US20050261230A1 - Method for the prophylaxis and/or treatment of medical disorders - Google Patents

Method for the prophylaxis and/or treatment of medical disorders Download PDF

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US20050261230A1
US20050261230A1 US11/090,351 US9035105A US2005261230A1 US 20050261230 A1 US20050261230 A1 US 20050261230A1 US 9035105 A US9035105 A US 9035105A US 2005261230 A1 US2005261230 A1 US 2005261230A1
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igf
nucleic acid
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Christopher Wraight
George Werther
Stephanie Edmondson
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • 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.
  • 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.
  • IGFs insulin-like growth factors
  • 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].
  • GH growth hormone
  • IGFBPs IGF binding proteins
  • 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].
  • IGF-I and other growth promoting molecules are responsible for or at least participate in a range of skin cell activities.
  • 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.
  • these molecules may also be used to facilitate apoptosis of target cells and may be useful as adjunctive therapy for epidermal hyperplasia.
  • sequence identifier i.e. ( ⁇ 400>1), ( ⁇ 400>2), etc.
  • a sequence listing is provided after the claims.
  • 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.
  • a medical disorder such as a proliferative and/or inflammatory skin disorder in a mammal
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • FIG. 1 is a representation of the nucleotide sequence of IGFBP-2.
  • IGFBP-2 insulin-like growth factor binding protein
  • ACCESSION X16302 KEYWORDS insulin-like growth factor binding protein.
  • SOURCE human ORGANISM Homo sapiens Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia;
  • FIG. 2 is a representation of the nucleotide sequence of IGFBP-3.
  • ORGANISM Homo sapiens Eukaryota; Animalia; Chordata; Vertebrata; Mammalia; Theria;
  • misc_feature 2240 .
  • 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 ⁇ M and 5 ⁇ M; * no oligonucleotide added.
  • BP3AS2, BP3AS3 and BP3S phosphorothioate oligonucleotides
  • 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 ⁇ M compared with several control oligonucleotides at 0.5 ⁇ M.
  • oligonucleotide BP3AS2NS oligonucleotide BP3AS4
  • oligonucleotide BP3AS4NS oligonucleotide BP3AS4NS
  • 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 ⁇ g/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 ⁇ g/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.
  • FIG. 13 is a graphical representation of an IGF-1 receptor mRNA in ODN treated (30 nM) HaCaT cells (2 ⁇ g/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.
  • 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 ⁇ g/ml GSV or with GSV alone in DMEM containing 10% v/v FCS with solutions replaced every 24 hours.
  • ODN Oligodeoxynucleotide
  • GSV Cytofectin GSV
  • 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 ⁇ l, 10 ⁇ M). 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 ⁇ l, Donor JH, #50 treated, 50 ⁇ l, 10 ⁇ M.
  • FIG. 18 ( b ) is a photographical representation of Donor LB, pregraft, Donor LB, PBS treated (50 ⁇ l), Donor LB, #74 treated (50 ⁇ l, 10 ⁇ M).
  • FIG. 18 ( c ) is a photographical representation of Donor PW, pregraft, Donor PW, R451 treated (50 ⁇ l, 10 ⁇ M), Donor LB, #74 treated (50 ⁇ l, 10 ⁇ M).
  • FIG. 18 ( d ) is a photographical representation of Donor GM, pregraft, Donor GB, R451 treated (50 ⁇ l, 10 ⁇ M), Donor GM, #27 treated (50 ⁇ l, 10 ⁇ M).
  • FIG. 19 ( a ) is a photographical representation showing Donor JH pregraft, Donor JH PBS treated 50, ⁇ l, Donor JH #50 treated 50 ⁇ l, 10 ⁇ M.
  • FIG. 19 ( b ) is a photographical representation Donor LB pregraft, Donor LB PBS treated 50 ⁇ l, Donor LB #74 treated 50 ⁇ l, 10 ⁇ M.
  • FIG. 19 ( c ) is a photographical representational showing Donor PW pregraft, Donor PW R451 treated 50 ⁇ l, 10 ⁇ M, Donor PW #74 treated 50 ⁇ l, 10 ⁇ M.
  • FIG. 19 ( d ) is a photographical representation showing Donor GM pregraft, Donor GM R451 treated 50, ⁇ l, 10 ⁇ M, Donor #27 treated 50, ⁇ l, 10 ⁇ M.
  • FIG. 20 is a graphical representation showing suppression of psoriasis after treatment with oligonucleotide (quantification).
  • FIG. 21 is a photographic representation of ⁇ hKi-67 imunobiological binding.
  • FIG. 22 is a photographical representation showing penetration of oligonucleotide into human skin after topical treatment. Fluorescently labelled oligonucleotide (10 ⁇ M R451) was applied topically after formulation with cytofectin GSV (10 ⁇ g/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 ⁇ M R451) was applied topically after complexing with cytofectin GSV (10 ⁇ g/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 ⁇ g/ml GSV).
  • FIG. 25 ( b ) is a graphical representation showing IGFBP-3 mRNA levels of AON treated (100 nm) HaCaT cells (2 ⁇ g/ml GSV).
  • FIG. 25 ( c ) is a graphical representation showing IGFBP-3 mRNA in AON treated (30 nM) HaCaT cells (2 ⁇ g/ml GSV).
  • FIG. 25 ( d ) is a graphical representation showing IGFBP-3 mRNA in AON treated (30 nM) HaCaT cells (2 ⁇ g/ml GSV).
  • FIG. 26 ( a ) is a graphical representation showing IGFBP-3 mRNA in ODN treated (30 nM) HaCaT cells (2 ⁇ g/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.
  • FIG. 26 ( b ) is a graphical representation showing IGFBP-3 mRNA in ODN treated (100 nM) HaCaT cells (2 ⁇ g/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.
  • 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 ⁇ g/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).
  • IGFR IGF-I receptor
  • GAPDH GAPDH
  • 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).
