; Primates; Haplorhini; Catarrhini; Hominidae. REFERENCE 1 (bases 1 to 2474) AUTHORS Wood,W.I., Cachianes,G., Henzel,W.J., Winslow,G.A., Spencer,S.A., Hellmiss,R., Martin,J.L. and Baxter,R.C.
TITLE Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein JOURNAL Mol. Endocrinol. 2, 1176-1185 (1988) STANDARD full automatic 30 COMMENT NCBI gi: 183115
FEATURES Location/Qualifiers
mRNA <1. .2474
/note=“GFIBP mRNA”
CDS 110. .985
/gene=“IGFBP1”
/note=“insulin-like growth factor-
binding protein; NCBI
gi: 183116.”
/codon_start=1
/translation=“MQRARPTLWAAALTLLVLLRGPP
VARAGASSGGLGPVVRCEPCDARALAQCAPPPAVCAE
LVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPS
PDEARPLQALLDGRGLCVNASAVSRLRAYLLPAPPAP
GNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSK
IIIIKKGHAXDSQRYKVDYESQSTDTQNFSSESKRET
EYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKKGF
YKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKED
VHCYSMQSK” (<400>24>)
source 1. .2474
/organism=“Homo sapiens”
BASECOUNT 597 a 646 c 651 g 580 t
ORIGIN
misc_feature 1343. .1351
/note=“pot.N-linked glycosylation
site (AA 408-410)”
misc_feature 1631. .1639
/note=“pot.N-linked glycostlation
site (AA 504-506)”
misc_feature 1850. .1858
/note=“pot.N-linked glycosylation
site (AA 577-579)”
misc_feature 1895. .1903
/note=“pot.N-linked glycosylation
site (AA 592-594)”
misc_feature 1949. .1957
/note=“pot.N-linked glycosylation
site (AA 610-612)”
misc_feature 2240. .2251
/note=“putative proreceptor
processing site (AA 707-710)
misc_feature 2252. .4132
/note=“beta-subunit (AA 711-1337)”
misc_feature 2270. .2278
/note=“pot.N-linked glycosylation
site (AA 717-719]”
misc_feature 2297. .2305
/note=“pot.N-linked glycosylation
site (AA 726-728)”
misc_feature 2321. .2329
/note=“pot.N-linked glycosylation
site (AA 734-736)”
misc_feature 2729. .2737
/note=“pot.N-linked glycosylation
site (AA 870-872)”
misc_feature 2768. .2776
/note=“pot.N-linked glycosylation
site (AA 883-885)”
misc_feature 2837. .2908
/note=“transmembrane region
(AA 906-929)”
misc_feature 2918. .2926
/note=“pot.N-linked glycosylation
site (AA 933-935)”
misc_feature 3047. .3049
/note=“pot.ATP binding site (AA 976)”
misc_feature 3053. .3055
/note=“pot.ATP binding site (AA 978)”
misc_feature 3062. .3064
/note=“pot.ATP binding site (AA 981)”
misc_feature 3128. .3130
/note=“pot.ATP binding site
(AA 1003)”
CDS 32. .4132
/product=“IGF-I receptor”
/note=“50 stops when translation
attempted, frame 1, code 0”
BASE COUNT 1216 a 1371 c 1320 g 1082 t
ORIGIN
HSIGFIRR Length: 4989 May 11, 1994 12:10 Type: N
Check: 133 . .
FIG. 4A is a photographic representation of a Western ligand blot of HaCaT conditioned medium showing IGFBP-3 secreted in 24 hours after 7 day treatment with phosphorothioate oligonucleotides (BP3AS2, BP3AS3 and BP3S) at 0.5 μM and 5 μM; * no oligonucleotide added.
FIG. 4B is a graphical representation of a scanning imaging desitometry of Western ligand blot (FIG. 4A), showing relative band intensities of IGFBP-3 and the 24 kDa IGFBP-4 after treatment with phosphorothioate oligonucleotides; * no oligonucleotide added.
FIG. 5A is a photographic representation of a Western ligand blot of HaCaT conditioned medium showing IGFBP-3 secreted in 24 hours after 7 day treatment with phosophorothioate oligonucleotide BP3AS2 at 0.5 μM compared with several control oligonucleotides at 0.5 μM. (a) oligonucleotide BP3AS2NS; (b) oligonucleotide BP3AS4; (c) oligonucleotide BP3AS4NS; and (untreated), no oligonucleotide added.
FIG. 5B is a graphical representation of a scanning imaging densitometry of Western ligand blot (FIG. 5A), showing relative band intensities of IGFBP-3 after treatment with phosphorothioate oligonucleotides as in FIG. 5A, showing IGFBP-3 band intensities expressed as a percentage of the average band intensity from conditioned medium of cells not treated with oligonucleotide.
FIG. 6 is a graphical representation showing inhibition of IGF-I binding by antisense oligonucleotides to IGF-I receptor. IGFR.AS: antisense; IGFR.S: sense.
FIG. 7 is a graphical representation showing inhibition of IGFBP-3 production in culture medium following initial treatment with antisense oligonucleotides once daily over a 2 day period.
FIG. 8 is a graphical representation showing optimization of IGFBP-3 antisense oligonucleotide concentration as determined by relative IGFBP-3 concentration in culture medium.
FIG. 9 is a diagramatic representation of a map of IGF-1 Receptor mRNA and position of target ODNs.
FIG. 10 is a photographical representation showing Lipid-mediated uptake of oligonucleotide in keratinocytes. HaCaT keratinocytes were incubated for 24 hours in medium 10 (DMEM plus 10% v/v FCS) containing fluorescently labelled ODN (R451, 30 nM) and cytofectin GSV (2 μg/ml). The cells were then transferred to ODN-free medium and fluorescence microscopy (a) and phase contrast (b) images of the cells were obtained.
FIG. 11 is a graphical representation of uptake (A) and toxicity (B) of ODN/lipid complexes in keratinocytes. Confluence HaCaT keratinocytes were incubated in DMEM containing fluoresently labelled ODN (R451) plus liposome over 120 hours, viewed using fluorescene microscopy and trypan blue stained and counted.
FIG. 12 is a graphical representation of an IGF-1 Receptor mRNA in ODN treated (30 nM) HaCaT cells (2 μg/ml GSV). HaCaT keratinocytes were treated for 96 hours with C-5 propynyl, dU, dC ODNs complexed with cytofectin GSV. Cells were treated with ODNs complementary to the human IGF-I receptor mRNA (27, 32, 74 and 78), 2 randomised sequence ODNs (R451) and R766), liposome alone (GSV) or were left untreated (UT). Total RNA was isolated then analysed for IGF-I receptor mRNA and GAPDH mRNA levels by RNase Protection and Phosphorlmager quantitiation.
(A) Electrophoretic analysis of IGF-I receptor and GAPDH mRNA fragments after RNase Protection. Molecular weight markers are shown on the right hand side. Full length probe is shown on the left hand side (G-probe and I-probe). GAPDH protected fragments (G) are seen at 316 bases and IGF-I receptor protected fragments (1) are seen at 276 bases.
(B) Relative level of IGF-I receptor mRNA following each treatment is shown.
FIG. 13 is a graphical representation of an IGF-1 receptor mRNA in ODN treated (30 nM) HaCaT cells (2 μg/ml GSV). Summary of IGF-I receptor ODN screening data. HaCaT keratinocytes were treated for 96 hours with C-5 propynyl, dU, dC ODNs complexed with cytofectin GSV. Total RNA was isolated then analysed for IGF-I receptor mRNA and GAPDH mRNA levels by RNase protection and phosphorImager quantitiation. Relative level of IGF-I receptor mRNA is shown after treatment with ODNs complementary to the human IGF-I receptor mRNA, 4 randomised sequence ODNs and liposome alone. (26-86=IGF-I receptor ODNs; R1, R4, R7 and R9=randomised ODNs (R1=R121, R4=R451, R7=R766, R9=R961); GSV=liposome alone; UT=untreated). *indicates a significant difference in relative IGF-I receptor mRNA from GSV treated cells (n=4-10, p<0.05).
FIG. 14 is a graphical representation of the effect of antisense oligonucleotides on IGF-1 receptor levels on the surface of keratinocytes. HaCaT cells were grown to confluence in 24-well plates in DMEM containing 10% v/v FCS. Oligodeoxynucleotide (ODN) and Cytofectin GSV (GSV, Glen Research) were mixed together in serum-free DMEM, incubated at room temperature for 10 minutes before being diluted ten-fold in medium and placed on the cells. Cells were incubated for 72 hours with 30 nM random sequence or antisense ODN and 2 μg/ml GSV or with GSV alone in DMEM containing 10% v/v FCS with solutions replaced every 24 hours. This was followed by incubation with ODN/GSV in serum-free DMEM for 48 hours. All incubations were performed at 37° C. Wells were washed twice with 1 ml cold PBS. Serum-free DMEM containing 10−10M 125I-IGF-I was added with or without the IGF-I analogue, des (1-3) IGF-I, at 10−10M to 10−7M. Cells were incubated at 4° C. for 17 hours with gentle shaking then washed three times with 1 ml cold PBS and lysed in 250 μl 0.5M NaOH/0.1% v/v Triton X-100 at room temperature for 4 hours. Specific binding of the solubilised cell extract was measured using a γ counter.
FIG. 15 is a graphical representation of the effect of antisense oligonucleotides on IGF-1 receptor levels on the surface of keratinocytes.
FIG. 16 is a photographical representation of H & E stained sections of (A) psoriatic skin biopsy prior to grafting and (B) 49 day old psoriatic skin graft using skin from the same donor.
FIG. 17 is a photographical representation of uptake of oligonucleotide after intradermal injection into psoriatic skin graft on a nude mouse. Psoriatic skin graft was intradermally injected with ODN (R451, 50 μl, 10 μM). The graft was removed and sectioned after 24 hours, then viewed using confocal microscopy.
FIG. 18(a) is a photographical representation of Pregraft, Donor JH, Donor JH, PBS treated, 50 μl, Donor JH, #50 treated, 50 μl, 10 μM.
FIG. 18(b) is a photographical representation of Donor LB, pregraft, Donor LB, PBS treated (50 μl), Donor LB, #74 treated (50 μl, 10 μM).
FIG. 18(c) is a photographical representation of Donor PW, pregraft, Donor PW, R451 treated (50 μl, 10 μM), Donor LB, #74 treated (50 μl, 10 μM).
FIG. 18(d) is a photographical representation of Donor GM, pregraft, Donor GB, R451 treated (50 μl, 10 μM), Donor GM, #27 treated (50 μl, 10 μM).
FIG. 19(a) is a photographical representation showing Donor JH pregraft, Donor JH PBS treated 50, μl, Donor JH #50 treated 50 μl, 10 μM.
FIG. 19(b) is a photographical representation Donor LB pregraft, Donor LB PBS treated 50 μl, Donor LB #74 treated 50 μl, 10 μM.
FIG. 19(c) is a photographical representational showing Donor PW pregraft, Donor PW R451 treated 50 μl, 10 μM, Donor PW #74 treated 50 μl, 10 μM.
FIG. 19(d) is a photographical representation showing Donor GM pregraft, Donor GM R451 treated 50, μl, 10 μM, Donor #27 treated 50, μl, 10 μM.
FIG. 20 is a graphical representation showing suppression of psoriasis after treatment with oligonucleotide (quantification). Oligonucleotide (50 μl, 10 μM) was injected every two days for 20 days, as were control treatments. Skin thickness was measured by removing the skin and using computer software (MCID analysis) to measure the exact thickness of each graft. N=3-4 for each treatment. *indicates a significant difference from the pregraft value (ANOVA, P<0.05)
FIG. 21 is a photographic representation of αhKi-67 imunobiological binding.
FIG. 22 is a photographical representation showing penetration of oligonucleotide into human skin after topical treatment. Fluorescently labelled oligonucleotide (10 μM R451) was applied topically after formulation with cytofectin GSV (10 μg/ml) and viewed using confocal microscopy.
FIG. 23 is a photographical representation showing penetration of oligonucleotide into human skin after application of topical gel formation. Fluorescently labelled oligonucleotide (10 μM R451) was applied topically after complexing with cytofectin GSV (10 μg/ml) and formulation into 3% methylcellulose gel. Image was obtained using confocal microscopy.
FIG. 24 is a graphical representation showing IGFBP-3 mRNA.
FIG. 25(a) is a graphical representation showing IGFBP-3 mRNA in AON treated (10 nM) HaCaT cells (2 μg/ml GSV).
FIG. 25(b) is a graphical representation showing IGFBP-3 mRNA levels of AON treated (100 nm) HaCaT cells (2 μg/ml GSV).
FIG. 25(c) is a graphical representation showing IGFBP-3 mRNA in AON treated (30 nM) HaCaT cells (2 μg/ml GSV).
FIG. 25(d) is a graphical representation showing IGFBP-3 mRNA in AON treated (30 nM) HaCaT cells (2 μg/ml GSV).
FIG. 26(a) is a graphical representation showing IGFBP-3 mRNA in ODN treated (30 nM) HaCaT cells (2 μg/ml). HaCaT keratinocytes were treated for 51 hours with C-5 propynl, dU, dC ODNs complexed with cytofectin GSV. Total RNA was isolated then analysed for IGFBP-3 mRNA and GAPDH mRNA levels by Northern analysis and phosphorimager quantitation. Relative level of IGFBP-3 mRNA is shown after treatment with ODNs complementary to the human IGFBP-3 mRNA, 4 randomised sequence ODNs and lipsome alone. (1-24=IGFBP-3 ODNs; R1, R4, R7 and R9=randomised ODNs (R1=R121, R4=R451, R7=R766, R9 R961); GS=liposome alone; UT=untreated). *indicates a significant different in relative IGFBP-3 mRNA from GSV treated cells (n-5-8, p<0.01), **indicates a significant difference in relative IGFBP-3 mRNA from GSV treated cells (n=5-8, p<0.05).
FIG. 26(b) is a graphical representation showing IGFBP-3 mRNA in ODN treated (100 nM) HaCaT cells (2 μg/ml GSV). HaCaT keratinocytes were treated for 51 hours with C-5 propynl, dU, dC ODNs complexed with cytofectin GSV. Total RNA was isolated then analysed for IGFBP-3 mRNA and GAPDH mRNA levels by Northern analysis and phosphorimager quantitation. Relative level of IGFBP-3 mRNA is shown after treatment with ODNs complementary to the human IGFBP-3 mRNA, 4 randomised sequence ODNs and liposome alone. (1-24=IGFBP-3 ODNs; R1, R4, R7 and R9=randomised ODNs (R1-R121, R4=R451, R7=R766, R9-R961), GS=lipsome alone; UT=untreated). *indicates a significant difference in relative IGFBP-3 mRNA from GSV treated cells (n-6-8, p<0.01).
FIG. 27 is a representation showing a reduction in IGF-I receptor mRNA in HaCaT cells following treatment with antisense oligonucleotides. Confluent HaCaT cells were treated every 24 h for 4 days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM IGF-I receptor specific oligonucleotides (#26 to #86) or random sequence oligonucleotides (R121, R451 and R766). Total RNA was isolated and analysed for IGF-I receptor and GAPDH mRNA by RNase protection assay. (a). Representative RNase protection assay gel showing IGF-I receptor (IGFR) and GAPDH mRNA in untreated or treated HaCaT cells. In this example, a reduction in IGFR band intensity relative to GAPDH can be seen with AON #27 and #78, but not with #32, #74 or the controls (R4, R7, random oligonucleotides R451 and R766, respectively; G, GSV lipid; UT, untreated).
(b) Densitometric quantitation of IGF-I receptor mRNA (normalised to GAPDH mRNA) in HaCaT cells following treatment with IGF-I receptor specific oligonucleotides (solid black), random sequence oligonucleotides (horizontal striped bar) or GSV alone (shaded bar) compared to untreated cells (UT, vertical striped bar). Each oligonucleotide was assayed in duplicate in at least two separate experiments.
Results are presented as mean±SEM. A one-way ANOVA followed by Tukey's (▴) test was performed; ▴ indicates a significant difference between cells treated with IGF-I receptor specific AONs and all of the control treatments (p<0.05). n=4 except for #27 and #32 (n=6), #28 and #68 (n=3), R766 (n=9), and R451, GSV and untreated (n=10).
FIG. 28 is a representation showing a reduction in total cellular IGF-I receptor protein following antisense oligonucleotide treatment. Confluent HaCaT cells were treated every 24 h for 4 days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM IGF-I receptor specific AONs (#27, #50 and #64) or the random sequence oligonucleotide, R451. Total cellular protein was isolated and analysed for IGF-I receptor by SDS PAGE followed by western blotting with an antibody specific for the human IGF-I receptor. (a) Duplicate treated cellular extracts showing the IGF-I receptor at the predicted size of 110 kD
(b) Densitometric quantitation of IGF-I receptor protein. Results are presented as mean±SEM of four different experiments each performed in duplicate. A one-way ANOVA followed by a Dunnett's test was performed; * indicates a significant difference from GSV treated cells (p<0.01). GSV, GSV lipid alone; UT, untreated; R451, random sequence oligonucleotide; 64, 50, 27, IGF-I receptor-specific AONs.
FIG. 29 is a representation showing a reduction in IGF-I receptor numbers on the keratinocyte cell surface after antisense oligonucleotide treatment. HaCaT cells were transfected with IGF-I receptor specific AONs #27 (-▴-), #50 (-x-), #64 (---▪---), a random sequence oligonucleotide R451 (-o-), or treated with GSV lipid alone (--□--) every 24 h for four days (untreated cells, --*--). Competition binding assays using 125I-IGF-I and the receptor-specific analogue, des(1-3)IGF-I, were performed (inset); plotted values are means ±standard error. The mean values were then subjected to Scatchard analysis.
FIG. 30 is a representation showing a reduction in keratinocyte cell number following antisense oligonucleotide treatment. HaCaT cells, initially at 40% confluence, were transfected with the IGF-I receptor specific AON #64, control sequences R451 and 6416, or treated with GSV lipid alone every 24 h for 2 days (UT, untreated cells). Cell number was measured in the culture wells using a dye binding assay (Experimental protocol). Results are presented as mean±SD. A one-way ANOVA was performed, followed by a Tukey's multiple comparison test. ▴ indicates a significant difference between cells treated with AON #64 and all of the control treatments (p<0.001).
FIG. 31 is a representation showing a reversal of epidermal hyperplasia in psoriatic human skin grafts on nude mice following intradermal injection with antisense oligonucleotides
Grafted psoriasis lesions were injected with IGF-I receptor specific AONs, a random sequence oligonucleotide in PBS, or with PBS alone, every 2 days for 20 days, then analysed histologically. (a) Donor A graft treated with AON #50 showing epidermal thinning compared
with pregraft and control (PBS) treated graft, and Donor B graft treated with AON #27 showing epidermal thinning compared with pregraft and control (R451) treated graft. E, epidermis; Scale bar, 400 mm; all pictures are at the same magnification. (b) Mean epidermal cross-sectional area over the full width of grafts was determined by digital image analysis. Results are presented as mean±SEM. Shaded bars, control treatments: R451, random oligonucleotide sequence; solid bars, treatments with oligonucleotides that inhibited IGF-I receptor expression in vitro. * indicates a significant difference from the vehicle treated graft (p<0.01, n=5-7), ++ indicates a significant difference from the random sequence (R451) treated graft (p<0.01, n=5-7). (c) Parakeratosis (arrow) was absent in grafts treated with IGF-I receptor AONs (AON #50) but persisted in pregraft and control (PBS) treated graft. Scale bar, 100 mm.
FIG. 32 is a representation showing a reversal of epidermal hyperplasia correlates with reduced IGF-I receptor mRNA in grafted psoriasis lesions treated with antisense oligonucleotides (a) A psoriasis lesion prior to grafting, and after grafting and treatment with IGF-I receptor specific oligonucleotide #27 (AON #27) or random sequence (R451) was immunostained with antibodies to Ki67 to identify proliferating cells. Proliferating cells are indicated by a dark brown nucleus (arrows). Scale bar, 250 mm; all pictures are at the same magnification. (b) The same lesion prior to grafting and after oligonucleotide treatment as in (a) was subjected to in situ hybridisation with a 35S-labeled cRNA probe complementary to the human IGF-I receptor mRNA. The presence of IGF-I receptor mRNA is indicated by silver grains (tiny black speckles), which are almost eliminated in the epidermis of the lesion treated with the IGF-I receptor-specific oligonucleotide #27 (AON #27). Arrows indicate the basal layer of the epidermis with dermis underneath. Scale bar, 50 μm.
FIG. 33 is a representation showing a reduction in IGF-I receptor mRNA in HaCaT keratinocytes following treatment with oligonucleotides. HaCaT cell monolayers grown to 90% confluence in DMEM contianing 10% v/v fetal calf serum were treated with 24 h for two days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM oligonucleotide. Total RNA was isolated and analysed for IGF-I receptor and GAPDH mRNA using a commercially availble ribonuclease protection assay kit (RPAII, Ambicon Inc, Austin, Tex.). Band intensity was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).
FIG. 34 is a representation showing a reduction in IGF-I receptor protein in HaCaT keratinocytes following treatment with oligonucleotides. HaCaT cell monolayers grown to 90% confluence in DMEM containing 10% v/v fetal calf serum were treated every 24 h for four days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM oligonucleotide. Cells were lyased in a buffer containing 50 mM HEPES, 150 mM NaCl, 10% v/v gycerol, 1% v/v Triton X-100 and 100 μg/ml aprotinin on ice for 30 mins, then 30 μg of lysate was loaded onto a denaturing 7% w/v polyacrylamide gel followed by transfer onto an Immobilon-P membrane (Millipore, Bedford, Mass.). Membranes were incubated with the anti-IGF-I receptor antibody C20 (Sanra Cruz Biotechnology Inc., Santa Cruz, Calif., 25 ng/ml in 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% v/v Tween 20) for 1 h at room temperature and developed using the Vistra ECF western blotting kit (Amersham, Buckinghamshire, England). Band intensity was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).
FIG. 35 is a representation showing a reduction in HaCaT keratinocyte cell number following treatment with oligonucleotides. HaCaT cell monolayers grown to 40% confluence in DMEM containing 10% fetal calf serum were treated every 24 h for three days with 2 μg/ml GSV lipid alone (GSV) or complexed with 15 nM oligonucleotide. Cell number was measured every 24 h using the amido black dye binding assay [32].
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated in part on the use of molecules and in particular genetic molecules and more particularly antisense molecules to down-regulate a growth factor, its receptor and/or growth factor expression facilitating sequences.
Accordingly, one aspect of the present invention contemplates a method for ameliorating the effects of a medical disorder such as a proliferative and/or inflammatory skin disorder in a mammal, said method comprising contacting the proliferating and/or inflamed skin or skin capable of proliferation and/or inflammation or a cell otherwise involved in the said medical disorder with an effective amount of a nucleic acid molecule or chemical analogue thereof capable of inhibiting or otherwise reducing a growth factor mediated cell proliferation and/or inflammation and/or other medical disorder.
Growth factor mediated cell proliferation and inflammation are also referred to as epidermal hyperplasias and these and other medical disorders may be mediated by any number of molecules such as but not limited to IGF-I, keratinocyte growth factor (KGF), transforming growth factor-α (TGFα), tumour necrosis factor-α (TNFα), interleukin-1, -4, -6 and 8 (IL-1, IL-4, IL-6 and IL-8, respectively), basic fibroblast growth factor (bFGF) or a combination of one or more of the above. The present invention is particularly described and exemplified with reference to IGF-I and its receptor (IGF-I receptor) and to IGF-I facilitating molecules, IGFBPs, since targeting these molecules according to the methods contemplated herein provides the best results to date. This is done, however, with the understanding that the present invention extends to any growth factor or cytokine-like molecule, a receptor thereof or a facilitating molecule like the IGFBPs involved in skin cell proliferation such as those molecules contemplated above and/or their receptors and/or facilitating molecules therefor.
According to this preferred embodiment, there is provided a method for ameliorating the effects of a medical disorder such as a proliferative and/or inflammatory skin disorder in a mammal, said method comprising contacting the proliferating and/or inflamed skin or skin capable of proliferation and/or inflammation or a cell otherwise involved with said medical disorder with an effective amount of a nucleic acid molecule or chemical analogue thereof capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation and/or inflammation and/or other medical disorder.
The present invention is particularly described by psoriasis as the proliferative skin disorder. However, the subject invention extends to a range of proliferative and/or inflammatory skin disorders or epidermal hyperplasias such as but not limited to psoriasis, ichthyosis, pityriasis rubra pilaris (“PRP”), seborrhoea, keloids, keratoses, neoplasias and scleroderma, warts, benign growths and cancers of the skin. The present invention extends to a range of other disorders such as neovascularization conditions such as but not limited to hyperneovasularization such as neovascularization of the retina, lining of the brain, skin, hyperproliferation of the inside of blood vessels, kidney disease, atherosclerotic disease, hyperplasias of the gut epithelium or growth factor mediated malignancies such as IGF1-mediated malignancies.
Furthermore, down-regulation of IGF-I receptor is useful as adjunctive therapy for epidermal hyperplasia. In accordance with this aspect of the present invention it is known that IGF-I receptor elicits separate intracellular signals which prevent apoptosis [19]. In keratinocytes, IGF-I receptor activation has been shown to protect UV-irradiated cells from apoptosis [20]. In another cell type, a number of IGF-I receptors expressed by the cells correlated with tumorigenicity and apoptotic resistance [21]. Consequently, in accordance with the present invention, by inactivating IGF-I receptor on cells such as epidermal keratinocytes will achieve three important outcomes:
-
- (i) Acute epidermal hyperplasia following UV has been suggested to increase the risk of keratinocyte carcinogenic transformation [22]. By reducing IGF-I receptor expression in the epidermis, the incidence of epidermal hyperplasia following UV exposure is likely to be reduced leading to an overall acceleration in normalization of the lesion and reduced carcinogenic risk.
- (ii) Inhibition of anti-apoptotic action of IGF-I receptor will enhance the reversal of epidermal thickening and accelerate normalization of differentiation. Topical or injected IGF-I receptor antisense as adjunctive treatment will increase apoptosis in the epidermal layer thereby enhancing the reduction in acanthosis observed in UV treatments.
- (iii) Survival of keratinocytes, ie. those which evade apoptosis is likely to occur when cells have damaged DNA. Such mutations may be in the tumor suppressor region. Consequently, the use of antisense therapy will result in less frequent selection of mutated keratinocytes and therefore reduced incidence of basal cell carcinomas and squamous.
According to this embodiment, there is provided a method for ameliorating the effects of a proliferative and/or inflammatory skin disorder such as psoriasis said method comprising contacting the proliferating and/or inflamed skin or skin capable of proliferation and/or inflammation with effective amounts of UV treatment and a nucleic acid molecule or chemical analogue thereof capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation and/or inflammation.
The UV treatment and nucleic acid molecule or its chemical analogue may be administered in any order or may be done simultaneously. This method is particularly useful in treating psoriasis by combination of UV and antisense therapy. Preferably the antisense therapy is directed to the IGF-I receptor.
In a preferred embodiment, the present invention is directed to a method for ameliorating the effects of psoriasis or other medical disorder, said method comprising contacting proliferating skin or skin capable of proliferation or cells associated with said disorder with an effective amount of a nucleic acid molecule or chemical analogue thereof capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation or ameliorating the medical disorder.
The present invention extends to any mammal such as but not limited to humans, livestock animals (e.g. horses, sheep, cows, goats, pigs, donkeys), laboratory test animals (e.g. rabbits, mice, guinea pigs), companion animals (e.g. cats, dogs) and captive wild animals. However, the instant invention is particularly directed to proliferative and/or inflammatory skin disorders such as psoriasis in humans as well as medical disorders contemplated above.
The aspects of the subject invention instantly contemplated are particularly directed to the topical application of one or more suitable nucleic molecules capable of inhibiting, reducing or otherwise interfering with IGF-mediated cell proliferation and/or inflammation. More particularly, the nucleic acid molecule targets IGF-I interaction with its receptor. Conveniently, therefore, the nucleic acid molecule is an antagonist of IGF-I interaction with its receptor. Most conveniently, the nucleic acid molecule antagonist is an antisense molecule to the IGF-I receptor, to IGF-I itself or to a molecule capable of facilitating IGF-I interaction with its receptor such as but not limited to an IGFBP.
Insofar as the invention relates to IGFBPs, the preferred molecules are IGFBP-2, -3, -4, -5 and -6. The most preferred molecules are IGFBP-2 and IGFBP-3.
The nucleotide sequences of IGFBP-2 and IGFBP-3 are set forth in FIGS. 1 (<400>1) and 2 (<400 >2), respectively. According to a particularly preferred aspect of the present invention, there is provided a nucleic acid molecule comprising at least about ten nucleotides capable of hybridising to, forming a heteroduplex or otherwise interacting with an mRNA molecule directed from a gene corresponding to a genomic form of <400>1 and/or <400>2 and which thereby reduces or inhibits translation of said mRNA molecule. Preferably, the nucleic acid molecule is at least about 15 nucleotides in length and more preferably at least about 20-25 nucleotides in length. However, the instant invention extends to any length nucleic acid molecule including a molecule of 100-200 nucleotides in length to correspond to the full length of or near full length of the subject genes.
The nucleotide sequence of the antisense molecules may correspond exactly to a region or portion of <400>1 or <400>2 or may differ by one or more nucleotide substitutions, deletions and/or additions. It is a requirement, however, that the nucleic acid molecule interact with an mRNA molecule to thereby reduce its translation into active protein.
Examples of potential antisense molecules for IGFBP-2 and IGFBP-3 are those capable of interacting with sequences selected from the lists in Examples 6 and 7, respectively.
The nucleic acid molecules in the form of an antisense molecule may be linear or covalently closed circular and single stranded or partially double stranded. A double stranded molecule may form a triplex with target mRNA or a target gene. The molecule may also be protected from, for example, nucleases, by any number of means such as using a nonionic backbone or a phosphorothioate linkage. A convenient nonionic backbone contemplated herein is ethylphosphotriester linkage or a 2′-O-methylribosyl derivative. A particularly useful modification modifies the DNA backbone by introducing phosphorothioate internucleotide linkages. Alternatively or in addition to the pyrimidine bases are modified by inclusion of a C-5 propyne substitution which modification is proposed to enhance duplex stability [23]. The present invention extends to any chemical modification to the bases and/or RNA or DNA backbone. Reference to a “chemical analogue” of a nucleic acid molecule includes reference to a modified base, nucleotide, nucleoside or phosphate backbone.
Examples of suitable oligonucleotide analogues are conveniently described in Ts'O et al [7]. Further suitable examples of oligonucleotide analogues and chemical modifications are described in references 25 to 31.
Alternatively, the antisense molecules of the present invention may target the IGF-I gene itself or its receptor or a multivalent antisense molecule may be constructed or separate molecules administered which target at least two or an IGFBP, IGF-I and/or IGF-I-receptor. Examples of suitable antisense molecules capable of targetting the IGF-I receptor are those capable of interacting with sequences selected from the list in Example 8. One particularly useful antisense molecule is 5′-ATCTCTCCGCTTCCTTTC-3′ (<400>10).
Other particularly useful antisense molecules are:
#27 UCCGGAGCCAGACUU
#64 CACAGUUGCUGCAAG
#78 UCUCCGCUUCCUUUC
#28 AGCCCCCACAGCGAG
#32 GCCUUGGAGAUGAGC
#40 UAACAGAGGUCAGCA
#42 GGAUCAGGGACCAGU
#46 CGGCAAGCUACACAG
#50 GGCAGGCAGGCACAC
Particularly useful molecules are selected from #27, #64 and #78. In a preferred embodiment these molecules comprise a C-5 propynyl dU, dC phosphorothioate modification.
A particularly preferred embodiment of the present invention contemplates a method of ameliorating the effects of psoriasis or other medical disorder, said method comprising contacting proliferating skin or skin capable of proliferation or cells associated with said medical disorder with an effective amount of one or more nucleic acid molecules or chemical analogues thereof capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation or ameliorating the medical disorder wherein said one or more molecules comprises a polynucleotide capable of interacting with mRNA directed from an IGF-I gene, an IGF-I receptor gene or a gene encoding an IGFBP such as IGFBP-2 and/or IGFBP-3.
Preferably, the nucleic acid molecule are antisense molecules. Particularly useful antisense molecules are:
#27 UCCGGAGCCAGACUU
#64 CACAGUUGCUGCAAG
#78 UCUCCGCUUCCUUUC
#28 AGCCCCCACAGCGAG
#32 GCCUUGGAGAUGAGC
#40 UAACAGAGGUCAGCA
#42 GGAUCAGGGACCAGU
#46 CGGCAAGCUACACAG
#50 GGCAGGCAGGCACAC
Even more particularly useful molecules are selected from #27, #64 and #78.
In accordance with one aspect of the present invention the nucleic acid molecule is topically applied in aqueous solution or in conjunction with a cream, ointment, oil or other suitable carrier and/or diluent. A single application may be sufficient depending on the severity or exigencies of the condition although more commonly, multiple applications are required ranging from hourly, multi-hourly, daily, multi-daily, weekly or monthly, or in some other suitable time interval. The treatment might comprise solely the application of the nucleic acid molecule or this may be applied in conjunction with other treatments for the skin proliferation and/or inflammatory disorder being treated or for other associated conditions including microbial infection, bleeding and the formation of a variety of rashes.
As an alternative to or in conjunction with antisense therapy, the subject invention extends to the nucleic acid molecule as, or incorporating, a ribozyme including a minizyme to, for example, IGF-I, its receptor or to molecules such as IGFBPs and in particular IGFBP-2 and -3. Ribozymes are synthetic nucleic acid molecules which possess highly specific endoribonuclease activity. In particular, they comprise a hybridising region which is complementary in nucleotide sequence to at least part of a target RNA. Ribozymes are well described by Haseloff and Gerlach [8] and in International Patent Application No. WO 89/05852. The present invention extends to ribozymes which target mRNA specified by genes encoding IGF-I, its receptor or one or more IGFBPs such as IGFBP-2 and/or IGFBP-3.
According to this embodiment, there is provided in a particularly preferred aspect a ribozyme comprising a hybridising region and a catalytic region wherein the hybridising region is capable of hybridising to at least part of a target mRNA sequence transcribed from a genomic gene corresponding to (<400>1) or (<400>2) wherein said catalytic domain is capable of cleaving said target mRNA sequence to reduce or inhibit IGF-I mediated cell proliferation and/or inflammation and/or other medical disorders.
Yet another aspect of the present invention contemplates co-suppression to reduce expression or to inhibit translation of an endogenous gene encoding, for example, IGF-I, its receptor, or IGFBPs such as IGFBP-2 and/or -3. In co-suppression, a second copy of an endogenous gene or a substantially similar copy or analogue of an endogenous gene is introduced into a cell following topical administration. As with antisense molecules, nucleic acid molecules defining a ribozyme or nucleic acid molecules useful in co-suppression may first be protected such as by using a nonionic backbone.
The efficacy of the nucleic acid molecules of the present invention can be conveniently tested and screened using an in vitro system comprising a basal keratinocyte cell line. A particularly useful system comprises the HaCaT cell line described by Boukamp et al [9]. In one assay, IGF-I is added to an oligonucleotide treated HaCaT cell line. Alternatively, growth of oligonucleotide treated HaCaT cells is observed on a feeder layer of irradiated 3T3 fibroblasts. Using such in vitro assays, it is observed that antisense oligonucleotides to IGFBP-3, for example, inhibit production of IGFBP-3 by HaCaT cells. Other suitable animal models include the nude mouse/human skin graft model (15; 16) and the “flaky skin” mouse model (17; 18). In the nude mouse model, microdermatome biopsies of psoriasis lesions are taken under local anaesthetic from volunteers then transplanted to congenital athymic (nude) mice. These transplanted human skin grafts maintain the characteristic hyperproliferating epidermis for 6-8 weeks. They are an established model for testing the efficacy of topically applied therapies for psoriasis. In the “flaky skin” mouse model, the fsn/fsn mutation produces mice with skin resembling human psoriasis. This mouse, or another mutant mouse with a similar phenotype is a further in vivo model to test the efficacy of topically applied therapies for psoriasis.
Another aspect of the present invention contemplates a pharmaceutical composition for topical administration which comprises a nucleic acid molecule capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation such as psoriasis and one or more pharmaceutically acceptable carriers and/or diluents. Preferably, the nucleic acid molecule is an antisense molecule to IGF-I, the IGF-I receptor or an IGFBP such as IGFBP-2 and/or IGFBP-3 or comprises a ribozyme to one or more of these targets or is a molecule suitable for co-suppression of one or more of these targets. The composition may comprise a single species of a nucleic acid molecule capable of targeting one of IGF-I, its receptor or an IGFBP, such as IGFBP-2 or IGFBP-3 or may be a multi-valent molecule capable of targeting two or more of IGF-I, its receptor or an IGFBP, such as IGFBP-2 and/or IGFBP-3.
The nucleic acid molecules may be administered in dispersions prepared in creams, ointments, oil or other suitable carrier and/or diluent such as glycerol, liquid polyethylene glycols and/or mixtures thereof. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for topical use include sterile aqueous solutions (where water soluble) or dispersions and powders for the extemporaneous preparation of topical solutions or dispersions. In all cases, the form is preferably sterile although this is not an absolute requirement and is stable under the conditions of manufacture and storage. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganism can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Topical solutions are prepared by incorporating the nucleic acid molecule compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by where necessary filter sterilization.
The active agent may alternatively be administered by intravenous, subcutaneous, nasal drip, suppository, implant means amongst other suitable routes of administration including intraperitoneal, intramuscular, absorption through epithelial or mucocutaneous linings for example via nasal, oral, vaginal, rectal or gastrointestinal administration. Reference may conveniently be made to reference 24.
As used herein “pharmaceutically acceptable carriers and/or diluents” include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. Conveniently, the nucleic acid molecules of the present invention are stored in freeze-dried form and are reconstituted prior to use.
Yet another aspect of the present invention contemplates the use of a nucleic acid molecule in the manufacture of a medicament for the treatment of proliferative and/or inflammatory skin disorders or other medical disorders mediated by a growth factor. The proliferative and/or inflammatory skin disorder is generally psoriasis or other medical disorders as described above and the nucleic acid molecule targets IGF-I, the IGF-I receptor and/or an IGFBP such as IGFBP-2 and/or IGFBP-3.
Still a further aspect of the present invention contemplates an agent comprising a nucleic acid molecule as hereinbefore defined useful in the treatment of proliferative and/or inflammatory skin disorders, such as psoriasis or other medical disorder.
The present invention further contemplates the use of the genetic molecules and in particular the antisense molecules to inhibit the anti-apoptotic activity of IGF-I receptor. Such a use is appropriate for the treatment of certain cancers and as adjunct therapy for epidermal hyperplasia such as in combination with UV treatment.
The present invention is further described by the following non-limiting Examples.
EXAMPLE 1 The differentiated human keratinocyte cell line, HaCaT [9] was used in the in vitro assay. Cells at passage numbers 33 to 36 were maintained as monolayer cultures in 5% V/V CO2 at 37° C. in Keratinocyte-SFM (Gibco) containing EGF and bovine pituitary extract as supplied. Media containing foetal calf serum were avoided because of the high content of IGF-I binding proteins in serum.
Feeder layer plates of lethally irradiated 3T3 fibroblasts were prepared exactly as described by Rheinwald and Green [10].
EXAMPLE 2 Cells were grown to 4 days post confluence in 2 cm2 wells with daily medium changes of Keratinocyte-SFM, then the medium was changed to DMEM (Cytosystems, Australia), with the following additions: 25 mM Hepes, 0.19% w/v, sodium bicarbonate, 0.03% w/v glutamine (Sigma Chemical Co, USA), 50IU/ml penicillin and 50 μg/ml streptomycin (Flow Laboratories). After 24 hours, IGF-I or tIGF-I was added to triplicate wells, at the concentrations indicated, in 0.5 ml fresh DMEM containing 0.02% v/v bovine serum albumin (Sigma molecular biology grade) and incubated for a further 21 hours. [3H]-Thymidine (0.1 μCi/well) was then added and the cells incubated for a further 3 hours. The medium was then aspirated and the cells washed once with ice-cold PBS and twice with ice-cold 10% v/v TCA. The TCA-precipitated monolayers were then solubilized with 0.25M NaOH (200 μl/well), transferred to scintillation vials and radioactivity determined by liquid scintillation counting (Pharmacia Wallac 1410 liquid scintillation counter).
EXAMPLE 3 HaCaT conditioned medium (250 μl) was concentrated by adding 750 μl cold ethanol, incubating at −20° C. for 2 hours and centrifuging at 16,000 g for 20 min at 4° C. The resulting pellet was air dried, resuspended thoroughly in non-reducing Laemmli sample buffer, heated to 90° C. for 5 minutes and separated on 12% w/v SDS-PAGE according to the method of Laemmli (1970).
Separated proteins were electrophoretically transferred to nitrocellulose membrane (0.45 mm, Schleicher and Schuell, Dassel, Germany) in a buffer containing 25 mM Tris, 192 mM glycine and 20% v/v methanol. IGFBPs were then visualised by the procedure of Hossenlopp et al [11], using [125I]-IGF-I, followed by autoradiography. Autoradiographs were scanned in a BioRad Model GS-670 Imaging Densitometer and band densities were determined using the Molecular Analyst program.
EXAMPLE 4 Phosphorothioate oligodeoxynucleotides were synthesised by Bresatec, Adelaide, South Australia, Australia. The following antisense sequences were used: BP3AS2, 5′-GCG CCC GCT GCA TGA CGC CTG CAA C-3′ (<400>4), a 25 mer complementary to the start codon region of the human IGFBP-3 mRNA; BP3AS3, 5′-CGG GCG GCT CAC CTG GAG CTG GCG-3′ (<400>5), a 24 mer complementary to the exon 1/intron 1 splice site; BP3AS4, 5′-AGG CGG CTG ACG GCA CTA-3′(<400>6), an 18 mer complementary to a region of the coding sequence lacking RNA secondary structure and oligonucleotide-dimer formation (using the computer software “OLIGO for PC”). Since BP3AS4 was found to be ineffective at inhibiting IGFBP-3 synthesis, it was used as a control. The following additional control oligonucleotide sequences were used: BP3S, 5′-CAG GCG TCA TGC AGC GGG C-3′ (<400>7), an 18 mer sense control sequence equivalent to the start codon region; BP3AS2NS, 5′-CGG AGA TGC CGC ATG CCA GCG CAG G-3′ (<400>8), a 25 mer randomised sequence with the same GC content as BP3AS2; BP3AS4NS, 5′-GAC AGC GTC GGA GCG ATC-3′ (<400>9), an 18 mer randomised sequence with the same GC content as BP3AS4NS. Design of the oligonucleotides was based on the human IGFBP-3 cDNA sequence of Spratt et al [12].
Cells were grown to one day post confluence in 2 cm2 wells with daily medium changes of 0.5 ml Keratinocyte-SFM, then subjected to daily medium changes of Keratinocyte-SFM for a further 4 days. Daily additions of 0.5 ml fresh Keratinocyte-SFM were then continued for a further 7 days, except that at the time of medium addition, 5 μl oligonucleotide in PBS was added to give the final concentrations indicated, then the wells were shaken to mix the oligonucleotide. After the final addition, cells were incubated for 24 hours and the medium collected for assay of IGFBPs. Cells were then counted after trypsinisation in a Coulter Industrial D Counter, Coulter Bedfordshire, UK. Cell numbers after oligonucleotide treatment differed by less than 10%.
EXAMPLE 5 HaCaT cells secrete mainly IGFBP-3 (>95%), with the only other IGFBP detectable in HaCaT conditioned medium being IGFBP-4 (<5%). The effect on IGFBP-3 and IGFBP-4 synthesis of antisense oligonucleotides at two concentrations, 5 μM and 0.5 μM, was tested. Two oligonucleotides were used, BP3AS2 and BP3AS3, directed against the start site and the intron 1/exon 1 splice site, respectively of the IGFBP-3 mRNA. As a control, a sense oligonucleotide corresponding to the start site was used. As shown in FIGS. 4A and 4B, all oligonucleotides at 5 μM caused a significant reduction of IGFBP-3 synthesis compared with untreated cells, however, the two antisense oligonucleotides inhibited IGFBP-3 synthesis of approximately 50% compared to the sense control (FIG. 4B). The antisense oligonucleotide directed to the start codon appeared to be more effective of the two, the difference being more apparent at the lower concentration of 0.5 μM. The cells of IGFBP-4 secreted by the HaCaT cells make photographic reproduction of the bands on Western ligand blots difficult, however densitometry measurements provide adequate relative quantitation. This resulted in the significant observation that IGFBP-4 levels were unaffected by oligonucleotide addition to the cells, suggesting that the observed inhibitory effects on IGFBP-3 are specific.
To further investigate the inhibitory effects of the more effective of the two antisense oligonucleotides, BP3AS2, inhibition by this oligonucleotide at 0.5 μM was compared with a number of control oligonucleotides, including one antisense oligonucleotide to IGFBP-3 that had proved to be ineffective at 0.5 μM. As shown in FIGS. 5A and 5B, BP3AS2 was again inhibitory, resulting in levels of IGFBP-3 of approximately 50% of the most non-specifically inhibitory control oligonucleotide, the randomised equivalent of BP3AS2. The other control oligonucleotides caused no reduction in IGFBP-3 levels at 0.5 μM, compared to untreated cells.
Of possible significance is the fact that this control oligonucleotide, BP3AS2NS, like BP3AS2 itself, has the highest potential Tm of the three control oligonucleotides used in this experiment, enhancing the probability of non-specific base pairing with non-target mRNAs. However, the lack of inhibition of IGFBP-4 secretion by BP3AS2 suggests that this oligonucleotide is selective even compared with the most closely related protein likely to be present in this cell line.