  • 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 ⁇ g/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.
  • 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 125 I-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
  • 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 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.
  • 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 ⁇ g/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 ⁇ g/ml GSV lipid alone (GSV) or complexed with 30 nM oligonucleotide.
  • GSV GSV lipid alone
  • 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 ⁇ g/ml aprotinin on ice for 30 mins, then 30 ⁇ g 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 ⁇ g/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].
  • 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.
  • 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.
  • a medical disorder such as a proliferative and/or inflammatory skin disorder in a mammal
  • 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- ⁇ (TGF ⁇ ), tumour necrosis factor- ⁇ (TNF ⁇ ), 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.
  • IGF-I keratinocyte growth factor
  • TGF ⁇ transforming growth factor- ⁇
  • TGF ⁇ tumour necrosis factor- ⁇
  • TNF ⁇ tumour necrosis factor- ⁇
  • IL-1, -4, -6 and 8 interleukin-1, -4, -6 and 8
  • bFGF basic fibroblast growth factor
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • IGF-I receptor elicits separate intracellular signals which prevent apoptosis [19].
  • IGF-I receptor activation has been shown to protect UV-irradiated cells from apoptosis [20].
  • 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:
  • 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.
  • the antisense therapy is directed to the IGF-I receptor.
  • 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.
  • 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
  • nucleic acid molecule targets IGF-I interaction with its receptor.
  • nucleic acid molecule is an antagonist of IGF-I interaction with its receptor.
  • 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.
  • the preferred molecules are IGFBP-2, -3, -4, -5 and -6.
  • the most preferred molecules are IGFBP-2 and IGFBP-3.
  • 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.
  • the nucleic acid molecule is at least about 15 nucleotides in length and more preferably at least about 20-25 nucleotides in length.
  • 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.
  • 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.
  • oligonucleotide analogues examples 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.
  • 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.
  • 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).
  • 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.
  • the nucleic acid molecule are antisense molecules.
  • antisense molecules are: #27 UCCGGAGCCAGACUU #64 CACAGUUGCUGCAAG #78 UCUCCGCUUCCUUUC #28 AGCCCCCACAGCGAG #32 GCCUUGGAGAUGAGC #40 UAACAGAGGUCAGCA #42 GGAUCAGGGACCAGU #46 CGGCAAGCUACACAG #50 GGCAGGCAGGCACAC
  • 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.
  • 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.
  • 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.
  • 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.
  • 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].
  • IGF-I is added to an oligonucleotide treated HaCaT cell line.
  • growth of oligonucleotide treated HaCaT cells is observed on a feeder layer of irradiated 3T3 fibroblasts.
  • antisense oligonucleotides to IGFBP-3 for example, inhibit production of IGFBP-3 by HaCaT cells.
  • nude mouse/human skin graft model 15; 16
  • flaky skin 17; 18
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • microorganism can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • 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.
  • 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.
  • 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.
  • 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 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 CO 2 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].
  • [ 3 H]-Thymidine (0.1 ⁇ Ci/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 ⁇ l/well), transferred to scintillation vials and radioactivity determined by liquid scintillation counting (Pharmacia Wallac 1410 liquid scintillation counter).
  • HaCaT conditioned medium 250 ⁇ l was concentrated by adding 750 ⁇ l 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).
  • IGFBPs were then visualised by the procedure of Hossenlopp et al [11], using [ 125 I]-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.
  • Phosphorothioate oligodeoxynucleotides were synthesised by Bresatec, Sydney, 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”).
  • 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 cm 2 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 ⁇ l 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%.
  • HaCaT cells secrete mainly IGFBP-3 (>95%), with the only other IGFBP detectable in HaCaT conditioned medium being IGFBP-4 ( ⁇ 5%).
  • 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.
  • a sense oligonucleotide corresponding to the start site was used. As shown in FIGS.
  • BP3AS2 inhibition by this oligonucleotide at 0.5 ⁇ M was compared with a number of control oligonucleotides, including one antisense oligonucleotide to IGFBP-3 that had proved to be ineffective at 0.5 ⁇ M.
  • 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 ⁇ M, compared to untreated cells.
  • this control oligonucleotide BP3AS2NS, like BP3AS2 itself, has the highest potential T m of the three control oligonucleotides used in this experiment, enhancing the probability of non-specific base pairing with non-target mRNAs.