EXAMPLE 6 Antisense oligonucleotides to IGFBP2 may be selected from molecules capable of interacting with one or more of the following sense oligonucleotides:
ATTCGGGGCGAGGGA AGGAGGCGGCTCCCG CACCTGCCCGCCCGC
TTCGGGGCGAGGGAG GGAGGCGGCTCCCGC ACCTGCCCGCCCGCC
TCGGGGCGAGGGAGG GAGGCGGCTCCCGCT CCTGCCCGCCCGCCC
CGGGGCGAGGGAGGA AGGCGGCTCCCGCTC CTGCCCGCCCGCCCG
GGGGCGAGGGAGGAG GGCGGCTCCCGCTCG TGCCCGCCCGCCCGC
GGGCGAGGGAGGAGG GCGGCTCCCGCTCGC GCCCGCCCGCCCGCT
GGCGAGGGAGGAGGA CGGCTCCCGCTCGCA CCCGCCCGCCCGCTC
GCGAGGGAGGAGGAA GGCTCCCGCTCGCAG CCGCCCGCCCGCTCG
CGAGGGAGGAGGAAG GCTCCCGCTCGCAGG CGCCCGCCCGCTCGC
GAGGGAGGAGGAAGA CTCCCGCTCGCAGGG GCCCGCCCGCTCGCT
AGGGAGGAGGAAGAA TCCCGCTCGCAGGGC CCCGCCCGCTCGCTC
GGGAGGAGGAAGAAG CCCGCTCGCAGGGCC CCGCCCGCTCGCTCG
GGAGGAGGAAGAAGC CCGCTCGCAGGGCCG CGCCCGCTCGCTCGC
GAGGAGGAAGAAGCG CGCTCGCAGGGCCGT GCCCGCTCGCTCGCT
AGGAGGAAGAAGCGG GCTCGCAGGGCCGTG CCCGCTCGCTCGCTC
GGAGGAAGAAGCGGA CTCGCAGGGCCGTGC CCGCTCGCTCGCTCG
GAGGAAGAAGCGGAG TCGCAGGGCCGTGCA CGCTCGCTCGCTCGC
AGGAAGAAGCGGAGG CGCAGGGCCGTGCAC GCTCGCTCGCTCGCC
GGAAGAAGCGGAGGA GCAGGGCCGTGCACC CTCGCTCGCTCGCCC
GAAGAAGCGGAGGAG CAGGGCCGTGCACCT TCGCTCGCTCGCCCG
AAGAAGCGGAGGAGG AGGGCCGTGCACCTG CGCTCGCTCGCCCGC
AGAAGCGGAGGAGGC GGGCCGTGCACCTGC GCTCGCTCGCCCGCC
GAAGCGGAGGAGGCG GGCCGTGCACCTGCC CTCGCTCGCCCGCCG
AAGCGGAGGAGGCGG GCCGTGCACCTGCCC TCGCTCGCCCGCCGC
AGCGGAGGAGGCGGC CCGTGCACCTGCCCG CGCTCGCCCGCCGCG
GCGGAGGAGGCGGCT CGTGCACCTGCCCGC GCTCGCCCGCCGCGC
CGGAGGAGGCGGCTC GTGCACCTGCCCGCC CTCGCCCGCCGCGCC
GGAGGAGGCGGCTCC TGCACCTGCCCGCCC TCGCCCGCCGCGCCG
GAGGAGGCGGCTCCC GCACCTGCCCGCCCG CGCCCGCCGCGCCGC
GCCCGCCGCGCCGCG GGGCTGCCCCGCGCT TGCTGCCGCTGCTGC
CCCGCCGCGCCGCGC GGCTGCCCCGCGCTG GCTGCCGCTGCTGCT
CCGCCGCGCCGCGCT GCTGCCCCGCGCTGC CTGCCGCTGCTGCTG
CGCCGCGCCGCGCTG CTGCCCCGCGCTGCC TGCCGCTGCTGCTGC
GCCGCGCCGCGCTGC TGCCCCGCGCTGCCG GCCGCTGCTGCTGCT
CCGCGCCGCGCTGCC GCCCCGCGCTGCCGC CCGCTGCTGCTGCTG
CGCGCCGCGCTGCCG CCCCGCGCTGCCGCT CGCTGCTGCTGCTGC
GCGCCGCGCTGCCGA CCCGCGCTGCCGCTG GCTGCTGCTGCTGCT
CGCCGCGCTGCCGAC CCGCGCTGCCGCTGC CTGCTGCTGCTGCTA
GCCGCGCTGCCGACC CGCGCTGCCGCTGCC TGCTGCTGCTGCTAC
CCGCGCTGCCGACCG GCGCTGCCGCTGCCG GCTGCTGCTGCTACT
CGCGCTGCCGACCGC CGCTGCCGCTGCCGC CTGCTGCTGCTACTG
GCGCTGCCGACCGCC GCTGCCGCTGCCGCC TGCTGCTGCTACTGG
CGCTGCCGACCGCCA CTGCCGCTGCCGCCG GCTGCTGCTACTGGG
GCTGCCGACCGCCAG TGCCGCTGCCGCCGC CTGCTGCTACTGGGC
CTGCCGACCGCCAGC GCCGCTGCCGCCGCC TGCTGCTACTGGGCG
TGCCGACCGCCAGCA CCGCTGCCGCCGCCG GCTGCTACTGGGCGC
GCCGACCGCCAGCAT CGCTGCCGCCGCCGC CTGCTACTGGGCGCG
CCGACCGCCAGCATG GCTGCCGCCGCCGCC TGCTACTGGGCGCGA
CGACCGCCAGCATGC CTGCCGCCGCCGCCG GCTACTGGGCGCGAG
GACCGCCAGCATGCT TGCCGCCGCCGCCGC CTACTGGGCGCGAGT
ACCGCCAGCATGCTG GCCGCCGCCGCCGCT TACTGGGCGCGAGTG
CCGCCAGCATGCTGC CCGCCGCCGCCGCTG ACTGGGCGCGAGTGG
CGCCAGCATGCTGCC CGCCGCCGCCGCTGC CTGGGCGCGAGTGGC
GCCAGCATGCTGCCG GCCGCCGCCGCTGCT TGGGCGCGAGTGGCG
CCAGCATGCTGCCGA CCGCCGCCGCTGCTG GGGCGCGAGTGGCGG
CAGCATGCTGCCGAG CGCCGCCGCTGCTGC GGCGCGAGTGGCGGC
AGCATGCTGCCGAGA GCCGCCGCTGCTGCC GCGCGAGTGGCGGCG
GCATGCTGCCGAGAG CCGCCGCTGCTGCCG CGCGAGTGGCGGCGG
CATGCTGCCGAGAGT CGCCGCTGCTGCCGC GCGAGTGGCGGCGGC
ATGCTGCCGAGAGTG GCCGCTGCTGCCGCT CGAGTGGCGGCGGCG
TGCTGCCGAGAGTGG CCGCTGCTGCCGCTG GAGTGGCGGCGGCGG
GCTGCCGAGAGTGGG CGCTGCTGCCGCTGC AGTGGCGGCGGCGGC
CTGCCGAGAGTGGGC GCTGCTGCCGCTGCT GTGGCGGCGGCGGCG
TGCCGAGAGTGGGCT CTGCTGCCGCTGCTG TGGCGGCGGCGGCGG
GCCGAGAGTGGGCTG TGCTGCCGCTGCTGC GGCGGCGGCGGCGGG
CCGAGAGTGGGCTGC GCTGCCGCTGCTGCC GCGGCGGCGGCGGGG
CGAGAGTGGGCTGCC CTGCCGCTGCTGCCG CGGCGGCGGCGGGGC
GAGAGTGGGCTGCCC TGCCGCTGCTGCCGC GGCGGCGGCGGGGCG
AGAGTGGGCTGCCCC GCCGCTGCTGCCGCT GCGGCGGCGGGGCGC
GAGTGGGCTGCCCCG CCGCTGCTGCCGCTG CGGCGGCGGGGCGCG
AGTGGGCTGCCCCGC CGCTGCTGCCGCTGC GGCGGCGGGGCGCGC
GTGGGCTGCCCCGCG GCTGCTGCCGCTGCT GCGGCGGGGCGCGCG
TGGGCTGCCCCGCGC CTGCTGCCGCTGCTG CGGCGGGGCGCGCGC
GGCGGGGCGCGCGCG ACCCGAGCGCCTGGC CCGCCGCGGTGGCCG
GCGGGGCGCGCGCGG CCCGAGCGCCTGGCC CGCCGCGGTGGCCGC
CGGGGCGCGCGCGGA CCGAGCGCCTGGCCG GCCGCGGTGGCCGCA
GGGGCGCGCGCGGAG CGAGCGCCTGGCCGC CCGCGGTGGCCGCAG
GGGCGCGCGCGGAGG GAGCGCCTGGCCGCC CGCGGTGGCCGCAGT
GGCGCGCGCGGAGGT AGCGCCTGGCCGCCT GCGGTGGCCGCAGTG
GCGCGCGCGGAGGTG GCGCCTGGCCGCCTG CGGTGGCCGCAGTGG
CGCGCGCGGAGGTGC CGCCTGGCCGCCTGC GGTGGCCGCAGTGGC
GCGCGCGGAGGTGCT GCCTGGCCGCCTGCG GTGGCCGCAGTGGCC
CGCGCGGAGGTGCTG CCTGGCCGCCTGCGG TGGCCGCAGTGGCCG
GCGCGGAGGTGCTGT CTGGCCGCCTGCGGG GGCCGCAGTGGCCGG
CGCGGAGGTGCTGTT TGGCCGCCTGCGGGC GCCGCAGTGGCCGGA
GCGGAGGTGCTGTTC GGCCGCCTGCGGGCC CCGCAGTGGCCGGAG
CGGAGGTGCTGTTCC GCCGCCTGCGGGCCC CGCAGTGGCCGGAGG
GGAGGTGCTGTTCCG CCGCCTGCGGGCCCC GCAGTGGCCGGAGGC
GAGGTGCTGTTCCGC CGCCTGCGGGCCCCC CAGTGGCCGGAGGCG
AGGTGCTGTTCCGCT GCCTGCGGGCCCCCG AGTGGCCGGAGGCGC
GGTGCTGTTCCGCTG CCTGCGGGCCCCCGC GTGGCCGGAGGCGCC
GTGCTGTTCCGCTGC CTGCGGGCCCCCGCC TGGCCGGAGGCGCCC
TGCTGTTCCGCTGCC TGCGGGCCCCCGCCG GGCCGGAGGCGCCCG
GCTGTTCCGCTGCCC GCGGGCCCCCGCCGG GCCGGAGGCGCCCGC
CTGTTCCGCTGCCCG CGGGCCCCCGCCGGT CCGGAGGCGCCCGCA
TGTTCCGCTGCCCGC GGGCCCCCGCCGGTT CGGAGGCGCCCGCAT
GTTCCGCTGCCCGCC GGCCCCCGCCGGTTG GGAGGCGCCCGCATG
TTCCGCTGCCCGCCC GCCCCCGCCGGTTGC GAGGCGCCCGCATGC
TCCGCTGCCCGCCCT CCCCCGCCGGTTGCG AGGCGCCCGCATGCC
CCGCTGCCCGCCCTG CCCCGCCGGTTGCGC GGCGCCCGCATGCCA
CGCTGCCCGCCCTGC CCCGCCGGTTGCGCC GCGCCCGCATGCCAT
GCTGCCCGCCCTGCA CCCCCGGTTGCGCCG CGCCCGCATGCCATG
CTGCCCGCCCTGCAC CGCCGGTTGCGCCGC GCCCGCATGCCATGC
TGCCCGCCCTGCACA GCCGGTTGCGCCGCC CCCGCATGCCATGCG
GCCCGCCCTGCACAC CCGGTTGCGCCGCCC CCGCATGCCATGCGC
CCCGCCCTGCACACC CGGTTGCGCCGCCCG CGCATGCCATGCGCG
CCGCCCTGCACACCC GGTTGCGCCGCCCGC GCATGCCATGCGCGG
CGCCCTGCACACCCG GTTGCGCCGCCCGCC CATGCCATGCGCGGA
GCCCTGCACACCCGA TTGCGCCGCCCGCCG ATGCCATGCGCGGAG
CCCTGCACACCCGAG TGCGCCGCCCGCCGC TGCCATGCGCGGAGC
CCTGCACACCCGAGC GCGCCGCCCGCCGCG GCCATGCGCGGAGCT
CTGCACACCCGAGCG CGCCGCCCGCCGCGG CCATGCGCGGAGCTC
TGCACACCCGAGCGC GCCGCCCGCCGCGGT CATGCGCGGAGCTCG
GCACACCCGAGCGCC CCGCCCGCCGCGGTG ATGCGCGGAGCTCGT
CACACCCGAGCGCCT CGCCCGCCGCGGTGG TGCGCGGAGCTCGTC
ACACCCGAGCGCCTG GCCCGCCGCGGTGGC GCGCGGAGCTCGTCC
CACCCGAGCGCCTGG CCCGCCGCGGTGGCC CGCGGAGCTCGTCCG
GCGGAGCTCGTCCGG CGCCCGGCTGGAGGG GCGGCCAGGGGCTGC
CGGAGCTCGTCCGCG GCCCGGCTGGAGGGC CGGCCAGGGGCTGCG
GGAGCTCGTCCGGGA CCCGGCTGGAGGGCG GGCCAGGGGCTGCGC
GAGCTCGTCCGGGAG CCGGCTGGAGGGCGA GCCAGGGGCTGCGCT
AGCTCGTCCGGGAGC CGGCTGGAGGGCGAG CCAGGGGCTGCGCTG
GCTCGTCCGGGAGCC GGCTGGAGGGCGAGG CAGGGGCTGCGCTGC
CTCGTCCGGGAGCCG GCTGGAGGGCGAGGC AGGGGCTGCGCTGCT
TCGTCCGGGAGCCGG CTGGAGGGCGAGGCG GGGGCTGCGCTGCTA
CGTCCGGGAGCCGGG TGGAGGGCGAGGCGT GGGCTGCGCTGCTAT
GTCCGGGAGCCGGGC GGAGGGCGAGGCGTG GGCTGCGCTGCTATC
TCCGGGAGCCGGGCT GAGGGCGAGGCGTGC GCTGCGCTGCTATCC
CCGGGAGCCGGGCTG AGGGCGAGGCGTGCG CTGCGCTGCTATCCC
CGGGAGCCGGGCTGC GGGCGAGGCGTGCGG TGCGCTGCTATCCCC
GGGAGCCGGGCTGCG GGCGAGGCGTGCGGC GCGCTGCTATCCCCA
GGAGCCGGGCTGCGG GCGAGGCGTGCGGCG CGCTGCTATCCCCAC
GAGCCGGGCTGCGGC CGAGGCGTGCGGCGT GCTGCTATCCCCACC
AGCCGGGCTGCGGCT GAGGCGTGCGGCGTC CTGCTATCCCCACCC
GCCGGGCTGCGGCTG AGGCGTGCGGCGTCT TGCTATCCCCACCCG
CCGGGCTGCGGCTGC GGCGTGCGGCGTCTA GCTATCCCCACCCGG
CGGGCTGCGGCTGCT GCGTGCGGCGTCTAC CTATCCCCACCCGGG
GGGCTGCGGCTGCTG CGTGCGGCGTCTACA TATCCCCACCCGGGC
GGCTGCGGCTGCTGC GTGCGGCGTCTACAC ATCCCCACCCGGGCT
GCTGCGGCTGCTGCT TGCGGCGTCTACACC TCCCCACCCGGGCTC
CTGCGGCTGCTGCTC GCGGCGTCTACACCC CCCCACCCGGGCTCC
TGCGGCTGCTGCTCG CGGCGTCTACACCCC CCCACCCGGGCTCCG
GCGGCTGCTGCTCGG GGCGTCTACACCCCG CCACCCGGGCTCCGA
CGGCTGCTGCTCGGT GCGTCTACACCCCGC CACCCGGGCTCCGAG
GGCTGCTGCTCGGTG CGTCTACACCCCGCG ACCCGGGCTCCGAGC
GCTGCTGCTCGGTGT GTCTACACCCCGCGC CCCGGGCTCCGAGCT
CTGCTGCTCGGTGTG TCTACACCCCGCGCT CCGGGCTCCGAGCTG
TGCTGCTCGGTGTGC CTACACCCCGCGCTG CGGGCTCCGAGCTGC
GCTGCTCGGTGTGCG TACACCCCGCGCTGC GGGCTCCGAGCTGCC
CTGCTCGGTGTGCGC ACACCCCGCGCTGCG GGCTCCGAGCTGCCC
TGCTCGGTGTGCGCC CACCCCGCGCTGCGG GCTCCGAGCTGCCCC
GCTCGGTGTGCGCCC ACCCCGCGCTGCGGC CTCCGAGCTGCCCCT
CTCGGTGTGCGCCCG CCCCGCGCTGCGGCC TCCGAGCTGCCCCTG
TCGGTGTGCGCCCGG CCCGCGCTGCGGCCA CCGAGCTGCCCCTGC
CGGTGTGCGCCCGGC CCGCGCTGCGGCCAG CGAGCTGCCCCTGCA
GGTGTGCGCCCGGCT CGCGCTGCGGCCAGG GAGCTGCCCCTGCAG
GTGTGCGCCCGGCTG GCGCTGCGGCCAGGG AGCTGCCCCTGCAGG
TGTGCGCCCGGCTGG CGCTGCGGCCAGGGG GCTGCCCCTGCAGGC
GTGCGCCCGGCTGGA GCTGCGGCCAGGGGC CTGCCCCTGCAGGCG
TGCGCCCGGCTGGAG CTGCGGCCAGGGGCT TGCCCCTGCAGGCGC
GCGCCCGGCTGGAGG TGCGGCCAGGGGCTG GCCCCTGCAGGCGCT
CCCCTGCAGGCGCTG CCGGGACGCCGAGTA ATGGCGATGACCACT
CCCTGCAGGCGCTGG CGGGACGCCGAGTAT TGGCGATGACCACTC
CCTGCAGGCGCTGGT GGGACGCCGAGTATG GGCGATGACCACTCA
CTGCAGGCGCTGGTC GGACGCCGAGTATGG GCGATGACCACTCAG
TGCAGGCGCTGGTCA GACGCCGAGTATGGC CGATGACCACTCAGA
GCAGGCGCTGGTCAT ACGCCGAGTATGGCG GATGACCACTCAGAA
CAGGCGCTGGTCATG CGCCGAGTATGGCGC ATGACCACTCAGAAG
AGGCGCTGGTCATGG GCCGAGTATGGCGCC TGACCACTCAGAAGG
GGCGCTGGTCATGGG CCGAGTATGGCGCCA GACCACTCAGAAGGA
GCGCTGGTCATGGGC CGAGTATGGCGCCAG ACCACTCAGAAGGAG
CGCTGGTCATGGGCG GAGTATGGCGCCAGC CCACTCAGAAGGAGG
GCTGGTCATGGGCGA AGTATGGCGCCAGCC CACTCAGAAGGAGGC
CTGGTCATGGGCGAG GTATGGCGCCAGCCC ACTCAGAAGGAGGCC
TGGTCATGGGCGAGG TATGGCGCCAGCCCG CTCAGAAGGAGGCCT
GGTCATGGGCGAGGG ATGGCGCCAGCCCGG TCAGAAGGAGGCCTG
GTCATGGGCGAGGGC TGGCGCCAGCCCGGA CAGAAGGAGGCCTGG
TCATGGGCGAGGGCA GGCGCCAGCCCGGAG AGAAGGAGGCCTGGT
CATGGGCGAGGGCAC GCGCCAGCCCGGAGC GAAGGAGGCCTGGTG
ATGGGCGAGGGCACT CGCCAGCCCGGAGCA AAGGAGGCCTGGTGG
TGGGCGAGGGCACTT GCCAGCCCGGAGCAG AGGAGGCCTGGTGGA
GGGCGAGGGCACTTG CCAGCCCGGAGCAGG GGAGGCCTGGTGGAG
GGCGAGGGCACTTGT CAGCCCGGAGCAGGT GAGGCCTGGTGGAGA
GCGAGGGCACTTGTG AGCCCGGAGCAGGTT AGGCCTGGTGGAGAA
CGAGGGCACTTGTGA GCCCGGAGCAGGTTG GGCCTGGTGGAGAAC
GAGGGCACTTGTGAG CCCGGAGCAGGTTGC GCCTGGTGGAGAACC
AGGGCACTTGTGAGA CCGGAGCAGGTTGCA CCTGGTGGAGAACCA
GGGCACTTGTGAGAA CGGAGCAGGTTGCAG CTGGTGGAGAACCAC
GGCACTTGTGAGAAG GGAGCAGGTTGCAGA TGGTGGAGAACCACG
GCACTTGTGAGAAGC GAGCAGGTTGCAGAC GGTGGAGAACCACGT
CACTTGTGAGAAGCG AGCAGGTTGCAGACA GTGGAGAACCACGTG
ACTTGTGAGAAGCGC GCAGGTTGCAGACAA TGGAGAACCACGTGG
CTTGTGAGAAGCGCC CAGGTTGCAGACAAT GGAGAACCACGTGGA
TTGTGAGAAGCGCCG AGGTTGCAGACAATG GAGAACCACGTGGAC
TGTGAGAAGCGCCGG GGTTGCAGACAATGG AGAACCACGTGGACA
GTGAGAAGCGCCGGG GTTGCAGACAATGGC GAACCACGTGGACAG
TGAGAAGCGCCGGGA TTGCAGACAATGGCG AACCACGTGGACAGC
GAGAAGCGCCGGGAC TGCAGACAATGGCGA ACCACGTGGACAGCA
AGAAGCGCCGGGACG GCAGACAATGGCGAT CCACGTGGACAGCAC
GAAGCGCCGGGACGC CAGACAATGGCGATG CACGTGGACAGCACC
AAGCGCCGGGACGCC AGACAATGGCGATGA ACGTGGACAGCACCA
AGCGCCGGGACGCCG GACAATGGCGATGAC CGTGGACAGCACCAT
GCGCCGGGACGCCGA ACAATGGCGATGACC GTGGACAGCACCATG
CGCCGGGACGCCGAG CAATGGCGATGACCA TGGACAGCACCATGA
GCCGGGACGCCGAGT AATGGCGATGACCAC GGACAGCACCATGAA
GACAGCACCATGAAC GAAGCCCCTCAAGTC AGAAGGTCACTGAGC
ACAGCACCATGAACA AAGCCCCTCAAGTCG GAAGGTCACTGAGCA
CAGCACCATGAACAT AGCCCCTCAAGTCGG AAGGTCACTGAGCAG
AGCACCATGAACATG GCCCCTCAAGTCGGG AGGTCACTGAGCAGC
GCACCATGAACATGT CCCCTCAAGTCGGGT GGTCACTGAGCAGCA
CACCATGAACATGTT CCCTCAAGTCGGGTA GTCACTGAGCAGCAC
ACCATGAACATGTTG CCTCAAGTCGGGTAT TCACTGAGCAGCACC
CCATGAACATGTTGG CTCAAGTCGGGTATG CACTGAGCAGCACCG
CATGAACATGTTGGG TCAAGTCGGGTATGA ACTGAGCAGCACCGG
ATGAACATGTTGGGC CAAGTCGGGTATGAA CTGAGCAGCACCGGC
TGAACATGTTGGGCG AAGTCGGGTATGAAG TGAGCAGCACCGGCA
GAACATGTTGGGCGG AGTCGGGTATGAAGG GAGCAGCACCGGCAG
AACATGTTGGGCGGG GTCGGGTATGAAGGA AGCAGCACCGGCAGA
ACATGTTGGGCGGGG TCGGGTATGAAGGAG GCAGCACCGGCAGAT
CATGTTGGGCGGGGG CGGGTATGAAGGAGC CAGCACCGGCAGATG
ATGTTGGGCGGGGGA GGGTATGAAGGAGCT AGCACCGGCAGATGG
TGTTGGGCGGGGGAG GGTATGAAGGAGCTG GCACCGGCAGATGGG
GTTGGGCGGGGGAGG GTATGAAGGAGCTGG CACCGGCAGATGGGC
TTGGGCGGGGGAGGC TATGAAGGAGCTGGC ACCGGCAGATGGGCA
TGGGCGGGGGAGGCA ATGAAGGAGCTGGCC CCGGCAGATGGGCAA
GGGCGGGGGAGGCAG TGAAGGAGCTGGCCG CGGCAGATGGGCAAG
GGCGGGGGAGGCAGT GAAGGAGCTGGCCGT GGCAGATGGGCAAGG
GCGGGGGAGGCAGTG AAGGAGCTGGCCGTG GCAGATGGGCAAGGG
CGGGGGAGGCAGTGC AGGAGCTGGCCGTGT CAGATGGGCAAGGGT
GGGGGAGGCAGTGCT GGAGCTGGCCGTGTT AGATGGGCAAGGGTG
GGGGAGGCAGTGCTG GAGCTGGCCGTGTTC GATGGGCAAGGGTGG
GGGAGGCAGTGCTGG AGCTGGCCGTGTTCC ATGGGCAAGGGTGGC
GGAGGCAGTGCTGGC GCTGGCCGTGTTCCG TGGGCPAGGGTGGCA
GAGGCAGTGCTGGCC CTGGCCGTGTTCCGG GGGCAAGGGTGGCAA
AGGCAGTGCTGGCCG TGGCCGTGTTCCGGG GGCAAGGGTGGCAAG
GGCAGTGCTGGCCGG GGCCGTGTTCCGGGA GCAAGGGTGGCAAGC
GCAGTGCTGGCCGGA GCCGTGTTCCGGGAG CAAGGGTGGCAAGCA
CAGTGCTGGCCGGAA CCGTGTTCCGGGAGA AAGGGTGGCAAGCAT
AGTGCTGGCCGGAAG CGTGTTCCGGGAGAA AGGGTGGCAAGCATC
GTGCTGGCCGGAAGC GTGTTCCGGGAGAAG GGGTGGCAAGCATCA
TGCTGGCCGGAAGCC TGTTCCGGGAGAAGG GGTGGCAAGCATCAC
GCTGGCCGGAAGCCC GTTCCGGGAGAAGGT GTGGCAAGCATCACC
CTGGCCGGAAGCCCC TTCCGGGAGAAGGTC TGGCAAGCATCACCT
TGGCCGGAAGCCCCT TCCGGGAGAAGGTCA GGCAAGCATCACCTT
GGCCGGAAGCCCCTC CCGGGAGAAGGTCAC GCAAGCATCACCTTG
GCCGGAAGCCCCTCA CGGGAGAAGGTCACT CAAGCATCACCTTGG
CCGGAAGCCCCTCAA GGGAGAAGGTCACTG AAGCATCACCTTGGC
CGGAAGCCCCTCAAG GGAGAAGGTCACTGA AGCATCACCTTGGCC
GGAAGCCCCTCAAGT GAGAAGGTCACTGAG GCATCACCTTGGCCT
CATCACCTTGGCCTG TGCCAGGACTCCCTG GGATCTCCACCATGC
ATCACCTTGGCCTGG GCCAGGACTCCCTGC GATCTCCACCATGCG
TCACCTTGGCCTCGA CCAGGACTCCCTGCC ATCTCCACCATGCGC
CACCTTGGCCTGGAG CAGGACTCCCTGCCA TCTCCACCATGCGCC
ACCTTGGCCTGGAGG AGGACTCCCTGCCAA CTCCACCATGCGCCT
CCTTGGCCTGGAGGA GGACTCCCTGCCAAC TCCACCATGCGCCTT
CTTGGCCTGGAGGAG GACTCCCTGCCAACA CCACCATGCGCCTTC
TTGGCCTGGAGGAGC ACTCCCTGCCAACAG CACCATGCGCCTTCC
TGGCCTGGAGGAGCC CTCCCTGCCAACAGG ACCATGCGCCTTCCG
GGCCTGGAGGAGCCC TGCCTGCCAACAGGA GGATGCGCCTTCCGG
GCCTGGAGGACCCCA GCCTGCCAACAGGAA CATGCGCCTTCCGGA
CCTGGAGGAGCCCAA CCTGCCAACAGGAAC ATGCGCCTTCCGGAT
CTGGAGGAGCCCAAG CTGCCAACAGGAACT TGCGCCTTCCGGATG
TGGAGGAGCCCAAGA TGCCAACAGGAACTG GCGCCTTCCGGATGA
GGAGGAGCCCAAGAA GCCAACAGGAACTGG CGCCTTCCGGATGAG
GAGGAGCCCAAGAAG CCAACAGGAACTGGA GCCTTCCGGATGAGC
AGGAGCCCAAGAAGC CAACAGGAACTGGAC CCTTCCGGATGAGCG
GGAGCCCAAGAAGCT AACAGGAACTGGACC CTTCCGGATGAGCGG
GAGCCCAAGAAGCTG ACAGGAACTGGACCA TTCCGGATGAGCGGG
AGCCCAAGAAGCTGC CAGGAACTGGACCAG TCCGGATGAGCGGGG
GCCCAAGAAGCTGCG AGGAACTGGACCAGG CCGGATGAGCGGGGC
CCCAAGAAGCTGCGA GGAACTGGACCAGGT CGGATGAGCGGGGCC
CCAAGAAGCTGCGAC GAACTGGACCAGGTC GGATGAGCGGGGCCC
CAAGAAGCTGCGACC AACTGGACCAGGTCC GATGAGCGGGGCCCT
AAGAAGCTGCGACCA ACTGGACCAGGTCCT ATGAGCGGGGCCCTC
AGAAGCTGCGACCAC CTGGACCAGGTCCTG TGAGCGGGGCCCTCT
GAAGCTGCGACCACC TGGACCAGGTCCTGG GAGCGGGGCCCTCTG
AAGCTGCGACCACCC GGACCAGGTCCTGGA AGCGGGGCCCTCTGG
AGCTGCGACCACCCC GACCAGGTCCTGGAG GCGGGGCCCTCTGGA
GCTGCGACCACCCCC ACCAGGTCCTGGAGC CGGGGCCCTCTGGAG
CTGCGACCACCCCCT CCAGGTCCTGGAGCG GGGGCCCTCTGGAGC
TGCGACCACCCCCTG CAGGTCCTGGAGCGG GGGCCCTCTGGAGCA
GCGACCACCCCCTGC AGGTCCTGGAGCGGA GGCCCTCTGGAGCAC
CGACCACCCCCTGCC GGTCCTGGAGCGGAT GCCCTCTGGAGCACC
GACCACCCCCTGCCA GTCCTGGAGCGGATC CCCTCTGGAGCACCT
ACCACCCCCTGCCAG TCCTGGAGCGGATCT CCTCTGGAGCACCTC
CCACCCCCTGCCAGG CCTGGAGCGGATCTC CTCTGGAGCACCTCT
CACCCCCTGCCAGGA CTGGAGCGGATCTCC TCTGGAGCACCTCTA
ACCCCCTGCCAGGAC TGGAGCGGATCTCCA CTGGAGCACCTCTAC
CCCCCTGCCAGGACT GGAGCGGATCTCCAC TGGAGCACCTCTACT
CCCCTGCCAGGACTC GAGCGGATCTCCACC GGAGCACCTCTACTC
CCCTGCCAGGACTCC AGCGGATCTCCACCA GAGCACCTCTACTCC
CCTGCCAGGACTCCC GCGGATCTCCACCAT AGCACCTCTACTCCC
CTGCCAGGACTCCCT CGGATCTCCACCATG GCACCTCTACTCCCT
CACCTCTACTCCCTG GTACAACCTCAAACA GGGAGTGCTGGTGTG
ACCTCTACTCCCTGC TACAACCTCAAACAG GGAGTGCTGGTGTGT
CCTCTACTCCCTGCA ACAACCTCAAACAGT GAGTGCTGGTGTGTG
CTCTACTCCCTGCAC CAACCTCAAACAGTG AGTGCTGGTGTGTGA
TCTACTCCCTGCACA AACCTCAAACAGTGC GTGCTGGTGTGTGAA
CTACTCCCTGCACAT ACCTCAAACAGTGCA TGCTGGTGTGTGAAC
TACTCCCTGCACATC CCTCAAACAGTGCAA GCTGGTGTGTGAACC
ACTCCCTGCACATCC CTCAAACAGTGCAAG CTGGTGTGTGAACCC
CTCCCTGCACATCCC TCAAACAGTGCAAGA TGGTGTGTGAACCCC
TCCCTGCACATCCCC CAAACAGTGCAAGAT GGTGTGTGAACCCCA
CCCTGCACATCCCCA AAACAGTGCAAGATG GTGTGTGAACCCCAA
CCTGCACATCCCCAA AACAGTGCAAGATGT TGTGTGAACCCCAAC
CTGCACATCCCCAAC ACAGTGCAAGATGTC GTGTGAACCCCAACA
TGCACATCCCCAACT CAGTGCAAGATGTCT TGTGAACCCCAACAC
GCACATCCCCAACTG AGTGCAAGATGTCTC GTGAACCCCAACACC
CACATCCCCAACTGT GTGCAAGATGTCTCT TGAACCCCAACACCG
ACATCCCCAACTGTG TGCAAGATGTCTCTG GAACCCCAACACCGG
CATCCCCAACTGTGA GCAAGATGTCTCTGA AACCCCAACACCGGG
ATCCCCAACTGTGAC CAAGATGTCTCTGAA ACCCCAACACCGGGA
TCCCCAACTGTGACA AAGATGTCTCTGAAC CCCCAACACCGGGAA
CCCCAACTGTGACAA AGATGTCTCTGAACG CCCAACACCGGGAAG
CCCAACTGTGACAAG GATGTCTCTGAACGG CCAACACCGGGAAGC
CCAACTGTGACAAGC ATGTCTCTGAACGGG CAACACCGGGAAGCT
CAACTGTGACAAGCA TGTCTCTGAACGGGC AACACCGGGAAGCTG
AACTGTGACAAGCAT GTCTCTGAACGGGCA ACACCGGGAAGCTGA
ACTGTGACAAGCATG TCTCTGAACGGGCAG CACCGGGAAGCTGAT
CTGTGACAAGCATGG CTCTGAACGGGCAGC ACCGGGAAGCTGATC
TGTGACAAGCATGGC TCTGAACGGGCAGCG CCGGGAAGCTGATCC
GTGACAAGCATGGCC CTGAACGGGCAGCGT CGGGAAGCTGATCCA
TGACAAGCATGGCCT TGAACGGGCAGCGTG GGGAAGCTGATCCAG
GACAAGCATGGCCTG GAACGGGCAGCGTGG GGAAGCTGATCCAGG
ACAAGCATGGCCTGT AACGGGCAGCGTGGG GAAGCTGATCCAGGG
CAAGCATGGCCTGTA ACGGGCAGCGTGGGG AAGCTGATCCAGGGA
AAGCATGGCCTGTAC CGGGCAGCGTGGGGA AGCTGATCCAGGGAG
AGCATGGCCTGTACA GGGCAGCGTGGGGAG GCTGATCCAGGGAGC
GCATGGCCTGTACAA GGCAGCGTGGGGAGT CTGATCCAGGGAGCC
CATGGCCTGTACAAC GCAGCGTGGGGAGTG TGATCCAGGGAGCCC
ATGGCCTGTACAACC CAGCGTGGGGAGTGC GATCCAGGGAGCCCC
TGGCCTGTACAACCT AGCGTGGGGAGTGCT ATCCAGGGAGCCCCC
GGCCTGTACAACCTC GCGTGGGGAGTGCTG TCCAGGGAGCCCCCA
GCCTGTACAACCTCA CGTGGGGAGTGCTGG CCAGGGAGCCCCCAC
CCTGTACAACCTCAA GTGGGGAGTGCTGGT CAGGGAGCCCCCACC
CTGTACAACCTCAAA TGGGGAGTGCTGGTG AGGGAGCCCCCACCA
TGTACAACCTCAAAC GGGGAGTGCTGGTGT GGGAGCCCCCACCAT
GGAGCCCCCACCATC CAATGAGCAGCAGGA AGTAGACCGCAGCCA
GAGCCCCCACCATCC AATGAGCAGCAGGAG GTAGACCGCAGCCAG
AGCCCCCACCATCCG ATGAGCAGCAGGAGG TAGACCGCAGCCAGC
GCCCCCACCATCCGG TGAGCAGCAGGAGGC AGACCGCAGCCAGCC
CCCCCACCATCCGGG GAGCAGCAGGAGGCT GACCGCAGCCAGCCG
CCCCACCATCCGGGG AGCAGCAGGAGGCTT ACCGCAGCCAGCCGG
CCCACCATCCGGGGG GCAGCAGGAGGCTTG CCGCAGCCAGCCGGT
CCACCATCCGGGGGG CAGCAGGAGGCTTGC CGCAGCCAGCCGGTG
CACCATCCGGGGGGA AGCAGGAGGCTTGCG GCAGCCAGCCGGTGC
ACCATCCGGGGGGAC GCAGGAGGCTTGCGG CAGCCAGCCGGTGCC
CCATCCGGGGGGACC CAGGAGGCTTGCGGG AGCCAGCCGGTGCCT
CATCCGGGGGGACCC AGGAGGCTTGCGGGG GCCAGCCGGTGCCTG
ATCCGGGGGGACCCC GGAGGCTTGCGGGGT CCAGCCGGTGCCTGG
TCCGGGGGGACCCCG GAGGCTTGCGGGGTG CAGCCGGTGCCTGGC
CCGGGGGGACCCCGA AGGCTTGCGGGGTGC AGCCGGTGCCTGGCG
CGGGGGGACCCCGAG GGCTTGCGGGGTGCA GCCGGTGCCTGGCGC
GGGGGGACCCCGAGT GCTTGCGGGGTGCAC CCGGTGCCTGGCGCC
GGGGGACCCCGAGTG CTTGCGGGGTGCACA CGGTGCCTGGCGCCC
GGGGACCCCGAGTGT TTGCGGGGTGCACAC GGTGCCTGGCGCCCC
GGGACCCCGAGTGTC TGCGGGGTGCACACC GTGCCTGGCGCCCCT
GGACCCCGAGTGTCA GCGGGGTGCACACCC TGCCTGGCGCCCCTG
GACCCCGAGTGTCAT CGGGGTGCACACCCA GCCTGGCGCCCCTGC
ACCCCGAGTGTCATC GGGGTGCACACCCAG CCTGGCGCCCCTGCC
CCCCGAGTGTCATCT GGGTGCACACCCAGC CTGGCGCCCCTGCCC
CCCGAGTGTCATCTC GGTGCACACCCAGCG TGGCGCCCCTGCCCC
CCGAGTGTCATCTCT GTGCACACCCAGCGG GGCGCCCCTGCCCCC
CGAGTGTCATCTCTT TGCACACCCAGCGGA GCGCCCCTGCCCCCC
GAGTGTCATCTCTTC GCACACCCAGCGGAT CGCCCCTGCCCCCCG
AGTGTCATCTCTTCT CACACCCAGCGGATG GCCCCTGCCCCCCGC
GTGTCATCTCTTCTA ACACCCAGCGGATGC CCCCTGCCCCCCGCC
TGTCATCTCTTCTAC CACCCAGCGGATGCA CCCTGCCCCCCGCCC
GTCATCTCTTCTACA ACCCAGCGGATGCAG CCTGCCCCCCGCCCC
TCATCTCTTCTACAA CCCAGCGGATGCAGT CTGCCCCCCGCCCCT
CATCTCTTCTACAAT CCAGCGGATGCAGTA TGCCCCCCGCCCCTC
ATCTCTTCTACAATG CAGCGGATGCAGTAG GCCCCCCGCCCCTCT
TCTCTTCTACAATGA AGCGGATGCAGTAGA CCCCCCGCCCCTCTC
CTCTTCTACAATGAG GCGGATGCAGTAGAC CCCCCGCCCCTCTCC
TCTTCTACAATGAGC CGGATGCAGTAGACC CCCCGCCCCTCTCCA
CTTCTACAATGAGCA GGATGCAGTAGACCG CCCGCCCCTCTCCAA
TTCTACAATGAGCAG GATGCAGTAGACCGC CCGCCCCTCTCCAAA
TCTACAATGAGCAGC ATGCAGTAGACCGCA CGCCCCTCTCCAAAC
CTACAATGAGCAGCA TGCAGTAGACCGCAG GCCCCTCTCCAAACA
TACAATGAGCAGCAG GCAGTAGACCGCAGC CCCCTCTCCAAACAC
ACAATGAGCAGCAGG CAGTAGACCGCAGCC CCCTCTCCAAACACC
CCTCTCCAAACACCG GTGCTGGAGGATTTT AAAGAGACCAGCACC
CTCTCCAAACACCGG TGCTGGAGGATTTTC AAGAGACCAGCACCG
TCTCCAAACACCGGC GCTGGAGGATTTTCC AGAGACCAGCACCGA
CTCCAAACACCGGCA CTGGAGGATTTTCCA GAGACCAGCACCGAG
TCCAAACACCGGCAG TGGAGGATTTTCCAG AGACCAGCACCGAGC
CCAAACACCGGCAGA GGAGGATTTTCCAGT GACCAGCACCGAGCT
CAAACACCGGCAGAA GAGGATTTTCCAGTT ACCAGCACCGAGCTC
AAACACCGGCAGAAA AGGATTTTCCAGTTC CCAGCACCGAGCTCG
AACACCGGCAGAAAA GGATTTTCCAGTTCT CAGCACCGAGCTCGG
ACACCGGCAGAAAAC GATTTTCCAGTTCTG AGCACCGAGCTCGGC
CACCGGCAGAAAACG ATTTTCCAGTTCTGA GCACCGAGCTCGGCA
ACCGGCAGAAAACGG TTTTCCAGTTCTGAC CACCGAGCTCGGCAC
CCGGCAGAAAACGGA TTTCCAGTTCTGACA ACCGAGCTCGGCACC
CGGCAGAAAACGGAG TTCCAGTTCTGACAC CCGAGCTCGGCACCT
GGCAGAAAACGGAGA TCCAGTTCTGACACA CGAGCTCGGCACCTC
GCAGAAAACGGAGAG CCAGTTCTGACACAC GAGCTCGGCACCTCC
CAGAAAACGGAGAGT CAGTTCTGACACACG AGCTCGGCACCTCCC
AGAAAACGGAGAGTG AGTTCTGACACACGT GCTCGGCACCTCCCC
GAAAACGGAGAGTGC GTTCTGACACACGTA CTCGGCACCTCCCCG
AAAACGGAGAGTGCT TTCTGACACACGTAT TCGGCACCTCCCCGG
AAACGGAGAGTGCTT TCTGACACACGTATT CGGCACCTCCCCGGC
AACGGAGAGTGCTTG CTGACACACGTATTT GGCACCTCCCCGGCC
ACGGAGAGTGCTTGG TGACACACGTATTTA GCACCTCCCCGGCCT
CGGAGAGTGCTTGGG GACACACGTATTTAT CACCTCCCCGGCCTC
GGAGAGTGCTTGGGT ACACACGTATTTATA ACCTCCCCGGCCTCT
GAGAGTGCTTGGGTG CACACGTATTTATAT CCTCCCCGGCCTCTC
AGAGTGCTTGGGTGG ACACGTATTTATATT CTCCCCGGCCTCTCT
GAGTGCTTGGGTGGT CACGTATTTATATTT TCCCCGGCCTCTCTC
AGTGCTTGGGTGGTG ACGTATTTATATTTG CCCCGGCCTCTCTCT
GTGCTTGGGTGGTGG CGTATTTATATTTGG CCCGGCCTCTCTCTT
TGCTTGGGTGGTGGG GTATTTATATTTGGA CCGGCCTCTCTCTTC
GCTTGGGTGGTGGGT TATTTATATTTGGAA CGGCCTCTCTCTTCC
CTTGGGTGGTGGGTG ATTTATATTTGGAAA GGCCTCTCTCTTCCC
TTGGGTGGTGGGTGC TTTATATTTGGAAAG GCCTCTCTCTTCCCA
TGGGTGGTGGGTGCT TTATATTTGGAAAGA CCTCTCTCTTCCCAG
GGGTGGTGGGTGCTG TATATTTGGAAAGAG CTCTCTCTTCCCAGC
GGTGGTGGGTGCTGG ATATTTGGAAAGAGA TCTCTCTTCCCAGCT
GTGGTGGGTGCTGGA TATTTGGAAAGAGAC CTCTCTTCCCAGCTG
TGGTGGGTGCTGGAG ATTTGGAAAGAGACC TCTCTTCCCAGCTGC
GGTGGGTGCTGGAGG TTTGGAAAGAGACCA CTCTTCCCAGCTGCA
GTGGGTGCTGGAGGA TTGGAAAGAGACCAG TCTTCCCAGCTGCAG
TGGGTGCTGGAGGAT TGGAAAGAGACCAGC CTTCCCAGCTGCAGA
GGGTGCTGGAGGATT GGAAAGAGACCAGCA TTCCCAGCTGCAGAT
GGTGCTGGAGGATTT GAAAGAGACCAGCAC TCCCAGCTGCAGATG
CCCAGCTGCAGATGC GGAGGAAGGGGGTTG GAGGGGGAAGAGAAA
CCAGCTGCAGATGCC GAGGAAGGGGGTTGT AGGGGGAAGAGAAAT
CAGCTGCAGATGCCA AGGAAGGGGGTTGTG GGGGGAAGAGAAATT
AGCTGCAGATGCCAC GGAAGGGGGTTGTGG GGGGAAGAGAAATTT
GCTGCAGATGCCACA GAAGGGGGTTGTGGT GGGAAGAGAAATTTT
CTGCAGATGCCACAC AAGGGGGTTGTGGTC GGAAGAGAAATTTTT
TGCAGATGCCACACC AGGGGGTTGTGGTCG GAAGAGAAATTTTTA
GCAGATGCCACACCT GGGGGTTGTGGTCGG AAGAGAAATTTTTAT
CAGATGCCACACCTG GGGGTTGTGGTCGGG AGAGAAATTTTTATT
AGATGCCACACCTGC GGGTTGTGGTCGGGG GAGAAATTTTTATTT
GATGCCACACCTGCT GGTTGTGGTCGGGGA AGAAATTTTTATTTT
ATGCCACACCTGCTC GTTGTGGTCGGGGAG GAAATTTTTATTTTT
TGCCACACCTGCTCC TTGTGGTCGGGGAGC AAATTTTTATTTTTG
GCCACACCTGCTCCT TGTGGTCGGGGAGCT AATTTTTATTTTTGA
CCACACCTGCTCCTT GTGGTCGGGGAGCTG ATTTTTATTTTTGAA
CACACCTGCTCCTTC TGGTCGGGGAGCTGG TTTTTATTTTTGAAC
ACACCTGCTCCTTCT GGTCGGGGAGCTGGG TTTTATTTTTGAACC
CACCTGCTCCTTCTT GTCGGGGAGCTGGGG TTTATTTTTGAACCC
ACCTGCTCCTTCTTG TCGGGGAGCTGGGGT TTATTTTTGAACCCC
CCTGCTCCTTCTTGC CGGGGAGCTGGGGTA TATTTTTGAACCCCT
CTGCTCCTTCTTGCT GGGGAGCTGGGGTAC ATTTTTGAACCCCTG
TGCTCCTTCTTGCTT GGGAGCTGGGGTACA TTTTTGAACCCCTGT
GCTCCTTCTTGCTTT GGAGCTGGGGTACAG TTTTGAACCCCTGTG
CTCCTTCTTGCTTTC GAGCTGGGGTACAGG TTTGAACCCCTGTGT
TCCTTCTTGCTTTCC AGCTGGGGTACAGGT TTGAACCCCTGTGTC
CCTTCTTGCTTTCCC GCTGGGGTACAGGTT TGAACCCCTGTGTCC
CTTCTTGCTTTCCCC CTGGGGTACAGGTTT GAACCCCTGTGTCCC
TTCTTGCTTTCCCCG TGGGGTACAGGTTTG AACCCCTGTGTCCCT
TCTTGCTTTCCCCGG GGGGTACAGGTTTGG ACCCCTGTGTCCCTT
CTTGCTTTCCCCGGG GGGTACAGGTTTGGG CCCCTGTGTCCCTTT
TTGCTTTCCCCGGGG GGTACAGGTTTGGGG CCCTGTGTCCCTTTT
TGCTTTCCCCGGGGG GTACAGGTTTGGGGA CCTGTGTCCCTTTTG
GCTTTCCCCGGGGGA TACAGGTTTGGGGAG CTGTGTCCCTTTTGC
CTTTCCCCGGGGGAG ACAGGTTTGGGGAGG TGTGTCCCTTTTGCA
TTTCCCCGGGGGAGG CAGGTTTGGGGAGGG GTGTCCCTTTTGCAT
TTCCCCGGGGGAGGA AGGTTTGGGGAGGGG TGTCCCTTTTGCATA
TCCCCGGGGGAGGAA GGTTTGGGGAGGGGG GTCCCTTTTGCATAA
CCCCGGGGGAGGAAG GTTTGGGGAGGGGGA TCCCTTTTGCATAAG
CCCGGGGGAGGAAGG TTTGGGGAGGGGGAA CCCTTTTGCATAAGA
CCGGGGGAGGAAGGG TTGGGGAGGGGGAAG CCTTTTGCATAAGAT
CGGGGGAGGAAGGGG TGGGGAGGGGGAAGA CTTTTGCATAAGATT
GGGGGAGGAAGGGGG GGGGAGGGGGAAGAG TTTTGCATAAGATTA
GGGGAGGAAGGGGGT GGGAGGGGGAAGAGA TTTGCATAAGATTAA
GGGAGGAAGGGGGTT GGAGGGGGAAGAGAA TTGCATAAGATTAAA
TGCATAAGATTAAAG
GCATAAGATTAAAGG
CATAAGATTAAAGGA
ATAAGATTAAAGGAA
TAAGATTAAAGGAAG
AAGATTAAAGGAAGG
AGATTAAAGGAAGGA
GATTAAAGGAAGGAA
ATTAAAGGAAGGAAA
TTAAAGGAAGGAAAA
TAAAGGAAGGAAAAG
AAAGGAAGGAAAAGT
EXAMPLE 7 Antisense oligonucleotides to IGFBP3 may be selected from molecules capable of interacting with one or more of the following sense oligonucleotides:
CTCAGCGCCCAGCCG TGGATTCCACAGCTT TACTGTCGCCCCATC
TCAGCGCCCAGCCGC GGATTCCACAGCTTC ACTGTCGCCCCATCC
CAGCGCCCAGCCGCT GATTCCACAGCTTCG CTGTCGCCCCATCCC
AGCGCCCAGCCGCTT ATTCCACAGCTTCGC TGTCGCCCCATCCCT
GCGCCCAGCCGCTTC TTCCACAGCTTCGCG GTCGCCCCATCCCTG
CGCCCAGCCGCTTCC TCCACAGCTTCGCGC TCGCCCCATCCCTGC
GCCCAGCCGCTTCCT CCACAGCTTCGCGCC CGCCCCATCCCTGCG
CCCAGCCGCTTCCTG CACAGCTTCGCGCCG GCCCCATCCCTGCGC
CCAGCCGCTTCCTGC ACAGCTTCGCGCCGT CCCCATCCCTGCGCG
CAGCCGCTTCCTGCC CAGCTTCGCGCCGTG CCCATCCCTGCGCGC
AGCCGCTTCCTGCCT AGCTTCGCGCCGTGT CCATCCCTGCGCGCC
GCCGCTTCCTGCCTG GCTTCGCGCCGTGTA CATCCCTGCGCGCCC
CCGCTTCCTGCCTGG CTTCGCGCCGTGTAC ATCCCTGCGCGCCCA
CGCTTCCTGCCTGGA TTCGCGCCGTGTACT TCCCTGCGCGCCCAG
GCTTCCTGCCTGGAT TCGCGCCGTGTACTG CCCTGCGCGCCCAGC
CTTCCTGCCTGGATT CGCGCCGTGTACTGT CCTGCGCGCCCAGCC
TTCCTGCCTGGATTC GCGCCGTGTACTGTC CTGCGCGCCCAGCCT
TCCTGCCTGGATTCC CGCCGTGTACTGTCG TGCGCGCCCAGCCTG
CCTGCCTGGATTCCA GCCGTGTACTGTCGC GCGCGCCCAGCCTGC
CTGCCTGGATTCCAC CCGTGTACTGTCGCC CGCGCCCAGCCTGCC
TGCCTGGATTCCACA CGTGTACTGTCGCCC GCGCCCAGCCTGCCA
GCCTGGATTCCACAG GTGTACTGTCGCCCC CGCCCAGCCTGCCAA
CCTGGATTCCACAGC TGTACTGTCGCCCCA GCCCAGCCTGCCAAG
CTGGATTCCACAGCT GTACTGTCGCCCCAT CCCAGCCTGCCAAGC
CCAGCCTGCCAAGCA GGGCGCGACCCACGC CTGCTCCGCGGGCCG
CAGCCTGCCAAGCAG GGCGCGACCCACGCT TGCTCCGCGGGCCGC
AGCCTGCCAAGCAGC GCGCGACCCACGCTC GCTCCGCGGGCCGCC
GCCTGCCAAGCAGCG CGCGACCCACGCTCT CTCCGCGGGCCGCCG
CCTGCCAAGCAGCGT GCGACCCACGCTCTG TCCGCGGGCCGCCGG
CTGCCAAGCAGCGTG CGACCCACGCTCTGG CCGCGGGCCGCCGGT
TGCCAAGCAGCGTGC GACCCACGCTCTGGG CGCGGGCCGCCGGTG
GCCAAGCAGCGTGCC ACCCACGCTCTGGGC GCGGGCCGCCGGTGG
CCAAGCAGCGTGCCC CCCACGCTCTGGGCC CGGGCCGCCGGTGGC
CAAGCAGCGTGCCCC CCACGCTCTGGGCCG GGGCCGCCGGTGGCG
AAGCAGCGTGCCCCG CACGCTCTGGGCCGC GGCCGCCGGTGGCGC
AGCAGCGTGCCCCGG ACGCTCTGGGCCGCT GCCGCCGGTGGCGCG
GCAGCGTGCCCCGGT CGCTCTGGGCCGCTG CCGCCGGTGGCGCGG
CAGCGTGCCCCGGTT GCTCTGGGCCGCTGC CGCCGGTGGCGCGGG
AGCGTGCCCCGGTTG CTCTGGGCCGCTGCG GCCGGTGGCGCGGGC
GCGTGCCCCGGTTGC TCTGGGCCGCTGCGC CCGGTGGCGCGGGCT
CGTGCCCCGGTTGCA CTGGGCCGCTGCGCT CGGTGGCGCGGGCTG
GTGCCCCGGTTGCAG TGGGCCGCTGCGCTG GGTGGCGCGGGCTGG
TGCCCCGGTTGCAGG GGGCCGCTGCGCTGA GTGGCGCGGGCTGGC
GCCCCGGTTGCAGGC GGCCGCTGCGCTGAC TGGCGCGGGCTGGCG
CCCCGGTTGCAGGCG GCCGCTGCGCTGACT GGCGCGGGCTGGCGC
CCCGGTTGCAGGCGT CCGCTGCGCTGACTC GCGCGGGCTGGCGCG
CCGGTTGCAGGCGTC CGCTGCGCTGACTCT CGCGGGCTGGCGCGA
CGGTTGCAGGCGTCA GCTGCGCTGACTCTG GCGGGCTGGCGCGAG
GGTTGCAGGCGTCAT CTGCGCTGACTCTGC CGGGCTGGCGCGAGC
GTTGCAGGCGTCATG TGCGCTGACTCTGCT GGGCTGGCGCGAGCT
TTGCAGGCGTCATGC GCGCTGACTCTGCTG GGCTGGCGCGAGCTC
TGCAGGCGTCATGCA CGCTGACTCTGCTGG GCTGGCGCGAGCTCG
GCAGGCGTCATGCAG GCTGACTCTGCTGGT CTGGCGCGAGCTCGG
CAGGCGTCATGCAGC CTGACTCTGCTGGTG TGGCGCGAGCTCGGG
AGGCGTCATGCAGCG TGACTCTGCTGGTGC GGCGCGAGCTCGGGG
GGCGTCATGCAGCGG GACTCTGCTGGTGCT GCGCGAGCTCGGGGG
GCGTCATGCAGCGGG ACTCTGCTGGTGCTG CGCGAGCTCGGGGGG
CGTCATGCAGCGGGC CTCTGCTGGTGCTGC GCGAGCTCGGGGGGC
GTCATGCAGCGGGCG TCTGCTGGTGCTGCT CGAGCTCGGGGGGCT
TCATGCAGCGGGCGC CTGCTGGTGCTGCTC GAGCTCGGGGGGCTT
CATGCAGCGGGCGCG TGCTGGTGCTGCTCC AGCTCGGGGGGCTTG
ATGCAGCGGGCGCGA GCTGGTGCTGCTCCG GCTCGGGGGGCTTGG
TGCAGCGGGCGCGAC CTGGTGCTGCTCCGC CTCGGGGGGCTTGGG
GCAGCGGGCGCGACC TGGTGCTGCTCCGCG TCGGGGGGCTTGGGT
CAGCGGGCGCGACCC GGTGCTGCTCCGCGG CGGGGGGCTTGGGTC
AGCGGGCGCGACCCA GTGCTGCTCCGCGGG GGGGGGCTTGGGTCC
GCGGGCGCGACCCAC TGCTGCTCCGCGGGC GGGGGCTTGGGTCCC
CGGGCGCGACCCACG GCTGCTCCGCGGGCC GGGGCTTGGGTCCCG
GGGCTTGGGTCCCGT CACTGGCCCAGTGCG GTGCGCGAGCCGGGC
GGCTTGGGTCCCGTG ACTGGCCCAGTGCGC TGCGCGAGCCGGGCT
GCTTGGGTCCCGTGG CTGGCCCAGTGCGCG GCGCGAGCCGGGCTG
CTTGGGTCCCGTGGT TGGCCCAGTGCGCGC CGCGAGCCGGGCTGC
TTGGGTCCCGTGGTG GGCCCAGTGCGCGCC GCGAGCCGGGCTGCG
TGGGTCCCGTGGTGC GCCCAGTGCGCGCCT CGAGCCGGGCTGCGG
GGGTCCCGTGGTGCG CCCAGTGCGCGCCTC GAGCCGGGCTGCGGC
GGTCCCGTGGTGCGC CCAGTGCGCGCCTCC AGCCGGGCTGCGGCT
GTCCCGTGGTGCGCT CAGTGCGCGCCTCCG GCCGGGCTGCGGCTG
TCCCGTGGTGCGCTG AGTGCGCGCCTCCGC CCGGGCTGCGGCTGC
CCCGTGGTGCGCTGC GTGCGCGCCTCCGCC CGGGCTGCGGCTGCT
CCGTGGTGCGCTGCG TGCGCGCCTCCGCCC GGGCTGCGGCTGCTG
CGTGGTGCGCTGCGA GCGCGCCTCCGCCCG GGCTGCGGCTGCTGC
GTGGTGCGCTGCGAG CGCGCCTCCGCCCGC GCTGCGGCTGCTGCC
TGGTGCGCTGCGAGC GCGCCTCCGCCCGCC CTGCGGCTGCTGCCT
GGTGCGCTGCGAGCC CGCCTCCGCCCGCCG TGCGGCTGCTGCCTG
GTGCGCTGCGAGCCG GCCTCCGCCCGCCGT GCGGCTGCTGCCTGA
TGCGCTGCGAGCCGT CCTCCGCCCGCCGTG CGGCTGCTGCCTGAC
GCGCTGCGAGCCGTG CTCCGCCCGCCGTGT GGCTGCTGCCTGACG
CGCTGCGAGCCGTGC TCCGCCCGCCGTGTG GCTGCTGCCTGACGT
GCTGCGAGCCGTGCG CCGCCCGCCGTGTGC CTGCTGCCTGACGTG
CTGCGAGCCGTGCGA CGCCCGCCGTGTGCG TGCTGCCTGACGTGC
TGCGAGCCGTGCGAC GCCCGCCGTGTGCGC GCTGCCTGACGTGCG
GCGAGCCGTGCGACG CCCGCCGTGTGCGCG CTGCCTGACGTGCGC
CGAGCCGTGCGACGC CCGCCGTGTGCGCGG TGCCTGACGTGCGCA
GAGCCGTGCGACGCG CGCCGTGTGCGCGGA GCCTGACGTGCGCAC
AGCCGTGCGACGCGC GCCGTGTGCGCGGAG CCTGACGTGCGCACT
GCCGTGCGACGCGCG CCGTGTGCGCGGAGC CTGACGTGCGCACTG
CCGTGCGACGCGCGT CGTGTGCGCGGAGCT TGACGTGCGCACTGA
CGTGCGACGCGCGTG GTGTGCGCGGAGCTG GACGTGCGCACTGAG
GTGCGACGCGCGTGC TGTGCGCGGAGCTGG ACGTGCGCACTGAGC
TGCGACGCGCGTGCA GTGCGCGGAGCTGGT CGTGCGCACTGAGCG
GCGACGCGCGTGCAC TGCGCGGAGCTGGTG GTGCGCACTGAGCGA
CGACGCGCGTGCACT GCGCGGAGCTGGTGC TGCGCACTGAGCGAG
GACGCGCGTGCACTG CGCGGAGCTGGTGCG GCGCACTGAGCGAGG
ACGCGCGTGCACTGG GCGGAGCTGGTGCGC CGCACTGAGCGAGGG
CGCGCGTGCACTGGC CGGAGCTGGTGCGCG GCACTGAGCGAGGGC
GCGCGTGCACTGGCC GGAGCTGGTGCGCGA CACTGAGCGAGGGCC
CGCGTGCACTGGCCC GAGCTGGTGCGCGAG ACTGAGCGAGGGCCA
GCGTGCACTGGCCCA AGCTGGTGCGCGAGC CTGAGCGAGGGCCAG
CGTGCACTGGCCCAG GCTGGTGCGCGAGCC TGAGCGAGGGCCAGC
GTGCACTGGCCCAGT CTGGTGCGCGAGCCG GAGCGAGGGCCAGCC
TGCACTGGCCCAGTG TGGTGCGCGAGCCGG AGCGAGGGCCAGCCG
GCACTGGCCCAGTGC GGTGCGCGAGCCGGG GCGAGGGCCAGCCGT
CGAGGGCCAGCCGTG GCCTTCGCTGCCAGC GCGCTGCTGGACGGC
GAGGGCCAGCCGTGC CCTTCGCTGCCAGCC CGCTGCTGGACGGCC
AGGGCCAGCCGTGCG CTTCGCTGCCAGCCG GCTGCTGGACGGCCG
GGGCCAGCCGTGCGG TTCGCTGCCAGCCGT CTGCTGGACGGCCGC
GGCCAGCCGTGCGGC TCGCTGCCAGCCGTC TGCTGGACGGCCGCG
GCCAGCCGTGCGGCA CGCTGCCAGCCGTCG GCTGGACGGCCGCGG
CCAGCCGTGCGGCAT GCTGCCAGCCGTCGC CTGGACGGCCGCGGG
CAGCCGTGCGGCATC CTGCCAGCCGTCGCC TGGACGGCCGCGGGC
AGCCGTGCGGCATCT TGCCAGCCGTCGCCC GGACGGCCGCGGGCT
CCCGTGCGGCATCTA GCCAGCCGTCGCCCG GACGGCCGCGGGCTC
CCGTGCGGCATCTAC CCAGCCGTCGCCCGA ACGGCCGCGGGCTCT
CGTGCGGCATCTACA CAGCCGTCGCCCGAC CGGCCGCGGGCTCTG
GTGCGGCATCTACAC AGCCGTCGCCCGACG GGCCGCGGGCTCTGC
TGCGGCATCTACACC GCCGTCGCCCGACGA GCCGCGGGCTCTGCG
GCGGCATCTACACCG CCGTCGCCCGACGAG CCGCGGGCTCTGCGT
CGGCATCTACACCGA CGTCGCCCGACGAGG CGCGGGCTCTGCGTC
GGCATCTACACCGAG GTCGCCCGACGAGGC GCGGGCTCTGCGTCA
GCATCTACACCGAGC TCGCCCGACGAGGCG CGGGCTCTGCGTCAA
CATCTACACCGAGCG CGCCCGACGAGGCGC GGGCTCTGCGTCAAC
ATCTACACCGAGCGC GCCCGACGAGGCGCG GGCTCTGCGTCAACG
TCTACACCGAGCGCT CCCGACGAGGCGCGA GCTCTGCGTCAACGC
CTACACCGAGCGCTG CCGACGAGGCGCGAC CTCTGCGTCAACGCT
TACACCGAGCGCTGT CGACGAGGCGCGACC TCTGCGTCAACGCTA
ACACCGAGCGCTGTG GACGAGGCGCGACCG CTGCGTCAACGCTAG
CACCGAGCGCTGTGG ACGAGGCGCGACCGC TGCGTCAACGCTAGT
ACCGAGCGCTGTGGC CGAGGCGCGACCGCT GCGTCAACGCTAGTG
CCGAGCGCTGTGGCT GAGGCGCGACCGCTG CGTCAACGCTAGTGC
CGAGCGCTGTGGCTC AGGCGCGACCGCTGC GTCAACGCTAGTGCC
GAGCGCTGTGGCTCC GGCGCGACCGCTGCA TCAACGCTAGTGCCG
AGCGCTGTGGCTCCG GCGCGACCGCTGCAG CAACGCTAGTGCCGT
GCGCTGTGGCTCCGG CGCGACCGCTGCAGG AACGCTAGTGCCGTC
CGCTGTGGCTCCGGC GCGACCGCTGCAGGC ACGCTAGTGCCGTCA
GCTGTGGCTCCGGCC CGACCGCTGCAGGCG CGCTAGTGCCGTCAG
CTGTGGCTCCGGCCT GACCGCTGCAGGCGC GCTAGTGCCGTCAGC
TGTGGCTCCGGCCTT ACCGCTGCAGGCGCT CTAGTGCCGTCAGCC
GTGGCTCCGGCCTTC CCGCTGCAGGCGCTG TAGTGCCGTCAGCCG
TGGCTCCGGCCTTCG CGCTGCAGGCGCTGC AGTGCCGTCAGCCGC
GGCTCCGGCCTTCGC GCTGCAGGCGCTGCT GTGCCGTCAGCCGCC
GCTCCGGCCTTCGCT CTGCAGGCGCTGCTG TGCCGTCAGCCGCCT
CTCCGGCCTTCGCTG TGCAGGCGCTGCTGG GCCGTCAGCCGCCTG
TCCGGCCTTCGCTGC GCAGGCGCTGCTGGA CCGTCAGCCGCCTGC
CCGGCCTTCGCTGCC CAGGCGCTGCTGGAC CGTCAGCCGCCTGCG
CGGCCTTCGCTGCCA AGGCGCTGCTGGACG GTCAGCCGCCTGCGC
GGCCTTCGCTGCCAG GGCGCTGCTGGACGG TCAGCCGCCTGCGCG
CAGCCGCCTGCGCGC GAAATGCTAGTGAGT GAGAGCCCGTCCGTC
AGCCGCCTGCGCGCC AAATGCTAGTGAGTC AGAGCCCGTCCGTCT
GCCGCCTGCGCGCCT AATGCTAGTGAGTCG GAGCCCGTCCGTCTC
CCGCCTGCGCGCCTA ATGCTAGTGAGTCGG AGCCCGTCCGTCTCC
CGCCTGCGCGCCTAC TGCTAGTGAGTCGGA GCCCGTCCGTCTCCA
GCCTGCGCGCCTACC GCTAGTGAGTCGGAG CCCGTCCGTCTCCAG
CCTGCGCGCCTACCT CTAGTGAGTCGGAGG CCGTCCGTCTCCAGC
CTGCGCGCCTACCTG TAGTGAGTCGGAGGA CGTCCGTCTCCAGCA
TGCGCGCCTACCTGC AGTGAGTCGGAGGAA GTCCGTCTCCAGCAC
GCGCGCCTACCTGCT GTGAGTCGGAGGAAG TCCGTCTCCAGCACG
CGCGCCTACCTGCTG TGAGTCGGAGGAAGA CCGTCTCCAGCACGC
GCGCCTACCTGCTGC GAGTCGGAGGAAGAC CGTCTCCAGCACGCA
CGCCTACCTGCTGCC AGTCGGAGGAAGACC GTCTCCAGCACGCAC
GCCTACCTGCTGCCA GTCGGAGGAAGACCG TCTCCAGCACGCACC
CCTACCTGCTGCCAG TCGGAGGAAGACCGC CTCCAGCACGCACCG
CTACCTGCTGCCAGC CGGAGGAAGACCGCA TCCAGCACGCACCGG
TACCTGCTGCCAGCG GGAGGAAGACCGCAG CCAGCACGCACCGGG
ACCTGCTGCCAGCGC GAGGAAGACCGCAGC CAGCACGCACCGGGT
CCTGCTGCCAGCGCC AGGAAGACCGCAGCG AGCACGCACCGGGTG
CTGCTGCCAGCGCCG GGAAGACCGCAGCGC GCACGCACCGGGTGT
TGCTGCCAGCGCCGC GAAGACCGCAGCGCC CACGCACCGGGTGTC
GCTGCCAGCGCCGCC AAGACCGCAGCGCCG ACGCACCGGGTGTCT
CTGCCAGCGCCGCCA AGACCGCAGCGCCGG CGCACCGGGTGTCTG
TGCCAGCGCCGCCAG GACCGCAGCGCCGGC GCACCGGGTGTCTGA
GCCAGCGCCGCCAGC ACCGCAGCGCCGGCA CACCGGGTGTCTGAT
CCAGCGCCGCCAGCT CCGCAGCGCCGGCAG ACCGGGTGTCTGATC
CAGCGCCGCCAGCTC CGCAGCGCCGGCAGT CCGGGTGTCTGATCC
AGCGCCGCCAGCTCC GCAGCGCCGGCAGTG CGGGTGTCTGATCCC
GCGCCGCCAGCTCCA CAGCGCCGGCAGTGT GGGTGTCTGATCCCA
CGCCGCCAGCTCCAG AGCGCCGGCAGTGTG GGTGTCTGATCCCAA
GCCGCCAGCTCCAGG GCGCCGGCAGTGTGG GTGTCTGATCCCAAG
CCGCCAGCTCCAGGA CGCCGGCAGTGTGGA TGTCTGATCCCAAGT
CGCCAGCTCCAGGAA GCCGGCAGTGTGGAG GTCTGATCCCAAGTT
GCCAGCTCCAGGAAA CCGGCAGTGTGGAGA TCTGATCCCAAGTTC
CCAGCTCCAGGAAAT CGGCAGTGTGGAGAG CTGATCCCAAGTTCC
CAGCTCCAGGAAATG GGCAGTGTGGAGAGC TGATCCCAAGTTCCA
AGCTCCAGGAAATGC GCAGTGTGGAGAGCC GATCCCAAGTTCCAC
GCTCCAGGAAATGCT CAGTGTGGAGAGCCC ATCCCAAGTTCCACC
CTCCAGGAAATGCTA AGTGTGGAGAGCCCG TCCCAAGTTCCACCC
TCCAGGAAATGCTAG GTGTGGAGAGCCCGT CCCAAGTTCCACCCC
CCAGGAAATGCTAGT TGTGGAGAGCCCGTC CCAAGTTCCACCCCC
CAGGAAATGCTAGTG GTGGAGAGCCCGTCC CAAGTTCCACCCCCT
AGGAAATGCTAGTGA TGGAGAGCCCGTCCG AAGTTCCACCCCCTC
GGAAATGCTAGTGAG GGAGAGCCCGTCCGT AGTTCCACCCCCTCC
GTTCCACCCCCTCCA ATGCTAAAGACAGCC AGCACAGATACCCAG
TTCCACCCCCTCCAT TGCTAAAGACAGCCA GCACAGATACCCAGA
TCCACCCCCTCCATT GCTAAAGACAGCCAG CACAGATACCCAGAA
CCACCCCCTCCATTC CTAAAGACAGCCAGC ACAGATACCCAGAAC
CACCCCCTCCATTCA TAAAGACAGCCAGCG CAGATACCCAGAACT
ACCCCCTCCATTCAA AAAGACAGCCAGCGC AGATACCCAGAACTT
CCCCCTCCATTCAAA AAGACAGCCAGCGCT GATACCCAGAACTTC
CCCCTCCATTCAAAG AGACAGCCAGCGCTA ATACCCAGAACTTCT
CCCTCCATTCAAAGA GACAGCCAGCGCTAC TACCCAGAACTTCTC
CCTCCATTCAAAGAT ACAGCCAGCGCTACA ACCCAGAACTTCTCC
CTCCATTCAAAGATA CAGCCAGCGCTACAA CCCAGAACTTCTCCT
TCCATTCAAAGATAA AGCCAGCGCTACAAA CCAGAACTTCTCCTC
CCATTCAAAGATAAT GCCAGCGCTACAAAG CAGAACTTCTCCTCC
CATTCAAAGATAATC CCAGCGCTACAAAGT AGAACTTCTCCTCCG
ATTCAAAGATAATCA CAGCGCTACAAAGTT GAACTTCTCCTCCGA
TTCAAAGATAATCAT AGCGCTACAAAGTTG AACTTCTCCTCCGAG
TCAAAGATAATCATC GCGCTACAAAGTTGA ACTTCTCCTCCGAGT
CAAAGATAATCATCA CGCTACAAAGTTGAC CTTCTCCTCCGAGTC
AAAGATAATCATCAT GCTACAAAGTTGACT TTCTCCTCCGAGTCC
AAGATAATCATCATC CTACAAAGTTGACTA TCTCCTCCGAGTCCA
AGATAATCATCATCA TACAAAGTTGACTAC CTCCTCCGAGTCCAA
GATAATCATCATCAA ACAAAGTTGACTACG TCCTCCGAGTCCAAG
ATAATCATCATCAAG CAAAGTTGACTACGA CCTCCGAGTCCAAGC
TAATCATCATCAAGA AAAGTTGACTACGAG CTCCGAGTCCAAGCG
AATCATCATCAAGAA AAGTTGACTACGAGT TCCGAGTCCAAGCGG
ATCATCATCAAGAAA AGTTGACTACGAGTC CCGAGTCCAAGCGGG
TCATCATCAAGAAAG GTTGACTACGAGTCT CGAGTCCAAGCGGGA
CATCATCAAGAAAGG TTGACTACGAGTCTC GAGTCCAAGCGGGAG
ATCATCAAGAAAGGG TGACTACGAGTCTCA AGTCCAAGCGGGAGA
TCATCAAGAAAGGGC GACTACGAGTCTCAG GTCCAAGCGGGAGAC
CATCAAGAAAGGGCA ACTACGAGTCTCAGA TCCAAGCGGGAGACA
ATCAAGAAAGGGCAT CTACGAGTCTCAGAG CCAAGCGGGAGACAG
TCAAGAAAGGGCATG TACGAGTCTCAGAGC CAAGCGGGAGACAGA
CAAGAAAGGGCATGC ACGAGTCTCAGAGCA AAGCGGGAGACAGAA
AAGAAAGGGCATGCT CGAGTCTCAGAGCAC AGCGGGAGACAGAAT
AGAAAGGGCATGCTA GAGTCTCAGAGCACA GCGGGAGACAGAATA
GAAAGGGCATGCTAA AGTCTCAGAGCACAG CGGGAGACAGAATAT
AAAGGGCATGCTAAA GTCTCAGAGCACAGA GGGAGACAGAATATG
AAGGGCATGCTAAAG TCTCAGAGCACAGAT GGAGACAGAATATGG
AGGGCATGCTAAAGA CTCAGAGCACAGATA GAGACAGAATATGGT
GGGCATGCTAAAGAC TCAGAGCACAGATAC AGACAGAATATGGTC
GGCATGCTAAAGACA CAGAGCACAGATACC GACAGAATATGGTCC
GCATGCTAAAGACAG AGAGCACAGATACCC ACAGAATATGGTCCC
CATGCTAAAGACAGC GAGCACAGATACCCA CAGAATATGGTCCCT
AGAATATGGTCCCTG ACCTGAAGTTCCTCA CCCAACTGTGACAAG
GAATATGGTCCCTGC CCTGAAGTTCCTCAA CCAACTGTGACAAGA
AATATGGTCCCTGCC CTGAAGTTCCTCAAT CAACTGTGACAAGAA
ATATGGTCCCTGCCG TGAAGTTCCTCAATG AACTGTGACAAGAAC
TATGGTCCCTGCCGT GAAGTTCCTCAATGT ACTGTGACAAGAAGG
ATGGTCCCTGCCGTA AAGTTCCTCAATGTG CTGTGACAAGAAGGG
TGGTCCCTGCCGTAG AGTTCCTCAATGTGC TGTGACAAGAAGGGA
GGTCCCTGCCGTAGA GTTCCTCAATGTGCT GTGACAAGAAGGGAT
GTCCCTGCCGTAGAG TTCCTCAATGTGCTG TGACAAGAAGGGATT
TCCCTGCCGTAGAGA TCCTCAATGTGCTGA GACAAGAAGGGATTT
CCCTGCCGTAGAGAA CCTCAATGTGCTGAG ACAAGAAGGGATTTT
CCTGCCGTAGAGAAA CTCAATGTGCTGAGT CAAGAAGGGATTTTA
CTGCCGTAGAGAAAT TCAATGTGCTGAGTC AAGAAGGGATTTTAT
TGCCGTAGAGAAATG CAATGTGCTGAGTCC AGPAGGGATTTTATA
GCCGTAGAGAAATGG AATGTGCTGAGTCCC GAAGGGATTTTATAA
CCGTAGAGAAATGGA ATGTGCTGAGTCCCA AAGGGATTTTATAAG
CGTAGAGAAATGGAA TGTGCTGAGTCCCAG AGGGATTTTATAAGA
GTAGAGAAATGGAAG GTGCTGAGTCCCAGG GGGATTTTATAAGAA
TAGAGAAATGGAAGA TGCTGAGTCCCAGGG GGATTTTATAAGAAA
AGAGAAATGGAAGAC GCTGAGTCCCAGGGG GATTTTATAAGAAAA
GAGAAATGGAAGACA CTGAGTCCCAGGGGT ATTTTATAAGAAAAA
AGAAATGGAAGACAC TGAGTCCCAGGGGTG TTTTATAAGAAAAAG
GAAATGGAAGACACA GAGTCCCAGGGGTGT TTTATAAGAAAAAGC
AAATGGAAGACACAC AGTCCCAGGGGTGTA TTATAAGAAAAAGCA
AATGGAAGACACACT GTCCCAGGGGTGTAC TATAAGAAAAAGCAG
ATGGAAGACACACTG TCCCAGGGGTGTACA ATAAGAAAAAGCAGT
TGGAAGACACACTGA CCCAGGGGTGTACAC TAAGAAAAAGCAGTG
GGAAGACACACTGAA CCAGGGGTGTACACA AAGAAAAAGCAGTGT
GAAGACACACTGAAT CAGGGGTGTACACAT AGAAAAAGCAGTGTC
AAGACACACTGAATC AGGGGTGTACACATT GAAAAAGCAGTGTCG
AGACACACTGAATCA GGGGTGTACACATTC AAAAAGCAGTGTCGC
GACACACTGAATCAC GGGTGTACACATTCC AAAAGCAGTGTCGCC
ACACACTGAATCACC GGTGTACACATTCCC AAAGCAGTGTCGCCC
CACACTGAATCACCT GTGTACACATTCCCA AAGCAGTGTCGCCCT
ACACTGAATCACCTG TGTACACATTCCCAA AGCAGTGTCGCCCTT
CACTGAATCACCTGA GTACACATTCCCAAC GCAGTGTCGCCCTTC
ACTGAATCACCTGAA TACACATTCCCAACT CAGTGTCGCCCTTCC
CTGAATCACCTGAAG ACACATTCCCAACTG AGTGTCGCCCTTCCA
TGAATCACCTGAAGT CACATTCCCAACTGT GTGTCGCCCTTCCAA
GAATCACCTGAAGTT ACATTCCCAACTGTG TGTCGCCCTTCCAAA
AATCACCTGAAGTTC CATTCCCAACTGTGA GTCGCCCTTCCAAAG
ATCACCTGAAGTTCC ATTCCCAACTGTGAC TCGCCCTTCCAAAGG
TCACCTGAAGTTCCT TTCCCAACTGTGACA CGCCCTTCCAAAGGC
CACCTGAAGTTCCTC TCCCAACTGTGACAA GCCCTTCCAAAGGCA
CCCTTCCAAAGGCAG AGTATGGGCAGCCTC GACGTGCACTGCTAC
CCTTCCAAAGGCAGG GTATGGGCAGCCTCT ACGTGCACTGCTACA
CTTCCAAAGGCAGGA TATGGGCAGCCTCTC CGTGCACTGCTACAG
TTCCAAAGGCAGGAA ATGGGCAGCCTCTCC GTGCACTGCTACAGC
TCCAAAGGCAGGAAG TGGGCAGCCTCTCCC TGCACTGCTACAGCA
CCAAAGGCAGGAAGC GGGCAGCCTCTCCCA GCACTGCTACAGCAT
CAAAGGCAGGAAGCG GGCAGCCTCTCCCAG CACTGCTACAGCATG
AAAGGCAGGAAGCGG GCAGCCTCTCCCAGG ACTGCTACAGCATGC
AAGGCAGGAAGCGGG CAGCCTCTCCCAGGC CTGCTACAGCATGCA
AGGCAGGAAGCGGGG AGCCTCTCCCAGGCT TGCTACAGCATGCAG
GGCAGGAAGCGGGGC GCCTCTCCCAGGCTA GCTACAGCATGCAGA
GCAGGAAGCGGGGCT CCTCTCCCAGGCTAC CTACAGCATGCAGAG
CAGGAAGCGGGGCTT CTCTCCCAGGCTACA TACAGCATGCAGAGC
AGGAAGCGGGGCTTC TCTCCCAGGCTACAC ACAGCATGCAGAGCA
GGAAGCGGGGCTTCT CTCCCAGGCTACACC CAGCATGCAGAGCAA
GAAGCGGGGCTTCTG TCCCAGGCTACACCA AGCATGCAGAGCAAG
AAGCGGGGCTTCTGC CCCAGGCTACACCAC GCATGCAGAGCAAGT
AGCGGGGCTTCTGCT CCAGGCTACACCACC CATGCAGAGCAAGTA
GCGGGGCTTCTGCTG CAGGCTACACCACCA ATGCAGAGCAAGTAG
CGGGGCTTCTGCTGG AGGCTACACCACCAA TGCAGAGCAAGTAGA
GGGGCTTCTGCTGGT GGCTACACCACCAAG GCAGAGCAAGTAGAC
GGGCTTCTGCTGGTG GCTACACCACCAAGG CAGAGCAAGTAGACG
GGCTTCTGCTGGTGT CTACACCACCAAGGG AGAGCAAGTAGACGC
GCTTCTGCTGGTGTG TACACCACCAAGGGG GAGCAAGTAGACGCC
CTTCTGCTGGTGTGT ACACCACCAAGGGGA AGCAAGTAGACGCCT
TTCTGCTGGTGTGTG CACCACCAAGGGGAA GCAAGTAGACGCCTG
TCTGCTGGTGTGTGG ACCACCAAGGGGAAG CAAGTAGACGCCTGC
CTGCTGGTGTGTGGA CCACCAAGGGGAAGG AAGTAGACGCCTGCC
TGCTGGTGTGTGGAT CACCAAGGGGAAGGA AGTAGACGCCTGCCG
GCTGGTGTGTGGATA ACCAAGGGGAAGGAG GTAGACGCCTGCCGC
CTGGTGTGTGGATAA CCAAGGGGAAGGAGG TAGACGCCTGCCGCA
TGGTGTGTGGATAAG CAAGGGGAAGGAGGA AGACGCCTGCCGCAA
GGTGTGTGGATAAGT AAGGGGAAGGAGGAC GACGCCTGCCGCAAG
GTGTGTGGATAAGTA AGGGGAAGGAGGACG ACGCCTGCCGCAAGT
TGTGTGGATAAGTAT GGGGAAGGAGGACGT CGCCTGCCGCAAGTT
GTGTGGATAAGTATG GGGAAGGAGGACGTG GCCTGCCGCAAGTTA
TGTGGATAAGTATGG GGAAGGAGGACGTGC CCTGCCGCAAGTTAA
GTGGATAAGTATGGG GAAGGAGGACGTGCA CTGCCGCAAGTTAAT
TGGATAAGTATGGGC AAGGAGGACGTGCAC TGCCGCAAGTTAATG
GGATAAGTATGGGCA AGGAGGACGTGCACT GCCGCAAGTTAATGT
GATAAGTATGGGCAG GGAGGACGTGCACTG CCGCAAGTTAATGTG
ATAAGTATGGGCAGC GAGGACGTGCACTGC CGCAAGTTAATGTGG
TAAGTATGGGCAGCC AGGACGTGCACTGCT GCAAGTTAATGTGGA
AAGTATGGGCAGCCT GGACGTGCACTGCTA CAAGTTAATGTGGAG
AAGTTAATGTGGAGC TGCCAAGGACATGAC TTCTGTTTGTGGTGA
AGTTAATGTGGAGCT GCCAAGGACATGACC TCTGTTTGTGGTGAA
GTTAATGTGGAGCTC CCAAGGACATGACCA CTGTTTGTGGTGAAC
TTAATGTGGAGCTCA CAAGGACATGACCAG TGTTTGTGGTGAACT
TAATGTGGAGCTCAA AAGGACATGACCAGC GTTTGTGGTGAACTG
AATGTGGAGCTCAAA AGGACATGACCAGCA TTTGTGGTGAACTGA
ATGTGGAGCTCAAAT GGACATGACCAGCAG TTGTGGTGAACTGAT
TGTGGAGCTCAAATA GACATGACCAGCAGC TGTGGTGAACTGATT
GTGGAGCTCAAATAT ACATGACCAGCAGCT GTGGTGAACTGATTT
TGGAGCTCAAATATG CATGACCAGCAGCTG TGGTGAACTGATTTT
GGAGCTCAAATATGC ATGACCAGCAGCTGG GGTGAACTGATTTTT
GAGCTCAAATATGCC TGACCAGCAGCTGGC GTGAACTGATTTTTT
AGCTCAAATATGCCT GACCAGCAGCTGGCT TGAACTGATTTTTTT
GCTCAAATATGCCTT ACCAGCAGCTGGCTA GAACTGATTTTTTTT
CTCAAATATGCCTTA CCAGCAGCTGGCTAC AACTGATTTTTTTTA
TCAAATATGCCTTAT CAGCAGCTGGCTACA ACTGATTTTTTTTAA
CAAATATGCCTTATT AGCAGCTGGCTACAG CTGATTTTTTTTAAA
AAATATGCCTTATTT GCAGCTGGCTACAGC TGATTTTTTTTAAAC
AATATGCCTTATTTT CAGCTGGCTACAGCC GATTTTTTTTAAACC
ATATGCCTTATTTTG AGCTGGCTACAGCCT ATTTTTTTTAAACCA
TATGCCTTATTTTGC GCTGGCTACAGCCTC TTTTTTTTAAACCAA
ATGCCTTATTTTGCA CTGGCTACAGCCTCG TTTTTTTAAACCAAA
TGCCTTATTTTGCAC TGGCTACAGCCTCGA TTTTTTAAACCAAAG
GCCTTATTTTGCACA GGCTACAGCCTCGAT TTTTTAAACCAAAGT
CCTTATTTTGCACAA GCTACAGCCTCGATT TTTTAAACCAAAGTT
CTTATTTTGCACAAA CTACAGCCTCGATTT TTTAAACCAAAGTTT
TTATTTTGCACAAAA TACAGCCTCGATTTA TTAAACCAAAGTTTA
TATTTTGCACAAAAG ACAGCCTCGATTTAT TAAACCAAAGTTTAG
ATTTTGCACAAAAGA CAGCCTCGATTTATA AAACCAAAGTTTAGA
TTTTGCACAAAAGAC AGCCTCGATTTATAT AACCAAAGTTTAGAA
TTTGCACAAAAGACT GCCTCGATTTATATT ACCAAAGTTTAGAAA
TTGCACAAAAGACTG CCTCGATTTATATTT CCAAAGTTTAGAAAG
TGCACAAAAGACTGC CTCGATTTATATTTC CAAAGTTTAGAAAGA
GCACAAAAGACTGCC TCGATTTATATTTCT AAAGTTTAGAAAGAG
CACAAAAGACTGCCA CGATTTATATTTCTG AAGTTTAGAAAGAGG
ACAAAAGACTGCCAA GATTTATATTTCTGT AGTTTAGAAAGAGGT
CAAAAGACTGCCAAG ATTTATATTTCTGTT GTTTAGAAAGAGGTT
AAAAGACTGCCAAGG TTTATATTTCTGTTT TTTAGAAAGAGGTTT
AAAGACTGCCAAGGA TTATATTTCTGTTTG TTAGAAAGAGGTTTT
AAGACTGCCAAGGAC TATATTTCTGTTTGT TAGAAAGAGGTTTTT
AGACTGCCAAGGACA ATATTTCTGTTTGTG AGAAAGAGGTTTTTG
GACTGCCAAGGACAT TATTTCTGTTTGTGG GAAAGAGGTTTTTGA
ACTGCCAAGGACATG ATTTCTGTTTGTGGT AAAGAGGTTTTTGAA
CTGCCAAGGACATGA TTTCTGTTTGTGGTG AAGAGGTTTTTGAAA
AGAGGTTTTTGAAAT AGCATCTTTTCACTT TTTCTTGTCGCTTCC
GAGGTTTTTGAAATG GCATCTTTTCACTTT TTCTTGTCGCTTCCT
AGGTTTTTGAAATGC CATCTTTTCACTTTC TCTTGTCGCTTCCTA
GGTTTTTGAAATGCC ATCTTTTCACTTTCC CTTGTCGCTTCCTAT
GTTTTTGAAATGCCT TCTTTTCACTTTCCA TTGTCGCTTCCTATC
TTTTTGAAATGCCTA CTTTTCACTTTCCAG TGTCGCTTCCTATCA
TTTTGAAATGCCTAT TTTTCACTTTCCAGT GTCGCTTCCTATCAA
TTTGAAATGCCTATG TTTCACTTTCCAGTA TCGCTTCCTATCAAA
TTGAAATGCCTATGG TTCACTTTCCAGTAG CGCTTCCTATCAAAA
TGAAATGCCTATGGT TCACTTTCCAGTAGT GCTTCCTATCAAAAT
GAAATGCCTATGGTT CACTTTCCAGTAGTC CTTCCTATCAAAATA
AAATGCCTATGGTTT ACTTTCCAGTAGTCA TTCCTATCAAAATAT
AATGCCTATGGTTTC CTTTCCAGTAGTCAG TCCTATCAAAATATT
ATGCCTATGGTTTCT TTTCCAGTAGTCAGC CCTATCAAAATATTC
TGCCTATGGTTTCTT TTCCAGTAGTCAGCA CTATCAAAATATTCA
GCCTATGGTTTCTTT TCCAGTAGTCAGCAA TATCAAAATATTCAG
CCTATGGTTTCTTTG CCAGTAGTCAGCAAA ATCAAAATATTCAGA
CTATGGTTTCTTTGA CAGTAGTCAGCAAAG TCAAAATATTCAGAG
TATGGTTTCTTTGAA AGTAGTCAGCAAAGA CAAAATATTCAGAGA
ATGGTTTCTTTGAAT GTAGTCAGCAAAGAG AAAATATTCAGAGAC
TGGTTTCTTTGAATG TAGTCAGCAAAGAGC AAATATTCAGAGACT
GGTTTCTTTGAATGG AGTCAGCAAAGAGCA AATATTCAGAGACTC
GTTTCTTTGAATGGT GTCAGCAAAGAGCAG ATATTCAGAGACTCG
TTTCTTTGAATGGTA TCAGCAAAGAGCAGT TATTCAGAGACTCGA
TTCTTTGAATGGTAA CAGCAAAGAGCAGTT ATTCAGAGACTCGAG
TCTTTGAATGGTAAA AGCAAAGAGCAGTTT TTCAGAGACTCGAGC
CTTTGAATGGTAAAC GCAAAGAGCAGTTTG TCAGAGACTCGAGCA
TTTGAATGGTAAACT CAAAGAGCAGTTTGA CAGAGACTCGAGCAC
TTGAATGGTAAACTT AAAGAGCAGTTTGAA AGAGACTCGAGCACA
TGAATGGTAAACTTG AAGAGCAGTTTGAAT GAGACTCGAGCACAG
GAATGGTAAACTTGA AGAGCAGTTTGAATT AGACTCGAGCACAGC
AATGGTAAACTTGAG GAGCAGTTTGAATTT GACTCGAGCACAGCA
ATGGTAAACTTGAGC AGCAGTTTGAATTTT ACTCGAGCACAGCAC
TGGTAAACTTGAGCA GCAGTTTGAATTTTC CTCGAGCACAGCACC
GGTAAACTTGAGCAT CAGTTTGAATTTTCT TCGAGCACAGCACCC
GTAAACTTGAGCATC AGTTTGAATTTTCTT CGAGCACAGCACCCA
TAAACTTGAGCATCT GTTTGAATTTTCTTG GAGCACAGCACCCAG
AAACTTGAGCATCTT TTTGAATTTTCTTGT AGCACAGCACCCAGA
AACTTGAGCATCTTT TTGAATTTTCTTGTC GCACAGCACCCAGAC
ACTTGAGCATCTTTT TGAATTTTCTTGTCG CACAGCACCCAGACT
CTTGAGCATCTTTTC GAATTTTCTTGTCGC ACAGCACCCAGACTT
TTGAGCATCTTTTCA AATTTTCTTGTCGCT CAGCACCCAGACTTC
TGAGCATCTTTTCAC ATTTTCTTGTCGCTT AGCACCCAGACTTCA
GAGCATCTTTTCACT TTTTCTTGTCGCTTC GCACCCAGACTTCAT
CACCCAGACTTCATG CGAAGCGGCCGACCA CCTATGTAGAGAACA
ACCCAGACTTCATGC GAAGCGGCCGACCAC CTATGTAGAGAACAC
CCCAGACTTCATGCG AAGCGGCCGACCACT TATGTAGAGAACACG
CCAGACTTCATGCGC AGCGGCCGACCACTG ATGTAGAGAACACGC
CAGACTTCATGCGCC GCGGCCGACCACTGA TGTAGAGAACACGCT
AGACTTCATGCGCCC CGGCCGACCACTGAC GTAGAGAACACGCTT
GACTTCATGCGCCCG GGCCGACCACTGACT TAGAGAACACGCTTC
ACTTCATGCGCCCGT GCCGACCACTGACTT AGAGAACACGCTTCA
CTTCATGCGCCCGTG CCGACCACTGACTTT GAGAACACGCTTCAC
TTCATGCGCCCGTGG CGACCACTGACTTTG AGAACACGCTTCACC
TCATGCGCCCGTGGA GACCACTGACTTTGT GAACACGCTTCACCC
CATGCGCCCGTGGAA ACCACTGACTTTGTG AACACGCTTCACCCC
ATGCGCCCGTGGAAT CCACTGACTTTGTGA ACACGCTTCACCCCC
TGCGCCCGTGGAATG CACTGACTTTGTGAC CACGCTTCACCCCCA
GCGCCCGTGGAATGC ACTGACTTTGTGACT ACGCTTCACCCCCAC
CGCCCGTGGAATGCT CTGACTTTGTGACTT CGCTTCACCCCCACT
GCCCGTGGAATGCTC TGACTTTGTGACTTA GCTTCACCCCCACTC
CCCGTGGAATGCTCA GACTTTGTGACTTAG CTTCACCCCCACTCC
CCGTGGAATGCTCAC ACTTTGTGACTTAGG TTCACCCCCACTCCC
CGTGGAATGCTCACC CTTTGTGACTTAGGC TCACCCCCACTCCCC
GTGGAATGCTCACCA TTTGTGACTTAGGCG CACCCCCACTCCCCG
TGGAATGCTCACCAC TTGTGACTTAGGCGG ACCCCCACTCCCCGT
GGAATGCTCACCACA TGTGACTTAGGCGGC CCCCCACTCCCCGTA
GAATGCTCACCACAT GTGACTTAGGCGGCT CCCCACTCCCCGTAC
AATGCTCACCACATG TGACTTAGGCGGCTG CCCACTCCCCGTACA
ATGCTCACCACATGT GACTTAGGCGGCTGT CCACTCCCCGTACAG
TGCTCACCACATGTT ACTTAGGCGGCTGTG CACTCCCCGTACAGT
GCTCACCACATGTTG CTTAGGCGGCTGTGT ACTCCCCGTACAGTG
CTCACCACATGTTGG TTAGGCGGCTGTGTT CTCCCCGTACAGTGC
TCACCACATGTTGGT TAGGCGGCTGTGTTG TCCCCGTACAGTGCG
CACCACATGTTGGTC AGGCGGCTGTGTTGC CCCCGTACAGTGCGC
ACCACATGTTGGTCG GGCGGCTGTGTTGCC CCCGTACAGTGCGCA
CCACATGTTGGTCGA GCGGCTGTGTTGCCT CCGTACAGTGCGCAC
CACATGTTGGTCGAA CGGCTGTGTTGCCTA CGTACAGTGCGCACA
ACATGTTGGTCGAAG GGCTGTGTTGCCTAT GTACAGTGCGCACAG
CATGTTGGTCGAAGC GCTGTGTTGCCTATG TACAGTGCGCACAGG
ATGTTGGTCGAAGCG CTGTGTTGCCTATGT ACAGTGCGCACAGGC
TGTTGGTCGAAGCGG TGTGTTGCCTATGTA CAGTGCGCACAGGCT
GTTGGTCGAAGCGGC GTGTTGCCTATGTAG AGTGCGCACAGGCTT
TTGGTCGAAGCGGCC TGTTGCCTATGTAGA GTGCGCACAGGCTTT
TGGTCGAAGCGGCCG GTTGCCTATGTAGAG TGCGCACAGGCTTTA
GGTCGAAGCGGCCGA TTGCCTATGTAGAGA GCGCACAGGCTTTAT
GTCGAAGCGGCCGAC TGCCTATGTAGAGAA CGCACAGGCTTTATC
TCGAAGCGGCCGACC GCCTATGTAGAGAAC GCACAGGCTTTATCG
CACAGGCTTTATCGA CCGGACATCCCAACG GTGTCATTTCTGAAA
ACAGGCTTTATCGAG CGGACATCCCAACGC TGTCATTTCTGAAAC
CAGGCTTTATCGAGA GGACATCCCAACGCA GTCATTTCTGAAACA
AGGCTTTATCGAGAA GACATCCCAACGCAT TCATTTCTGAAACAA
GGCTTTATCGAGAAT ACATCCCAACGCATG CATTTCTGAAACAAG
GCTTTATCGAGAATA CATCCCAACGCATGC ATTTCTGAAACAAGG
CTTTATCGAGAATAG ATCCCAACGCATGCT TTTCTGAAACAAGGG
TTTATCGAGAATAGG TCCCAACGCATGCTC TTCTGAAACAAGGGC
TTATCGAGAATAGGA CCCAACGCATGCTCC TCTGAAACAAGGGCG
TATCGAGAATAGGAA CCAACGCATGCTCCT CTGAAACAAGGGCGT
ATCGAGAATAGGAAA CAACGCATGCTCCTG TGAAACAAGGGCGTG
TCGAGAATAGGAAAA AACGCATGCTCCTGG GAAACAAGGGCGTGG
CGAGAATAGGAAAAC ACGCATGCTCCTGGA AAACAAGGGCGTGGA
GAGAATAGGAAAACC CGCATGCTCCTGGAG AACAAGGGCGTGGAT
AGAATAGGAAAACCT GCATGCTCCTGGAGC ACAAGGGCGTGGATC
GAATAGGAAAACCTT CATGCTCCTGGAGCT CAAGGGCGTGGATCC
AATAGGAAAACCTTT ATGCTCCTGGAGCTC AAGGGCGTGGATCCC
ATAGGAAAACCTTTA TGCTCCTGGAGCTCA AGGGCGTGGATCCCT
TAGGAAAACCTTTAA GCTCCTGGAGCTCAC GGGCGTGGATCCCTC
AGGAAAACCTTTAAA CTCCTGGAGCTCACA GGCGTGGATCCCTCA
GGAAAACCTTTAAAC TCCTGGAGCTCACAG GCGTGGATCCCTCAA
GAAAACCTTTAAACC CCTGGAGCTCACAGC CGTGGATCCCTCAAC
AAAACCTTTAAACCC CTGGAGCTCACAGCC GTGGATCCCTCAACC
AAACCTTTAAACCCC TGGAGCTCACAGCCT TGGATCCCTCAACCA
AACCTTTAAACCCCG GGAGCTCACAGCCTT GGATCCCTCAACCAA
ACCTTTAAACCCCGG GAGCTCACAGCCTTC GATCCCTCAACCAAG
CCTTTAAACCCCGGT AGCTCACAGCCTTCT ATCCCTCAACCAAGA
CTTTAAACCCCGGTC GCTCACAGCCTTCTG TCCCTCAACCAAGAA
TTTAAACCCCGGTCA CTCACAGCCTTCTGT CCCTCAACCAAGAAG
TTAAACCCCGGTCAT TCACAGCCTTCTGTG CCTCAACCAAGAAGA
TAAACCCCGGTCATC CACAGCCTTCTGTGG CTCAACCAAGAAGAA
AAACCCCGGTCATCC ACAGCCTTCTGTGGT TCAACCAAGAAGAAT
AACCCCGGTCATCCG CAGCCTTCTGTGGTG CAACCAAGAAGAATG
ACCCCGGTCATCCGG AGCCTTCTGTGGTGT AACCAAGAAGAATGT
CCCCGGTCATCCGGA GCCTTCTGTGGTGTC ACCAAGAAGAATGTT
CCCGGTCATCCGGAC CCTTCTGTGGTGTCA CCAAGAAGAATGTTT
CCGGTCATCCGGACA CTTCTGTGGTGTCAT CAAGAAGAATGTTTA
CGGTCATCCGGACAT TTCTGTGGTGTCATT AAGAAGAATGTTTAT
GGTCATCCGGACATC TCTGTGGTGTCATTT AGAAGAATGTTTATG
GTCATCCGGACATCC CTGTGGTGTCATTTC GAAGAATGTTTATGT
TCATCCGGACATCCC TGTGGTGTCATTTCT AAGAATGTTTATGTC
CATCCGGACATCCCA GTGGTGTCATTTCTG AGAATGTTTATGTCT
ATCCGGACATCCCAA TGGTGTCATTTCTGA GAATGTTTATGTCTT
TCCGGACATCCCAAC GGTGTCATTTCTGAA AATGTTTATGTCTTC
ATGTTTATGTCTTCA AGAAAATAAGGTGGA AGAATGTTCTAGGGC
TGTTTATGTCTTCAA GAAAATAAGGTGGAG GAATGTTCTAGGGCA
GTTTATGTCTTCAAG AAAATAAGGTGGAGT AATGTTCTAGGGCAC
TTTATGTCTTCAAGT AAATAAGGTGGAGTC ATGTTCTAGGGCACT
TTATGTCTTCAAGTG AATAAGGTGGAGTCC TGTTCTAGGGCACTC
TATGTCTTCAAGTGA ATAAGGTGGAGTCCT GTTCTAGGGCACTCT
ATGTCTTCAAGTGAC TAAGGTGGAGTCCTA TTCTAGGGCACTCTG
TGTCTTCAAGTGACC AAGGTGGAGTCCTAC TCTAGGGCACTCTGG
GTCTTCAAGTGACCT AGGTGGAGTCCTACT CTAGGGCACTCTGGG
TCTTCAAGTGACCTG GGTGGAGTCCTACTT TAGGGCACTCTGGGA
CTTCAAGTGACCTGT GTGGAGTCCTACTTG AGGGCACTCTGGGAA
TTCAAGTGACCTGTA TGGAGTCCTACTTGT GGGCACTCTGGGAAC
TCAAGTGACCTGTAC GGAGTCCTACTTGTT GGCACTCTGGGAACC
CAAGTGACCTGTACT GAGTCCTACTTGTTT GCACTCTGGGAACCT
AAGTGACCTGTACTG AGTCCTACTTGTTTA CACTCTGGGAACCTA
AGTGACCTGTACTGC GTCCTACTTGTTTAA ACTCTGGGAACCTAT
GTGACCTGTACTGCT TCCTACTTGTTTAAA CTCTGGGAACCTATA
TGACCTGTACTGCTT CCTACTTGTTTAAAA TCTGGGAACCTATAA
GACCTGTACTGCTTG CTACTTGTTTAAAAA CTGGGAACCTATAAA
ACCTGTACTGCTTGG TACTTGTTTAAAAAA TGGGAACCTATAAAG
CCTGTACTGCTTGGG ACTTGTTTAAAAAAT GGGAACCTATAAAGG
CTGTACTGCTTGGGG CTTGTTTAAAAAATA GGAACCTATAAAGGC
TGTACTGCTTGGGGA TTGTTTAAAAAATAT GAACCTATAAAGGCA
GTACTGCTTGGGGAC TGTTTAAAAAATATG AACCTATAAAGGCAG
TACTGCTTGGGGACT GTTTAAAAAATATGT ACCTATAAAGGCAGG
ACTGCTTGGGGACTA TTTAAAAAATATGTA CCTATAAAGGCAGGT
CTGCTTGGGGACTAT TTAAAAAATATGTAT CTATAAAGGCAGGTA
TGCTTGGGGACTATT TAAAAAATATGTATC TATAAAGGCAGGTAT
GCTTGGGGACTATTG AAAAAATATGTATCT ATAAAGGCAGGTATT
CTTGGGGACTATTGG AAAAATATGTATCTA TAAAGGCAGGTATTT
TTGGGGACTATTGGA AAAATATGTATCTAA AAAGGCAGGTATTTC
TGGGGACTATTGGAG AAATATGTATCTAAG AAGGCAGGTATTTCG
GGGGACTATTGGAGA AATATGTATCTAAGA AGGCAGGTATTTCGG
GGGACTATTGGAGAA ATATGTATCTAAGAA GGCAGGTATTTCGGG
GGACTATTGGAGAAA TATGTATCTAAGAAT GCAGGTATTTCGGGC
GACTATTGGAGAAAA ATGTATCTAAGAATG CAGGTATTTCGGGCC
ACTATTGGAGAAAAT TGTATCTAAGAATGT AGGTATTTCGGGCCC
CTATTGGAGAAAATA GTATCTAAGAATGTT GGTATTTCGGGCCCT
TATTGGAGAAAATAA TATCTAAGAATGTTC GTATTTCGGGCCCTC
ATTGGAGAAAATAAG ATCTAAGAATGTTCT TATTTCGGGCCCTCC