  • T m the lowest potential of the three control oligonucleotides used in this experiment.
  • 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.
  • 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 CCTGCCCGCCC CGGGGCGAGGGAGGA AGGCGGCTCCCGCTC CTGCCCGCCCGCCCG GGGGCGAGGGAGGAG GGCGGCTCCCGCTCG TGCCCGCCCGCCCGC GGGCGAGGGAGGAGG GCGGCTCCCGCTCGC GCCCGCCCGCT GGCGAGGGAGGAGGA CGGCTCCCGCTCGCA CCCGCCCGCCCGCTC GCGAGGGAGGAGGAA GGCTCCCGCTCGCAG CCGCCCGCTCGCTCGCTCGCA CCCGCCCGCCCGCTC GCGAGGGAGGAGGAA GGCTCCCGC
  • 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 CACAGCTTCGCCG GCCCCATCCCTGC CCAGCCGCTTCCTGCTTCGCCCATCCCTGCG CCCAGCCGCTTCCTG
  • Antisense oligonucleotides to IGF-I may be selected from molecules capable of interacting with one or more of the following sense oligonucleotides: TTTTTTTTTTTTG ATTTCATCCCAAATA AAGTCTGGCTCCGGA TTTTTTTTTTTGA TTTCATCCCAAATAA AGTCTGGCTCCGGAG TTTTTTTTTTTTGAG TTCATCCCAAATAAA GTCTGGCTCCGGAGG TTTTTTTTTTTGAGA TCATCCCAAATAAAA TCTGGCTCCGGAGGA TTTTTTTTTTGAGAA CATCCCAAATAAAAG CTGGCTCCGGAGGAG TTTTTTTTTGAGAAA ATCCCAAATAAAAGG TGGCTCCGGAGGAGG TTTTTTTTGAGAAATAAAAGGA GGCTCCGGAGGAGGG TTTTTTTGAAAGG CCCAAATAAAAGGAA GCTCCGGAGGAGGGT TTTTTTGAGGG CCAAATAAAAGGAAT CTCCGGAGGAGGG
  • 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 125 I-IGF-I.
  • IGFR.AS antisense: 5′-ATCTCTCCGCTTCCTTTC-3′; ( ⁇ 400>10); ref 13
  • IGFR.S sense control: 5′-GAAAGGAAGCGGAGAGAT-3′; ( ⁇ 400>11); ref 13
  • IGF-I binding to the cell monolayers was then measured as 125 I-IGF-I.
  • 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,
  • 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 ⁇ l of conditioned medium was applied to nitrocellulose filters with a vacuum dot-blot apparatus.
  • relative amounts of IGFBP are determined by 125 I-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 125 I-IGF-I (30,000 cpm/well) at 4° C.
  • 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 11 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 11 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 11 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.
  • the 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.
  • 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 .
  • 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.
  • 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 ⁇ g/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).
  • IGF-I receptor mRNA and GAPDH mRNA levels were simultaneously determined by a ribonuclease (RNase) protection assay.
  • RNase ribonuclease
  • 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).
  • 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
  • oligonucleotide #27 also significantly reduced cell surface IGF-I receptor levels relative to untreated keratinocytes or treatment with liposome alone or random nucleotide R451.
  • 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.
  • 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.
  • FIG. 16 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).
  • H&E hemotoxylin and eosin
  • oligonucleotide uptake was measured in epidermal keratinocytes in vivo after intradermal injection.
  • Fluorescently labelled oligonucleotide (R451, 50 ⁇ l, 10 ⁇ M 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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).
  • the ⁇ Ki67 antibody (DAKO, Glostrup, Denmark) recognizes the Ki67 antigen transiently expressed in nuclei of proliferating cells during late G 1 , S, M and G 2 phases of the cycle and thsu provides a marker for proliferation.
  • Pregraft and graft sections were immunohistochemically processed by standard methods using ⁇ Ki67 (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.
  • 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 ⁇ M R451) complexed with cytofectin GSV (10 ⁇ g/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.
  • oligonucleotide/GSV complexes 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.
  • FIG. 24 attached hereto is a schematic diagram indicating the position of the thirteen oligonucleotides relative to the IGFBP-3 mRNA structure.
  • 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 ⁇ g/ml) and added to cells in complete KSFM to allow oligonucleotides to enter the nucleus before removal of EGF.
  • 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.
  • 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 .
  • 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:
  • antisense therapy especially against IGF-I-receptor is useful in combination with UV therapy in the treatment of epidermal hyperplasia.
  • 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).
  • 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.
  • IGF-I receptor numbers were 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 125 I-IGF-I and the receptor-specific analogue, des(1-3)IGF-I were performed. Results are shown in FIG. 29 .
  • 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.
  • the anti-apoptotic effect is interrupted and apoptosis results in the reduction in keratinocyte cell number. Results are shown in FIG. 30 .
  • 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.
  • 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.
  • 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.
  • 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.
  • IGF-I receptor mRNA 35 S-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.
  • 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 ⁇ g/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.
  • 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 ⁇ g/ml GSV lipid alone (GSV) or complexed with 30 nM oligonucleotide.
  • GSV GSV lipid alone
  • 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 ⁇ g/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.