TTGGAGAAAATAAGG TCTAAGAATGTTCTA ATTTCGGGCCCTCCT
TGGAGAAAATAAGGT CTAAGAATGTTCTAG TTTCGGGCCCTCCTC
GGAGAAAATAAGGTG TAAGAATGTTCTAGG TTCGGGCCCTCCTCT
GAGAAAATAAGGTGG AAGAATGTTCTAGGG TCGGGCCCTCCTCTT
CGGGCCCTCCTCTTC GAAGGCCCAGGATGG GACAGAGAGACGGGA
GGGCCCTCCTCTTCA AAGGCCCAGGATGGC ACAGAGAGACGGGAG
GGCCCTCCTCTTCAG AGGCCCAGGATGGCT CAGAGAGACGGGAGA
GCCCTCCTCTTCAGG GGCCCAGGATGGCTT AGAGAGACGGGAGAG
CCCTCCTCTTCAGGA GCCCAGGATGGCTTT GAGAGACGGGAGAGT
CCTCCTCTTCACGAA CCCAGGATGGCTTTT AGAGACGGGAGAGTC
CTCCTCTTCAGGAAT CCAGGATGGCTTTTG GAGACGGGAGAGTCA
TCCTCTTCAGGAATC CAGGATGGCTTTTGC AGACGGGAGAGTCAG
CCTCTTCAGGAATCT AGGATGGCTTTTGCT GACGGGAGAGTCAGC
CTCTTCAGGAATCTT GGATGGCTTTTGCTG ACGGGAGAGTCAGCC
TCTTCAGGAATCTTC GATGGCTTTTGCTGC CGGGAGAGTCAGCCT
CTTCAGGAATCTTCC ATGGCTTTTGCTGCG GGGAGAGTCAGCCTC
TTCAGGAATCTTCCT TGGCTTTTGCTGCGG GGAGAGTCAGCCTCC
TCAGGAATCTTCCTG GGCTTTTGCTGCGGC GAGAGTCAGCCTCCA
CAGGAATCTTCCTGA GCTTTTGCTGCGGCC AGAGTCAGCCTCCAC
AGGAATCTTCCTGAA CTTTTGCTGCGGCCC GAGTCAGCCTCCACA
GGAATCTTCCTGAAG TTTTGCTGCGGCCCC AGTCAGCCTCCACAT
GAATCTTCCTGAAGA TTTGCTGCGGCCCCG GTCAGCCTCCACATT
AATCTTCCTGAAGAC TTGCTGCGGCCCCGT TCAGCCTCCACATTC
ATCTTCCTGAAGACA TGCTGCGGCCCCGTG CAGCCTCCACATTCA
TCTTCCTGAAGACAT GCTGCGGCCCCGTGG AGCCTCCACATTCAG
CTTCCTGAAGACATG CTGCGGCCCCGTGGG GCCTCCACATTCAGA
TTCCTGAAGACATGG TGCGGCCCCGTGGGG CCTCCACATTCAGAG
TCCTGAAGACATGGC GCGGCCCCGTGGGGT CTCCACATTCAGAGG
CCTGAAGACATGGCC CGGCCCCGTGGGGTA TCCACATTCAGAGGC
CTGAAGACATGGCCC GGCCCCGTGGGGTAG CCACATTCAGAGGCA
TGAAGACATGGCCCA GCCCCGTGGGGTAGG CACATTCAGAGGCAT
GAAGACATGGCCCAG CCCCGTGGGGTAGGA ACATTCAGAGGCATC
AAGACATGGCCCAGT CCCGTGGGGTAGGAG CATTCAGAGGCATCA
AGACATGGCCCAGTC CCGTGGGGTAGGAGG ATTCAGAGGCATCAC
GACATGGCCCAGTCG CGTGGGGTAGGAGGG TTCAGAGGCATCACA
ACATGGCCCAGTCGA GTGGGGTAGGAGGGA TCAGAGGCATCACAA
CATGGCCCAGTCGAA TGGGGTAGGAGGGAC CAGAGGCATCACAAG
ATGGCCCAGTCGAAG GGGGTAGGAGGGACA AGAGGCATCACAAGT
TGGCCCAGTCGAAGG GGGTAGGAGGGACAG GAGGCATCACAAGTA
GGCCCAGTCGAAGGC GGTAGGAGGGACAGA AGGCATCACAAGTAA
GCCCAGTCGAAGGCC GTAGGAGGGACAGAG GGCATCACAAGTAAT
CCCAGTCGAAGGCCC TAGGAGGGACAGAGA GCATCACAAGTAATG
CCAGTCGAAGGCCCA AGGAGGGACAGAGAG CATCACAAGTAATGG
CAGTCGAAGGCCCAG GGAGGGACAGAGAGA ATCACAAGTAATGGC
AGTCGAAGGCCCAGG GAGGGACAGAGAGAC TCACAAGTAATGGCA
GTCGAAGGCCCAGGA AGGGACAGAGAGACG CACAAGTAATGGCAC
TCGAAGGCCCAGGAT GGGACAGAGAGACGG ACAAGTAATGGCACA
CGAAGGCCCAGGATG GGACAGAGAGACGGG CAAGTAATGGCACAA
AAGTAATGGCACAAT TGTAGTTCAACAACT TCTTTAAAGGCAAAG
AGTAATGGCACAATT GTAGTTCAACAACTC CTTTAAAGGCAAAGC
GTAATGGCACAATTC TAGTTCAACAACTCA TTTAAAGGCAAAGCT
TAATGGCACAATTCT AGTTCAACAACTCAA TTAAAGGCAAAGCTT
AATGGCACAATTCTT GTTCAACAACTCAAG TAAAGGCAAAGCTTT
ATGGCACAATTCTTC TTCAACAACTCAAGA AAAGGCAAAGCTTTA
TGGCACAATTCTTCG TCAACAACTCAAGAC AAGGCAAAGCTTTAT
GGCACAATTCTTCGG CAACAACTCAAGACG AGGCAAAGCTTTATT
GCACAATTCTTCGGA AACAACTCAAGACGA GGCAAAGCTTTATTT
CACAATTCTTCGGAT ACAACTCAAGACGAA GCAAAGCTTTATTTT
ACAATTCTTCGGATG CAACTCAAGACGAAG CAAAGCTTTATTTTC
CAATTCTTCGGATGA AACTCAAGACGAAGC AAAGCTTTATTTTCA
AATTCTTCGGATGAC ACTCAAGACGAAGCT AAGCTTTATTTTCAT
ATTCTTCGGATGACT CTCAAGACGAAGCTT AGCTTTATTTTCATC
TTCTTCGGATGACTG TCAAGACGAAGCTTA GCTTTATTTTCATCT
TCTTCGGATGACTGC CAAGACGAAGCTTAT CTTTATTTTCATCTC
CTTCGGATGACTGCA AAGACGAAGCTTATT TTTATTTTCATCTCT
TTCGGATGACTGCAG AGACGAAGCTTATTT TTATTTTCATCTCTC
TCGGATGACTGCAGA GACGAAGCTTATTTC TATTTTCATCTCTCA
CGGATGACTGCAGAA ACGAAGCTTATTTCT ATTTTCATCTCTCAT
GGATGACTGCAGAAA CGAAGCTTATTTCTG TTTTCATCTCTCATC
GATGACTGCAGAAAA GAAGCTTATTTCTGA TTTCATCTCTCATCT
ATGACTGCAGAAAAT AAGCTTATTTCTGAG TTCATCTCTCATCTT
TGACTGCAGAAAATA AGCTTATTTCTGAGG TCATCTCTCATCTTT
GACTGCAGAAAATAG GCTTATTTCTGAGGA CATCTCTCATCTTTT
ACTGCAGAAAATAGT CTTATTTCTGAGGAT ATCTCTCATCTTTTG
CTGCAGAAAATAGTG TTATTTCTGAGGATA TCTCTCATCTTTTGT
TGCAGAAAATAGTGT TATTTCTGAGGATAA CTCTCATCTTTTGTC
GCAGAAAATAGTGTT ATTTCTGAGGATAAG TCTCATCTTTTGTCC
CAGAAAATAGTGTTT TTTCTGAGGATAAGC CTCATCTTTTGTCCT
AGAAAATAGTGTTTT TTCTGAGGATAAGCT TCATCTTTTGTCCTC
GAAAATAGTGTTTTG TCTGAGGATAAGCTC CATCTTTTGTCCTCC
AAAATAGTGTTTTGT CTGAGGATAAGCTCT ATCTTTTGTCCTCCT
AAATAGTGTTTTGTA TGAGGATAAGCTCTT TCTTTTGTCCTCCTT
AATAGTGTTTTGTAG GAGGATAAGCTCTTT CTTTTGTCCTCCTTA
ATAGTGTTTTGTAGT AGGATAAGCTCTTTA TTTTGTCCTCCTTAG
TAGTGTTTTGTAGTT GGATAAGCTCTTTAA TTTGTCCTCCTTAGC
AGTGTTTTGTAGTTC GATAAGCTCTTTAAA TTGTCCTCCTTAGCA
GTGTTTTGTAGTTCA ATAAGCTCTTTAAAG TGTCCTCCTTAGCAC
TGTTTTGTAGTTCAA TAAGCTCTTTAAAGG GTCCTCCTTAGCACA
GTTTTGTAGTTCAAC AAGCTCTTTAAAGGC TCCTCCTTAGCACAA
TTTTGTAGTTCAACA AGCTCTTTAAAGGCA CCTCCTTAGCACAAT
TTTGTAGTTCAACAA GCTCTTTAAAGGCAA CTCCTTAGCACAATG
TTGTAGTTCAACAAC CTCTTTAAAGGCAAA TCCTTAGCACAATGT
CCTTAGCACAATGTA GAGGAATGGCTTGCT ATAGAGATTCACCCA
CTTAGCACAATGTAA AGGAATGGCTTGCTG TAGAGATTCACCCAT
TTAGCACAATGTAAA GGAATGGCTTGCTGG AGAGATTCACCCATG
TAGCACAATGTAAAA GAATGGCTTGCTGGG GAGATTCACCCATGT
AGCACAATGTAAAAA AATGGCTTGCTGGGG AGATTCACCCATGTT
GCACAATGTAAAAAA ATGGCTTGCTGGGGA GATTCACCCATGTTT
CACAATGTAAAAAAG TGGCTTGCTGGGGAG ATTCACCCATGTTTG
ACAATGTAAAAAAAG GGCTTGCTGGGGAGC TTCACCCATGTTTGT
CAATGTAAAAAAGAA GCTTGCTGGGGAGCC TCACCCATGTTTGTT
AATGTAAAAAAGAAT CTTGCTGGGGAGCCC CACCCATGTTTGTTG
ATGTAAAAAAGAATA TTGCTGGGGAGCCCA ACCCATGTTTGTTGA
TGTAPAAAAGAATAG TGCTGGGGAGCCCAT CCCATGTTTGTTGAA
GTAAAAAAGAATAGT GCTGGGGAGCCCATC CCATGTTTGTTGAAC
TAAAAAAGAATAGTA CTGGGGAGCCCATCC CATGTTTGTTGAACT
AAAAAAGAATAGTAA TGGGGAGCCCATCCA ATGTTTGTTGAACTT
AAAAAGAATAGTAAT GGGGAGCCCATCCAG TGTTTGTTGAACTTA
AAAAGAATAGTAATA GGGAGCCCATCCAGG GTTTGTTGAACTTAG
AAAGAATAGTAATAT GGAGCCCATCCAGGA TTTGTTGAACTTAGA
AAGAATAGTAATATC GAGCCCATCCAGGAC TTGTTGAACTTAGAG
AGAATAGTAATATCA AGCCCATCCAGGACA TGTTGAACTTAGAGT
GAATAGTAATATCAG GCCCATCCAGGACAC GTTGAACTTAGAGTC
AATAGTAATATCAGA CCCATCCAGGACACT TTGAACTTAGAGTCA
ATAGTAATATCAGAA CCATCCAGGACACTG TGAACTTAGAGTCAT
TAGTAATATCAGAAC CATCCAGGACACTGG GAACTTAGAGTCATT
AGTAATATCAGAACA ATCCAGGACACTGGG AACTTAGAGTCATTC
GTAATATCAGAACAG TCCAGGACACTGGGA ACTTAGAGTCATTCT
TAATATCAGAACAGG CCAGGACACTGGGAG CTTAGAGTCATTCTC
AATATCAGAACAGGA CAGGACACTGGGAGC TTAGAGTCATTCTCA
ATATCAGAACAGGAA AGGACACTGGGAGCA TAGAGTCATTCTCAT
TATCAGAACAGGAAG GGACACTGGGAGCAC AGAGTCATTCTCATG
ATCAGAACAGGAAGG GACACTGGGAGCACA GAGTCATTCTCATGC
TCAGAACAGGAAGGA ACACTGGGAGCACAT AGTCATTCTCATGCT
CAGAACAGGAAGGAG CACTGGGAGCACATA GTCATTCTCATGCTT
AGAACAGGAAGGAGG ACTGGGAGCACATAG TCATTCTCATGCTTT
GAACAGGAAGGAGGA CTGGGAGCACATAGA CATTCTCATGCTTTT
AACAGGAAGGAGGAA TGGGAGCACATAGAG ATTCTCATGCTTTTC
ACAGGAAGGAGGAAT GGGAGCACATAGAGA TTCTCATGCTTTTCT
CAGGAAGGAGGAATG GGAGCACATAGAGAT TCTCATGCTTTTCTT
AGGAAGGAGGAATGG GAGCACATAGAGATT CTCATGCTTTTCTTT
GGAAGGAGGAATGGC AGCACATAGAGATTC TCATGCTTTTCTTTA
GAAGGAGGAATGGCT GCACATAGAGATTCA CATGCTTTTCTTTAT
AAGGAGGAATGGCTT CACATAGAGATTCAC ATGCTTTTCTTTATA
AGGAGGAATGGCTTG ACATAGAGATTCACC TGCTTTTCTTTATAA
GGAGGAATGGCTTGC CATAGAGATTCACCC GCTTTTCTTTATAAT
CTTTTCTTTATAATT TGTTAACATTGTATA TACTAGATAATCCTA
TTTTCTTTATAATTC GTTAACATTGTATAC ACTAGATAATCCTAG
TTTCTTTATAATTCA TTAACATTGTATACA CTAGATAATCCTAGA
TTCTTTATAATTCAC TAACATTGTATACAA TAGATAATCCTAGAT
TCTTTATAATTCACA AACATTGTATACAAC AGATAATCCTAGATG
CTTTATAATTCACAC ACATTGTATACAACA GATAATCCTAGATGA
TTTATAATTCACACA CATTGTATACAACAT ATAATCCTAGATGAA
TTATAATTCACACAT ATTGTATACAACATA TAATCCTAGATGAAA
TATAATTCACACATA TTGTATACAACATAG AATCCTAGATGAAAT
ATAATTCACACATAT TGTATACAACATAGC ATCCTAGATGAAATG
TAATTCACACATATA GTATACAACATAGCC TCCTAGATGAAATGT
AATTCACACATATAT TATACAACATAGCCC CCTAGATGAAATGTT
ATTCACACATATATG ATACAACATAGCCCC CTAGATGAAATGTTA
TTCACACATATATGC TACAACATAGCCCCA TAGATGAAATGTTAG
TCACACATATATGCA ACAACATAGCCCCAA AGATGAAATGTTAGA
CACACATATATGCAG CAACATAGCCCCAAA GATGAAATGTTAGAG
ACACATATATGCAGA AACATAGCCCCAAAT ATGAAATGTTAGAGA
CACATATATGCAGAG ACATAGCCCCAAATA TGAAATGTTAGAGAT
ACATATATGCAGAGA CATAGCCCCAAATAT GAAATGTTAGAGATG
CATATATGCAGAGAA ATAGCCCCAAATATA AAATGTTAGAGATGC
ATATATGCAGAGAAG TAGCCCCAAATATAG AATGTTAGAGATGCT
TATATGCAGAGAAGA AGCCCCAAATATAGT ATGTTAGAGATGCTA
ATATGCAGAGAAGAT GCCCCAAATATAGTA TGTTAGAGATGCTAT
TATGCAGAGAAGATA CCCCAAATATAGTAA GTTAGAGATGCTATA
ATGCAGAGAAGATAT CCCAAATATAGTAAG TTAGAGATGCTATAT
TGCAGAGAAGATATG CCAAATATAGTAAGA TAGAGATGCTATATG
GCAGAGAAGATATGT CAAATATAGTAAGAT AGAGATGCTATATGA
CAGAGAAGATATGTT AAATATAGTAAGATC GAGATGCTATATGAT
AGAGAAGATATGTTC AATATAGTAAGATCT AGATGCTATATGATA
GAGAAGATATGTTCT ATATAGTAAGATCTA GATGCTATATGATAC
AGAAGATATGTTCTT TATAGTAAGATCTAT ATGCTATATGATACA
GAAGATATGTTCTTG ATAGTAAGATCTATA TGCTATATGATACAA
AAGATATGTTCTTGT TAGTAAGATCTATAC GCTATATGATACAAC
AGATATGTTCTTGTT AGTAAGATCTATACT CTATATGATACAACT
GATATGTTCTTGTTA GTAAGATCTATACTA TATATGATACAACTG
ATATGTTCTTGTTAA TAAGATCTATACTAG ATATGATACAACTGT
TATGTTCTTGTTAAC AAGATCTATACTAGA TATGATACAACTGTG
ATGTTCTTGTTAACA AGATCTATACTAGAT ATGATACAACTGTGG
TGTTCTTGTTAACAT GATCTATACTAGATA TGATACAACTGTGGC
GTTCTTGTTAACATT ATCTATACTAGATAA GATACAACTGTGGCC
TTCTTGTTAACATTG TCTATACTAGATAAT ATACAACTGTGGCCA
TCTTGTTAACATTGT CTATACTAGATAATC TACAACTGTGGCCAT
CTTGTTAACATTGTA TATACTAGATAATCC ACAACTGTGGCCATG
TTGTTAACATTGTAT ATACTAGATAATCCT CAACTGTGGCCATGA
AACTGTGGCCATGAC GGCTGCTCTCCCGGA AGGCTCAGGGAGACT
ACTGTGGCCATGACT GCTGCTCTCCCGGAG GGCTCAGGGAGACTC
CTGTGGCCATGACTG CTGCTCTCCCGGAGG GCTCAGGGAGACTCT
TGTGGCCATGACTGA TGCTCTCCCGGAGGC CTCAGGGAGACTCTG
GTGGCCATGACTGAG GCTCTCCCGGAGGCC TCAGGGAGACTCTGC
TGGCCATGACTGAGG CTCTCCCGGAGGCCA CAGGGAGACTCTGCC
GGCCATGACTGAGGA TCTCCCGGAGGCCAA AGGGAGACTCTGCCC
GCCATGACTGAGGAA CTCCCGGAGGCCAAA GGGAGACTCTGCCCT
CCATGACTGAGGAAA TCCCGGAGGCCAAAC GGAGACTCTGCCCTG
CATGACTGAGGAAAG CCCGGAGGCCAAACC GAGACTCTGCCCTGC
ATGACTGAGGAAAGG CCGGAGGCCAAACCC AGACTCTGCCCTGCT
TGACTGAGGAAAGGA CGGAGGCCAAACCCA GACTCTGCCCTGCTG
GACTGAGGAAAGGAG GGAGGCCAAACCCAA ACTCTGCCCTGCTGC
ACTGAGGAAAGGAGC GAGGCCAAACCCAAG CTCTGCCCTGCTGCA
CTGAGGAAAGGAGCT AGGCCAAACCCAAGA TCTGCCCTGCTGCAG
TGAGGAAAGGAGCTC GGCCAAACCCAAGAA CTGCCCTGCTGCAGA
GAGGAAAGGAGCTCA GCCAAACCCAAGAAG TGCCCTGCTGCAGAC
AGGAAAGGAGCTCAC CCAAACCCAAGAAGG GCCCTGCTGCAGACC
GGAAAGGAGCTCACG CAAACCCAAGAAGGT CCCTGCTGCAGACCT
GAAAGGAGCTCACGC AAACCCAAGAAGGTC CCTGCTGCAGACCTC
AAAGGAGCTCACGCC AACCCAAGAAGGTCT CTGCTGCAGACCTCG
AAGGAGCTCACGCCC ACCCAAGAAGGTCTG TGCTGCAGACCTCGG
AGGAGCTCACGCCCA CCCAAGAAGGTCTGG GCTGCAGACCTCGGT
GGAGCTCACGCCCAG CCAAGAAGGTCTGGC CTGCAGACCTCGGTG
GAGCTCACGCCCAGA CAAGAAGGTCTGGCA TGCAGACCTCGGTGT
AGCTCACGCCCAGAG AAGAAGGTCTGGCAA GCAGACCTCGGTGTG
GCTCACGCCCAGAGA AGAAGGTCTGGCAAA CAGACCTCGGTGTGG
CTCACGCCCAGAGAC GAAGGTCTGGCAAAG AGACCTCGGTGTGGA
TCACGCCCAGAGACT AAGGTCTGGCAAAGT GACCTCGGTGTGGAC
CACGCCCAGAGACTG AGGTCTGGCAAAGTC ACCTCGGTGTGGACA
ACGCCCAGAGACTGG GGTCTGGCAAAGTCA CCTCGGTGTGGACAC
CGCCCAGAGACTGGG GTCTGGCAAAGTCAG CTCGGTGTGGACACA
GCCCAGAGACTGGGC TCTGGCAAAGTCAGG TCGGTGTGGACACAC
CCCAGAGACTGGGCT CTGGCAAAGTCAGGC CGGTGTGGACACACG
CCAGAGACTGGGCTG TGGCAAAGTCAGGCT GGTGTGGACACACGC
CAGAGACTGGGCTGC GGCAAAGTCAGGCTC GTGTGGACACACGCT
AGAGACTGGGCTGCT GCAAAGTCAGGCTCA TGTGGACACACGCTG
GAGACTGGGCTGCTC CAAAGTCAGGCTCAG GTGGACACACGCTGC
AGACTGGGCTGCTCT AAAGTCAGGCTCAGG TGGACACACGCTGCA
GACTGGGCTGCTCTC AAGTCAGGCTCAGGG GGACACACGCTGCAT
ACTGGGCTGCTCTCC AGTCAGGCTCAGGGA GACACACGCTGCATA
CTGGGCTGCTCTCCC GTCAGGCTCAGGGAG ACACACGCTGCATAG
TGGGCTGCTCTCCCG TCAGGCTCAGGGAGA CACACGCTGCATAGA
GGGCTGCTCTCCCGG CAGGCTCAGGGAGAC ACACGCTGCATAGAG
CACGCTGCATAGAGC ATTCTGCCTACCTAT GGGGGAAAAGTATTT
ACGCTGCATAGAGCT TTCTGCCTACCTATT GGGGAAAAGTATTTT
CGCTGCATAGAGCTC TCTGCCTACCTATTA GGGAAAAGTATTTTT
GCTGCATAGAGCTCT CTGCCTACCTATTAG GGAAAAGTATTTTTG
CTGCATAGAGCTCTC TGCCTACCTATTAGC GAAAAGTATTTTTGA
TGCATAGAGCTCTCC GCCTACCTATTAGCT AAAAGTATTTTTGAG
GCATAGAGCTCTCCT CCTACCTATTAGCTT AAAGTATTTTTGAGA
CATAGAGCTCTCCTT CTACCTATTAGCTTT AAGTATTTTTGAGAA
ATAGAGCTCTCCTTG TACCTATTAGCTTTT AGTATTTTTGAGAAG
TAGAGCTCTCCTTGA ACCTATTAGCTTTTC GTATTTTTGAGAAGT
AGAGCTCTCCTTGAA CCTATTAGCTTTTCT TATTTTTGAGAAGTT
GAGCTCTCCTTGAAA CTATTAGCTTTTCTT ATTTTTGAGAAGTTT
AGCTCTCCTTGAAAA TATTAGCTTTTCTTT TTTTTGAGAAGTTTG
GCTCTCCTTGAAAAC ATTAGCTTTTCTTTA TTTTGAGAAGTTTGT
CTCTCCTTGAAAACA TTAGCTTTTCTTTAT TTTGAGAAGTTTGTC
TCTCCTTGAAAACAG TAGCTTTTCTTTATT TTGAGAAGTTTGTCT
CTCCTTGAAAACAGA AGCTTTTCTTTATTT TGAGAAGTTTGTCTT
TCCTTGAAAACAGAG GCTTTTCTTTATTTT GAGAAGTTTGTCTTG
CCTTGAAAACAGAGG CTTTTCTTTATTTTT AGAAGTTTGTCTTGC
CTTGAAAACAGAGGG TTTTCTTTATTTTTT GAAGTTTGTCTTGCA
TTGAAAACAGAGGGG TTTCTTTATTTTTTT AAGTTTGTCTTGCAA
TGAAAACAGAGGGGT TTCTTTATTTTTTTA AGTTTGTCTTGCAAT
GAAAACAGAGGGGTC TCTTTATTTTTTTAA GTTTGTCTTGCAATG
AAAACAGAGGGGTCT CTTTATTTTTTTAAC TTTGTCTTGCAATGT
AAACAGAGGGGTCTC TTTATTTTTTTAACT TTGTCTTGCAATGTA
AACAGAGGGGTCTCA TTATTTTTTTAACTT TGTCTTGCAATGTAT
ACAGAGGGGTCTCAA TATTTTTTTAACTTT GTCTTGCAATGTATT
CAGAGGGGTCTCAAG ATTTTTTTAACTTTT TCTTGCAATGTATTT
AGAGGGGTCTCAAGA TTTTTTTAACTTTTT CTTGCAATGTATTTA
GAGGGGTCTCAAGAC TTTTTTAACTTTTTG TTGCAATGTATTTAT
AGGGGTCTCAAGACA TTTTTAACTTTTTGG TGCAATGTATTTATA
GGGGTCTCAAGACAT TTTTAACTTTTTGGG GCAATGTATTTATAA
GGGTCTCAAGACATT TTTAACTTTTTGGGG CAATGTATTTATAAA
GGTCTCAAGACATTC TTAACTTTTTGGGGG AATGTATTTATAAAT
GTCTCAAGACATTCT TAACTTTTTGGGGGG ATGTATTTATAAATA
TCTCAAGACATTCTG AACTTTTTGGGGGGA TGTATTTATAAATAG
CTCAAGACATTCTGC ACTTTTTGGGGGGAA GTATTTATAAATAGT
TCAAGACATTCTGCC CTTTTTGGGGGGAAA TATTTATAAATAGTA
CAAGACATTCTGCCT TTTTTGGGGGGAAAA ATTTATAAATAGTAA
AAGACATTCTGCCTA TTTTGGGGGGAAAAG TTTATAAATAGTAAA
AGACATTCTGCCTAC TTTGGGGGGAAAAGT TTATAAATAGTAAAT
GACATTCTGCCTACC TTGGGGGGAAAAGTA TATAAATAGTAAATA
ACATTCTGCCTACCT TGGGGGGAAAAGTAT ATAAATAGTAAATAA
CATTCTGCCTACCTA GGGGGGAAAAGTATT TAAATAGTAAATAAA
AAATAGTAAATAAAG
AATAGTAAATAAAGT
ATAGTAAATAAAGTT
TAGTAAATAAAGTTT
AGTAAATAAAGTTTT
GTAAATAAAGTTTTT
TAAATAAAGTTTTTA
AAATAAAGTTTTTAC
AATAAAGTTTTTACC
ATAAAGTTTTTACCA
TAAAGTTTTTACCAT
AAAGTTTTTACCATT
EXAMPLE 8 Antisense oligonucleotides to IGF-I may be selected from molecules capable of interacting with one or more of the following sense oligonucleotides:
TTTTTTTTTTTTTTG ATTTCATCCCAAATA AAGTCTGGCTCCGGA
TTTTTTTTTTTTTGA TTTCATCCCAAATAA AGTCTGGCTCCGGAG
TTTTTTTTTTTTGAG TTCATCCCAAATAAA GTCTGGCTCCGGAGG
TTTTTTTTTTTGAGA TCATCCCAAATAAAA TCTGGCTCCGGAGGA
TTTTTTTTTTGAGAA CATCCCAAATAAAAG CTGGCTCCGGAGGAG
TTTTTTTTTGAGAAA ATCCCAAATAAAAGG TGGCTCCGGAGGAGG
TTTTTTTTGAGAAAG TCCCAAATAAAAGGA GGCTCCGGAGGAGGG
TTTTTTTGAGAAAGG CCCAAATAAAAGGAA GCTCCGGAGGAGGGT
TTTTTTGAGAAAGGG CCAAATAAAAGGAAT CTCCGGAGGAGGGTC
TTTTTGAGAAAGGGA CAAATAAAAGGAATG TCCGGAGGAGGGTCC
TTTTGAGAAAGGGAA AAATAAAAGGAATGA CCGGAGGAGGGTCCC
TTTGAGAAAGGGAAT AATAAAAGGAATGAA CGGAGGAGGGTCCCC
TTGAGAAAGGGAATT ATAAAAGGAATGAAG GGAGGAGGGTCCCCG
TGAGAAAGGGAATTT TAAAAGGAATGAAGT GAGGAGGGTCCCCGA
GAGAAAGGGAATTTC AAAAGGAATGAAGTC AGGAGGGTCCCCGAC
AGAAAGGGAATTTCA AAAGGAATGAAGTCT GGAGGGTCCCCGACC
GAAAGGGAATTTCAT AAGGAATGAAGTCTG GAGGOTCCCCGACCT
AAAGGGAATTTCATC AGGAATGAAGTCTGG AGGGTCCCCGACCTC
AAGGGAATTTCATCC GGAATGAAGTCTGGC GGGTCCCCGACCTCG
AGGGAATTTCATCCC GAATGAAGTCTGGCT GGTCCCCGACCTCGC
GGGAATTTCATCCCA AATGAAGTCTGGCTC GTCCCCGACCTCGCT
GGAATTTCATCCCAA ATGAAGTCTGGCTCC TCCCCGACCTCGCTG
GAATTTCATCCCAAA TGAAGTCTGGCTCCG CCCCGACCTCGCTGT
AATTTCATCCCAAAT GAAGTCTGGCTCCGG CCCGACCTCGCTGTG
CCGACCTCGCTGTGG GCTCTGGCCGACGAG TCCGCAACGACTATC
CGACCTCGCTGTGGG CTCTGGCCGACGAGT CCGCAACGACTATCA
GACCTCGCTGTGGGG TCTGGCCGACGAGTG CGCAACGACTATCAG
ACCTCGCTGTGGGGG CTGGCCGACGAGTGG GCAACGACTATCAGC
CCTCGCTGTGGGGGC TGGCCGACGAGTGGA CAACGACTATCAGCA
CTCGCTGTGGGGGCT GGCCGACGAGTGGAG AACGACTATCAGCAG
TCGCTGTGGGGGCTC GCCGACGAGTGGAGA ACGACTATCAGCAGC
CGCTGTGGGGGCTCC CCGACGAGTGGAGAA CGACTATCAGCAGCT
GCTGTGGGGGCTCCT CGACGAGTGGAGATA GACTATCAGCAGCTG
CTGTGGGGGCTCCTG GACGAGTGGAGAAAT ACTATCAGCAGCTGA
TGTGGGGGCTCCTGT ACGAGTGGAGAAATC CTATCAGCAGCTGAA
GTGGGGGCTCCTGTT CGAGTGGAGAAATCT TATCAGCAGCTGAAG
TGGGGGCTCCTGTTT GAGTGGAGAAATCTG ATCAGCAGCTGAAGC
GGGGGCTCCTGTTTC AGTGGAGAAATCTGC TCAGCAGCTGAAGCG
GGGGCTCCTGTTTCT GTGGAGAAATCTGCG CAGCAGCTGAAGCGC
GGGCTCCTGTTTCTC TGGAGAAATCTGCGG AGCAGCTGAAGCGCC
GGCTCCTGTTTCTCT GGAGAAATCTGCGGG GCAGCTGAAGCGCCT
GCTCCTGTTTCTCTC GAGAAATCTGCGGGC CAGCTGAAGCGCCTG
CTCCTGTTTCTCTCC AGAAATCTGCGGGCC AGCTGAAGCGCCTGG
TCCTGTTTCTCTCCG GAAATCTGCGGGCCA GCTGAAGCGCCTGGA
CCTGTTTCTCTCCGC AAATCTGCGGGCCAG CTGAAGCGCCTGGAG
CTGTTTCTCTCCGCC AATCTGCGGGCCAGG TGAAGCGCCTGGAGA
TGTTTCTCTCCGCCG ATCTGCGGGCCAGGC GAAGCGCCTGGAGAA
GTTTCTCTCCGCCGC TCTGCGGGCCAGGCA AAGCGCCTGGAGAAC
TTTCTCTCCGCCGCG CTGCGGGCCAGGCAT AGCGCCTGGAGAACT
TTCTCTCCGCCGCGC TGCGGGCCAGGCATC GCGCCTGGAGAACTG
TCTCTCCGCCGCGCT GCGGGCCAGGCATCG CGCCTGGAGAACTGC
CTCTCCGCCGCGCTC CGGGCCAGGCATCGA GCCTGGAGAACTGCA
TCTCCGCCGCGCTCT GGGCCAGGCATCGAC CCTGGAGAACTGCAC
CTCCGCCGCGCTCTC GGCCAGGCATCGACA CTGGAGAACTGCACG
TCCGCCGCGCTCTCG GCCAGGCATCGACAT TGGAGAACTGCACGG
CCGCCGCGCTCTCGC CCAGGCATCGACATC GGAGAACTGCACGGT
CGCCGCGCTCTCGCT CAGGCATCGACATCC GAGAACTGCACGGTG
GCCGCGCTCTCGCTC AGGCATCGACATCCG AGAACTGCACGGTGA
CCGCGCTCTCGCTCT GGCATCGACATCCGC GAACTGCACGGTGAT
CGCGCTCTCGCTCTG GCATCGACATCCGCA AACTGCACGGTGATC
GCGCTCTCGCTCTGG CATCGACATCCGCAA ACTGCACGGTGATCG
CGCTCTCGCTCTGGC ATCGACATCCGCAAC CTGCACGGTGATCGA
GCTCTCGCTCTGGCC TCGACATCCGCAACG TGCACGGTGATCGAG
CTCTCGCTCTGGCCG CGACATCCGCAACGA GCACGGTGATCGAGG
TCTCGCTCTGGCCGA GACATCCGCAACGAC CACGGTGATCGAGGG
CTCGCTCTGGCCGAC ACATCCGCAACGACT ACGGTGATCGAGGGC
TCGCTCTGGCCGACG CATCCGCAACGACTA CGGTGATCGAGGGCT
CGCTCTGGCCGACGA ATCCGCAACGACTAT GGTGATCGAGGGCTA
GTGATCGAGGGCTAC GGACTACCGCAGCTA AGTACTTGCTGCTGT
TGATCGAGGGCTACC GACTACCGCAGCTAC GTACTTGCTGCTGTT
GATCGAGGGCTACCT ACTACCGCAGCTACC TACTTGCTGCTGTTC
ATCGAGGGCTACCTC CTACCGCAGCTACCG ACTTGCTGCTGTTCC
TCGAGGGCTACCTCC TACCGCAGCTACCGC CTTGCTGCTGTTCCG
CGAGGGCTACCTCCA ACCGCAGCTACCGCT TTGCTGCTGTTCCGA
GAGGGCTACCTCCAC CCGCAGCTACCGCTT TGCTGCTGTTCCGAG
AGGGCTACCTCCACA CGCAGCTACCGCTTC GCTGCTGTTCCGAGT
GGGCTACCTCCACAT GCAGCTACCGCTTCC CTGCTGTTCCGAGTG
GGCTACCTCCACATC CAGCTACCGCTTCCC TGCTGTTCCGAGTGG
GCTACCTCCACATCC AGCTACCGCTTCCCC GCTGTTCCGAGTGGC
CTACCTCCACATCCT GCTACCGCTTCCCCA CTGTTCCGAGTGGCT
TACCTCCACATCCTG CTACCGCTTCCCCAA TGTTCCGAGTGGCTG
ACCTCCACATCCTGC TACCGCTTCCCCAAG GTTCCGAGTGGCTGG
CCTCCACATCCTGCT ACCGCTTCCCCAAGC TTCCGAGTGGCTGGC
CTCCACATCCTGCTC CCGCTTCCCCAAGCT TCCGAGTGGCTGGCC
TCCACATCCTGCTCA CGCTTCCCCAAGCTC CCGAGTGGCTGGCCT
CCACATCCTGCTCAT GCTTCCCCAAGCTCA CGAGTGGCTGGCCTC
CACATCCTGCTCATC CTTCCCCAAGCTCAC GAGTGGCTGGCCTCG
ACATCCTGCTCATCT TTCCCCAAGCTCACG AGTGGCTGGCCTCGA
CATCCTGCTCATCTC TCCCCAAGCTCACGG GTGGCTGGCCTCGAG
ATCCTGCTCATCTCC CCCCAAGCTCACGGT TGGCTGGCCTCGAGA
TCCTGCTCATCTCCA CCCAAGCTCACGGTC GGCTGGCCTCGAGAG
CCTGCTCATCTCCAA CCAAGCTCACGGTCA GCTGGCCTCGAGAGC
CTGCTCATCTCCAAG CAAGCTCACGGTCAT CTGGCCTCGAGAGCC
TGCTCATCTCCAAGG AAGCTCACGGTCATT TGGCCTCGAGAGCCT
GCTCATCTCCAAGGC AGCTCACGGTCATTA GGCCTCGAGAGCCTC
CTCATCTCCAAGGCC GCTCACGGTCATTAC GCCTCGAGAGCCTCG
TCATCTCCAAGGCCG CTCACGGTCATTACC CCTCGAGAGCCTCGG
CATCTCCAAGGCCGA TCACGGTCATTACCG CTCGAGAGCCTCGGA
ATCTCCAAGGCCGAG CACGGTCATTACCGA TCGAGAGCCTCGGAG
TCTCCAAGGCCGAGG ACGGTCATTACCGAG CGAGAGCCTCGGAGA
CTCCAAGGCCGAGGA CGGTCATTACCGAGT GAGAGCCTCGGAGAC
TCCAAGGCCGAGGAC GGTCATTACCGAGTA AGAGCCTCGGAGACC
CCAAGGCCGAGGACT GTCATTACCGAGTAC GAGCCTCGGAGACCT
CAAGGCCGAGGACTA TCATTACCGAGTACT AGCCTCGGAGACCTC
AAGGCCGAGGACTAC CATTACCGAGTACTT GCCTCGGAGACCTCT
AGGCCGAGGACTACC ATTACCGAGTACTTG CCTCGGAGACCTCTT
GGCCGAGGACTACCG TTACCGAGTACTTGC CTCGGAGACCTCTTC
GCCGAGGACTACCGC TACCGAGTACTTGCT TCGGAGACCTCTTCC
CCGAGGACTACCGCA ACCGAGTACTTGCTG CGGAGACCTCTTCCC
CGAGGACTACCGCAG CCGAGTACTTGCTGC GGAGACCTCTTCCCC
GAGGACTACCGCAGC CGAGTACTTGCTGCT GAGACCTCTTCCCCA
AGGACTACCGCAGCT GAGTACTTGCTGCTG AGACCTCTTCCCCAA
GACCTCTTCCCCAAC CTACAACTACGCCCT ATATTGGGCTTTACA
ACCTCTTCCCCAACC TACAACTACGCCCTG TATTGGGCTTTACAA
CCTCTTCCCCAACCT ACAACTACGCCCTGG ATTGGGCTTTACAAC
CTCTTCCCCAACCTC CAACTACGCCCTGGT TTGGGCTTTACAACC
TCTTCCCCAACCTCA AACTACGCCCTGGTC TGGGCTTTACAACCT
CTTCCCCAACCTCAC ACTACGCCCTGGTCA GGGCTTTACAACCTG
TTCCCCAACCTCACG CTACGCCCTGGTCAT GGCTTTACAACCTGA
TCCCCAACCTCACGG TACGCCCTGGTCATC GCTTTACAACCTGAG
CCCCAACCTCACGGT ACGCCCTGGTCATCT CTTTACAACCTGAGG
CCCAACCTCACGGTC CGCCCTGGTCATCTT TTTACAACCTGAGGA
CCAACCTCACGGTCA GCCCTGGTCATCTTC TTACAACCTGAGGAA
CAACCTCACGGTCAT CCCTGGTCATCTTCG TACAACCTGAGGAAC
AACCTCACGGTCATC CCTGGTCATCTTCGA ACAACCTGAGGAACA
ACCTCACGGTCATCC CTGGTCATCTTCGAG CAACCTGAGGAACAT
CCTCACGGTCATCCG TGGTCATCTTCGAGA AACCTGAGGAACATT
CTCACGGTCATCCGC GGTCATCTTCGAGAT ACCTGAGGAACATTA
TCACGGTCATCCGCG GTCATCTTCGAGATG CCTGAGGAACATTAC
CACGGTCATCCGCGG TCATCTTCGAGATGA CTGAGGAACATTACT
ACGGTCATCCGCGGC CATCTTCGAGATGAC TGAGGAACATTACTC
CGGTCATCCGCGGCT ATCTTCGAGATGACC GAGGAACATTACTCG
GGTCATCCGCGGCTG TCTTCGAGATGACCA AGGAACATTACTCGG
GTCATCCGCGGCTGG CTTCGAGATGACCAA GGAACATTACTCGGG
TCATCCGCGGCTGGA TTCGAGATGACCAAT GAACATTACTCGGGG
CATCCGCGGCTGGAA TCGAGATGACCAATC AACATTACTCGGGGG
ATCCGCGGCTGGAAA CGAGATGACCAATCT ACATTACTCGGGGGG
TCCGCGGCTGGAAAC GAGATGACCAATCTC CATTACTCGGGGGGC
CCGCGGCTGGAAACT AGATGACCAATCTCA ATTACTCGGGGGGCC
CGCGGCTGGAAACTC GATGACCAATCTCAA TTACTCGGGGGGCCA
GCGGCTGGAAACTCT ATGACCAATCTCAAG TACTCGGGGGGCCAT
CGGCTGGAAACTCTT TGACCAATCTCAAGG ACTCGGGGGGCCATC
GGCTGGAAACTCTTC GACCAATCTCAAGGA CTCGGGGGGCCATCA
GCTGGAAACTCTTCT ACCAATCTCAAGGAT TCGGGGGGCCATCAG
CTGGAAACTCTTCTA CCAATCTCAAGGATA CGGGGGGCCATCAGG
TGGAAACTCTTCTAC CAATCTCAAGGATAT GGGGGGCCATCAGGA
GGAAACTCTTCTACA AATCTCAAGGATATT GGGGGCCATCAGGAT
GAAACTCTTCTACAA ATCTCAAGGATATTG GGGGCCATCAGGATT
AAACTCTTCTACAAC TCTCAAGGATATTGG GGGCCATCAGGATTG
AACTCTTCTACAACT CTCAAGGATATTGGG GGCCATCAGGATTGA
ACTCTTCTACAACTA TCAAGGATATTGGGC GCCATCAGGATTGAG
CTCTTCTACAACTAC CAAGGATATTGGGCT CCATCAGGATTGAGA
TCTTCTACAACTACG AAGGATATTGGGCTT CATCAGGATTGAGAA
CTTCTACAACTACGC AGGATATTGGGCTTT ATCAGGATTGAGAAA
TTCTACAACTACGCC GGATATTGGGCTTTA TCAGGATTGAGAAAA
TCTACAACTACGCCC GATATTGGGCTTTAC CAGGATTGAGAAAAA
AGGATTGAGAAAAAT CTGGTCCCTGATCCT GGAATAAGCCCCCAA
GGATTGAGAAAAATG TGGTCCCTGATCCTG GAATAAGCCCCCAAA
GATTGAGAAAAATGC GGTCCCTGATCCTGG AATAAGCCCCCAAAG
ATTGAGAAAAATGCT GTCCCTGATCCTGGA ATAAGCCCCCAAAGG
TTGAGAAAAATGCTG TCCCTGATCCTGGAT TAAGCCCCCAAAGGA
TGAGAAAAATGCTGA CCCTGATCCTGGATG AAGCCCCCAAAGGAA
GAGAAAAATGCTGAC CCTGATCCTGGATGC AGCCCCCAAAGGAAT
AGAAAAATGCTGACC CTGATCCTGGATGCG GCCCCCAAAGGAATG
GAAAAATGCTGACCT TGATCCTGGATGCGG CCCCCAAAGGAATGT
AAAAATGCTGACCTC GATCCTGGATGCGGT CCCCAAAGGAATGTG
AAAATGCTGACCTCT ATCCTGGATGCGGTG CCCAAAGGAATGTGG
AAATGCTGACCTCTG TCCTGGATGCGGTGT CCAAAGGAATGTGGG
AATGCTGACCTCTGT CCTGGATGCGGTGTC CAAAGGAATGTGGGG
ATGCTGACCTCTGTT CTGGATGCGGTGTCC AAAGGAATGTGGGGA
TGCTGACCTCTGTTA TGGATGCGGTGTCCA AAGGAATGTGGGGAC
GCTGACCTCTGTTAC GGATGCGGTGTCCAA AGGAATGTGGGGACC
CTGACCTCTGTTACC GATGCGGTGTCCAAT GGAATGTGGGGACCT
TGACCTCTGTTACCT ATGCGGTGTCCAATA GAATGTGGGGACCTG
GACCTCTGTTACCTC TGCGGTGTCCAATAA AATGTGGGGACCTGT
ACCTCTGTTACCTCT GCGGTGTCCAATAAC ATGTGGGGACCTGTG
CCTCTGTTACCTCTC CGGTGTCCAATAACT TGTGGGGACCTGTGT
CTCTGTTACCTCTCC GGTGTCCAATAACTA GTGGGGACCTGTGTC
TCTGTTACCTCTCCA GTGTCCAATAACTAC TGGGGACCTGTGTCC
CTGTTACCTCTCCAC TGTCCAATAACTACA GGGGACCTGTGTCCA
TGTTACCTCTCCACT GTCCAATAACTACAT GGGACCTGTGTCCAG
GTTACCTCTCCACTG TCCAATAACTACATT GGACCTGTGTCCAGG
TTACCTCTCCACTGT CCAATAACTACATTG GACCTGTGTCCAGGG
TACCTCTCCACTGTG CAATAACTACATTGT ACCTGTGTCCAGGGA
ACCTCTCCACTGTGG AATAACTACATTGTG CCTGTGTCCAGGGAC
CCTCTCCACTGTGGA ATAACTACATTGTGG CTGTGTCCAGGGACC
CTCTCCACTGTGGAC TAACTACATTGTGGG TGTGTCCAGGGACCA
TCTCCACTGTGGACT AACTACATTGTGGGG GTGTCCAGGGACCAT
CTCCACTGTGGACTG ACTACATTGTGGGGA TGTCCAGGGACCATG
TCCACTGTGGACTGG CTACATTGTGGGGAA GTCCAGGGACCATGG
CCACTGTGGACTGGT TACATTGTGGGGAAT TCCAGGGACCATGGA
CACTGTGGACTGGTC ACATTGTGGGGAATA CCAGGGACCATGGAG
ACTGTGGACTGGTCC CATTGTGGGGAATAA CAGGGACCATGGAGG
CTGTGGACTGGTCCC ATTGTGGGGAATAAG AGGGACCATGGAGGA
TGTGGACTGGTCCCT TTGTGGGGAATAAGC GGGACCATGGAGGAG
GTGGACTGGTCCCTG TGTGGGGAATAAGCC GGACCATGGAGGAGA
TGGACTGGTCCCTGA GTGGGGAATAAGCCC GACCATGGAGGAGAA
GGACTGGTCCCTGAT TGGGGAATAAGCCCC ACCATGGAGGAGAAG
GACTGGTCCCTGATC GGGGAATAAGCCCCC CCATGGAGGAGAAGC
ACTGGTCCCTGATCC GGGAATAAGCCCCCA CATGGAGGAGAAGCC
ATGGAGGAGAAGCCG GTACAACTACCGCTG GCCCAAGCACGTGTG
TGGAGGAGAAGCCGA TACAACTACCGCTGC CCCAAGCACGTGTGG
GGAGGAGAAGCCGAT ACAACTACCGCTGCT CCAAGCACGTGTGGG
GAGGAGAAGCCGATG CAACTACCGCTGCTG CAAGCACGTGTGGGA
AGGAGAAGCCGATGT AACTACCGCTGCTGG AAGCACGTGTGGGAA
GGAGAAGCCGATGTG ACTACCGCTGCTGGA AGCACGTGTGGGAAG
GAGAAGCCGATGTGT CTACCGCTGCTGGAC GCACGTGTGGGAAGC
AGAAGCCGATGTGTG TACCGCTGCTGGACC CACGTGTGGGAAGCG
GAAGCCGATGTGTGA ACCGCTGCTGGACCA ACGTGTGGGAAGCGG
AAGCCGATGTGTGAG CCGCTGCTGGACCAC CGTGTGGGAAGCGGG
AGCCGATGTGTGAGA CGCTGCTGGACCACA GTGTGGGAAGCGGGC
GCCGATGTGTGAGAA GCTGCTGGACCACAA TGTGGGAAGCGGGCG
CCGATGTGTGAGAAG CTGCTGGACCACAAA GTGGGAAGCGGGCGT
CGATGTGTGAGAAGA TGCTGGACCACAAAC TGGGAAGCGGGCGTG
GATGTGTGAGAAGAC GCTGGACCACAAACC GGGAAGCGGGCGTGC
ATGTGTGAGAAGACC CTGGACCACAAACCG GGAAGCGGGCGTGCA
TGTGTGAGAAGACCA TGGACCACAAACCGC GAAGCGGGCGTGCAC
GTGTGAGAAGACCAC GGACCACAAACCGCT AAGCGGGCGTGCACC
TGTGAGAAGACCACC GACCACAAACCGCTG AGCGGGCGTGCACCG
GTGAGAAGACCACCA ACCACAAACCGCTGC GCGGGCGTGCACCGA
TGAGAAGACCACCAT CCACAAACCGCTGCC CGGGCGTGCACCGAG
GAGAAGACCACCATC CACAAACCGCTGCCA GGGCGTGCACCGAGA
AGAAGACCACCATCA ACAAACCGCTGCCAG GGCGTGCACCGAGAA
GAAGACCACCATCAA CAAACCGCTGCCAGA GCGTGCACCGAGAAC
AAGACCACCATCAAC AAACCGCTGCCAGAA CGTGCACCGAGAACA
AGACCACCATCAACA AACCGCTGCCAGAAA GTGCACCGAGAACAA
GACCACCATCAACAA ACCGCTGCCAGAAAA TGCACCGAGAACAAT
ACCACCATCAACAAT CCGCTGCCAGAAAAT GCACCGAGAACAATG
CCACCATCAACAATG CGCTGCCAGAAAATG CACCGAGAACAATGA
CACCATCAACAATGA GCTGCCAGAAAATGT ACCGAGAACAATGAG
ACCATCAACAATGAG CTGCCAGAAAATGTG CCGAGAACAATGAGT
CCATCAACAATGAGT TGCCAGAAAATGTGC CGAGAACAATGAGTG
CATCAACAATGAGTA GCCAGAAAATGTGCC GAGAACAATGAGTGC
ATCAACAATGAGTAC CCAGAAAATGTGCCC AGAACAATGAGTGCT
TCAACAATGAGTACA CAGAAAATGTGCCCA GAACAATGAGTGCTG
CAACAATGAGTACAA AGAAAATGTGCCCAA AACAATGAGTGCTGC
AACAATGAGTACAAC GAAAATGTGCCCAAG ACAATGAGTGCTGCC
ACAATGAGTACAACT AAAATGTGCCCAAGC CAATGAGTGCTGCCA
CAATGAGTACAACTA AAATGTGCCCAAGCA AATGAGTGCTGCCAC
AATGAGTACAACTAC AATGTGCCCAAGCAC ATGAGTGCTGCCACC
ATGAGTACAACTACC ATGTGCCCAAGCACG TGAGTGCTGCCACCC
TGAGTACAACTACCG TGTGCCCAAGCACGT GAGTGCTGCCACCCC
GAGTACAACTACCGC GTGCCCAAGCACGTG AGTGCTGCCACCCCG
AGTACAACTACCGCT TGCCCAAGCACGTGT GTGCTGCCACCCCGA
TGCTGCCACCCCGAG CGACACGGCCTGTGT TCTGTGTGCCTGCCT
GCTGCCACCCCGAGT GACACGGCCTGTGTA CTGTGTGCCTGCCTG
CTGCCACCCCGAGTG ACACGGCCTGTGTAG TGTGTGCCTGCCTGC
TGCCACCCCGAGTGC CACGGCCTGTGTAGC GTGTGCCTGCCTGCC
GCCACCCCGAGTGCC ACGGCCTGTGTAGCT TGTGCCTGCCTGCCC
CCACCCCGAGTGCCT CGGCCTGTGTAGCTT GTGCCTGCCTGCCCG
CACCCCGAGTGCCTG GGCCTGTGTAGCTTG TGCCTGCCTGCCCGC
ACCCCGAGTGCCTGG GCCTGTGTAGCTTGC GCCTGCCTGCCCGCC
CCCCGAGTGCCTGGG CCTGTGTAGCTTGCC CCTGCCTGCCCGCCC
CCCGAGTGCCTGGGC CTGTGTAGCTTGCCG CTGCCTGCCCGCCCA
CCGAGTGCCTGGGCA TGTGTAGCTTGCCGC TGCCTGCCCGCCCAA
CGAGTGCCTGGGCAG GTGTAGCTTGCCGCC GCCTGCCCGCCCAAC
GAGTGCCTGGGCAGC TGTAGCTTGCCGCCA CCTGCCCGCCCAACA
AGTGCCTGGGCAGCT GTAGCTTGCCGCCAC CTGCCCGCCCAACAC
GTGCCTGGGCAGCTG TAGCTTGCCGCCACT TGCCCGCCCAACACC
TGCCTGGGCAGCTGC AGCTTGCCGCCACTA GCCCGCCCAACACCT
GCCTGGGCAGCTGCA GCTTGCCGCCACTAC CCCGCCCAACACCTA
CCTGGGCAGCTGCAG CTTGCCGCCACTACT CCGCCCAACACCTAC
CTGGGCAGCTGCAGC TTGCCGCCACTACTA CGCCCAACACCTACA
TGGGCAGCTGCAGCG TGCCGCCACTACTAC GCCCAACACCTACAG
GGGCAGCTGCAGCGC GCCGCCACTACTACT CCCAACACCTACAGG
GGCAGCTGCAGCGCG CCGCCACTACTACTA CCAACACCTACAGGT
GCAGCTGCAGCGCGC CGCCACTACTACTAT CAACACCTACAGGTT
CAGCTGCAGCGCGCC GCCACTACTACTATG AACACCTACAGGTTT
AGCTGCAGCGCGCCT CCACTACTACTATGC ACACCTACAGGTTTG
GCTGCAGCGCGCCTG CACTACTACTATGCC CACCTACAGGTTTGA
CTGCAGCGCGCCTGA ACTACTACTATGCCG ACCTACAGGTTTGAG
TGCAGCGCGCCTGAC CTACTACTATGCCGG CCTACAGGTTTGAGG
GCAGCGCGCCTGACA TACTACTATGCCGGT CTACAGGTTTGAGGG
CAGCGCGCCTGACAA ACTACTATGCCGGTG TACAGGTTTGAGGGC
AGCGCGCCTGACAAC CTACTATGCCGGTGT ACAGGTTTGAGGGCT
GCGCGCCTGACAACG TACTATGCCGGTGTC CAGGTTTGAGGGCTG
CGCGCCTGACAACGA ACTATGCCGGTGTCT AGGTTTGAGGGCTGG
GCGCCTGACAACGAC CTATGCCGGTGTCTG GGTTTGAGGGCTGGC
CGCCTGACAACGACA TATGCCGGTGTCTGT GTTTGAGGGCTGGCG
GCCTGACAACGACAC ATGCCGGTGTCTGTG TTTGAGGGCTGGCGC
CCTGACAACGACACG TGCCGGTGTCTGTGT TTGAGGGCTGGCGCT
CTGACAACGACACGG GCCGGTGTCTGTGTG TGAGGGCTGGCGCTG
TGACAACGACACGGC CCGGTGTCTGTGTGC GAGGGCTGGCGCTGT
GACAACGACACGGCC CGGTGTCTGTGTGCC AGGGCTGGCGCTGTG
ACAACGACACGGCCT GGTGTCTGTGTGCCT GGGCTGGCGCTGTGT
CAACGACACGGCCTG GTGTCTGTGTGCCTG GGCTGGCGCTGTGTG
AACGACACGGCCTGT TGTCTGTGTGCCTGC GCTGGCGCTGTGTGG
ACGACACGGCCTGTG GTCTGTGTGCCTGCC CTGGCGCTGTGTGGA
TGGCGCTGTGTGGAC CGAGAGCAGCGACTC GCATGCAGGAGTGCC
GGCGCTGTGTGGACC GAGAGCAGCGACTCC CATGCAGGAGTGCCC
GCGCTGTGTGGACCG AGAGCAGCGACTCCG ATGCAGGAGTGCCCC
CGCTGTGTGGACCGT GAGCAGCGACTCCGA TGCAGGAGTGCCCCT
GCTGTGTGGACCGTG AGCAGCGACTCCGAG GCAGGAGTGCCCCTC
CTGTGTGGACCGTGA GCAGCGACTCCGAGG CAGGAGTGCCCCTCG
TGTGTGGACCGTGAC CAGCGACTCCGAGGG AGGAGTGCCCCTCGG
GTGTGGACCGTGACT AGCGACTCCGAGGGG GGAGTGCCCCTCGGG
TGTGGACCGTGACTT GCGACTCCGAGGGGT GAGTGCCCCTCGGGC
GTGGACCGTGACTTC CGACTCCGAGGGGTT AGTGCCCCTCGGGCT
TGGACCGTGACTTCT GACTCCGAGGGGTTT GTGCCCCTCGGGCTT
GGACCGTGACTTCTG ACTCCGAGGGGTTTG TGCCCCTCGGGCTTC
GACCGTGACTTCTGC CTCCGAGGGGTTTGT GCCCCTCGGGCTTCA
ACCGTGACTTCTGCG TCCGAGGGGTTTGTG CCCCTCGGGCTTCAT
CCGTGACTTCTGCGC CCGAGGGGTTTGTGA CCCTCGGGCTTCATC
CGTGACTTCTGCGCC CGAGGGGTTTGTGAT CCTCGGGCTTCATCC
GTGACTTCTGCGCCA GAGGGGTTTGTGATC CTCGGGCTTCATCCG
TGACTTCTGCGCCAA AGGGGTTTGTGATCC TCGGGCTTCATCCGC
GACTTCTGCGCCAAC GGGGTTTGTGATCCA CGGGCTTCATCCGCA
ACTTCTGCGCCAACA GGGTTTGTGATCCAC GGGCTTCATCCGCAA
CTTCTGCGCCAACAT GGTTTGTGATCCACG GGCTTCATCCGCAAC
TTCTGCGCCAACATC GTTTGTGATCCACGA GCTTCATCCGCAACG
TCTGCGCCAACATCC TTTGTGATCCACGAC CTTCATCCGCAACGG
CTGCGCCAACATCCT TTGTGATCCACGACG TTCATCCGCAACGGC
TGCGCCAACATCCTC TGTGATCCACGACGG TCATCCGCAACGGCA
GCGCCAACATCCTCA GTGATCCACGACGGC CATCCGCAACGGCAG
CGCCAACATCCTCAG TGATCCACGACGGCG ATCCGCAACGGCAGC
GCCAACATCCTCAGC GATCCACGACGGCGA TCCGCAACGGCAGCC
CCAACATCCTCAGCG ATCCACGACGGCGAG CCGCAACGGCAGCCA
CAACATCCTCAGCGC TCCACGACGGCGAGT CGCAACGGCAGCCAG
AACATCCTCAGCGCC CCACGACGGCGAGTG GCAACGGCAGCCAGA
ACATCCTCAGCGCCG CACGACGGCGAGTGC CAACGGCAGCCAGAG
CATCCTCAGCGCCGA ACGACGGCGAGTGCA AACGGCAGCCAGAGC
ATCCTCAGCGCCGAG CGACGGCGAGTGCAT ACGGCAGCCAGAGCA
TCCTCAGCGCCGAGA GACGGCGAGTGCATG CGGCAGCCAGAGCAT
CCTCAGCGCCGAGAG ACGGCGAGTGCATGC GGCAGCCAGAGCATG
CTCAGCGCCGAGAGC CGGCGAGTGCATGCA GCAGCCAGAGCATGT
TCAGCGCCGAGAGCA GGCGAGTGCATGCAG CAGCCAGAGCATGTA
CAGCGCCGAGAGCAG GCGAGTGCATGCAGG AGCCAGAGCATGTAC
AGCGCCGAGAGCAGC CGAGTGCATGCAGGA GCCAGAGCATGTACT
GCGCCGAGAGCAGCG GAGTGCATGCAGGAG CCAGAGCATGTACTG
CGCCGAGAGCAGCGA AGTGCATGCAGGAGT CAGAGCATGTACTGC
GCCGAGAGCAGCGAC GTGCATGCAGGAGTG AGAGCATGTACTGCA
CCGAGAGCAGCGACT TGCATGCAGGAGTGC GAGCATGTACTGCAT
AGCATGTACTGCATC TGAGGAAGAAAAGAA CTCAGATGCTCCAAG
GCATGTACTGCATCC GAGGAAGAAAAGAAA TCAGATGCTCCAAGG
CATGTACTGCATCCC AGGAAGAAAAGAAAA CAGATGCTCCAAGGA
ATGTACTGCATCCCT GGAAGAAAAGAAAAC AGATGCTCCAAGGAT
TGTACTGCATCCCTT GAAGAAAAGAAAACA GATGCTCCAAGGATG
GTACTGCATCCCTTG AAGAAAAGAAAACAA ATGCTCCAAGGATGC
TACTGCATCCCTTGT AGAAAAGAAAACAAA TGCTCCAAGGATGCA
ACTGCATCCCTTGTG GAAAAGAAAACAAAG GCTCCAAGGATGCAC
CTGCATCCCTTGTGA AAAAGAAAACAAAGA CTCCAAGGATGCACC
TGCATCCCTTGTGAA AAAGAAAACAAAGAC TCCAAGGATGCACCA
GCATCCCTTGTGAAG AAGAAAACAAAGACC CCAAGGATGCACCAT
CATCCCTTGTGAAGG AGAAAACAAAGACCA CAAGGATGCACCATC
ATCCCTTGTGAAGGT GAAAACAAAGACCAT AAGGATGCACCATCT
TCCCTTGTGAAGGTC AAAACAAAGACCATT AGGATGCACCATCTT
CCCTTGTGAAGGTCC AAACAAAGACCATTG GGATGCACCATCTTC
CCTTGTGAAGGTCCT AACAAAGACCATTGA GATGCACCATCTTCA
CTTGTGAAGGTCCTT ACAAAGACCATTGAT ATGCACCATCTTCAA
TTGTGAAGGTCCTTG CAAAGACCATTGATT TGCACCATCTTCAAG
TGTGAAGGTCCTTGC AAAGACCATTGATTC GCACCATCTTCAAGG
GTGAAGGTCCTTGCC AAGACCATTGATTCT CACCATCTTCAAGGG
TGAAGGTCCTTGCCC AGACCATTGATTCTG ACCATCTTCAAGGGC
GAAGGTCCTTGCCCG GACCATTGATTCTGT CCATCTTCAAGGGCA
AAGGTCCTTGCCCGA ACCATTGATTCTGTT CATCTTCAAGGGCAA
AGGTCCTTGCCCGAA CCATTGATTCTGTTA ATCTTCAAGGGCAAT
GGTCCTTGCCCGAAG CATTGATTCTGTTAC TCTTCAAGGGCAATT
GTCCTTGCCCGAAGG ATTGATTCTGTTACT CTTCAAGGGCAATTT
TCCTTGCCCGAAGGT TTGATTCTGTTACTT TTCAAGGGCAATTTG
CCTTGCCCGAAGGTC TGATTCTGTTACTTC TCAAGGGCAATTTGC
CTTGCCCGAAGGTCT GATTCTGTTACTTCT CAAGGGCAATTTGCT
TTGCCCGAAGGTCTG ATTCTGTTACTTCTG AAGGGCAATTTGCTC
TGCCCGAAGGTCTGT TTCTGTTACTTCTGC AGGGCAATTTGCTCA
GCCCGAAGGTCTGTG TCTGTTACTTCTGCT GGGCAATTTGCTCAT
CCCGAAGGTCTGTGA CTGTTACTTCTGCTC GGCAATTTGCTCATT
CCGAAGGTCTGTGAG TGTTACTTCTGCTCA GCAATTTGCTCATTA
CGAAGGTCTGTGAGG GTTACTTCTGCTCAG CAATTTGCTCATTAA
GAAGGTCTGTGAGGA TTACTTCTGCTCAGA AATTTGCTCATTAAC
AAGGTCTGTGAGGAA TACTTCTGCTCAGAT ATTTGCTCATTAACA
AGGTCTGTGAGGAAG ACTTCTGCTCAGATG TTTGCTCATTAACAT
GGTCTGTGAGGAAGA CTTCTGCTCAGATGC TTGCTCATTAACATC
GTCTGTGAGGAAGAA TTCTGCTCAGATGCT TGCTCATTAACATCC
TCTGTGAGGAAGAAA TCTGCTCAGATGCTC GCTCATTAACATCCG
CTGTGAGGAAGAAAA CTGCTCAGATGCTCC CTCATTAACATCCGA
TGTGAGGAAGAAAAG TGCTCAGATGCTCCA TCATTAACATCCGAC
GTGAGGAAGAAAAGA GCTCAGATGCTCCAA CATTAACATCCGACG
ATTAACATCCGACGG CTTCATGGGGCTCAT GCCATTCTCATGCCT
TTAACATCCGACGGG TTCATGGGGCTCATC CCATTCTCATGCCTT
TAACATCCGACGGGG TCATGGGGCTCATCG CATTCTCATGCCTTG
AACATCCGACGGGGG CATGGGGCTCATCGA ATTCTCATGCCTTGG
ACATCCGACGGGGGA ATGGGGCTCATCGAG TTCTCATGCCTTGGT
CATCCGACGGGGGAA TGGGGCTCATCGAGG TCTCATGCCTTGGTC
ATCCGACGGGGGAAT GGGGCTCATCGAGGT CTCATGCCTTGGTCT
TCCGACGGGGGAATA GGGCTCATCGAGGTG TCATGCCTTGGTCTC
CCGACGGGGGAATAA GGCTCATCGAGGTGG CATGCCTTGGTCTCC
CGACGGGGGAATAAC GCTCATCGAGGTGGT ATGCCTTGGTCTCCT
GACGGGGGAATAACA CTCATCGAGGTGGTG TGCCTTGGTCTCCTT
ACGGGGGAATAACAT TCATCGAGGTGGTGA GCCTTGGTCTCCTTG
CGGGGGAATAACATT CATCGAGGTGGTGAC CCTTGGTCTCCTTGT
GGGGGAATAACATTG ATCGAGGTGGTGACG CTTGGTCTCCTTGTC
GGGGAATAACATTGC TCGAGGTGGTGACGG TTGGTCTCCTTGTCC
GGGAATAACATTGCT CGAGGTGGTGACGGG TGGTCTCCTTGTCCT
GGAATAACATTGCTT GAGGTGGTGACGGGC GGTCTCCTTGTCCTT
GAATAACATTGCTTC AGGTGGTGACGGGCT GTCTCCTTGTCCTTC
AATAACATTGCTTCA GGTGGTGACGGGCTA TCTCCTTGTCCTTCC
ATAACATTGCTTCAG GTGGTGACGGGCTAC CTCCTTGTCCTTCCT
TAACATTGCTTCAGA TGGTGACGGGCTACG TCCTTGTCCTTCCTA
AACATTGCTTCAGAG GGTGACGGGCTACGT CCTTGTCCTTCCTAA
ACATTGCTTCAGAGC GTGACGGGCTACGTG CTTGTCCTTCCTAAA
CATTGCTTCAGAGCT TGACGGGCTACGTGA TTGTCCTTCCTAAAA
ATTGCTTCAGAGCTG GACGGGCTACGTGAA TGTCCTTCCTAAAAA
TTGCTTCAGAGCTGG ACGGGCTACGTGAAG GTCCTTCCTAAAAAA
TGCTTCAGAGCTGGA CGGGCTACGTGAAGA TCCTTCCTAAAAAAC
GCTTCAGAGCTGGAG GGGCTACGTGAAGAT CCTTCCTAAAAAACC
CTTCAGAGCTGGAGA GGCTACGTGAAGATC CTTCCTAAAAAACCT
TTCAGAGCTGGAGAA GCTACGTGAAGATCC TTCCTAAAAAACCTT
TCAGAGCTGGAGAAC CTACGTGAAGATCCG TCCTAAAAAACCTTC
CAGAGCTGGAGAACT TACGTGAAGATCCGC CCTAAAAAACCTTCG
AGAGCTGGAGAACTT ACGTGAAGATCCGCC CTAAAAAACCTTCGC
GAGCTGGAGAACTTC CGTGAAQATCCGCCA TAAAAAACCTTCGCC
AGCTGGAGAACTTCA GTGAAGATCCGCCAT AAAAAACCTTCGCCT
GCTGGAGAACTTCAT TGAAGATCCGCCATT AAAAACCTTCGCCTC
CTGGAGAACTTCATG GAAGATCCGCCATTC AAAACCTTCGCCTCA
TGGAGAACTTCATGG AAGATCCGCCATTCT AAACCTTCGCCTCAT
GGAGAACTTCATGGG AGATCCGCCATTCTC AACCTTCGCCTCATC
GAGAACTTCATGGGG GATCCGCCATTCTCA ACCTTCGCCTCATCC
AGAACTTCATGGGGC ATCCGCCATTCTCAT CCTTCGCCTCATCCT
GAACTTCATGGGGCT TCCGCCATTCTCATG CTTCGCCTCATCCTA
AACTTCATGGGGCTC CCGCCATTCTCATGC TTCGCCTCATCCTAG
ACTTCATGGGGCTCA CGCCATTCTCATGCC TCGCCTCATCCTAGG
CGCCTCATCCTAGGA CTACGTCCTCGACAA ACCACCGCAACCTGA
GCCTCATCCTAGGAG TACGTCCTCGACAAC CCACCGCAACCTGAC
CCTCATCCTAGGAGA ACGTCCTCGACAACC CACCGCAACCTGACC
CTCATCCTAGGAGAG CGTCCTCGACAACCA ACCGCAACCTGACCA
TCATCCTAGGAGAGG GTCCTCGACAACCAG CCGCAACCTGACCAT
CATCCTAGGAGAGGA TCCTCGACAACCAGA CGCAACCTGACCATC
ATCCTAGGAGAGGAG CCTCGACAACCAGAA GCAACCTGACCATCA
TCCTAGGAGAGGAGC CTCGACAACCAGAAC CAACCTGACCATCAA
CCTAGGAGAGGAGCA TCGACAACCAGAACT AACCTGACCATCAAA
CTAGGAGAGGAGCAG CGACAACCAGAACTT ACCTGACCATCAAAG
TAGGAGAGGAGCAGC GACAACCAGAACTTG CCTGACCATCAAAGC
AGGAGAGGAGCAGCT ACAACCAGAACTTGC CTGACCATCAAAGCA
GGAGAGGAGCAGCTA CAACCAGAACTTGCA TGACCATCAAAGCAG
GAGAGGAGCAGCTAG AACCAGAACTTGCAG GACCATCAAAGCAGG
AGAGGAGCAGCTAGA ACCAGAACTTGCAGC ACCATCAAAGCAGGG
GAGGAGCAGCTAGAA CCAGAACTTGCAGCA CCATCAAAGCAGGGA
AGGAGCAGCTAGAAG CAGAACTTGCAGCAA CATCAAAGCAGGGAA
GGAGCAGCTAGAAGG AGAACTTGCAGCAAC ATCAAAGCAGGGAAA
GAGCAGCTAGAAGGG GAACTTGCAGCAACT TCAAAGCAGGGAAAA
AGCAGCTAGAAGGGA AACTTGCAGCAACTG CAAAGCAGGGAAAAT
GCAGCTAGAAGGGAA ACTTGCAGCAACTGT AAAGCAGGGAAAATG
CAGCTAGAAGGGAAT CTTGCAGCAACTGTG AAGCAGGGAAAATGT
AGCTAGAAGGGAATT TTGCAGCAACTGTGG AGCAGGGAAAATGTA
GCTAGAAGGGAATTA TGCAGCAACTGTGGG GCAGGGAAAATGTAC
CTAGAAGGGAATTAC GCAGCAACTGTGGGA CAGGGAAAATGTACT
TAGAAGGGAATTACT CAGCAACTGTGGGAC AGGGAAAATGTACTT
AGAAGGGAATTACTC AGCAACTGTGGGACT GGGAAAATGTACTTT
GAAGGGAATTACTCC GCAACTGTGGGACTG GGAAAATGTACTTTG
AAGGGAATTACTCCT CAACTGTGGGACTGG GAAAATGTACTTTGC
AGGGAATTACTCCTT AACTGTGGGACTGGG AAAATGTACTTTGCT
GGGAATTACTCCTTC ACTGTGGGACTGGGA AAATGTACTTTGCTT
GGAATTACTCCTTCT CTGTGGGACTGGGAC AATGTACTTTGCTTT
GAATTACTCCTTCTA TGTGGGACTGGGACC ATGTACTTTGCTTTC
AATTACTCCTTCTAC GTGGGACTGGGACCA TGTACTTTGCTTTCA
ATTACTCCTTCTACG TGGGACTGGGACCAC GTACTTTGCTTTCAA
TTACTCCTTCTACGT GGGACTGGGACCACC TACTTTGCTTTCAAT
TACTCCTTCTACGTC GGACTGGGACCACCG ACTTTGCTTTCAATC
ACTCCTTCTACGTCC GACTGGGACCACCGC CTTTGCTTTCAATCC
CTCCTTCTACGTCCT ACTGGGACCACCGCA TTTGCTTTCAATCCC
TCCTTCTACGTCCTC CTGGGACCACCGCAA TTGCTTTCAATCCCA
CCTTCTACGTCCTCG TGGGACCACCGCAAC TGCTTTCAATCCCAA
CTTCTACGTCCTCGA GGGACCACCGCAACC GCTTTCAATCCCAAA
TTCTACGTCCTCGAC GGACCACCGCAACCT CTTTCAATCCCAAAT
TCTACGTCCTCGACA GACCACCGCAACCTG TTTCAATCCCAAATT
TTCAATCCCAAATTA AGTGACGGGGACTAA CCAGGAACAACGGGG
TCAATCCCAAATTAT GTGACGGGGACTAAA CAGGAACAACGGGGA
CAATCCCAAATTATG TGACGGGGACTAAAG AGGAACAACGGGGAG
AATCCCAAATTATGT GACGGGGACTAAAGG GGAACAACGGGGAGA
ATCCCAAATTATGTG ACGGGGACTAAAGGG GAACAACGGGGAGAG
TCCCAAATTATGTGT CGGGGACTAAAGGGC AACAACGGGGAGAGA
CCCAAATTATGTGTT GGGGACTAAAGGGCG ACAACGGGGAGAGAG
CCAAATTATGTGTTT GGGACTAAAGGGCGC CAACGGGGAGAGAGC
CAAATTATGTGTTTC GGACTAAAGGGCGCC AACGGGGAGAGAGCC
AAATTATGTGTTTCC GACTAAAGGGCGCCA ACGGGGAGAGAGCCT
AATTATGTGTTTCCG ACTAAAGGGCGCCAA CGGGGAGAGAGCCTC
ATTATGTGTTTCCGA CTAAAGGGCGCCAAA GGGGAGAGAGCCTCC
TTATGTGTTTCCGAA TAAAGGGCGCCAAAG GGGAGAGAGCCTCCT
TATGTGTTTCCGAAA AAAGGGCGCCAAAGC GGAGAGAGCCTCCTG
ATGTGTTTCCGAAAT AAGGGCGCCAAAGCA GAGAGAGCCTCCTGT
TGTGTTTCCGAAATT AGGGCGCCAAAGCAA AGAGAGCCTCCTGTG
GTGTTTCCGAAATTT GGGCGCCAAAGCAAA GAGAGCCTCCTGTGA
TGTTTCCGAAATTTA GGCGCCAAAGCAAAG AGAGCCTCCTGTGAA
GTTTCCGAAATTTAC GCGCCAAAGCAAAGG GAGCCTCCTGTGAAA
TTTCCGAAATTTACC CGCCAAAGCAAAGGG AGCCTCCTGTGAAAG
TTCCGAAATTTACCG GCCAAAGCAAAGGGG GCCTCCTGTGAAAGT
TCCGAAATTTACCGC CCAAAGCAAAGGGGA CCTCCTGTGAAAGTG
CCGAAATTTACCGCA CAAAGCAAAGGGGAC CTCCTGTGAAAGTGA
CGAAATTTACCGCAT AAAGCAAAGGGGACA TCCTGTGAAAGTGAC
GAAATTTACCGCATG AAGCAAAGGGGACAT CCTGTGAAAGTGACG
AAATTTACCGCATGG AGCAAAGGGGACATA CTGTGAAAGTGACGT
AATTTACCGCATGGA GCAAAGGGGACATAA TGTGAAAGTGACGTC
ATTTACCGCATGGAG CAAAGGGGACATAAA GTGAAAGTGACGTCC
TTTACCGCATGGAGG AAAGGGGACATAAAC TGAAAGTGACGTCCT
TTACCGCATGGAGGA AAGGGGACATAAACA GAAAGTGACGTCCTG
TACCGCATGGAGGAA AGGGGACATAAACAC AAAGTGACGTCCTGC
ACCGCATGGAGGAAG GGGGACATAAACACC AAGTGACGTCCTGCA
CCGCATGGAGGAAGT GGGACATAAACACCA AGTGACGTCCTGCAT
CGCATGGAGGAAGTG GGACATAAACACCAG GTGACGTCCTGCATT
GCATGGAGGAAGTGA GACATAAACACCAGG TGACGTCCTGCATTT
CATGGAGGAAGTGAC ACATAAACACCAGGA GACGTCCTGCATTTC
ATGGAGGAAGTGACG CATAAACACCAGGAA ACGTCCTGCATTTCA
TGGAGGAAGTGACGG ATAAACACCAGGAAC CGTCCTGCATTTCAC
GGAGGAAGTGACGGG TAAACACCAGGAACA GTCCTGCATTTCACC
GAGGAAGTGACGGGG AAACACCAGGAACAA TCCTGCATTTCACCT
AGGAAGTGACGGGGA AACACCAGGAACAAC CCTGCATTTCACCTC
GGAAGTGACGGGGAC ACACCAGGAACAACG CTGCATTTCACCTCC
GAAGTGACGGGGACT CACCAGGAACAACGG TGCATTTCACCTCCA
AAGTGACGGGGACTA ACCAGGAACAACGGG GCATTTCACCTCCAC
CATTTCACCTCCACC CTGGCACCGGTACCG TCACCGTTTACTACA
ATTTCACCTCCACCA TGGCACCGGTACCGG CACCGTTTACTACAA
TTTCACCTCCACCAC GGCACCGGTACCGGC ACCGTTTACTACAAG
TTCACCTCCACCACC GCACCGGTACCGGCC CCGTTTACTACAAGG
TCACCTCCACCACCA CACCGGTACCGGCCC CGTTTACTACAAGGA
CACCTCCACCACCAC ACCGGTACCGGCCCC GTTTACTACAAGGAA
ACCTCCACCACCACG CCGGTACCGGCCCCC TTTACTACAAGGAAG
CCTCCACCACCACGT CGGTACCGGCCCCCT TTACTACAAGGAAGC
CTCCACCACCACGTC GGTACCGGCCCCCTG TACTACAAGGAAGCA
TCCACCACCACGTCG GTACCGGCCCCCTGA ACTACAAGGAAGCAC
CCACCACCACGTCGA TACCGGCCCCCTGAC CTACAAGGAAGCACC
CACCACCACGTCGAA ACCGGCCCCCTGACT TACAAGGAAGCACCC
ACCACCACGTCGAAG CCGGCCCCCTGACTA ACAAGGAAGCACCCT
CCACCACGTCGAAGA CGGCCCCCTGACTAC CAAGGAAGCACCCTT
CACCACGTCGAAGAA GGCCCCCTGACTACA AAGGAAGCACCCTTT
ACCACGTCGAAGAAT GCCCCCTGACTACAG AGGAAGCACCCTTTA
CCACGTCGAAGAATC CCCCCTGACTACAGG GGAAGCACCCTTTAA
CACGTCGAAGAATCG CCCCTGACTACAGGG GAAGCACCCTTTAAG
ACGTCGAAGAATCGC CCCTGACTACAGGGA AAGCACCCTTTAAGA
CGTCGAAGAATCGCA CCTGACTACAGGGAT AGCACCCTTTAAGAA
GTCGAAGAATCGCAT CTGACTACAGGGATC GCACCCTTTAAGAAT
TCGAAGAATCGCATC TGACTACAGGGATCT CACCCTTTAAGAATG
CGAAGAATCGCATCA GACTACAGGGATCTC ACCCTTTAAGAATGT
GAAGAATCGCATCAT ACTACAGGGATCTCA CCCTTTAAGAATGTC
AAGAATCGCATCATC CTACAGGGATCTCAT CCTTTAAGAATGTCA
AGAATCGCATCATCA TACAGGGATCTCATC CTTTAAGAATGTCAC
GAATCGCATCATCAT ACAGGGATCTCATCA TTTAAGAATGTCACA
AATCGCATCATCATA CAGGGATCTCATCAG TTAAGAATGTCACAG
ATCGCATCATCATAA AGGGATCTCATCAGC TAAGAATGTCACAGA
TCGCATCATCATAAC GGGATCTCATCAGCT AAGAATGTCACAGAG
CGCATCATCATAACC GGATCTCATCAGCTT AGAATGTCACAGAGT
GCATCATCATAACCT GATCTCATCAGCTTC GAATGTCACAGAGTA
CATCATCATAACCTG ATCTCATCAGCTTCA AATGTCACAGAGTAT
ATCATCATAACCTGG TCTCATCAGCTTCAC ATGTCACAGAGTATG
TCATCATAACCTGGC CTCATCAGCTTCACC TGTCACAGAGTATGA
CATCATAACCTGGCA TCATCAGCTTCACCG GTCACAGAGTATGAT
ATCATAACCTGGCAC CATCAGCTTCACCGT TCACAGAGTATGATG
TCATAACCTGGCACC ATCAGCTTCACCGTT CACAGAGTATGATGG
CATAACCTGGCACCG TCAGCTTCACCGTTT ACAGAGTATGATGGG
ATAACCTGGCACCGG CAGCTTCACCGTTTA CAGAGTATGATGGGC
TAACCTGGCACCGGT AGCTTCACCGTTTAC AGAGTATGATGGGCA
AACCTGGCACCGGTA GCTTCACCGTTTACT GAGTATGATGGGCAG
ACCTGGCACCGGTAC CTTCACCGTTTACTA AGTATGATGGGCAGG
CCTGGCACCGGTACC TTCACCGTTTACTAC GTATGATGGGCAGGA
TATGATGGGCAGGAT GGACGTGGACCTCCC TACTACATGGGCTGA
ATGATGGGCAGGATG GACGTGGACCTCCCG ACTACATGGGCTGAA
TGATGGGCAGGATGC ACGTGGACCTCCCGC CTACATGGGCTGAAG
GATGGGCAGGATGCC CGTGGACCTCCCGCC TACATGGGCTGAAGC
ATGGGCAGGATGCCT GTGGACCTCCCGCCC ACATGGGCTGAAGCC
TGGGCAGGATGCCTG TGGACCTCCCGCCCA CATGGGCTGAAGCCC
GGGCAGGATGCCTGC GGACCTCCCGCCCAA ATGGGCTGAAGCCCT
GGCAGGATGCCTGCG GACCTCCCGCCCAAC TGGGCTGAAGCCCTG
GCAGGATGCCTGCGG ACCTCCCGCCCAACA GGGCTGAAGCCCTGG
CAGGATGCCTGCGGC CCTCCCGCCCAACAA GGCTGAAGCCCTGGA
AGGATGCCTGCGGCT CTCCCGCCCAACAAG GCTGAAGCCCTGGAC
GGATGCCTGCGGCTC TCCCGCCCAACAAGG CTGAAGCCCTGGACT
GATGCCTGCGGCTCC CCCGCCCAACAAGGA TGAAGCCCTGGACTC
ATGCCTGCGGCTCCA CCGCCCAACAAGGAC GAAGCCCTGGACTCA
TGCCTGCGGCTCCAA CGCCCAACAAGGACG AAGCCCTGGACTCAG
GCCTGCGGCTCCAAC GCCCAACAAGGACGT AGCCCTGGACTCAGT
CCTGCGGCTCCAACA CCCAACAAGGACGTG GCCCTGGACTCAGTA
CTGCGGCTCCAACAG CCAACAAGGACGTGG CCCTGGACTCAGTAC
TGCGGCTCCAACAGC CAACAAGGACGTGGA CCTGGACTCAGTACG
GCGGCTCCAACAGCT AACAAGGACGTGGAG CTGGACTCAGTACGC
CGGCTCCAACAGCTG ACAAGGACGTGGAGC TGGACTCAGTACGCC
GGCTCCAACAGCTGG CAAGGACGTGGAGCC GGACTCAGTACGCCG
GCTCCAACAGCTGGA AAGGACGTGGAGCCC GACTCAGTACGCCGT
CTCCAACAGCTGGAA AGGACGTGGAGCCCG ACTCAGTACGCCGTT
TCCAACAGCTGGAAC GGACGTGGAGCCCGG CTCAGTACGCCGTTT
CCAACAGCTGGAACA GACGTGGAGCCCGGC TCAGTACGCCGTTTA
CAACAGCTGGAACAT ACGTGGAGCCCGGCA CAGTACGCCGTTTAC
AACAGCTGGAACATG CGTGGAGCCCGGCAT AGTACGCCGTTTACG
ACAGCTGGAACATGG GTGGAGCCCGGCATC GTACGCCGTTTACGT
CAGCTGGAACATGGT TGGAGCCCGGCATCT TACGCCGTTTACGTC
AGCTGGAACATGGTG GGAGCCCGGCATCTT ACGCCGTTTACGTCA
GCTGGAACATGGTGG GAGCCCGGCATCTTA CGCCGTTTACGTCAA
CTGGAACATGGTGGA AGCCCGGCATCTTAC GCCGTTTACGTCAAG
TGGAACATGGTGGAC GCCCGGCATCTTACT CCGTTTACGTCAAGG
GGAACATGGTGGACG CCCGGCATCTTACTA CGTTTACGTCAAGGC
GAACATGGTGGACGT CCGGCATCTTACTAC GTTTACGTCAAGGCT
AACATGGTGGACGTG CGGCATCTTACTACA TTTACGTCAAGGCTG
ACATGGTGGACGTGG GGCATCTTACTACAT TTACGTCAAGGCTGT
CATGGTGGACGTGGA GCATCTTACTACATG TACGTCAAGGCTGTG
ATGGTGGACGTGGAC CATCTTACTACATGG ACGTCAAGGCTGTGA
TGGTGGACGTGGACC ATCTTACTACATGGG CGTCAAGGCTGTGAC
GGTGGACGTGGACCT TCTTACTACATGGGC GTCAAGGCTGTGACC
GTGGACGTGGACCTC CTTACTACATGGGCT TCAAGGCTGTGACCC
TGGACGTGGACCTCC TTACTACATGGGCTG CAAGGCTGTGACCCT
AAGGCTGTGACCCTC GGCCAAGAGTGAGAT CTTCCATTCCCTTGG
AGGCTGTGACCCTCA GCCAAGAGTGAGATC TTCCATTCCCTTGGA
GGCTGTGACCCTCAC CCAAGAGTGAGATCT TCCATTCCCTTGGAC
GCTGTGACCCTCACC CAAGAGTGAGATCTT CCATTCCCTTGGACG
CTGTGACCCTCACCA AAGAGTGAGATCTTG CATTCCCTTGGACGT
TGTGACCCTCACCAT AGAGTGAGATCTTGT ATTCCCTTGGACGTT
GTGACCCTCACCATG GAGTGAGATCTTGTA TTCCCTTGGACGTTC
TGACCCTCACCATGG AGTGAGATCTTGTAC TCCCTTGGACGTTCT
GACCCTCACCATGGT GTGAGATCTTGTACA CCCTTGGACGTTCTT
ACCCTCACCATGGTG TGAGATCTTGTACAT CCTTGGACGTTCTTT
CCCTCACCATGGTGG GAGATCTTGTACATT CTTGGACGTTCTTTC
CCTCACCATGGTGGA AGATCTTGTACATTC TTGGACGTTCTTTCA
CTCACCATGGTGGAG GATCTTGTACATTCG TGGACGTTCTTTCAG
TCACCATGGTGGAGA ATCTTGTACATTCGC GGACGTTCTTTCAGC
CACCATGGTGGAGAA TCTTGTACATTCGCA GACGTTCTTTCAGCA
ACCATGGTGGAGAAC CTTGTACATTCGCAC ACGTTCTTTCAGCAT
CCATGGTGGAGAACG TTGTACATTCGCACC CGTTCTTTCAGCATC
CATGGTGGAGAACGA TGTACATTCGCACCA GTTCTTTCAGCATCG
ATGGTGGAGAACGAC GTACATTCGCACCAA TTCTTTCAGCATCGA
TGGTGGAGAACGACC TACATTCGCACCAAT TCTTTCAGCATCGAA
GGTGGAGAACGACCA ACATTCGCACCAATG CTTTCAGCATCGAAC
GTGGAGAACGACCAT CATTCGCACCAATGC TTTCAGCATCGAACT
TGGAGAACGACCATA ATTCGCACCAATGCT TTCAGCATCGAACTC
GGAGAACGACCATAT TTCGCACCAATGCTT TCAGCATCGAACTCC
GAGAACGACCATATC TCGCACCAATGCTTC CAGCATCGAACTCCT
AGAACGACCATATCC CGCACCAATGCTTCA AGCATCGAACTCCTC
GAACGACCATATCCG GCACCAATGCTTCAG GCATCGAACTCCTCT
AACGACCATATCCGT CACCAATGCTTCAGT CATCGAACTCCTCTT
ACGACCATATCCGTG ACCAATGCTTCAGTT ATCGAACTCCTCTTC
CGACCATATCCGTGG CCAATGCTTCAGTTC TCGAACTCCTCTTCT
GACCATATCCGTGGG CAATGCTTCAGTTCC CGAACTCCTCTTCTC
ACCATATCCGTGGGG AATGCTTCAGTTCCT GAACTCCTCTTCTCA
CCATATCCGTGGGGC ATGCTTCAGTTCCTT AACTCCTCTTCTCAG
CATATCCGTGGGGCC TGCTTCAGTTCCTTC ACTCCTCTTCTCAGT
ATATCCGTGGGGCCA GCTTCAGTTCCTTCC CTCCTCTTCTCAGTT
TATCCGTGGGGCCAA CTTCAGTTCCTTCCA TCCTCTTCTCAGTTA
ATCCGTGGGGCCAAG TTCAGTTCCTTCCAT CCTCTTCTCAGTTAA
TCCGTGGGGCCAAGA TCAGTTCCTTCCATT CTCTTCTCAGTTAAT
CCGTGGGGCCAAGAG CAGTTCCTTCCATTC TCTTCTCAGTTAATC
CGTGGGGCCAAGAGT AGTTCCTTCCATTCC CTTCTCAGTTAATCG
GTGGGGCCAAGAGTG GTTCCTTCCATTCCC TTCTCAGTTAATCGT
TGGGGCCAAGAGTGA TTCCTTCCATTCCCT TCTCAGTTAATCGTG
GGGGCCAAGAGTGAG TCCTTCCATTCCCTT CTCAGTTAATCGTGA
GGGCCAAGAGTGAGA CCTTCCATTCCCTTG TCAGTTAATCGTGAA
CAGTTAATCGTGAAG CCTGAGTTACTACAT GCTACCTTTACCGGC
AGTTAATCGTGAAGT CTGAGTTACTACATT CTACCTTTACCGGCA
GTTAATCGTGAAGTG TGAGTTACTACATTG TACCTTTACCGGCAC
TTAATCGTGAAGTGG GAGTTACTACATTGT ACCTTTACCGGCACA
TAATCGTGAAGTGGA AGTTACTACATTGTG CCTTTACCGGCACAA
AATCGTGAAGTGGAA GTTACTACATTGTGC CTTTACCGGCACAAT
ATCGTGAAGTGGAAC TTACTACATTGTGCG TTTACCGGCACAATT
TCGTGAAGTGGAACC TACTACATTGTGCGC TTACCGGCACAATTA
CGTGAAGTGGAACCC ACTACATTGTGCGCT TACCGGCACAATTAC
GTGAAGTGGAACCCT CTACATTGTGCGCTG ACCGGCACAATTACT
TGAAGTGGAACCCTC TACATTGTGCGCTGG CCGGCACAATTACTG
GAAGTGGAACCCTCC ACATTGTGCGCTGGC CGGCACAATTACTGC
AAGTGGAACCCTCCC CATTGTGCGCTGGCA GGCACAATTACTGCT
AGTGGAACCCTCCCT ATTGTGCGCTGGCAG GCACAATTACTGCTC
GTGGAACCCTCCCTC TTGTGCGCTGGCAGC CACAATTACTGCTCC
TGGAACCCTCCCTCT TGTGCGCTGGCAGCG ACAATTACTGCTCCA
GGAACCCTCCCTCTC GTGCGCTGGCAGCGG CAATTACTGCTCCAA
GAACCCTCCCTCTCT TGCGCTGGCAGCGGC AATTACTGCTCCAAA
AACCCTCCCTCTCTG GCGCTGGCAGCGGCA ATTACTGCTCCAAAG
ACCCTCCCTCTCTGC CGCTGGCAGCGGCAG TTACTGCTCCAAAGA
CCCTCCCTCTCTGCC GCTGGCAGCGGCAGC TACTGCTCCAAAGAC
CCTCCCTCTCTGCCC CTGGCAGCGGCAGCC ACTGCTCCAAAGACA
CTCCCTCTCTGCCCA TGGCAGCGGCAGCCT CTGCTCCAAAGACAA
TCCCTCTCTGCCCAA GGCAGCGGCAGCCTC TGCTCCAAAGACAAA
CCCTCTCTGCCCAAC GCAGCGGCAGCCTCA GCTCCAAAGACAAAA
CCTCTCTGCCCAACG CAGCGGCAGCCTCAG CTCCAAAGACAAAAT
CTCTCTGCCCAACGG AGCGGCAGCCTCAGG TCCAAAGACAAAATC
TCTCTGCCCAACGGC GCGGCAGCCTCAGGA CCAAAGACAAAATCC
CTCTGCCCAACGGCA CGGCAGCCTCAGGAC CAAAGACAAAATCCC
TCTGCCCAACGGCAA GGCAGCCTCAGGACG AAAGACAAAATCCCC
CTGCCCAACGGCAAC GCAGCCTCAGGACGG AAGACAAAATCCCCA
TGCCCAACGGCAACC CAGCCTCAGGACGGC AGACAAAATCCCCAT
GCCCAACGGCAACCT AGCCTCAGGACGGCT GACAAAATCCCCATC
CCCAACGGCAACCTG GCCTCAGGACGGCTA ACAAAATCCCCATCA
CCAACGGCAACCTGA CCTCAGGACGGCTAC CAAAATCCCCATCAG
CAACGGCAACCTGAG CTCAGGACGGCTACC AAAATCCCCATCAGG
AACGGCAACCTGAGT TCAGGACGGCTACCT AAATCCCCATCAGGA
ACGGCAACCTGAGTT CAGGACGGCTACCTT AATCCCCATCAGGAA
CGGCAACCTGAGTTA AGGACGGCTACCTTT ATCCCCATCAGGAAG
GGCAACCTGAGTTAC GGACGGCTACCTTTA TCCCCATCAGGAAGT
GCAACCTGAGTTACT GACGGCTACCTTTAC CCCCATCAGGAAGTA
CAACCTGAGTTACTA ACGGCTACCTTTACC CCCATCAGGAAGTAT
AACCTGAGTTACTAC CGGCTACCTTTACCG CCATCAGGAAGTATG
ACCTGAGTTACTACA GGCTACCTTTACCGG CATCAGGAAGTATGC
ATCAGGAAGTATGCC AGAGAACCCCAAGAC GCTGCGCCTGCCCCA
TCAGGAAGTATGCCG GAGAACCCCAAGACT CTGCGCCTGCCCCAA
CAGGAAGTATGCCGA AGAACCCCAAGACTG TGCGCCTGCCCCAAA
AGGAAGTATGCCGAC GAACCCCAAGACTGA GCGCCTGCCCCAAAA
GGAAGTATGCCGACG AACCCCAAGACTGAG CGCCTGCCCCAAAAC
GAAGTATGCCGACGG ACCCCAAGACTGAGG GCCTGCCCCAAAACT
AAGTATGCCGACGGC CCCCAAGACTGAGGT CCTGCCCCAAAACTG
AGTATGCCGACGGCA CCCAAGACTGAGGTG CTGCCCCAAAACTGA
GTATGCCGACGGCAC CCAAGACTGAGGTGT TGCCCCAAAACTGAA
TATGCCGACGGCACC CAAGACTGAGGTGTG GCCCCAA.AACTGAAG
ATGCCGACGGCACCA AAGACTGAGGTGTGT CCCCAAAACTGAAGC
TGCCGACGGCACCAT AGACTGAGGTGTGTG CCCAAAACTGAAGCC
GCCGACGGCACCATC GACTGAGGTGTGTGG CCAAAACTGAAGCCG
CCGACGGCACCATCG ACTGAGGTGTGTGGT CAAAACTGAAGCCGA
CGACGGCACCATCGA CTGAGGTGTGTGGTG AAAACTGAAGCCGAG
GACGGCACCATCGAC TGAGGTGTGTGGTGG AAACTGAAGCCGAGA
ACGGCACCATCGACA GAGGTGTGTGGTGGG AACTGAAGCCGAGAA
CGGCACCATCGACAT AGGTGTGTGGTGGGG ACTGAAGCCGAGAAG
GGCACCATCGACATT GGTGTGTGGTGGGGA CTGAAGCCGAGAAGC
GCACCATCGACATTG GTGTGTGGTGGGGAG TGAAGCCGAGAAGCA
CACCATCGACATTGA TGTGTGGTGGGGAGA GAAGCCGAGAAGCAG
ACCATCGACATTGAG GTGTGGTGGGGAGAA AAGCCGAGAAGCAGG
CCATCGACATTGAGG TGTGGTGGGGAGAAA AGCCGAGAAGCAGGC
CATCGACATTGAGGA GTGGTGGGGAGAAAG GCCGAGAAGCAGGCC
ATCGACATTGAGGAG TGGTGGGGAGAAAGG CCGAGAAGCAGGCCG
TCGACATTGAGGAGG GGTGGGGAGAAAGGG CGAGAAGCAGGCCGA
CGACATTGAGGAGGT GTGGGGAGAAAGGGC GAGAAGCAGGCCGAG
GACATTGAGGAGGTC TGGGGAGAAAGGGCC AGAAGCAGGCCGAGA
ACATTGAGGAGGTCA GGGGAGAAAGGGCCT GAAGCAGGCCGAGAA
CATTGAGGAGGTCAC GGGAGAAAGGGCCTT AAGCAGGCCGAGAAG
ATTGAGGAGGTCACA GGAGAAAGGGCCTTG AGCAGGCCGAGAAGG
TTGAGGAGGTCACAG GAGAAAGGGCCTTGC GCAGGCCGAGAAGGA
TGAGGAGGTCACAGA AGAAAGGGCCTTGCT CAGGCCGAGAAGGAG
GAGGAGGTCACAGAG GAAAGGGCCTTGCTG AGGCCGAGAAGGAGG
AGGAGGTCACAGAGA AAAGGGCCTTGCTGC GGCCGAGAAGGAGGA
GGAGGTCACAGAGAA AAGGGCCTTGCTGCG GCCGAGAAGGAGGAG
GAGGTCACAGAGAAC AGGGCCTTGCTGCGC CCGAGAAGGAGGAGG
AGGTCACAGAGAACC GGGCCTTGCTGCGCC CGAGAAGGAGGAGGC
GGTCACAGAGAACCC GGCCTTGCTGCGCCT GAGAAGGAGGAGGCT
GTCACAGAGAACCCC GCCTTGCTGCGCCTG AGAAGGAGGAGGCTG
TCACAGAGAACCCCA CCTTGCTGCGCCTGC GAAGGAGGAGGCTGA
CACAGAGAACCCCAA CTTGCTGCGCCTGCC AAGGAGGAGGCTGAA
ACAGAGAACCCCAAG TTGCTGCGCCTGCCC AGGAGGAGGCTGAAT
CAGAGAACCCCAAGA TGCTGCGCCTGCCCC GGAGGAGGCTGAATA
GAGGAGGCTGAATAC CTCCATCTTCGTGCC TGCAAGTGGCCAACA
AGGAGGCTGAATACC TCCATCTTCGTGCCC GCAAGTGGCCAACAC
GGAGGCTGAATACCG CCATCTTCGTGCCCA CAAGTGGCCAACACC
GAGGCTGAATACCGC CATCTTCGTGCCCAG AAGTGGCCAACACCA
AGGCTGAATACCGCA ATCTTCGTGCCCAGA AGTGGCCAACACCAC
GGCTGAATACCGCAA TCTTCGTGCCCAGAC GTGGCCAACACCACC
GCTGAATACCGCAAA CTTCGTGCCCAGACC TGGCCAACACCACCA
CTGAATACCGCAAAG TTCGTGCCCAGACCT GGCCAACACCACCAT
TGAATACCGCAAAGT TCGTGCCCAGACCTG GCCAACACCACCATG
GAATACCGCAAAGTC CGTGCCCAGACCTGA CCAACACCACCATGT
AATACCGCAAAGTCT GTGCCCAGACCTCAA CAACACCACCATGTC
ATACCGCAAAGTCTT TGCCCAGACCTGAAA AACACCACCATGTCC
TACCGCAAAGTCTTT GCCCAGACCTGAAAG ACACCACCATGTCCA
ACCGCAAAGTCTTTG CCCAGACCTGAAAGG CACCACCATGTCCAG
CCGCAAAGTCTTTGA CCAGACCTGAAAGGA ACCACCATGTCCAGC
CGCAAAGTCTTTGAG CAGACCTGAAAGGAA CCACCATGTCCAGCC
GCAAAGTCTTTGAGA AGACCTGAAAGGAAG CACCATGTCCAGCCG
CAAAGTCTTTGAGAA GACCTGAAAGGAAGC ACCATGTCCAGCCGA
AAAGTCTTTGAGAAT ACCTGAAAGGAAGCG CCATGTCCAGCCGAA
AAGTCTTTGAGAATT CCTGAAAGGAAGCGG CATGTCCAGCCGAAG
AGTCTTTGAGAATTT CTGAAAGGAAGCGGA ATGTCCAGCCGAAGC
GTCTTTGAGAATTTC TGAAAGGAAGCGGAG TGTCCAGCCGAAGCA
TCTTTGAGAATTTCC GAAAGGAAGCGGAGA GTCCAGCCGAAGCAG
CTTTGAGAATTTCCT AAAGGAAGCGGAGAG TCCAGCCGAAGCAGG
TTTGAGAATTTCCTG AAGGAAGCGGAGAGA CCAGCCGAAGCAGGA
TTGAGAATTTCCTGC AGGAAGCGGAGAGAT CAGCCGAAGCAGGAA
TGAGAATTTCCTGCA GGAAGCGGAGAGATG AGCCGAAGCAGGAAC
GAGAATTTCCTGCAC GAAGCGGAGAGATGT GCCGAAGCAGGAACA
AGAATTTCCTGCACA AAGCGGAGAGATGTC CCGAAGCAGGAACAC
GAATTTCCTGCACAA AGCGGAGAGATGTCA CGAAGCAGGAACACC
AATTTCCTGCACAAC GCGGAGAGATGTCAT GAAGCAGGAACACCA
ATTTCCTGCACAACT CGGAGAGATGTCATG AAGCAGGAACACCAC
TTTCCTGCACAACTC GGAGAGATGTCATGC AGCAGGAACACCACG
TTCCTGCACAACTCC GAGAGATGTCATGCA GCAGGAACACCACGG
TCCTGCACAACTCCA AGAGATGTCATGCAA CAGGAACACCACGGC
CCTGCACAACTCCAT GAGATGTCATGCAAG AGGAACACCACGGCC
CTGCACAACTCCATC AGATGTCATGCAAGT GGAACACCACGGCCG
TGCACAACTCCATCT GATGTCATGCAAGTG GAACACCACGGCCGC
GCACAACTCCATCTT ATGTCATGCAAGTGG AACACCACGGCCGCA
CACAACTCCATCTTC TGTCATGCAAGTGGC ACACCACGGCCGCAG
ACAACTCCATCTTCG GTCATGCAAGTGGCC CACCACGGCCGCAGA
CAACTCCATCTTCGT TCATGCAAGTGGCCA ACCACGGCCGCAGAC
AACTCCATCTTCGTG CATGCAAGTGGCCAA CCACGGCCGCAGACA
ACTCCATCTTCGTGC ATGCAAGTGGCCAAC CACGGCCGCAGACAC
ACGGCCGCAGACACC GACAGAGTACCCTTT GAACTGTCATTTCTA
CGGCCGCAGACACCT ACAGAGTACCCTTTC AACTGTCATTTCTAA
GGCCGCAGACACCTA CAGAGTACCCTTTCT ACTGTCATTTCTAAC
GCCGCAGACACCTAC AGAGTACCCTTTCTT CTGTCATTTCTAACC
CCGCAGACACCTACA GAGTACCCTTTCTTT TGTCATTTCTAACCT
CGCAGACACCTACAA AGTACCCTTTCTTTG GTCATTTCTAACCTT
GCAGACACCTACAAC GTACCCTTTCTTTGA TCATTTCTAACCTTC
CAGACACCTACAACA TACCCTTTCTTTGAG CATTTCTAACCTTCG
AGACACCTACAACAT ACCCTTTCTTTGAGA ATTTCTAACCTTCGG
GACACCTACAACATC CCCTTTCTTTGAGAG TTTCTAACCTTCGGC
ACACCTACAACATCA CCTTTCTTTGAGAGC TTCTAACCTTCGGCC
CACCTACAACATCAC CTTTCTTTGAGAGCA TCTAACCTTCGGCCT
ACCTACAACATCACC TTTCTTTGAGAGCAG CTAACCTTCGGCCTT
CCTACAACATCACCG TTCTTTGAGAGCAGA TAACCTTCGGCCTTT
CTACAACATCACCGA TCTTTGAGAGCAGAG AACCTTCGGCCTTTC
TACAACATCACCGAC CTTTGAGAGCAGAGT ACCTTCGGCCTTTCA
ACAACATCACCGACC TTTGAGAGCAGAGTG CCTTCGGCCTTTCAC
CAACATCACCGACCC TTGAGAGCAGAGTGG CTTCGGCCTTTCACA
AACATCACCGACCCG TGAGAGCAGAGTGGA TTCGGCCTTTCACAT
ACATCACCGACCCGG GAGAGCAGAGTGGAT TCGGCCTTTCACATT
CATCACCGACCCGGA AGAGCAGAGTGGATA CGGCCTTTCACATTG
ATCACCGACCCGGAA GAGCAGAGTGGATAA GGCCTTTCACATTGT
TCACCGACCCGGAAG AGCAGAGTGGATAAC GCCTTTCACATTGTA
CACCGACCCGGAAGA GCAGAGTGGATAACA CCTTTCACATTGTAC
ACCGACCCGGAAGAG CAGAGTGGATAACAA CTTTCACATTGTACC
CCGACCCGGAAGAGC AGAGTGGATAACAAG TTTCACATTGTACCG
CGACCCGGAAGAGCT GAGTGGATAACAAGG TTCACATTGTACCGC
GACCCGGAAGAGCTG AGTGGATAACAAGGA TCACATTGTACCGCA
ACCCGGAAGAGCTGG GTGGATAACAAGGAG CACATTGTACCGCAT
CCCGGAAGAGCTGGA TGGATAACAAGGAGA ACATTGTACCGCATC
CCGGAAGAGCTGGAG GGATAACAAGGAGAG CATTGTACCGCATCG
CGGAAGAGCTGGAGA GATAACAAGGAGAGA ATTGTACCGCATCGA
GGAAGAGCTGGAGAC ATAACAAGGAGAGAA TTGTACCGCATCGAT
GAAGAGCTGGAGACA TAACAAGGAGAGAAC TGTACCGCATCGATA
AAGAGCTGGAGACAG AACAAGGAGAGAACT GTACCGCATCGATAT
AGAGCTGGAGACAGA ACAAGGAGAGAACTG TACCGCATCGATATC
GAGCTGGAGACAGAG CAAGGAGAGAACTGT ACCGCATCGATATCC
AGCTGGAGACAGAGT AAGGAGAGAACTGTC CCGCATCGATATCCA
GCTGGAGACAGAGTA AGGAGAGAACTGTCA CGCATCGATATCCAC
CTGGAGACAGAGTAC GGAGAGAACTGTCAT GCATCGATATCCACA
TGGAGACAGAGTACC GAGAGAACTGTCATT CATCGATATCCACAG
GGAGACAGAGTACCC AGAGAACTGTCATTT ATCGATATCCACAGC
GAGACAGAGTACCCT GAGAACTGTCATTTC TCGATATCCACAGCT
AGACAGAGTACCCTT AGAACTGTCATTTCT CGATATCCACAGCTG
GATATCCACAGCTGC CGCCTCCAACTTCGT CAGATGACATTCCTG
ATATCCACAGCTGCA GCCTCCAACTTCGTC AGATGACATTCCTGG
TATCCACAGCTGCAA CCTCCAACTTCGTCT GATGACATTCCTGGG
ATCCACAGCTGCAAC CTCCAACTTCGTCTT ATGACATTCCTGGGC
TCCACAGCTGCAACC TCCAACTTCGTCTTT TGACATTCCTGGGCC
CCACAGCTGCAACCA CCAACTTCGTCTTTG GACATTCCTGGGCCA
CACAGCTGCAACCAC CAACTTCGTCTTTGC ACATTCCTGGGCCAG
ACAGCTGCAACCACG AACTTCGTCTTTGCA CATTCCTGGGCCAGT
CAGCTGCAACCACGA ACTTCGTCTTTGCAA ATTCCTGGGCCAGTG
AGCTGCAACCACGAG CTTCGTCTTTGCAAG TTCCTGGGCCAGTGA
GCTGCAACCACGAGG TTCGTCTTTGCAAGG TCCTGGGCCAGTGAC
CTGCAACCACGAGGC TCGTCTTTGCAAGGA CCTGGGCCAGTGACC
TGCAACCACGAGGCT CGTCTTTGCAAGGAC CTGGGCCAGTGACCT
GCAACCACGAGGCTG GTCTTTGCAAGGACT TGGGCCAGTGACCTG
CAACCACGAGGCTGA TCTTTGCAAGGACTA GGGCCAGTGACCTGG
AACCACGAGGCTGAG CTTTGCAAGGACTAT GGCCAGTGACCTGGG
ACCACGAGGCTGAGA TTTGCAAGGACTATG GCCAGTGACCTGGGA
CCACGAGGCTGAGAA TTGCAAGGACTATGC CCAGTGACCTGGGAG
CACGAGGCTGAGAAG TGCAAGGACTATGCC CAGTGACCTGGGAGC
ACGAGGCTGAGAAGC GCAAGGACTATGCCC AGTGACCTGGGAGCC
CGAGGCTGAGAAGCT CAAGGACTATGCCCG GTGACCTGGGAGCCA
GAGGCTGAGAAGCTG AAGGACTATGCCCGC TGACCTGGGAGCCAA
AGGCTGAGAAGCTGG AGGACTATGCCCGCA GACCTGGGAGCCAAG
GGCTGAGAAGCTGGG GGACTATGCCCGCAG ACCTGGGAGCCAAGG
GCTGAGAAGCTGGGC GACTATGCCCGCAGA CCTGGGAGCCAAGGC
CTGAGAAGCTGGGCT ACTATGCCCGCAGAA CTGGGAGCCAAGGCC
TGAGAAGCTGGGCTG CTATGCCCGCAGAAG TGGGAGCCAAGGCCT
GAGAAGCTGGGCTGC TATGCCCGCAGAAGG GGGAGCCAAGGCCTG
AGAAGCTGGGCTGCA ATGCCCGCAGAAGGA GGAGCCAAGGCCTGA
GAAGCTGGGCTGCAG TGCCCGCAGAAGGAG GAGCCAAGGCCTGAA
AAGCTGGGCTGCAGC GCCCGCAGAAGGAGC AGCCAAGGCCTGAAA
AGCTGGGCTGCAGCG CCCGCAGAAGGAGCA GCCAAGGCCTGAAAA
GCTGGGCTGCAGCGC CCGCAGAAGGAGCAG CCAAGGCCTGAAAAC
CTGGGCTGCAGCGCC CGCAGAAGGAGCAGA CAAGGCCTGAAAACT
TGGGCTGCAGCGCCT GCAGAAGGAGCAGAT AAGGCCTGAAAACTC
GGGCTGCAGCGCCTC CAGAAGGAGCAGATG AGGCCTGAAAACTCC
GGCTGCAGCGCCTCC AGAAGGAGCAGATGA GGCCTGAAAACTCCA
GCTGCAGCGCCTCCA GAAGGAGCAGATGAC GCCTGAAAACTCCAT
CTGCAGCGCCTCCAA AAGGAGCAGATGACA CCTGAAAACTCCATC
TGCAGCGCCTCCAAC AGGAGCAGATGACAT CTGAAAACTCCATCT
GCAGCGCCTCCAACT GGAGCAGATGACATT TGAAAACTCCATCTT
CAGCGCCTCCAACTT GAGCAGATGACATTC GAAAACTCCATCTTT
AGCGCCTCCAACTTC AGCAGATGACATTCC AAAACTCCATCTTTT
GCGCCTCCAACTTCG GCAGATGACATTCCT AAACTCCATCTTTTT
AACTCCATCTTTTTA ATTGATTCTAATGTA ATCAGCGAGAATGTG
ACTCCATCTTTTTAA TTGATTCTAATGTAT TCAGCGAGAATGTGT
CTCCATCTTTTTAAA TGATTCTAATGTATG CAGCGAGAATGTGTG
TCCATCTTTTTAAAG GATTCTAATGTATGA AGCGAGAATGTGTGT
CCATCTTTTTAAAGT ATTCTAATGTATGAA GCGAGAATGTGTGTC
CATCTTTTTAAAGTG TTCTAATGTATGAAA CGAGAATGTGTGTCC
ATCTTTTTAAAGTGG TCTAATGTATGAAAT GAGAATGTGTGTCCA
TCTTTTTAAAGTGGC CTAATGTATGAAATA AGAATGTGTGTCCAG
CTTTTTAAAGTGGCC TAATGTATGAAATAA GAATGTGTGTCCAGA
TTTTTAAAGTGGCCG AATGTATGAAATAAA AATGTGTGTCCAGAC
TTTTAAAGTGGCCGG ATGTATGAAATAAAA ATGTGTGTCCAGACA
TTTAAAGTGGCCGGA TGTATGAAATAAAAT TGTGTGTCCAGACAG
TTAAAGTGGCCGGAA GTATGAAATAAAATA GTGTGTCCAGACAGG
TAAAGTGGCCGGAAC TATGAAATAAAATAC TGTGTCCAGACAGGA
AAAGTGGCCGGAACC ATGAAATAAAATACG GTGTCCAGACAGGAA
AAGTGGCCGGAACCT TGAAATAAAATACGG TGTCCAGACAGGAAT
AGTGGCCGGAACCTG GAAATAAAATACGGA GTCCAGACAGGAATA
GTGGCCGGAACCTGA AAATAAAATACGGAT TCCAGACAGGAATAC
TGGCCGGAACCTGAG AATAAAATACGGATC CCAGACAGGAATACA
GGCCGGAACCTGAGA ATAAAATACGGATCA CAGACAGGAATACAG
GCCGGAACCTGAGAA TAAAATACGGATCAC AGACAGGAATACAGG
CCGGAACCTGAGAAT AAAATACGGATCACA GACAGGAATACAGGA
CGGAACCTGAGAATC AAATACGGATCACAA ACAGGAATACAGGAA
GGAACCTGAGAATCC AATACGGATCACAAG CAGGAATACAGGAAG
GAACCTGAGAATCCC ATACGGATCACAAGT AGGAATACAGGAAGT
AACCTGAGAATCCCA TACGGATCACAAGTT GGAATACAGGAAGTA
ACCTGAGAATCCCAA ACGGATCACAAGTTG GAATACAGGAAGTAT
CCTGAGAATCCCAAT CGGATCACAAGTTGA AATACAGGAAGTATG
CTGAGAATCCCAATG GGATCACAAGTTGAG ATACAGGAAGTATGG
TGAGAATCCCAATGG GATCACAAGTTGAGG TACAGGAAGTATGGA
GAGAATCCCAATGGA ATCACAAGTTGAGGA ACAGGAAGTATGGAG
AGAATCCCAATGGAT TCACAAGTTGAGGAT CAGGAAGTATGGAGG
GAATCCCAATGGATT CACAAGTTGAGGATC AGGAAGTATGGAGGG
AATCCCAATGGATTG ACAAGTTGAGGATCA GGAAGTATGGAGGGG
ATCCCAATGGATTGA CAAGTTGAGGATCAG GAAGTATGGAGGGGC
TCCCAATGGATTGAT AAGTTGAGGATCAGC AAGTATGGAGGGGCC
CCCAATGGATTGATT AGTTGAGGATCAGCG AGTATGGAGGGGCCA
CCAATGGATTGATTC GTTGAGGATCAGCGA GTATGGAGGGGCCAA
CAATGGATTGATTCT TTGAGGATCAGCGAG TATGGAGGGGCCAAG
AATGGATTGATTCTA TGAGGATCAGCGAGA ATGGAGGGGCCAAGC
ATGGATTGATTCTAA GAGGATCAGCGAGAA TGGAGGGGCCAAGCT
TGGATTGATTCTAAT AGGATCAGCGAGAAT GGAGGGGCCAAGCTA
GGATTGATTCTAATG GGATCAGCGAGAATG GAGGGGCCAAGCTAA
GATTGATTCTAATGT GATCAGCGAGAATGT AGGGGCCAAGCTAAA
GGGGCCAAGCTAAAC GATTCAGGCCACATC CTGTGTTCTTCTATG
GGGCCAAGCTAAACC ATTCAGGCCACATCT TGTGTTCTTCTATGT
GGCCAAGCTAAACCG TTCAGGCCACATCTC GTGTTCTTCTATGTC
GCCAAGCTAAACCGG TCAGGCCACATCTCT TGTTCTTCTATGTCC
CCAAGCTAAACCGGC CAGGCCACATCTCTC GTTCTTCTATGTCCA
CAAGCTAAACCGGCT AGGCCACATCTCTCT TTCTTCTATGTCCAG
AAGCTAAACCGGCTA GGCCACATCTCTCTC TCTTCTATGTCCAGG
AGCTAAACCGGCTAA GCCACATCTCTCTCT CTTCTATGTCCAGGC
GCTAAACCGGCTAAA CCACATCTCTCTCTG TTCTATGTCCAGGCC
CTAAACCGGCTAAAC CACATCTCTCTCTGG TCTATGTCCAGGCCA
TAAACCGGCTAAACC ACATCTCTCTCTGGG CTATGTCCAGGCCAA
AAACCGGCTAAACCC CATCTCTCTCTGGGA TATGTCCAGGCCAAA
AACCGGCTAAACCCG ATCTCTCTCTGGGAA ATGTCCAGGCCAAAA
ACCGGCTAAACCCGG TCTCTCTCTGGGAAT TGTCCAGGCCAAAAC
CCGGCTAAACCCGGG CTCTCTCTGGGAATG GTCCAGGCCAAAACA
CGGCTAAACCCGGGG TCTCTCTGGGAATGG TCCAGGCCAAAACAG
GGCTAAACCCGGGGA CTCTCTGGGAATGGG CCAGGCCAAAACAGG
GCTAAACCCGGGGAA TCTCTGGGAATGGGT CAGGCCAAAACAGGA
CTAAACCCGGGGAAC CTCTGGGAATGGGTC AGGCCAAAACAGGAT
TAAACCCGGGGAACT TCTGGGAATGGGTCG GGCCAAAACAGGATA
AAACCCGGGGAACTA CTGGGAATGGGTCGT GCCAAAACAGGATAT
AACCCGGGGAACTAC TGGGAATGGGTCGTG CCAAAACAGGATATG
ACCCGGGGAACTACA GGGAATGGGTCGTGG CAAAACAGGATATGA
CCCGGGGAACTACAC GGAATGGGTCGTGGA AAAACAGGATATGAA
CCGGGGAACTACACA GAATGGGTCGTGGAC AAACAGGATATGAAA
CGGGGAACTACACAG AATGGGTCGTGGACA AACAGGATATGAAAA
GGGGAACTACACAGC ATGGGTCGTGGACAG ACAGGATATGAAAAC
GGGAACTACACAGCC TGGGTCGTGGACAGA CAGGATATGAAAACT
GGAACTACACAGCCC GGGTCGTGGACAGAT AGGATATGAAAACTT
GAACTACACAGCCCG GGTCGTGGACAGATC GGATATGAAAACTTC
AACTACACAGCCCGG GTCGTGGACAGATCC GATATGAAAACTTCA
ACTACACAGCCCGGA TCGTGGACAGATCCT ATATGAAAACTTCAT
CTACACAGCCCGGAT CGTGGACAGATCCTG TATGAAAACTTCATC
TACACAGCCCGGATT GTGGACAGATCCTGT ATGAAAACTTCATCC
ACACAGCCCGGATTC TGGACAGATCCTGTG TGAAAACTTCATCCA
CACAGCCCGGATTCA GGACAGATCCTGTGT GAAAACTTCATCCAT
ACAGCCCGGATTCAG GACAGATCCTGTGTT AAAACTTCATCCATC
CAGCCCGGATTCAGG ACAGATCCTGTGTTC AAACTTCATCCATCT
AGCCCGGATTCAGGC CAGATCCTGTGTTCT AACTTCATCCATCTG
GCCCGGATTCAGGCC AGATCCTGTGTTCTT ACTTCATCCATCTGA
CCCGGATTCAGGCCA GATCCTGTGTTCTTC CTTCATCCATCTGAT
CCGGATTCAGGCCAC ATCCTGTGTTCTTCT TTCATCCATCTGATC
CGGATTCAGGCCACA TCCTGTGTTCTTCTA TCATCCATCTGATCA
GGATTCAGGCCACAT CCTGTGTTCTTCTAT CATCCATCTGATCAT
ATCCATCTGATCATC GGGAGGGTTGGTGAT ATAACAGCAGGCTGG
TCCATCTGATCATCG GGAGGGTTGGTGATT TAACAGCAGGCTGGG
CCATCTGATCATCGC GAGGGTTGGTGATTA AACAGCAGGCTGGGG
CATCTGATCATCGCT AGGGTTGGTGATTAT ACAGCAGGCTGGGGA
ATCTGATCATCGCTC GGGTTGGTGATTATG CAGCAGGCTGGGGAA
TCTGATCATCGCTCT GGTTGGTGATTATGC AGCAGGCTGGGGAAT
CTGATCATCGCTCTG GTTGGTGATTATGCT GCAGGCTGGGGAATG
TGATCATCGCTCTGC TTGGTGATTATGCTG CAGGCTGGGGAATGG
GATCATCGCTCTGCC TGGTGATTATGCTGT AGGCTGGGGAATGGA
ATCATCGCTCTGCCC GGTGATTATGCTGTA GGCTGGGGAATGGAG
TCATCGCTCTGCCCG GTGATTATGCTGTAC GCTGGGGAATGGAGT
CATCGCTCTGCCCGT TGATTATGCTGTACG CTGGGGAATGGAGTG
ATCGCTCTGCCCGTC GATTATGCTGTACGT TGGGGAATGGAGTGC
TCGCTCTGCCCGTCG ATTATGCTGTACGTC GGGGAATGGAGTGCT
CGCTCTGCCCGTCGC TTATGCTGTACGTCT GGGAATGGAGTGCTG
GCTCTGCCCGTCGCT TATGCTGTACGTCTT GGAATGGAGTGCTGT
CTCTGCCCGTCGCTG ATGCTGTACGTCTTC GAATGGAGTGCTGTA
TCTGCCCGTCGCTGT TGCTGTACGTCTTCC AATGGAGTGCTGTAT
CTGCCCGTCGCTGTC GCTGTACGTCTTCCA ATGGAGTGCTGTATG
TGCCCGTCGCTGTCC CTGTACGTCTTCCAT TGGAGTGCTGTATGC
GCCCGTCGCTGTCCT TGTACGTCTTCCATA GGAGTGCTGTATGCC
CCCGTCGCTGTCCTG GTACGTCTTCCATAG GAGTGCTGTATGCCT
CCGTCGCTGTCCTGT TACGTCTTCCATAGA AGTGCTGTATGCCTC
CGTCGCTGTCCTGTT ACGTCTTCCATAGAA GTGCTGTATGCCTCT
GTCGCTGTCCTGTTG CGTCTTCCATAGAAA TGCTGTATGCCTCTG
TCGCTGTCCTGTTGA GTCTTCCATAGAAAG GCTGTATGCCTCTGT
CGCTGTCCTGTTGAT TCTTCCATAGAPAGA CTGTATGCCTCTGTG
GCTGTCCTGTTGATC CTTCCATAGAAAGAG TGTATGCCTCTGTGA
CTGTCCTGTTGATCG TTCCATAGAAAGAGA GTATGCCTCTGTGAA
TGTCCTGTTGATCGT TCCATAGAAAGAGAA TATGCCTCTGTGAAC
GTCCTGTTGATCGTG CCATAGAAAGAGAAA ATGCCTCTGTGAACC
TCCTGTTGATCGTGG CATAGAAAGAGAAAT TGCCTCTGTGAACCC
CCTGTTGATCGTGGG ATAGAAAGAGAAATA GCCTCTGTGAACCCG
CTGTTGATCGTGGGA TAGAAAGAGAAATAA CCTCTGTGAACCCGG
TGTTGATCGTGGGAG AGAAAGAGPAATAAC CTCTGTGAACCCGGA
GTTGATCGTGGGAGG GAAAGAGAAATAACA TCTGTGAACCCGGAG
TTGATCGTGGGAGGG AAAGAGAAATAACAG CTGTGAACCCGGAGT
TGATCGTGGGAGGGT AAGAGAAATAACAGC TGTGAACCCGGAGTA
GATCGTGGGAGGGTT AGAGAAATAACAGCA GTGAACCCGGAGTAC
ATCGTGGGAGGGTTG GAGAAATAACAGCAG TGAACCCGGAGTACT
TCGTGGGAGGGTTGG AGAAATAACAGCAGG GAACCCGGAGTACTT
CGTGGGAGGGTTGGT GAAATAACAGCAGGC AACCCGGAGTACTTC
GTGGGAGGGTTGGTG AAATAACAGCAGGCT ACCCGGAGTACTTCA
TGGGAGGGTTGGTGA AATAACAGCAGGCTG CCCGGAGTACTTCAG
CCGGAGTACTTCAGC GGAGGTGGCTCGGGA AGGGGTCGTTTGGGA
CGGAGTACTTCAGCG GAGGTGGCTCGGGAG GGGGTCGTTTGGGAT
GGAGTACTTCAGCGC AGGTGGCTCGGGAGA GGGTCGTTTGGGATG
GAGTACTTCAGCGCT GGTGGCTCGGGAGAA GGTCGTTTGGGATGG
AGTACTTCAGCGCTG GTGGCTCGGGAGAAG GTCGTTTGGGATGGT
GTACTTCAGCGCTGC TGGCTCGGGAGAAGA TCGTTTGGGATGGTC
TACTTCAGCGCTGCT GGCTCGGGAGAAGAT CGTTTGGGATGGTCT
ACTTCAGCGCTGCTG GCTCGGGAGAAGATC GTTTGGGATGGTCTA
CTTCAGCGCTGCTGA CTCGGGAGAAGATCA TTTGGGATGGTCTAT
TTCAGCGCTGCTGAT TCGGGAGAAGATCAC TTGGGATGGTCTATG
TCAGCGCTGCTGATG CGGGAGAAGATCACC TGGGATGGTCTATGA
CAGCGCTGCTGATGT GGGAGAAGATCACCA GGGATGGTCTATGAA
AGCGCTGCTGATGTG GGAGAAGATCACCAT GGATGGTCTATGAAG
GCGCTGCTGATGTGT GAGAAGATCACCATG GATGGTCTATGAAGG
CGCTGCTGATGTGTA AGAAGATCACCATGA ATGGTCTATGAAGGA
GCTGCTGATGTGTAC GAAGATCACCATGAG TGGTCTATGAAGGAG
CTGCTGATGTGTACG AAGATCACCATGAGC GGTCTATGAAGGAGT
TGCTGATGTGTACGT AGATCACCATGAGCC GTCTATGAAGGAGTT
GCTGATGTGTACGTT GATCACCATGAGCCG TCTATGAAGGAGTTG
CTGATGTGTACGTTC ATCACCATGAGCCGG CTATGAAGGAGTTGC
TGATGTGTACGTTCC TCACCATGAGCCGGG TATGAAGGAGTTGCC
GATGTGTACGTTCCT CACCATGAGCCGGGA ATGAAGGAGTTGCCA
ATGTGTACGTTCCTG ACCATGAGCCGGGAA TGAAGGAGTTGCCAA
TGTGTACGTTCCTGA CCATGAGCCGGGAAC GAAGGAGTTGCCAAG
GTGTACGTTCCTGAT CATGAGCCGGGAACT AAGGAGTTGCCAAGG
TGTACGTTCCTGATG ATGAGCCGGGAACTT AGGAGTTGCCAAGGG
GTACGTTCCTGATGA TGAGCCGGGAACTTG GGAGTTGCCAAGGGT
TACGTTCCTGATGAG GAGCCGGGAACTTGG GAGTTGCCAAGGGTG
ACGTTCCTGATGAGT AGCCGGGAACTTGGG AGTTGCCAAGGGTGT
CGTTCCTGATGAGTG GCCGGGAACTTGGGC GTTGCCAAGGGTGTG
GTTCCTGATGAGTGG CCGGGAACTTGGGCA TTGCCAAGGGTGTGG
TTCCTGATGAGTGGG CGGGAACTTGGGCAG TGCCAAGGGTGTGGT
TCCTGATGAGTGGGA GGGAACTTGGGCAGG GCCAAGGGTGTGGTG
CCTGATGAGTGGGAG GGAACTTGGGCAGGG CCAAGGGTGTGGTGA
CTGATGAGTGGGAGG GAACTTGGGCAGGGG CAAGGGTGTGGTGAA
TGATGAGTGGGAGGT AACTTGGGCAGGGGT AAGGGTGTGGTGAAA
GATGAGTGGGAGGTG ACTTGGGCAGGGGTC AGGGTGTGGTGAAAG
ATGAGTGGGAGGTGG CTTGGGCAGGGGTCG GGGTGTGGTGAAAGA
TGAGTGGGAGGTGGC TTGGGCAGGGGTCGT GGTGTGGTGAAAGAT
GAGTGGGAGGTGGCT TGGGCAGGGGTCGTT GTGTGGTGAAAGATG
AGTGGGAGGTGGCTC GGGCAGGGGTCGTTT TGTGGTGAAAGATGA
GTGGGAGGTGGCTCG GGCAGGGGTCGTTTG GTGGTGAAAGATGAA
TGGGAGGTGGCTCGG GCAGGGGTCGTTTGG TGGTGAAAGATGAAC
GGGAGGTGGCTCGGG CAGGGGTCGTTTGGG GGTGAAAGATGAACC
GTGAAAGATGAACCT CGAGGCCGCAAGCAT CTTCTGTGATGAAGG
TGAAAGATGAACCTG GAGGCCGCAAGCATG TTCTGTGATGAAGGA
GAAAGATGAACCTGA AGGCCGCAAGCATGC TCTGTGATGAAGGAG
AAAGATGAACCTGAA GGCCGCAAGCATGCG CTGTGATGAAGGAGT
AAGATGAACCTGAAA GCCGCAAGCATGCGT TGTGATGAAGGAGTT
AGATGAACCTGAAAC CCGCAAGCATGCGTG GTGATGAAGGAGTTC
GATGAACCTGAAACC CGCAAGCATGCGTGA TGATGAAGGAGTTCA
ATGAACCTGAAACCA GCAAGCATGCGTGAG GATGAAGGAGTTCAA
TGAACCTGAAACCAG CAAGCATGCGTGAGA ATGAAGGAGTTCAAT
GAACCTGAAACCAGA AAGCATGCGTGAGAG TGAAGGAGTTCAATT
AACCTGAAACCAGAG AGCATGCGTGAGAGG GAAGGAGTTCAATTG
ACCTGAAACCAGAGT GCATGCGTGAGAGGA AAGGAGTTCAATTGT
CCTGAAACCAGAGTG CATGCGTGAGAGGAT AGGAGTTCAATTGTC
CTGAAACCAGAGTGG ATGCGTGAGAGGATT GGAGTTCAATTGTCA
TGAAACCAGAGTGGC TGCGTGAGAGGATTG GAGTTCAATTGTCAC
GAAACCAGAGTGGCC GCGTGAGAGGATTGA AGTTCAATTGTCACC
AAACCAGAGTGGCCA CGTGAGAGGATTGAG GTTCAATTGTCACCA
AACCAGAGTGGCCAT GTGAGAGGATTGAGT TTCAATTGTCACCAT
ACCAGAGTGGCCATT TGAGAGGATTGAGTT TCAATTGTCACCATG
CCAGAGTGGCCATTA GAGAGGATTGAGTTT CAATTGTCACCATGT
CAGAGTGGCCATTAA AGAGGATTGAGTTTC AATTGTCACCATGTG
AGAGTGGCCATTAAA GAGGATTGAGTTTCT ATTGTCACCATGTGG
GAGTGGCCATTAAAA AGGATTGAGTTTCTC TTGTCACCATGTGGT
AGTGGCCATTAAAAC GGATTGAGTTTCTCA TGTCACCATGTGGTG
GTGGCCATTAAAACA GATTGAGTTTCTCAA GTCACCATGTGGTGC
TGGCCATTAAAACAG ATTGAGTTTCTCAAC TCACCATGTGGTGCG
GGCCATTAAAACAGT TTGAGTTTCTCAACG CACCATGTGGTGCGA
GCCATTAAAACAGTG TGAGTTTCTCAACGA ACCATGTGGTGCGAT
CCATTAAAACAGTGA GAGTTTCTCAACGAA CCATGTGGTGCGATT
CATTAAAACAGTGAA AGTTTCTCAACGAAG CATGTGGTGCGATTG
ATTAAAACAGTGAAC GTTTCTCAACGAAGC ATGTGGTGCGATTGC
TTAAAACAGTGAACG TTTCTCAACGAAGCT TGTGGTGCGATTGCT
TAAAACAGTGAACGA TTCTCAACGAAGCTT GTGGTGCGATTGCTG
AAAACAGTGAACGAG TCTCAACGAAGCTTC TGGTGCGATTGCTGG
AAACAGTGAACGAGG CTCAACGAAGCTTCT GGTGCGATTGCTGGG
AACAGTGAACGAGGC TCAACGAAGCTTCTG GTGCGATTGCTGGGT
ACAGTGAACGAGGCC CAACGAAGCTTCTGT TGCGATTGCTGGGTG
CAGTGAACGAGGCCG AACGAAGCTTCTGTG GCGATTGCTGGGTGT
AGTGAACGAGGCCGC ACGAAGCTTCTGTGA CGATTGCTGGGTGTG
GTGAACGAGGCCGCA CGAAGCTTCTGTGAT GATTGCTGGGTGTGG
TGAACGAGGCCGCAA GAAGCTTCTGTGATG ATTGCTGGGTGTGGT
GAACGAGGCCGCAAG AAGCTTCTGTGATGA TTGCTGGGTGTGGTG
AACGAGGCCGCAAGC AGCTTCTGTGATGAA TGCTGGGTGTGGTGT
ACGAGGCCGCAAGCA GCTTCTGTGATGAAG GCTGGGTGTGGTGTC
CTGGGTGTGGTGTCC ACTGATGACACGGGG GGCCAGAAATGGAGA
TGGGTGTGGTGTCCC CTGATGACACGGGGC GCCAGAAATGGAGAA
GGGTGTGGTGTCCCA TGATGACACGGGGCG CCAGAAATGGAGAAT
GGTGTGGTGTCCCAA GATGACACGGGGCGA CAGAAATGGAGAATA
GTGTGGTGTCCCAAG ATGACACGGGGCGAT AGAAATGGAGAATAA
TGTGGTGTCCCAAGG TGACACGGGGCGATC GAAATGGAGAATAAT
GTGGTGTCCCAAGGC GACACGGGGCGATCT AAATGGAGAATAATC
TGGTGTCCCAAGGCC ACACGGGGCGATCTC AATGGAGAATAATCC
GGTGTCCCAAGGCCA CACGGGGCGATCTCA ATGGAGAATAATCCA
GTGTCCCAAGGCCAG ACGGGGCGATCTCAA TGGAGAATAATCCAG
TGTCCCAAGGCCAGC CGGGGCGATCTCAAA GGAGAATAATCCAGT
GTCCCAAGGCCAGCC GGGGCGATCTCAAAA GAGAATAATCCAGTC
TCCCAAGGCCAGCCA GGGCGATCTCAAAAG AGAATAATCCAGTCC
CCCAAGGCCAGCCAA GGCGATCTCAAAAGT GAATAATCCAGTCCT
CCAAGGCCAGCCAAC GCGATCTCAAAAGTT AATAATCCAGTCCTA
CAAGGCCAGCCAACA CGATCTCAAAAGTTA ATAATCCAGTCCTAG
AAGGCCAGCCAACAC GATCTCAAAAGTTAT TAATCCAGTCCTAGC
AGGCCAGCCAACACT ATCTCAAAAGTTATC AATCCAGTCCTAGCA
GGCCAGCCAACACTG TCTCAAAAGTTATCT ATCCAGTCCTAGCAC
GCCAGCCAACACTGG CTCAAAAGTTATCTC TCCAGTCCTAGCACC
CCAGCCAACACTGGT TCAAAAGTTATCTCC CCAGTCCTAGCACCT
CAGCCAACACTGGTC CAAAAGTTATCTCCG CAGTCCTAGCACCTC
AGCCAACACTGGTCA AAAAGTTATCTCCGG AGTCCTAGCACCTCC
GCCAACACTGGTCAT AAAGTTATCTCCGGT GTCCTAGCACCTCCA
CCAACACTGGTCATC AAGTTATCTCCGGTC TCCTAGCACCTCCAA
CAACACTGGTCATCA AGTTATCTCCGGTCT CCTAGCACCTCCAAG
AACACTGGTCATCAT GTTATCTCCGGTCTC CTAGCACCTCCAAGC
ACACTGGTCATCATG TTATCTCCGGTCTCT TAGCACCTCCAAGCC
CACTGGTCATCATGG TATCTCCGGTCTCTG AGCACCTCCAAGCCT
ACTGGTCATCATGGA ATCTCCGGTCTCTGA GCACCTCCAAGCCTG
CTGGTCATCATGGAA TCTCCGGTCTCTGAG CACCTCCAAGCCTGA
TGGTCATCATGGAAC CTCCGGTCTCTGAGG ACCTCCAAGCCTGAG
GGTCATCATGGAACT TCCGGTCTCTGAGGC CCTCCAAGCCTGAGC
GTCATCATGGAACTG CCGGTCTCTGAGGCC CTCCAAGCCTGAGCA
TCATCATGGAACTGA CGGTCTCTGAGGCCA TCCAAGCCTGAGCAA
CATCATGGAACTGAT GGTCTCTGAGGCCAG CCAAGCCTGAGCAAG
ATCATGGAACTGATG GTCTCTGAGGCCAGA CAAGCCTGAGCAAGA
TCATGGAACTGATGA TCTCTGAGGCCAGAA AAGCCTGAGCAAGAT
CATGGAACTGATGAC CTCTGAGGCCAGAAA AGCCTGAGCAAGATG
ATGGAACTGATGACA TCTGAGGCCAGAAAT GCCTGAGCAAGATGA
TGGAACTGATGACAC CTGAGGCCAGAAATG CCTGAGCAAGATGAT
GGAACTGATGACACG TGAGGCCAGAAATGG CTGAGCAAGATGATT
GAACTGATGACACGG GAGGCCAGAAATGGA TGAGCAAGATGATTC
AACTGATGACACGGG AGGCCAGAAATGGAG GAGCAAGATGATTCA
AGCAAGATGATTCAG ATACCTCAACGCCAA GGAATTGCATGGTAG
GCAAGATGATTCAGA TACCTCAACGCCAAT GAATTGCATGGTAGC
CAAGATGATTCAGAT ACCTCAACGCCAATA AATTGCATGGTAGCC
AAGATGATTCAGATG CCTCAACGCCAATAA ATTGCATGGTAGCCG
AGATGATTCAGATGG CTCAACGCCAATAAG TTGCATGGTAGCCGA
GATGATTCAGATGGC TCAACGCCAATAAGT TGCATGGTAGCCGAA
ATGATTCAGATGGCC CAACGCCAATAAGTT GCATGGTAGCCGAAG
TGATTCAGATGGCCG AACGCCAATAAGTTC CATGGTAGCCGAAGA
GATTCAGATGGCCGG ACGCCAATAAGTTCG ATGGTAGCCGAAGAT
ATTCAGATGGCCGGA CGCCAATAAGTTCGT TGGTAGCCGAAGATT
TTCAGATGGCCGGAG GCCAATAAGTTCGTC GGTAGCCGAAGATTT
TCAGATGGCCGGAGA CCAATAAGTTCGTCC GTAGCCGAAGATTTC
CAGATGGCCGGAGAG CAATAAGTTCGTCCA TAGCCGAAGATTTCA
AGATGGCCGGAGAGA AATAAGTTCGTCCAC AGCCGAAGATTTCAC
GATGGCCGGAGAGAT ATAAGTTCGTCCACA GCCGAAGATTTCACA
ATGGCCGGAGAGATT TAAGTTCGTCCACAG CCGAAGATTTCACAG
TGGCCGGAGAGATTG AAGTTCGTCCACAGA CGAAGATTTCACAGT
GGCCGGAGAGATTGC AGTTCGTCCACAGAG GAAGATTTCACAGTC
GCCGGAGAGATTGCA GTTCGTCCACAGAGA AAGATTTCACAGTCA
CCGGAGAGATTGCAG TTCGTCCACAGAGAC AGATTTCACAGTCAA
CGGAGAGATTGCAGA TCGTCCACAGAGACC GATTTCACAGTCAAA
GGAGAGATTGCAGAC CGTCCACAGAGACCT ATTTCACAGTCAAAA
GAGAGATTGCAGACG GTCCACAGAGACCTT TTTCACAGTCAAAAT
AGAGATTGCAGACGG TCCACAGAGACCTTG TTCACAGTCAAAATC
GAGATTGCAGACGGC CCACAGAGACCTTGC TCACAGTCAAAATCG
AGATTGCAGACGGCA CACAGAGACCTTGCT CACAGTCAAAATCGG
GATTGCAGACGGCAT ACAGAGACCTTGCTG ACAGTCAAAATCGGA
ATTGCAGACGGCATG CAGAGACCTTGCTGC CAGTCAAAATCGGAG
TTGCAGACGGCATGG AGAGACCTTGCTGCC AGTCAAAATCGGAGA
TGCAGACGGCATGGC GAGACCTTGCTGCCC GTCAAAATCGGAGAT
GCAGACGGCATGGCA AGACCTTGCTGCCCG TCAAAATCGGAGATT
CAGACGGCATGGCAT GACCTTGCTGCCCGG CAAAATCGGAGATTT
AGACGGCATGGCATA ACCTTGCTGCCCGGA AAAATCGGAGATTTT
GACGGCATGGCATAC CCTTGCTGCCCGGAA AAATCGGAGATTTTG
ACGGCATGGCATACC CTTGCTGCCCGGAAT AATCGGAGATTTTGG
CGGCATGGCATACCT TTGCTGCCCGGAATT ATCGGAGATTTTGGT
GGCATGGCATACCTC TGCTGCCCGGAATTG TCGGAGATTTTGGTA
GCATGGCATACCTCA GCTGCCCGGAATTGC CGGAGATTTTGGTAT
CATGGCATACCTCAA CTGCCCGGAATTGCA GGAGATTTTGGTATG
ATGGCATACCTCAAC TGCCCGGAATTGCAT GAGATTTTGGTATGA
TGGCATACCTCAACG GCCCGGAATTGCATG AGATTTTGGTATGAC
GGCATACCTCAACGC CCCGGAATTGCATGG GATTTTGGTATGACG
GCATACCTCAACGCC CCGGAATTGCATGGT ATTTTGGTATGACGC
CATACCTCAACGCCA CGGAATTGCATGGTA TTTTGGTATGACGCG
TTTGGTATGACGCGA AGGAGGCAAAGGGCT CCCTCAAGGATGGAG
TTGGTATGACGCGAG GGAGGCAAAGGGCTG CCTCAAGGATGGAGT
TGGTATGACGCGAGA GAGGCAAAGGGCTGC CTCAAGGATGGAGTC
GGTATGACGCGAGAT AGGCAAAGGGCTGCT TCAAGGATGGAGTCT
GTATGACGCGAGATA GGCAAAGGGCTGCTG CAAGGATGGAGTCTT
TATGACGCGAGATAT GCAAAGGGCTGCTGC AAGGATGGAGTCTTC
ATGACGCGAGATATC CAAAGGGCTGCTGCC AGGATGGAGTCTTCA
TGACGCGAGATATCT AAAGGGCTGCTGCCC GGATGGAGTCTTCAC
GACGCGAGATATCTA AAGGGCTGCTGCCCG GATGGAGTCTTCACC
ACGCGAGATATCTAT AGGGCTGCTGCCCGT ATGGAGTCTTCACCA
CGCGAGATATCTATG GGGCTGCTGCCCGTG TGGAGTCTTCACCAC
GCGAGATATCTATGA GGCTGCTGCCCGTGC GGAGTCTTCACCACT
CGAGATATCTATGAG GCTGCTGCCCGTGCG GAGTCTTCACCACTT
GAGATATCTATGAGA CTGCTGCCCGTGCGC AGTCTTCACCACTTA
AGATATCTATGAGAC TGCTGCCCGTGCGCT GTCTTCACCACTTAC
GATATCTATGAGACA GCTGCCCGTGCGCTG TCTTCACCACTTACT
ATATCTATGAGACAG CTGCCCGTGCGCTGG CTTCACCACTTACTC
TATCTATGAGACAGA TGCCCGTGCGCTGGA TTCACCACTTACTCG
ATCTATGAGACAGAC GCCCGTGCGCTGGAT TCACCACTTACTCGG
TCTATGAGACAGACT CCCGTGCGCTGGATG CACCACTTACTCGGA
CTATGAGACAGACTA CCGTGCGCTGGATGT ACCACTTACTCGGAC
TATGAGACAGACTAT CGTGCGCTGGATGTC CCACTTACTCGGACG
ATGAGACAGACTATT GTGCGCTGGATGTCT CACTTACTCGGACGT
TGAGACAGACTATTA TGCGCTGGATGTCTC ACTTACTCGGACGTC
GAGACAGACTATTAC GCGCTGGATGTCTCC CTTACTCGGACGTCT
AGACAGACTATTACC CGCTGGATGTCTCCT TTACTCGGACGTCTG
GACAGACTATTACCG GCTGGATGTCTCCTG TACTCGGACGTCTGG
ACAGACTATTACCGG CTGGATGTCTCCTGA ACTCGGACGTCTGGT
CAGACTATTACCGGA TGGATGTCTCCTGAG CTCGGACGTCTGGTC
AGACTATTACCGGAA GGATGTCTCCTGAGT TCGGACGTCTGGTCC
GACTATTACCGGAAA GATGTCTCCTGAGTC CGGACGTCTGGTCCT
ACTATTACCGGAAAG ATGTCTCCTGAGTCC GGACGTCTGGTCCTT
CTATTACCGGAAAGG TGTCTCCTGAGTCCC GACGTCTGGTCCTTC
TATTACCGGAAAGGA GTCTCCTGAGTCCCT ACGTCTGGTCCTTCG
ATTACCGGAAAGGAG TCTCCTGAGTCCCTC CGTCTGGTCCTTCGG
TTACCGGAAAGGAGG CTCCTGAGTCCCTCA GTCTGGTCCTTCGGG
TACCGGAAAGGAGGC TCCTGAGTCCCTCAA TCTGGTCCTTCGGGG
ACCGGAAAGGAGGCA CCTGAGTCCCTCAAG CTGGTCCTTCGGGGT
CCGGAAAGGAGGCAA CTGAGTCCCTCAAGG TGGTCCTTCGGGGTC
CGGAAAGGAGGCAAA TGAGTCCCTCAAGGA GGTCCTTCGGGGTCG
GGAAAGGAGGCAAAG GAGTCCCTCAAGGAT GTCCTTCGGGGTCGT
GAAAGGAGGCAAAGG AGTCCCTCAAGGATG TCCTTCGGGGTCGTC
AAAGGAGGCAAAGGG GTCCCTCAAGGATGG CCTTCGGGGTCGTCC
AAGGAGGCAAAGGGC TCCCTCAAGGATGGA CTTCGGGGTCGTCCT
TTCGGGGTCGTCCTC CTACCAGGGCTTGTC AGGGCGGCCTTCTGG
TCGGGGTCGTCCTCT TACCAGGGCTTGTCC GGGCGGCCTTCTGGA
CGGGGTCGTCCTCTG ACCAGGGCTTGTCCA GGCGGCCTTCTGGAC
GGGGTCGTCCTCTGG CCAGGGCTTGTCCAA GCGGCCTTCTGGACA
GGGTCGTCCTCTGGG CAGGGCTTGTCCAAC CGGCCTTCTGGACAA
GGTCGTCCTCTGGGA AGGGCTTGTCCAACG GGCCTTCTGGACAAG
GTCGTCCTCTGGGAG GGGCTTGTCCAACGA GCCTTCTGGACAAGC
TCGTCCTCTGGGAGA GGCTTGTCCAACGAG CCTTCTGGACAAGCC
CGTCCTCTGGGAGAT GCTTGTCCAACGAGC CTTCTGGACAAGCCA
GTCCTCTGGGAGATC CTTGTCCAACGAGCA TTCTGGACAAGCCAG
TCCTCTGGGAGATCG TTGTCCAACGAGCAA TCTGGACAAGCCAGA
CCTCTGGGAGATCGC TGTCCAACGAGCAAG CTGGACAAGCCAGAC
CTCTGGGAGATCGCC GTCCAACGAGCAAGT TGGACAAGCCAGACA
TCTGGGAGATCGCCA TCCAACGAGCAAGTC GGACAAGCCAGACAA
CTGGGAGATCGCCAC CCAACGAGCAAGTCC GACAAGCCAGACAAC
TGGGAGATCGCCACA CAACGAGCAAGTCCT ACAAGCCAGACAACT
GGGAGATCGCCACAC AACGAGCAAGTCCTT CAAGCCAGACAACTG
GGAGATCGCCACACT ACGAGCAAGTCCTTC AAGCCAGACAACTGT
GAGATCGCCACACTG CGAGCAAGTCCTTCG AGCCAGACAACTGTC
AGATCGCCACACTGG GAGCAAGTCCTTCGC GCCAGACAACTGTCC
GATCGCCACACTGGC AGCAAGTCCTTCGCT CCAGACAACTGTCCT
ATCGCCACACTGGCC GCAAGTCCTTCGCTT CAGACAACTGTCCTG
TCGCCACACTGGCCG CAAGTCCTTCGCTTC AGACAACTGTCCTGA
CGCCACACTGGCCGA AAGTCCTTCGCTTCG GACAACTGTCCTGAC
GCCACACTGGCCGAG AGTCCTTCGCTTCGT ACAACTGTCCTGACA
CCACACTGGCCGAGC GTCCTTCGCTTCGTC CAACTGTCCTGACAT
CACACTGGCCGAGCA TCCTTCGCTTCGTCA AACTGTCCTGACATG
ACACTGGCCGAGCAG CCTTCGCTTCGTCAT ACTGTCCTGACATGC
CACTGGCCGAGCAGC CTTCGCTTCGTCATG CTGTCCTGACATGCT
ACTGGCCGAGCAGCC TTCGCTTCGTCATGG TGTCCTGACATGCTG
CTGGCCGAGCAGCCC TCGCTTCGTCATGGA GTCCTGACATGCTGT
TGGCCGAGCAGCCCT CGCTTCGTCATGGAG TCCTGACATGCTGTT
GGCCGAGCAGCCCTA GCTTCGTCATGGAGG CCTGACATGCTGTTT
GCCGAGCAGCCCTAC CTTCGTCATGGAGGG CTGACATGCTGTTTG
CCGAGCAGCCCTACC TTCGTCATGGAGGGC TGACATGCTGTTTGA
CGAGCAGCCCTACCA TCGTCATGGAGGGCG GACATGCTGTTTGAA
GAGCAGCCCTACCAG CGTCATGGAGGGCGG ACATGCTGTTTGAAC
AGCAGCCCTACCAGG GTCATGGAGGGCGGC CATGCTGTTTGAACT
GCAGCCCTACCAGGG TCATGGAGGGCGGCC ATGCTGTTTGAACTG
CAGCCCTACCAGGGC CATGGAGGGCGGCCT TGCTGTTTGAACTGA
AGCCCTACCAGGGCT ATGGAGGGCGGCCTT GCTGTTTGAACTGAT
GCCCTACCAGGGCTT TGGAGGGCGGCCTTC CTGTTTGAACTGATG
CCCTACCAGGGCTTG GGAGGGCGGCCTTCT TGTTTGAACTGATGC
CCTACCAGGGCTTGT GAGGGCGGCCTTCTG GTTTGAACTGATGCG
TTTGAACTGATGCGC GCCTTCCTTCCTGGA AGCCTGGCTTCCGGG
TTGAACTGATGCGCA CCTTCCTTCCTGGAG GCCTGGCTTCCGGGA
TGAACTGATGCGCAT CTTCCTTCCTGGAGA CCTGGCTTCCGGGAG
GAACTGATGCGCATG TTCCTTCCTGGAGAT CTGGCTTCCGGGAGG
AACTGATGCGCATGT TCCTTCCTGGAGATC TGGCTTCCGGGAGGT
ACTGATGCGCATGTG CCTTCCTGGAGATCA GGCTTCCGGGAGGTC
CTGATGCGCATGTGC CTTCCTGGAGATCAT GCTTCCGGGAGGTCT
TGATGCGCATGTGCT TTCCTGGAGATCATC CTTCCGGGAGGTCTC
GATGCGCATGTGCTG TCCTGGAGATCATCA TTCCGGGAGGTCTCC
ATGCGCATGTGCTGG CCTGGAGATCATCAG TCCGGGAGGTCTCCT
TGCGCATGTGCTGGC CTGGAGATCATCAGC CCGGGAGGTCTCCTT
GCGCATGTGCTGGCA TGGAGATCATCAGCA CGGGAGGTCTCCTTC
CGCATGTGCTGGCAG GGAGATCATCAGCAG GGGAGGTCTCCTTCT
GCATGTGCTGGCAGT GAGATCATCAGCAGC GGAGGTCTCCTTCTA
CATGTGCTGGCAGTA AGATCATCAGCAGCA GAGGTCTCCTTCTAC
ATGTGCTGGCAGTAT GATCATCAGCAGCAT AGGTCTCCTTCTACT
TGTGCTGGCAGTATA ATCATCAGCAGCATC GGTCTCCTTCTACTA
GTGCTGGCAGTATAA TCATCAGCAGCATCA GTCTCCTTCTACTAC
TGCTGGCAGTATAAC CATCAGCAGCATCAA TCTCCTTCTACTACA
GCTGGCAGTATAACC ATCAGCAGCATCAAA CTCCTTCTACTACAG
CTGGCAGTATAACCC TCAGCAGCATCAAAG TCCTTCTACTACAGC
TGGCAGTATAACCCC CAGCAGCATCAAAGA CCTTCTACTACAGCG
GGCAGTATAACCCCA AGCAGCATCAAAGAG CTTCTACTACAGCGA
GCAGTATAACCCCAA GCAGCATCAAAGAGG TTCTACTACAGCGAG
CAGTATAACCCCAAG CAGCATCAAAGAGGA TCTACTACAGCGAGG
AGTATAACCCCAAGA AGCATCAAAGAGGAG CTACTACAGCGAGGA
GTATAACCCCAAGAT GCATCAAAGAGGAGA TACTACAGCGAGGAG
TATAACCCCAAGATG CATCAAAGAGGAGAT ACTACAGCGAGGAGA
ATAACCCCAAGATGA ATCAAAGAGGAGATG CTACAGCGAGGAGAA
TAACCCCAAGATGAG TCAAAGAGGAGATGG TACAGCGAGGAGAAC
AACCCCAAGATGAGG CAAAGAGGAGATGGA ACAGCGAGGAGAACA
ACCCCAAGATGAGGC AAAGAGGAGATGGAG CAGCGAGGAGAACAA
CCCCAAGATGAGGCC AAGAGGAGATGGAGC AGCGAGGAGAACAAG
CCCAAGATGAGGCCT AGAGGAGATGGAGCC GCGAGGAGAACAAGC
CCAAGATGAGGCCTT GAGGAGATGGAGCCT CGAGGAGAACAAGCT
CAAGATGAGGCCTTC AGGAGATGGAGCCTG GAGGAGAACAAGCTG
AAGATGAGGCCTTCC GGAGATGGAGCCTGG AGGAGAACAAGCTGC
AGATGAGGCCTTCCT GAGATGGAGCCTGGC GGAGAACAAGCTGCC
GATGAGGCCTTCCTT AGATGGAGCCTGGCT GAGAACAAGCTGCCC
ATGAGGCCTTCCTTC GATGGAGCCTGGCTT AGAACAAGCTGCCCG
TGAGGCCTTCCTTCC ATGGAGCCTGGCTTC GAACAAGCTGCCCGA
GAGGCCTTCCTTCCT TGGAGCCTGGCTTCC AACAAGCTGCCCGAG
AGGCCTTCCTTCCTG GGAGCCTGGCTTCCG ACAAGCTGCCCGAGC
GGCCTTCCTTCCTGG GAGCCTGGCTTCCGG CAAGCTGCCCGAGCC
AAGCTGCCCGAGCCG GGAGAGCGTCCCCCT CACTGCCCGACAGAC
AGCTGCCCGAGCCGG GAGAGCGTCCCCCTG ACTGCCCGACAGACA
GCTGCCCGAGCCGGA AGAGCGTCCCCCTGG CTGCCCGACAGACAC
CTGCCCGAGCCGGAG GAGCGTCCCCCTGGA TGCCCGACAGACACT
TGCCCGAGCCGGAGG AGCGTCCCCCTGGAC GCCCGACAGACACTC
GCCCGAGCCGGAGGA GCGTCCCCCTGGACC CCCGACAGACACTCA
CCCGAGCCGGAGGAG CGTCCCCCTGGACCC CCGACAGACACTCAG
CCGAGCCGGAGGAGC GTCCCCCTGGACCCC CGACAGACACTCAGG
CGAGCCGGAGGAGCT TCCCCCTGGACCCCT GACAGACACTCAGGA
GAGCCGGAGGAGCTG CCCCCTGGACCCCTC ACAGACACTCAGGAC
AGCCGGAGGAGCTGG CCCCTGGACCCCTCG CAGACACTCAGGACA
GCCGGAGGAGCTGGA CCCTGGACCCCTCGG AGACACTCAGGACAC
CCGGAGGAGCTGGAC CCTGGACCCCTCGGC GACACTCAGGACACA
CGGAGGAGCTGGACC CTGGACCCCTCGGCC ACACTCAGGACACAA
GGAGGAGCTGGACCT TGGACCCCTCGGCCT CACTCAGGACACAAG
GAGGAGCTGGACCTG GGACCCCTCGGCCTC ACTCAGGACACAAGG
AGGAGCTGGACCTGG GACCCCTCGGCCTCC CTCAGGACACAAGGC
GGAGCTGGACCTGGA ACCCCTCGGCCTCCT TCAGGACACAAGGCC
GAGCTGGACCTGGAG CCCCTCGGCCTCCTC CAGGACACAAGGCCG
AGCTGGACCTGGAGC CCCTCGGCCTCCTCG AGGACACAAGGCCGA
GCTGGACCTGGAGCC CCTCGGCCTCCTCGT GGACACAAGGCCGAG
CTGGACCTGGAGCCA CTCGGCCTCCTCGTC GACACAAGGCCGAGA
TGGACCTGGAGCCAG TCGGCCTCCTCGTCC ACACAAGGCCGAGAA
GGACCTGGAGCCAGA CGGCCTCCTCGTCCT CACAAGGCCGAGAAC
GACCTGGAGCCAGAG GGCCTCCTCGTCCTC ACAAGGCCGAGAACG
ACCTGGAGCCAGAGA GCCTCCTCGTCCTCC CAAGGCCGAGAACGG
CCTGGAGCCAGAGAA CCTCCTCGTCCTCCC AAGGCCGAGAACGGC
CTGGAGCCAGAGAAC CTCCTCGTCCTCCCT AGGCCGAGAACGGCC
TGGAGCCAGAGAACA TCCTCGTCCTCCCTG GGCCGAGAACGGCCC
GGAGCCAGAGAACAT CCTCGTCCTCCCTGC GCCGAGAACGGCCCC
GAGCCAGAGAACATG CTCGTCCTCCCTGCC CCGAGAACGGCCCCG
AGCCAGAGAACATGG TCGTCCTCCCTGCCA CGAGAACGGCCCCGG
GCCAGAGAACATGGA CGTCCTCCCTGCCAC GAGAACGGCCCCGGC
CCAGAGAACATGGAG GTCCTCCCTGCCACT AGAACGGCCCCGGCC
CAGAGAACATGGAGA TCCTCCCTGCCACTG GAACGGCCCCGGCCC
AGAGAACATGGAGAG CCTCCCTGCCACTGC AACGGCCCCGGCCCT
GAGAACATGGAGAGC CTCCCTGCCACTGCC ACGGCCCCGGCCCTG
AGAACATGGAGAGCG TCCCTGCCACTGCCC CGGCCCCGGCCCTGG
GAACATGGAGAGCGT CCCTGCCACTGCCCG GGCCCCGGCCCTGGG
AACATGGAGAGCGTC CCTGCCACTGCCCGA GCCCCGGCCCTGGGG
ACATGGAGAGCGTCC CTGCCACTGCCCGAC CCCCGGCCCTGGGGT
CATGGAGAGCGTCCC TGCCACTGCCCGACA CCCGGCCCTGGGGTG
ATGGAGAGCGTCCCC GCCACTGCCCGACAG CCGGCCCTGGGGTGC
TGGAGAGCGTCCCCC CCACTGCCCGACAGA CGGCCCTGGGGTGCT
GGCCCTGGGGTGCTG GCCTTACGCCCACAT TGCCGCTGCCCCAGT
GCCCTGGGGTGCTGG CCTTACGCCCACATG GCCGCTGCCCCAGTC
CCCTGGGGTGCTGGT CTTACGCCCACATGA CCGCTGCCCCAGTCT
CCTGGGGTGCTGGTC TTACGCCCACATGAA CGCTGCCCCAGTCTT
CTGGGGTGCTGGTCC TACGCCCACATGAAC GCTGCCCCAGTCTTC
TGGGGTGCTGGTCCT ACGCCCACATGAACG CTGCCCCAGTCTTCG
GGGGTGCTGGTCCTC CGCCCACATGAACGG TGCCCCAGTCTTCGA
GGGTGCTGGTCCTCC GCCCACATGAACGGG GCCCCAGTCTTCGAC
GGTGCTGGTCCTCCG CCCACATGAACGGGG CCCCAGTCTTCGACC
GTGCTGGTCCTCCGC CCACATGAACGGGGG CCCAGTCTTCGACCT
TGCTGGTCCTCCGCG CACATGAACGGGGGC CCAGTCTTCGACCTG
GCTGGTCCTCCGCGC ACATGAACGGGGGCC CAGTCTTCGACCTGC
CTGGTCCTCCGCGCC CATGAACGGGGGCCG AGTCTTCGACCTGCT
TGGTCCTCCGCGCCA ATGAACGGGGGCCGC GTCTTCGACCTGCTG
GGTCCTCCGCGCCAG TGAACGGGGGCCGCA TCTTCGACCTGCTGA
GTCCTCCGCGCCAGC GAACGGGGGCCGCAA CTTCGACCTGCTGAT
TCCTCCGCGCCAGCT AACGGGGGCCGCAAG TTCGACCTGCTGATC
CCTCCGCGCCAGCTT ACGGGGGCCGCAAGA TCGACCTGCTGATCC
CTCCGCGCCAGCTTC CGGGGGCCGCAAGAA CGACCTGCTGATCCT
TCCGCGCCAGCTTCG GGGGGCCGCAAGAAC GACCTGCTGATCCTT
CCGCGCCAGCTTCGA GGGGCCGCAAGAACG ACCTGCTGATCCTTG
CGCGCCAGCTTCGAC GGGCCGCAAGAACGA CCTGCTGATCCTTGG
GCGCCAGCTTCGACG GGCCGCAAGAACGAG CTGCTGATCCTTGGA
CGCCAGCTTCGACGA GCCGCAAGAACGAGC TGCTGATCCTTGGAT
GCCAGCTTCGACGAG CCGCAAGAACGAGCG GCTGATCCTTGGATC
CCAGCTTCGACGAGA CGCAAGAACGAGCGG CTGATCCTTGGATCC
CAGCTTCGACGAGAG GCAAGAACGAGCGGG TGATCCTTGGATCCT
AGCTTCGACGAGAGA CAAGAACGAGCGGGC GATCCTTGGATCCTG
GCTTCGACGAGAGAC AAGAACGAGCGGGCC ATCCTTGGATCCTGA
CTTCGACGAGAGACA AGAACGAGCGGGCCT TCCTTGGATCCTGAA
TTCGACGAGAGACAG GAACGAGCGGGCCTT CCTTGGATCCTGAAT
TCGACGAGAGACAGC AACGAGCGGGCCTTG CTTGGATCCTGAATC
CGACGAGAGACAGCC ACGAGCGGGCCTTGC TTGGATCCTGAATCT
GACGAGAGACAGCCT CGAGCGGGCCTTGCC TGGATCCTGAATCTG
ACGAGAGACAGCCTT GAGCGGGCCTTGCCG GGATCCTGAATCTGT
CGAGAGACAGCCTTA AGCGGGCCTTGCCGC GATCCTGAATCTGTG
GAGAGACAGCCTTAC GCGGGCCTTGCCGCT ATCCTGAATCTGTGC
AGAGACAGCCTTACG CGGGCCTTGCCGCTG TCCTGAATCTGTGCA
GAGACAGCCTTACGC GGGCCTTGCCGCTGC CCTGAATCTGTGCAA
AGACAGCCTTACGCC GGCCTTGCCGCTGCC CTGAATCTGTGCAAA
GACAGCCTTACGCCC GCCTTGCCGCTGCCC TGAATCTGTGCAAAC
ACAGCCTTACGCCCA CCTTGCCGCTGCCCC GAATCTGTGCAAACA
CAGCCTTACGCCCAC CTTGCCGCTGCCCCA AATCTGTGCAAACAG
AGCCTTACGCCCACA TTGCCGCTGCCCCAG ATCTGTGCAAACAGT
TCTGTGCAAACAGTA GGGAGAGAGAGTTTT CAGTGGATCTTCAGT
CTGTGCAAACAGTAA GGAGAGAGAGTTTTA AGTGGATCTTCAGTT
TGTGCAAACAGTAAC GAGAGAGAGTTTTAA GTGGATCTTCAGTTC
GTGCAAACAGTAACG AGAGAGAGTTTTAAC TGGATCTTCAGTTCT
TGCAAACAGTAACGT GAGAGAGTTTTAACA GGATCTTCAGTTCTG
GCAAACAGTAACGTG AGAGAGTTTTAACAA GATCTTCAGTTCTGC
CAAACAGTAACGTGT GAGAGTTTTAACAAT ATCTTCAGTTCTGCC
AAACAGTAACGTGTG AGAGTTTTAACAATC TCTTCAGTTCTGCCC
AACAGTAACGTGTGC GAGTTTTAACAATCC CTTCAGTTCTGCCCT
ACAGTAACGTGTGCG AGTTTTAACAATCCA TTCAGTTCTGCCCTT
CAGTAACGTGTGCGC GTTTTAACAATCCAT TCAGTTCTGCCCTTG
AGTAACGTGTGCGCA TTTTAACAATCCATT CAGTTCTGCCCTTGC
GTAACGTGTGCGCAC TTTAACAATCCATTC AGTTCTGCCCTTGCT
TAACGTGTGCGCACG TTAACAATCCATTCA GTTCTGCCCTTGCTG
AACGTGTGCGCACGC TAACAATCCATTCAC TTCTGCCCTTGCTGC
ACGTGTGCGCACGCG AACAATCCATTCACA TCTGCCCTTGCTGCC
CGTGTGCGCACGCGC ACAATCCATTCACAA CTGCCCTTGCTGCCC
GTGTGCGCACGCGCA CAATCCATTCACAAG TGCCCTTGCTGCCCG
TGTGCGCACGCGCAG AATCCATTCACAAGC GCCCTTGCTGCCCGC
GTGCGCACGCGCAGC ATCCATTCACAAGCC CCCTTGCTGCCCGCG
TGCGCACGCGCAGCG TCCATTCACAAGCCT CCTTGCTGCCCGCGG
GCGCACGCGCAGCGG CCATTCACAAGCCTC CTTGCTGCCCGCGGG
CGCACGCGCAGCGGG CATTCACAAGCCTCC TTGCTGCCCGCGGGA
GCACGCGCAGCGGGG ATTCACAAGCCTCCT TGCTGCCCGCGGGAG
CACGCGCAGCGGGGT TTCACAAGCCTCCTG GCTGCCCGCGGGAGA
ACGCGCAGCGGGGTG TCACAAGCCTCCTGT CTGCCCGCGGGAGAC
CGCGCAGCGGGGTGG CACAAGCCTCCTGTA TGCCCGCGGGAGACA
GCGCAGCGGGGTGGG ACAAGCCTCCTGTAC GCCCGCGGGAGACAG
CGCAGCGGGGTGGGG CAAGCCTCCTGTACC CCCGCGGGAGACAGC
GCAGCGGGGTGGGGG AAGCCTCCTGTACCT CCGCGGGAGACAGCT
CAGCGGGGTGGGGGG AGCCTCCTGTACCTC CGCGGGAGACAGCTT
AGCGGGGTGGGGGGG GCCTCCTGTACCTCA GCGGGAGACAGCTTC
GCGGGGTGGGGGGGG CCTCCTGTACCTCAG CGGGAGACAGCTTCT
CGGGGTGGGGGGGGA CTCCTGTACCTCAGT GGGAGACAGCTTCTC
GGGGTGGGGGGGGAG TCCTGTACCTCAGTG GGAGACAGCTTCTCT
GGGTGGGGGGGGAGA CCTGTACCTCAGTGG GAGACAGCTTCTCTG
GGTGGGGGGGGAGAG CTGTACCTCAGTGGA AGACAGCTTCTCTGC
GTGGGGGGGGAGAGA TGTACCTCAGTGGAT GACAGCTTCTCTGCA
TGGGGGGGGAGAGAG GTACCTCAGTGGATC ACAGCTTCTCTGCAG
GGGGGGGGAGAGAGA TACCTCAGTGGATCT CAGCTTCTCTGCAGT
GGGGGGGAGAGAGAG ACCTCAGTGGATCTT AGCTTCTCTGCAGTA
GGGGGGAGAGAGAGT CCTCAGTGGATCTTC GCTTCTCTGCAGTAA
GGGGGAGAGAGAGTT CTCAGTGGATCTTCA CTTCTCTGCAGTAAA
GGGGAGAGAGAGTTT TCAGTGGATCTTCAG TTCTCTGCAGTAAAA
TCTCTGCAGTAAAAC CAAGCAGCTTTTTAT CCTTTAAGAACCTTA
CTCTGCAGTAAAACA AAGCAGCTTTTTATT CTTTAAGAACCTTAA
TCTGCAGTAAAACAC AGCAGCTTTTTATTC TTTAAGAACCTTAAT
CTGCAGTAAAACACA GCAGCTTTTTATTCC TTAAGAACCTTAATG
TGCAGTAAAACACAT CAGCTTTTTATTCCC TAAGAACCTTAATGA
GCAGTAAAACACATT AGCTTTTTATTCCCT AAGAACCTTAATGAC
CAGTAAAACACATTT GCTTTTTATTCCCTG AGAACCTTAATGACA
AGTAAAACACATTTG CTTTTTATTCCCTGC GAACCTTAATGACAA
GTAAAACACATTTGG TTTTTATTCCCTGCC AACCTTAATGACAAC
TAAAACACATTTGGG TTTTATTCCCTGCCC ACCTTAATGACAACA
AAAACACATTTGGGA TTTATTCCCTGCCCA CCTTAATGACAACAC
AAACACATTTGGGAT TTATTCCCTGCCCAA CTTAATGACAACACT
AACACATTTGGGATG TATTCCCTGCCCAAA TTAATGACAACACTT
ACACATTTGGGATGT ATTCCCTGCCCAAAC TAATGACAACACTTA
CACATTTGGGATGTT TTCCCTGCCCAAACC AATGACAACACTTAA
ACATTTGGGATGTTC TCCCTGCCCAAACCC ATGACAACACTTAAT
CATTTGGGATGTTCC CCCTGCCCAAACCCT TGACAACACTTAATA
ATTTGGGATGTTCCT CCTGCCCAAACCCTT GACAACACTTAATAG
TTTGGGATGTTCCTT CTGCCCAAACCCTTA ACAACACTTAATAGC
TTGGGATGTTCCTTT TGCCCAAACCCTTAA CAACACTTAATAGCA
TGGGATGTTCCTTTT GCCCAAACCCTTAAC AACACTTAATAGCAA
GGGATGTTCCTTTTT CCCAAACCCTTAACT ACACTTAATAGCAAC
GGATGTTCCTTTTTT CCAAACCCTTAACTG CACTTAATAGCAACA
GATGTTCCTTTTTTC CAAACCCTTAACTGA ACTTAATAGCAACAG
ATGTTCCTTTTTTCA AAACCCTTAACTGAC CTTAATAGCAACAGA
TGTTCCTTTTTTCAA AACCCTTAACTGACA TTAATAGCAACAGAG
GTTCCTTTTTTCAAT ACCCTTAACTGACAT TAATAGCAACAGAGC
TTCCTTTTTTCAATA CCCTTAACTGACATG AATAGCAACAGAGCA
TCCTTTTTTCAATAT CCTTAACTGACATGG ATAGCAACAGAGCAC
CCTTTTTTCAATATG CTTAACTGACATGGG TAGCAACAGAGCACT
CTTTTTTCAATATGC TTAACTGACATGGGC AGCAACAGAGCACTT
TTTTTTCAATATGCA TAACTGACATGGGCC GCAACAGAGCACTTG
TTTTTCAATATGCAA AACTGACATGGGCCT CAACAGAGCACTTGA
TTTTCAATATGCAAG ACTGACATGGGCCTT AACAGAGCACTTGAG
TTTCAATATGCAAGC CTGACATGGGCCTTT ACAGAGCACTTGAGA
TTCAATATGCAAGCA TGACATGGGCCTTTA CAGAGCACTTGAGAA
TCAATATGCAAGCAG GACATGGGCCTTTAA AGAGCACTTGAGAAC
CAATATGCAAGCAGC ACATGGGCCTTTAAG GAGCACTTGAGAACC
AATATGCAAGCAGCT CATGGGCCTTTAAGA AGCACTTGAGAACCA
ATATGCAAGCAGCTT ATGGGCCTTTAAGAA GCACTTGAGAACCAG
TATGCAAGCAGCTTT TGGGCCTTTAAGAAC CACTTGAGAACCAGT
ATGCAAGCAGCTTTT GGGCCTTTAAGAACC ACTTGAGAACCAGTC
TGCAAGCAGCTTTTT GGCCTTTAAGAACCT CTTGAGAACCAGTCT
GCAAGCAGCTTTTTA GCCTTTAAGAACCTT TTGAGAACCAGTCTC
TGAGAACCAGTCTCC CCTTTCTCTCTCCTC CAAGTCCAGCTGGGA
GAGAACCAGTCTCCT CTTTCTCTCTCCTCT AAGTCCAGCTGGGAA
AGAACCAGTCTCCTC TTTCTCTCTCCTCTC AGTCCAGCTGGGAAG
GAACCAGTCTCCTCA TTCTCTCTCCTCTCT GTCCAGCTGGGAAGC
AACCAGTCTCCTCAC TCTCTCTCCTCTCTG TCCAGCTGGGAAGCC
ACCAGTCTCCTCACT CTCTCTCCTCTCTGC CCAGCTGGGAAGCCC
CCAGTCTCCTCACTC TCTCTCCTCTCTGCT CAGCTGGGAAGCCCT
CAGTCTCCTCACTCT CTCTCCTCTCTGCTT AGCTGGGAAGCCCTT
AGTCTCCTCACTCTG TCTCCTCTCTGCTTC GCTGGGAAGCCCTTT
GTCTCCTCACTCTGT CTCCTCTCTGCTTCA CTGGGAAGCCCTTTT
TCTCCTCACTCTGTC TCCTCTCTGCTTCAT TGGGAAGCCCTTTTT
CTCCTCACTCTGTCC CCTCTCTGCTTCATA GGGAAGCCCTTTTTA
TCCTCACTCTGTCCC CTCTCTGCTTCATAA GGAAGCCCTTTTTAT
CCTCACTCTGTCCCT TCTCTGCTTCATAAC GAAGCCCTTTTTATC
CTCACTCTGTCCCTG CTCTGCTTCATAACG AAGCCCTTTTTATCA
TCACTCTGTCCCTGT TCTGCTTCATAACGG AGCCCTTTTTATCAG
CACTCTGTCCCTGTC CTGCTTCATAACGGA GCCCTTTTTATCAGT
ACTCTGTCCCTGTCC TGCTTCATAACGGAA CCCTTTTTATCAGTT
CTCTGTCCCTGTCCT GCTTCATAACGGAAA CCTTTTTATCAGTTT
TCTGTCCCTGTCCTT CTTCATAACGGAAAA CTTTTTATCAGTTTG
CTGTCCCTGTCCTTC TTCATAACGGAAAAA TTTTTATCAGTTTGA
TGTCCCTGTCCTTCC TCATAACGGAAAAAT TTTTATCAGTTTGAG
GTCCCTGTCCTTCCC CATAACGGAAAAATA TTTATCAGTTTGAGG
TCCCTGTCCTTCCCT ATAACGGAAAAATAA TTATCAGTTTGAGGA
CCCTGTCCTTCCCTG TAACGGAAAAATAAT TATCAGTTTGAGGAA
CCTGTCCTTCCCTGT AACGGAAAAATAATT ATCAGTTTGAGGAAG
CTGTCCTTCCCTGTT ACGGAAAAATAATTG TCAGTTTGAGGAAGT
TGTCCTTCCCTGTTC CGGAAAAATAATTGC CAGTTTGAGGAAGTG
GTCCTTCCCTGTTCT GGAAAAATAATTGCC AGTTTGAGGAAGTGG
TCCTTCCCTGTTCTC GAAAAATAATTGCCA GTTTGAGGAAGTGGC
CCTTCCCTGTTCTCC AAAAATAATTGCCAC TTTGAGGAAGTGGCT
CTTCCCTGTTCTCCC AAAATAATTGCCACA TTGAGGAAGTGGCTG
TTCCCTGTTCTCCCT AAATAATTGCCACAA TGAGGAAGTGGCTGT
TCCCTGTTCTCCCTT AATAATTGCCACAAG GAGGAAGTGGCTGTC
CCCTGTTCTCCCTTT ATAATTGCCACAAGT AGGAAGTGGCTGTCC
CCTGTTCTCCCTTTC TAATTGCCACAAGTC GGAAGTGGCTGTCCC
CTGTTCTCCCTTTCT AATTGCCACAAGTCC GAAGTGGCTGTCCCT
TGTTCTCCCTTTCTC ATTGCCACAAGTCCA AAGTGGCTGTCCCTG
GTTCTCCCTTTCTCT TTGCCACAAGTCCAG AGTGGCTGTCCCTGT
TTCTCCCTTTCTCTC TGCCACAAGTCCAGC GTGGCTGTCCCTGTG
TCTCCCTTTCTCTCT GCCACAAGTCCAGCT TGGCTGTCCCTGTGG
CTCCCTTTCTCTCTC CCACAAGTCCAGCTG GGCTGTCCCTGTGGC
TCCCTTTCTCTCTCC CACAAGTCCAGCTGG GCTGTCCCTGTGGCC
CCCTTTCTCTCTCCT ACAAGTCCAGCTGGG CTGTCCCTGTGGCCC
TGTCCCTGTGGCCCC CCGTGGGTCATTACA CTTTATCTTTCACCT
GTCCCTGTGGCCCCA CGTGGGTCATTACAA TTTATCTTTCACCTT
TCCCTGTGGCCCCAT GTGGGTCATTACAAA TTATCTTTCACCTTT
CCCTGTGGCCCCATC TGGGTCATTACAAAA TATCTTTCACCTTTC
CCTGTGGCCCCATCC GGGTCATTACAAAAA ATCTTTCACCTTTCT
CTGTGGCCCCATCCA GGTCATTACAAAAAA TCTTTCACCTTTCTA
TGTGGCCCCATCCAA GTCATTACAAAAAAA CTTTCACCTTTCTAG
GTGGCCCCATCCAAC TCATTACAAAAAAAC TTTCACCTTTCTAGG
TGGCCCCATCCAACC CATTACAAAAAAACA TTCACCTTTCTAGGG
GGCCCCATCCAACCA ATTACAAAAAAACAC TCACCTTTCTAGGGA
GCCCCATCCAACCAC TTACAAAAAAACACG CACCTTTCTAGGGAC
CCCCATCCAACCACT TACAAAAAAACACGT ACCTTTCTAGGGACA
CCCATCCAACCACTG ACAAAAAAACACGTG CCTTTCTAGGGACAT
CCATCCAACCACTGT CAAAAAAACACGTGG CTTTCTAGGGACATG
CATCCAACCACTGTA AAAAAAACACGTGGA TTTCTAGGGACATGA
ATCCAACCACTGTAC AAAAAACACGTGGAG TTCTAGGGACATGAA
TCCAACCACTGTACA AAAAACACGTGGAGA TCTAGGGACATGAAA
CCAACCACTGTACAC AAAACACGTGGAGAT CTAGGGACATGAAAT
CAACCACTGTACACA AAACACGTGGAGATG TAGGGACATGAAATT
AACCACTGTACACAC AACACGTGGAGATGG AGGGACATGAAATTT
ACCACTGTACACACC ACACGTGGAGATGGA GGGACATGAAATTTA
CCACTGTACACACCC CACGTGGAGATGGAA GGACATGAAATTTAC
CACTGTACACACCCG ACGTGGAGATGGAAA GACATGAAATTTACA
ACTGTACACACCCGC CGTGGAGATGGAAAT ACATGAAATTTACAA
CTGTACACACCCGCC GTGGAGATGGAAATT CATGAAATTTACAAA
TGTACACACCCGCCT TGGAGATGGAAATTT ATGAAATTTACAAAG
GTACACACCCGCCTG GGAGATGGAAATTTT TGAAATTTACAAAGG
TACACACCCGCCTGA GAGATGGAAATTTTT GAAATTTACAAAGGG
ACACACCCGCCTGAC AGATGGAAATTTTTA AAATTTACAAAGGGC
CACACCCGCCTGACA GATGGAAATTTTTAC AATTTACAAAGGGCC
ACACCCGCCTGACAC ATGGAAATTTTTACC ATTTACAAAGGGCCA
CACCCGCCTGACACC TGGAAATTTTTACCT TTTACAAAGGGCCAT
ACCCGCCTGACACCG GGAAATTTTTACCTT TTACAAAGGGCCATC
CCCGCCTGACACCGT GAAATTTTTACCTTT TACAAAGGGCCATCG
CCGCCTGACACCGTG AAATTTTTACCTTTA ACAAAGGGCCATCGT
CGCCTGACACCGTGG AATTTTTACCTTTAT CAAAGGGCCATCGTT
GCCTGACACCGTGGG ATTTTTACCTTTATC AAAGGGCCATCGTTC
CCTGACACCGTGGGT TTTTTACCTTTATCT AAGGGCCATCGTTCA
CTGACACCGTGGGTC TTTTACCTTTATCTT AGGGCCATCGTTCAT
TGACACCGTGGGTCA TTTACCTTTATCTTT GGGCCATCGTTCATC
GACACCGTGGGTCAT TTACCTTTATCTTTC GGCCATCGTTCATCC
ACACCGTGGGTCATT TACCTTTATCTTTCA GCCATCGTTCATCCA
CACCGTGGGTCATTA ACCTTTATCTTTCAC CCATCGTTCATCCAA
ACCGTGGGTCATTAC CCTTTATCTTTCACC CATCGTTCATCCAAG
ATCGTTCATCCAAGG GCCAAAATCCTGAAC CTCGTGTCCGGAGGC
TCGTTCATCCAAGGC CCAAAATCCTGAACT TCGTGTCCGGAGGCA
CGTTCATCCAAGGCT CAAAATCCTGAACTT CGTGTCCGGAGGCAT
GTTCATCCAAGGCTG AAAATCCTGAACTTT GTGTCCGGAGGCATG
TTCATCCAAGGCTGT AAATCCTGAACTTTC TGTCCGGAGGCATGG
TCATCCAAGGCTGTT AATCCTGAACTTTCT GTCCGGAGGCATGGG
CATCCAAGGCTGTTA ATCCTGAACTTTCTC TCCGGAGGCATGGGT
ATCCAAGGCTGTTAC TCCTGAACTTTCTCC CCGGAGGCATGGGTG
TCCAAGGCTGTTACC CCTGAACTTTCTCCC CGGAGGCATGGGTGA
CCAAGGCTGTTACCA CTGAACTTTCTCCCT GGAGGCATGGGTGAG
CAAGGCTGTTACCAT TGAACTTTCTCCCTC GAGGCATGGGTGAGC
AAGGCTGTTACCATT GAACTTTCTCCCTCA AGGCATGGGTGAGCA
AGGCTGTTACCATTT AACTTTCTCCCTCAT GGCATGGGTGAGCAT
GGCTGTTACCATTTT ACTTTCTCCCTCATC GCATGGGTGAGCATG
GCTGTTACCATTTTA CTTTCTCCCTCATCG CATGGGTGAGCATGG
CTGTTACCATTTTAA TTTCTCCCTCATCGG ATGGGTGAGCATGGC
TGTTACCATTTTAAC TTCTCCCTCATCGGC TGGGTGAGCATGGCA
GTTACCATTTTAACG TCTCCCTCATCGGCC GGGTGAGCATGGCAG
TTACCATTTTAACGC CTCCCTCATCGGCCC GGTGAGCATGGCAGC
TACCATTTTAACGCT TCCCTCATCGGCCCG GTGAGCATGGCAGCT
ACCATTTTAACGCTG CCCTCATCGGCCCGG TGAGCATGGCAGCTG
CCATTTTAACGCTGC CCTCATCGGCCCGGC GAGCATGGCAGCTGG
CATTTTAACGCTGCC CTCATCGGCCCGGCG AGCATGGCAGCTGGT
ATTTTAACGCTGCCT TCATCGGCCCGGCGC GCATGGCAGCTGGTT
TTTTAACGCTGCCTA CATCGGCCCGGCGCT CATGGCAGCTGGTTG
TTTAACGCTGCCTAA ATCGGCCCGGCGCTG ATGGCAGCTGGTTGC
TTAACGCTGCCTAAT TCGGCCCGGCGCTGA TGGCAGCTGGTTGCT
TAACGCTGCCTAATT CGGCCCGGCGCTGAT GGCAGCTGGTTGCTC
AACGCTGCCTAATTT GGCCCGGCGCTGATT GCAGCTGGTTGCTCC
ACGCTGCCTAATTTT GCCCGGCGCTGATTC CAGCTGGTTGCTCCA
CGCTGCCTAATTTTG CCCGGCGCTGATTCC AGCTGGTTGCTCCAT
GCTGCCTAATTTTGC CCGGCGCTGATTCCT GCTGGTTGCTCCATT
CTGCCTAATTTTGCC CGGCGCTGATTCCTC CTGGTTGCTCCATTT
TGCCTAATTTTGCCA GGCGCTGATTCCTCG TGGTTGCTCCATTTG
GCCTAATTTTGCCAA GCGCTGATTCCTCGT GGTTGCTCCATTTGA
CCTAATTTTGCCAAA CGCTGATTCCTCGTG GTTGCTCCATTTGAG
CTAATTTTGCCAAAA GCTGATTCCTCGTGT TTGCTCCATTTGAGA
TAATTTTGCCAAAAT CTGATTCCTCGTGTC TGCTCCATTTGAGAG
AATTTTGCCAAAATC TGATTCCTCGTGTCC GCTCCATTTGAGAGA
ATTTTGCCAAAATCC GATTCCTCGTGTCCG CTCCATTTGAGAGAC
TTTTGCCAAAATCCT ATTCCTCGTGTCCGG TCCATTTGAGAGACA
TTTGCCAAAATCCTG TTCCTCGTGTCCGGA CCATTTGAGAGACAC
TTGCCAAAATCCTGA TCCTCGTGTCCGGAG CATTTGAGAGACACG
TGCCAAAATCCTGAA CCTCGTGTCCGGAGG ATTTGAGAGACACGC
TTTGAGAGACACGCT CTGCTGTGCTGCTCA CTGACTAGATTATTA
TTGAGAGACACGCTG TGCTGTGCTGCTCAA TGACTAGATTATTAT
TGAGAGACACGCTGG GCTGTGCTGCTCAAG GACTAGATTATTATT
GAGAGACACGCTGGC CTGTGCTGCTCAAGG ACTAGATTATTATTT
AGAGACACGCTGGCG TGTGCTGCTCAAGGC CTAGATTATTATTTG
GAGACACGCTGGCGA GTGCTGCTCAAGGCC TAGATTATTATTTGG
AGACACGCTGGCGAC TGCTGCTCAAGGCCA AGATTATTATTTGGG
GACACGCTGGCGACA GCTGCTCAAGGCCAC GATTATTATTTGGGG
ACACGCTGGCGACAC CTGCTCAAGGCCACA ATTATTATTTGGGGG
CACGCTGGCGACACA TGCTCAAGGCCACAG TTATTATTTGGGGGA
ACGCTGGCGACACAC GCTCAAGGCCACAGG TATTATTTGGGGGAA
CGCTGGCGACACACT CTCAAGGCCACAGGC ATTATTTGGGGGAAC
GCTGGCGACACACTC TCAAGGCCACAGGCA TTATTTGGGGGAACT
CTGGCGACACACTCC CAAGGCCACAGGCAC TATTTGGGGGAACTG
TGGCGACACACTCCG AAGGCCACAGGCACA ATTTGGGGGAACTGG
GGCGACACACTCCGT AGGCCACAGGCACAC TTTGGGGGAACTGGA
GCGACACACTCCGTC GGCCACAGGCACACA TTGGGGGAACTGGAC
CGACACACTCCGTCC GCCACAGGCACACAG TGGGGGAACTGGACA
GACACACTCCGTCCA CCACAGGCACACAGG GGGGGAACTGGACAC
ACACACTCCGTCCAT CACAGGCACACAGGT GGGGAACTGGACACA
CACACTCCGTCCATC ACAGGCACACAGGTC GGGAACTGGACACAA
ACACTCCGTCCATCC CAGGCACACAGGTCT GGAACTGGACACAAT
CACTCCGTCCATCCG AGGCACACAGGTCTC GAACTGGACACAATA
ACTCCGTCCATCCGA GGCACACAGGTCTCA AACTGGACACAATAG
CTCCGTCCATCCGAC GCACACAGGTCTCAT ACTGGACACAATAGG
TCCGTCCATCCGACT CACACAGGTCTCATT CTGGACACAATAGGT
CCGTCCATCCGACTG ACACAGGTCTCATTG TGGACACAATAGGTC
CGTCCATCCGACTGC CACAGGTCTCATTGC GGACACAATAGGTCT
GTCCATCCGACTGCC ACAGGTCTCATTGCT GACACAATAGGTCTT
TCCATCCGACTGCCC CAGGTCTCATTGCTT ACACAATAGGTCTTT
CCATCCGACTGCCCC AGGTCTCATTGCTTC CACAATAGGTCTTTC
CATCCGACTGCCCCT GGTCTCATTGCTTCT ACAATAGGTCTTTCT
ATCCGACTGCCCCTG GTCTCATTGCTTCTG CAATAGGTCTTTCTC
TCCGACTGCCCCTGC TCTCATTGCTTCTGA AATAGGTCTTTCTCT
CCGACTGCCCCTGCT CTCATTGCTTCTGAC ATAGGTCTTTCTCTC
CGACTGCCCCTGCTG TCATTGCTTCTGACT TAGGTCTTTCTCTCA
GACTGCCCCTGCTGT CATTGCTTCTGACTA AGGTCTTTCTCTCAG
ACTGCCCCTGCTGTG ATTGCTTCTGACTAG GGTCTTTCTCTCAGT
CTGCCCCTGCTGTGC TTGCTTCTGACTAGA GTCTTTCTCTCAGTG
TGCCCCTGCTGTGCT TGCTTCTGACTAGAT TCTTTCTCTCAGTGA
GCCCCTGCTGTGCTG GCTTCTGACTAGATT CTTTCTCTCAGTGAA
CCCCTGCTGTGCTGC CTTCTGACTAGATTA TTTCTCTCAGTGAAG
CCCTGCTGTGCTGCT TTCTGACTAGATTAT TTCTCTCAGTGAAGG
CCTGCTGTGCTGCTC TCTGACTAGATTATT TCTCTCAGTGAAGGT
CTCTCAGTGAAGGTG
TCTCAGTGAAGGTGG
CTCAGTGAAGGTGGG
TCAGTGAAGGTGGGG
CAGTGAAGGTGGGGA
AGTGAAGGTGGGGAG
GTGAAGGTGGGGAGA
TGAAGGTGGGGAGAA
GAAGGTGGGGAGAAG
AAGGTGGGGAGAAGC
AGGTGGGGAGAAGCT
GGTGGGGAGAAGCTG
GTGGGGAGAAGCTGA
TGGGGAGAAGCTGAA
GGGGAGAAGCTGAAC
GGGAGAAGCTGAACC
GGAGAAGCTGAACCG
GAGAAGCTGAACCGG
AGAAGCTGAACCGGC
EXAMPLE 9 Sub-confluent HaCaT cells were treated as described above with phosphorothioate oligonucleotides IGFR.AS (antisense: 5′-ATCTCTCCGCTTCCTTTC-3′; (<400>10); ref 13) and IGFR.S (sense control: 5′-GAAAGGAAGCGGAGAGAT-3′; (<400>11); ref 13) IGF-I binding to the cell monolayers was then measured as 125I-IGF-I.
EXAMPLE 10 The results of this experiment are shown in FIGS. 7 and 8.
HaCaT cells were initially plated in DMEM with 10% v/v serum, then AS oligo experiments were performed in complete “Keratinocyte-SFM” (Gibco) to exclude the influence of exogenous IGFBPs. Oligos were synthesised as phosphorothioate (nuclease-resistant) derivatives (Bresatec, South Australia) and were as follows: antisense: AS2, 5′-GCGCCCGCTGCATGACGCCTGCAAC-3′ (IGFBP-3 start codon); controls: AS2NS, 5′-CGGAGATGCCGCATGCCAGCGCAGG-3′; AS4,
5′-AGGCGGCTGACGGCACTA-3′; AS4NS, 5′-GACAGCGTCGGAGCGATC-3′; IGFRAS, 5′-ATCTCTCCGCTTCCTTTC-3′;
IGFRS, 5′-GAAAGGAAGCGGAGAGAT-3′. Oligos to IGFBP-3 were based on the published sequence of Spratt et al [12]. AS oligos were added to HaCaT monolayers in 0.5 ml medium in 24-well plates at the concentrations and addition frequencies indicated. IGFBP-3 measured in cell-conditioned medium using a dot-blot assay, adapted from the Western ligand blot method of Hossenlopp et al [11], in which 100 μl of conditioned medium was applied to nitrocellulose filters with a vacuum dot-blot apparatus. After drying the membranes at 37° C., relative amounts of IGFBP are determined by 125I-IGF-I-binding, autoradiography and computerised imaging densitometry. Triplicate wells (except in FIG. 7, where duplicate wells were measured as shown) were analysed and corrected for changes in cell number per well. Relative cell number per well was determined using an amido black dye method, developed specifically for cultured monolayers of HaCaT cells [14]. Cell numbers differed by less than 10% after treatment. For oligos to the IGF receptor, receptor quantitation in intact HaCaT monolayers was by overnight incubation with 125I-IGF-I (30,000 cpm/well) at 4° C.
EXAMPLE 11 Experiments involving ribozymes are generally conducted as described in Internaitonal Patent Application No. WO 89/05852 and in Haselhoff and Gerlach [8]. Ribozymes are constructed with a hybridising region which is complementary in nucleotide sequence to at least part of a target RNA which, in this case, encodes IGFBP-2. Activity of ribozymes is measurable on, for example, Northern blots or using animal models such as in the nude mouse model (15; 16) or the “flaky skin” mouse model (17; 18).
EXAMPLE 12 The methods described in Example 11 are used for the screening of ribozymes which inhibit IGFBP-3 production. The activity of the ribozymes is determined as in Example 11.
EXAMPLE 13 The methods described in Example 11 are used for the screening of ribozymes which inhibit IGF-1 production. The activity of the ribozymes is determined as in Example 11.
EXAMPLE 14 The methods described in Example 11 are used for the screening of ribozymes which inhibit IGF-1 production. The activity of the ribozymes is determined as in Example 11.
EXAMPLE 15 Twenty-one antisense oligonucleotides targeted to mRNA sequences enducing the IGF-1 receptor, and four random oligonucleotides were synthesized. The antisense oligonucleotides are C5-propynyl-dU, dC 15 mer phosphorothioate oligodeoxyribonucleotides. In these oligonucleotides, a phosphorothioate backbone replaces the phosphodiester backbone of naturally occurring DNA. The positions of the 21 sequence specific antisense oligonucleotides relative to the IGF-1 receptor mRNA structure are shown in FIG. 9.
EXAMPLE 16 Experiments were performed to determine the uptake of the antisense oligonucleotides of Example 15 into keratinocytes. Cells of the differentiated human keratinocyte cell line, HaCaT, were incubated for 24 hours in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (w/v) fetal calf serum (FCS) containing fluorescently labelled oligonucleotide (R451, a randomized sequence oligonucleotide, 30 nM) and cytofectin GSV (2 μg/ml, Glen Research, 44901 Falcon Place, Sterling, Va. 20166, Cat. No. 70-3815-78). Cells were then transferred to oligonucleotide-free medium and fluorescence microcopy and phase contrast images of the cells were obtained. FIG. 10 shows fluorescence microscopy (Panel A) and phase contrast (Panel B) images of uptake of fluorescently labelled oligonucleotide in the majority of cells in a HaCaT monolayer. The degree of uptake obtained with the cationic lipid cytofectin was far greater than the uptake obtained with the next best lipid tried, Tfx-50.
A further experiment was performed to assess the uptake and toxicity associated with the use of cytofectin GSV over five days. Confluent HaCaT keratinocytes were incubated in DMEM containing fluorescently labelled oligonucleotide R451 (30 nM or 100 nM) plus cytofectin GSV (2 μg/ml or 5 μg/ml) over 120 hours, viewed by fluorescence microscopy, tryptan blue stained, and counted. The graphs in FIG. 11 depict uptake (Panel A) and toxicity (Panel B). The proportion of cells containing oligonucleotide remained high over the 120 hour period. The combination of 30 nM oligonucleotide and 2 μg/ml GSV provided optimal uptake and minimal toxicity.
EXAMPLE 17 The twenty-one oligonucleotides of Example 15 were then screened for their ability to inhibit IGF-I receptor mRNA levels in HaCaT cells, in accordance with the teachings herein. HaCaT cells were grown to 90% confluence in DMEM supplemented with 10% (v/v) FCS. Antisense oligonucleotides (30 nM) were completed with cytofectin GSV (2 μg/ml) and added to the cells in the presence of serum. HaCaT keratinocytes were treated with the oligonucleotide/GSV complexes or randomized sequence oligonucleotides (R451, R766), liposome alone (GSV), or were left untreated (UT). Duplicate treatments were performed. Repeat additions of the oligonucleotides/GSV complex were performed at 24, 48 and 76 hours following the first addition. Total RNA was isolated as per the RNAzolB protocol (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, Tex. 77033) 96 hours following the first addition.
IGF-I receptor mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were simultaneously determined by a ribonuclease (RNase) protection assay. The RNase Protection Assay kit, in vitro transcription kit, and IGF-I receptor and GAPDH DNA templates were obtained from Ambion, Inc. (2130 Woodward St., Houston, Tex. 78744). The amount of IGF-I receptor mRNA in any given sample was expressed as the amount of IGF-I receptor mRNA relative to the amount of GAPDH mRNA. Each oligonucleotide was tested in at least two separate experiments.
FIG. 12 depicts representative results of the screening process. Panel A shows an electrophoretic analysis of IGF-I receptor and GAPDH mRNA fragments after RNase protection. Molecular weight markers are shown on the right hand side. The full-length probe is shown on the left hand side; G-probe indicates the IGF-I receptor probe. GAPDH protected fragments (G) are seen at 316 bases and IGF-I protected fragments (I) are seen at 276 bases. Exhibit E, Panel B provides a graph indicating the relative level of IGF-I receptor mRNA following each treatment.
The results obtaining from the above screening assays are summarized in FIG. 13. The graph depicts the relative level of IGF-I receptor mRNA after treatment with oligonucleotides complementary to the human IGF-I receptor mRNA (26-86), four randomized sequence oligonucleotides (R1, R4, R7, R9), liposome alone (GSV), or no treatment (UT). Asterisks indicate a significant different in relative IGF-I receptor mRNA as compared to GSV treated cells (n=4-10, p<0.05).
As demonstrated in FIG. 13, treatment with eighteen of the twenty-one oligonucleotides resulted in a significant different in levels of IGF-I receptor mRNA relative to GSV treated cells. Three of the antisense oligonucleotides tested in the screening assay reduce IGF-I receptor mRNA to less than 35% of GSV-treated cells. These antisense oligonucleotides have the following sequences, presented in the 5′ to 3′ direction:
#27 UCCGGAGCCAGACUU
#64 CACAGUUGCUGCAAG
#78 UCUCCGCUUCCUUUC
As further demonstrated in FIG. 13, six of the antisense oligonucleotides tested in the screening assay reduce IGF-I receptor mRNA to between 35 and 50% of GSV-treated cells. These antisense oligonucleotides have the following sequences, presented in the 5′ to 3′ direction:
#28 AGCCCCCACAGCGAG
#32 GCCUUGGAGAUGAGC
#40 UAACAGAGGUCAGCA
#42 GGAUCAGGGACCAGU
#46 CGGCAAGCUACACAG
#50 GGCAGGCAGGCACAC
EXAMPLE 19 Another experiment was performed demonstrating that antisense oligonucleotides targeted to genetic sequences encoding the IGF0I receptor and that reduce IGF-I receptor mRNA levels also inhibit the IGF-I receptor level on the surface of the treated cultured keratinocytes. HaCaT cells were grown to confluence in 24-well plates in DMEM containing 10% (v/v) FCS. Oligodeoxynucleotide and cytofectin GSV were mixed together in serum-free DMEM, and incubated at room temperature for 10 minutes before being diluted ten-fold in medium and placed on the cells. Cells were incubated for 72 hours with 30 nM random sequence or antisense oligonucleotide and 2 μm/ml GSV, or with GSV alone in DMEM containing 10% (v/v) FCS with solutions replaced every 24 hours. This was followed by incubation with oligonucleotide/GSV in serum-free DMEM for 48 hours. All incubations were performed at 37° C. Cells were washed twice with 1 ml cold PBS. Serum-free DMEM containing 10−10M 125I-IGF-I was added with or without the IGF-I analogue, des (1-3) IGF-I, at 10−11M to 10−7M. Cells were incubated at 4° C. for 17 hours with gentle shaking, then washed three times with 1 ml cold PBS and lysed in 250 μl 0.5M NaOH/0.1% (v/v) Triton X-100 at room temperature for 4 hours. Specific binding of the solubilised cell extract was measured using a gamma counter. As shown in FIG. 14, treatment of HaCaT keratinocytes with oligonucleotide reduced cell surface IGF-I receptor levels to 30% of levels in untreated keratinocytes or in keratinocytes treated with liposome alone or a random oligonucleotide, R766. As shown in FIG. 15, treatment with oligonucleotide #27 also significantly reduced cell surface IGF-I receptor levels relative to untreated keratinocytes or treatment with liposome alone or random nucleotide R451. As demonstrated in Example 17, oligonucleotides #64 and #27 reduce IGF-I receptor mRNA levels in cultured keratinocytes to less than 35% of GSV-treated cells. Accordingly, the ability of an oligonucleotide to reduce IGF-I receptor mRNA levels in correlated with its ability to reduce cell surface IGF-I receptor levels.
The forgoing Examples demonstrate that antisense oligonucleotides targeted to the IGF-I receptor can be delivered to human keratinocytes in vitro, can inhibit IGF-I receptor mRNA levels in human keratinocytes in vitro, and that inhibition of mRNA levels is correlated with reduction of cell surface IGF-I receptor levels.
EXAMPLE 19 Further experiments demonstrated the efficacy of antisense oligonucleotides targeted to the IGF-I receptor in an in vivo model of psoriasis. An animal model of psoriasis is the human psoriatic skin xenograft model. The skin used in this model contains the true disease state. In this model, reduction in epidermal thickness of psoriatic grafts in response to treatment is positively correlated with efficacy of treatment. Both normal and psoriatic human skin were grated into a thymic (nude) mice in accordance with a thymic (nude) mice in accordance with the methods of Baker et al (1992) Brit. J. Dermatol. 126:105 and Nanney et al (1992) J. Invest. Dermatol, 92:296. Successful grafting was achieved, as demonstrated in FIG. 16, which shows hemotoxylin and eosin (H&E) stained sections of a 49-day old psoriatic human skin graft (Panel B) compared to the histology of the skin graft prior to grafting (Panel A). The histological features of psoriasis present in the pregraft section (e.g., parakeratosis, acanthosis and pronounced rete ridges) are present in the grafts more than seven weeks post grafting.
Using the model, oligonucleotide uptake was measured in epidermal keratinocytes in vivo after intradermal injection. Fluorescently labelled oligonucleotide (R451, 50 μl, 10 μM injection) was intradermally injected into psoriatic and normal skin grafts on a thymic mice. Live confocal microscopy and fluorescence microscopy of fixed sections was then employed.
Using both techniques, oligonucleotide was found to localize in the nucleus of over 90% of basal keratinocytes. FIG. 17 shows the nuclear localization of oligonucleotide in psoriatic skin cells using conventional fluorescence microscopy of a graft that was removed and sectioned after 24 hours.
After establishing oligonucleotide uptake in the in vivo model, a small number of pilots experiments were performed to determine a schedule for treatment of grated mice with antisense oligonucleotides targeted to genetic sequences encoding the IGF-I receptor. The treatment schedule was finalized as follows:
Volume
Graft of ODN Duration of
Number Treatment Injection Concentration Treatment
1-3 Vehicle (PBS) 50 μl — 20 days
4-6 RandomODN#R451 50 μl 10 μM 20 days
7-9 ODN#27 50 μl 10 μM 20 days
10-12 ODN#74 50 μl 10 μM 20 days
13-15 ODN#50 50 μl 10 μM 20 days
As determined above, oligonucleotide #27 (ODN #27) reduced IGF-I receptor mRNA in vitro to less than 35% of GSV-treated cells. Oligonucleotide #50 (ODN#50) reduced IGF-I receptor mRNA in vitro to between 35 and 50% of GSV-treated cells. Oligonucleotide #74 (ODN #74) was not inhibitory to IGF-I receptor mRNA in vitro. In the in vivo model, each mouse received two grafts. Random oligonucleotide or vehicle was injected intradermally in one graft and acted as a control. The second graft was injected with the targeted oligonucleotide. Each graft received an injection every second day for the duration of the treatment.
Histology of representative grafts from each treatment type are shown in FIGS. 18(a)-(d) and 19(a)-(d). Each sheet shows three images of H&E stained sections: the pregraft histology, the control treated graft, and the targeted oligonucleotide treated graft. FIGS. 18(a)-(d) are shown at 100× magnification; FIGS. 19(a)-(d) are shown at 400× magnification. The total cross sectional area of epidermis of each graft was assessed using MCID analysis software. The pooled results from all of the treated grafts are shown in FIG. 20.
As shown in FIGS. 18(a)-(d) and 19(a)-(d), the vehicle-treated (control) grafts were marginally thinner than the pregraft sections. The degree of regression in these experiments (ie., less than 10%) is not significant. A similar amount of marginal thinning of epidermis compared to pregraft also occurred in pilot experiments in which psoriatic grafts were not injected, and thsu it is unlikely that the vehicle itself has any effect. Histological features of psoriasis present in skin samples prior to grafting (clubbing of rete ridges, parakeratosis, acanthosis) were present in these grafts.
The random oliognucleotide treated grafts varied in epidermal thickness after 20 days of treatment. Grafts were either a similar thickness to the pregraft histology, or marginally thinner. Random oligonucleotide treated grafts were in each case significantly thicker than their targeted oligonucleotide treated pairs.
As shown in FIG. 20, the targeted oligonucleotide treated grafts were significantly thinner than the pregraft sections and showed less parakeratosis and clubbing of rete ridges. Antisense oligonucleotides which were effective at reducing IGF-I receptor mRNA levels in vitro (#27 and #50) produced greatere epidermal thinning than an oligonucleotide which was not inhibitory to IGF-I receptor mRNA in vitro (#74). Accordingly, there is a direct correlation between the ability of an oligonucleotide targeted to the IGF-I receptor to inhibit IGF-I receptor mRNA levels in vitro and the efficacy of the oligonucleotide as an anti-psoriasis agent in an in vivo model.
EXAMPLE 20 Another experiment demonstrated that treatment of psoriatic grafts with an oligonucleotide targeted to a genetic sequence encoding the IGF-I receptor results in inhibition of proliferation. Pregrafts from psoriatic patients, control grafts treated with R4541, and grafts treated with oligonucleotide #27 were obtained as described in Example 19. An antibody to the cell cycle-specific nuclear antigen Ki67 was used to immunohistochemically detect actively dividing cells and tereby assess proliferation. The αKi67 antibody (DAKO, Glostrup, Denmark) recognizes the Ki67 antigen transiently expressed in nuclei of proliferating cells during late G1, S, M and G2 phases of the cycle and thsu provides a marker for proliferation. Pregraft and graft sections were immunohistochemically processed by standard methods using αKi67 (according to the manufacturer's instructions), peroxidase-conjugated anti-rabbit second stage antibody, and a chromogenic peroxidase substrate.
The results of this experiment are presented in FIG. 21 as immunohistochemical sections at 100× magnification. The top panel of FIG. 21 depicts a pregraft section obtained from a psoriatic patient. The epidermis is thicker than normal and nucleic are evident in the stratum corneum. Ki67 positive cells, appearing as brown dots, are evidence in the basal and suprabasal layers, and indicate actively proliferating cells. The control (R450-treated) graft in the bottom panel of FIG. 21 also exhibits evidence of proliferation, including parakeratosis and Ki67-positive cells appearing as brown-staining nuclei. The center panel of FIG. 21 exhibits the oligonucleotide #27-treated graft. This graft exhibits significantly reduced proliferation as evidenced by normal (thin) epidermis, lack of invaginations, and substantial loss of Ki67-positive cells.
These results indicate that treatment of human psoriatic grafts with an oligonucleotide targeted to mRNA encoding the IGF-I receptor results in inhibition of epidermal proliferation.
EXAMPLE 21 Topical formulations of complexes of oligonucleotides with cytofectin GSV in aqueous or methylcellulose gel formulations were prepared and assessed for uptake of the oligonucleotide by keratinocytes in vivo. The topical formulations contained oligonucleotides complexed with cytofectin GSV in an aqueous solution or methylcellulose carrier, as taught herein. With both aqueous and methylcellulose gel formulations, locatlization of oligonucleotide R451 to nuclei and cytoplasm of keratinocytes in normal human skin grafts on nuce mice was observed. FIG. 22 shows an image from confocal microscopy demonstrating oligonucleotide locatlization in the nuclei and cytoplasm of keratinocytes in normal human skin grafts after topical application of fluroescently labeled oligonucleotide (10 μM R451) complexed with cytofectin GSV (10 μg/ml). FIG. 23 shows an image from confocal microscopy demonstrating that topical application of the same oligonucleotide/GSV concentrations in a 3% (w/v) methylcellulose gel produced similar uptake in the target keratinocyte population. Using an aqueous formulation of oligonucleotide/GSV complexes, penetration of oligonucleotide into the viable epidermis was observed, whereas application of formulations of oliognucleotide complexed with other cationic lipids resulted in localization of oligonucleotide in the stratum corneum.
EXAMPLE 22 Thirteen antisense oligonucleotides targeted to IGFBP-3 were synthesized. The antisense oligonucleotides are C5-propynyl-dU, Dc15 mer phosphorothioate oligodeoxyribonucleotides. FIG. 24 attached hereto is a schematic diagram indicating the position of the thirteen oligonucleotides relative to the IGFBP-3 mRNA structure.
These oligonucleotides were screened for their ability to inhibit IGFBP-3 mRNA levels of HaCaT cells in accordance with the teachings herein. HaCaT cells were grown to 90% confluence in DMEM supplemented with 10% (v/v) FCS, then placed in complete keratinocyte serum free medium (KSFM, Gibco), which has a defined amount of EGF, for 24 hours. Oligonucleotides (30 nM or 100 nM) were complexed with GSV cytofectin (2 μg/ml) and added to cells in complete KSFM to allow oligonucleotides to enter the nucleus before removal of EGF. Repeat additions were performed at three hours (in serum free DMEM, which releases the EGF inhibition of IGFBP-3 mRNA) and again after another 24 hours. HaCaT cells were also treated with randomized sequence oligonucleotides (R121, R451, R766 and R961), liposome alone (GSV) or were left untreated (UT). Total RNA was isolated as described in Example 17, 24 hours after the last treatment. Total RNA (15 μg) was analyzed by Northern analysis and phosphoroimager quantitation for IGFBP-3 and GADPH mRNA. IGFBP-3 mRNA is expressed as the amount of IGFBP-3 mRNA relative to the amount of GAPDH mRNA.
FIGS. 25(a)-(d) provide graphs which depict results in this screening process. In these graphs, R1 and R12 refer to R121; R4, R4(0) and R45 rfer to R451; R7, R7(0) and R76 refer to R766; and R9 and R96 refer to R961. The values were standardized to GSV-treated cells, and data was pooled and statistically analyzed by ANOVA followed by Domet's test to compare each treatment to GSV-treated cells. The pooled data are presented as a bar graph in FIG. 26. As demonstrated, at a concentration of 30 nM, treatment of HaCaT cells with 8 of the 12 targeted oligonucleotides tested resulted in a statistically significant reduction in levels of IGFBP-3 mRNA relative to GSV-treated cells. At a concentration of 100 nM, treatment with 9 fo the 13 targeted oligonucleotides tested resulted in a statistically significant reduction in levels of IGFBP-3 mRNA relative to GSV-treated cells.
These experiments demonstrate that antisense oligonucleotides targeted to genetic sequences encoding IGFBP-3 can inhibit IGFBP-3 mRNA levels in human keratinocytes in vitro.
EXAMPLE 23 IGF-I receptor is a potent mitotic signalling molecule for keratinocytes and the human receptor elicits separate intracellular signals that prevent apoptosis (19). It is proposed in accordance with the present invention that inactivation of IGF-I receptors in epidermal keratinocytes will achieve three important outcomes in subsequent UV treatment of lesions:
-
- (i) Acute epidermal hyperplasia following UV has been suggested to increase the risk of keratinocyte carcinogenic transformation (22). By reducing IGF-I receptor expression in the epidermis, the incidence of epidermal hyperplasia following UV exposure is likely to be reduced leading to an overall acceleration in normalization of the lesion and reduced carcinogenic risk.
- ii) Inhibition of anti-apoptotic action of IGF-I receptor will enhance the reversal of epidermal thickening and accelerate normalization of differentiation. Topical or injected IGF-I receptor antisense as adjunctive treatment will increase apoptosis in the epidermal layer thereby enhancing the reduction in acanthosis observed in UV treatments.
- iii) Survival of keratinocytes, ie. those which evade apoptosis is likely to occur when cells have damaged DNA. Such mutations may be in the tumor suppressor region. Consequently, the use of antisense therapy will result in less frequent selection of mutated keratinocytes and therefore reduced incidence of basal cell carcinomas and squamous.
Accordingly, antisense therapy, especially against IGF-I-receptor is useful in combination with UV therapy in the treatment of epidermal hyperplasia.
EXAMPLE 24 HaCaT cells were treated with antisense oligonucleotides directed to IGF-I receptor mRNA. Levels of IGF-I receptor mRNA were then monitored. In essence, confluent HaCaT cells were treated every 24 hours for four days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM IGF-I receptor specific oligonucleotides (#26 to #86) or random sequence oligonucleotides (R121, R451 and R766). FIG. 27(a) is a photographic representation showing representative RNase protection assay gel showing IGF-I receptor (IGFR) and GAPDH mRNA in untreated or treated HaCaT cells. FIG. 27(b) is a densitometric quantification of IGF-I receptor mRNA in a HaCaT cells following treatment with IGF-I receptor specific oligonucleotides (solid black) random sequence oligonucleotides (horizontal striped bar) or GSV alone (shaded bar) compared to untreated cells (UT, vertical striped bar).
EXAMPLE 25 In this example, reduction in total cellular IGF-I receptor protein was monitored following antisense oligonucleotide treatment. Confluence HaCaT cells were treated with 24 hours for 4 days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM IGF-I receptor specific AONS (#27, #50 and #64) or the random sequence oligonucleotide, R451. Total cellular protein was isolated and analysed for IGF-I receptor by SDS PAGE followed by western blotting with antibody specific for the human IGF-I receptor. FIG. 28(a) shows duplicate treated cellular extracts following the IGF-I receptor at the predicted size of 110 kD. FIG. 28(b) is a densitometric quantification of IGF-I receptor protein.
EXAMPLE 26 The reduction in IGF-I receptor numbers was determined on the keratinocyte cell surface after antisense oligonucleotide treatment. HaCaT cells were tranfected with IGF-I receptor specific AONs #27, #50, #64, a random sequence oligonucleotides (R451) or following treatment with GSV a lipid alone every 24 hours for 4 days. Competition binding assays using 125I-IGF-I and the receptor-specific analogue, des(1-3)IGF-I were performed. Results are shown in FIG. 29.
EXAMPLE 27 In this example, the apoptotic protecting effects of IGF-I receptor on keratinocyte cells was tested by following the reduction in keratino cell numbers following antisense oligonucleotide treatment. HaCaT cells, initially at 40% confluence, were transfected with the IGF-I receptor specific AON #64, control sequences R451 and 6414 or treated with GSV a lipid alone every 24 hours for 2 days. The cell number was measured in culture wells using a dye binding assay. The results are presented in FIG. 30. The results clearly confirm that the IGF-I receptor exhibits an anti-apoptotic effect. By reducing IGF-I receptor levels using antisense oligonucleotide treatment, the anti-apoptotic effect is interrupted and apoptosis results in the reduction in keratinocyte cell number. Results are shown in FIG. 30.
EXAMPLE 28 This example shows a reversal of epidermal hyperplasia in psoriatic human skin grafts on nude mice following intradermal injection with antisense oligonucleotides. Grafted psoriasis lesions were injected with IGF-I receptor specific AONs, a random sequence oligonucleotide in PBS, or with PBS alone, every 2 days for 20 days, then analysed histologically. The results are shown in FIG. 31. In FIG. 31(a), donor A graft treated with AON #50 showing epidermal thinning compared with the pregraft and control (PBS) treated graft and donor graft treated with AON #27 showing epidermal thinning compared with pregraft and control (R451) treated graft. In FIG. 31(b), the mean epidermal cross-sectional area over the full width of grafts is shown as determined by digital image analysis. The results show that epidermal hyperplasia is reversed following the intradermal injection of antisense oligonucleotides.
EXAMPLE 29 FIG. 32 shows the reversal of epidermal hyperplasia correlating with reduced IGF-I receptor mRNA in grafted psoriasis lesions treated with antisense oligonucleotides. FIG. 32(a) shows a psoriasis lesion prior to grafting and after grafting and treatment with IGF-I receptor specific oligonucleotide #27 (AON #27) or random sequence (R451) immunostained with antibodies to Ki67 to identify proliferating cells. Proliferating cells are indicated by a dark brown nucleus (arrows). FIG. 32(b) shows the same lesion prior to grafting and after oligonucleotide treatment as in FIG. 32(a) but subjected to in situ hybridisation with 35S-labelled cRNA probe complementary to the human IGF-I receptor mRNA. The presence of IGF-I receptor mRNA is indicated by silver grains which are almost eliminated in the epidermis of the lesion treated with IGF-I receptor specific oligonucleotide # 27 (AON #27). This experiment shows that reversal of epidermal hyperplasia correlates with reduced IGF-I receptor mRNA in grafted psoriasis lesions treated with antisense oligonucleotides.
EXAMPLE 30 FIG. 33 treatment with oligonucleotides. HaCaT cell monolayers were grown to 90% confluence in DMEM containing 10% fetal calf serum treated every 24 hours for two days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM oligonucleotide. Total RNA was isolated and analysed for IGF-I receptor and GAPDH mRNA using a commercially available ribonuclease protection assay kit. The results show a reduction in IGF-I receptor mRNA in the HaCaT keratinocyte cells.
EXAMPLE 31 FIG. 34 treatment with oligonucleotides. HaCaT cell monolayers were grown to 90% confluence in DMEM containing 10% fetal calf serum treated every 24 hours for 4 days with 2 μg/ml GSV lipid alone (GSV) or complexed with 30 nM oligonucleotide. Cells were lysed in a buffer containing 50 mM HEPES, 150 mM NaCl, 10% v/v glycerol, 1 v/v Trison X-100 and 100 μg/ml aprotinin on ice for 30 minutes, then 30 μg of lysate was loaded onto a denaturing 7% w/v polyacrylamide gel followed by transfer onto an Immobilon-P membrane. Membranes were then incubated with anti-IGF-I receptor antibodies C20 (available from Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) for 1 hour at room temperature and developed using the Vistra ECF western blotting kit (Amersham). The results shown in FIG. 34 confirm that IGF-I receptor protein is reduced in HaCaT keratinocytes following treatment with oligonucleotides.
EXAMPLE 32 This example shows a reduction in HaCaT keratinocyte cell number following treatment with oligonucleotides. The results are shown in FIG. 35. HaCaT cell monolayers were grown at 40% confluence in DMEM containing 10% fetal calf serum treated every 24 hours for 3 days with 2 μg/ml GSV lipid alone (GSV) or complexed with 15 nM oligonucleotide. Cell numbers were then measured every 24 hours using the amido black dye binding assay [32]. Results show that HaCaT keratino cells decrease in number following treatment with oligonucleotides due to a reduction in the anti-apoptotic effect of the IGF-I receptor.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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