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Abstract

The present invention relates generally to a method for the prophylaxis and/or treatment of skin 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 epidermal keratinocytes to inhibit, reduce or otherwise decrease stimulation of this layer of cells. The present invention contemplates, in a most preferred embodiment, a method for the prophylaxis and/or treatment of psoriasis.

Description

    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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Claims (10)

1-44. (canceled)
45. A method for ameliorating a proliferative or inflammatory skin disorder in a mammal, the method comprising contacting a cell in proliferating or inflamed skin with an effective amount of a nucleic acid molecule, or a chemical modification thereof, wherein the modification produces a modified nucleic acid molecule that has the same length and nucleotide sequence as the nucleic acid molecule prior to modification, and wherein the nucleic acid molecule or modified nucleic acid molecule inhibits growth-factor-mediated cell proliferation or inflammation.
46. The method of claim 45 wherein the proliferative or inflammatory skin disorder is mediated by a growth factor, wherein the growth factor is selected from the group consisting of insulin-like growth factor I (IGF), keratinocyte growth factor (KGF), transforming growth factor (TGF), tumor necrosis factor (TNF), interleukin (IL), basic fibroblast growth factor (bFGF), and a combination thereof.
47. The method of claim 45, wherein the proliferative or inflammatory skin disorder is selected from the group consisting of psoriasis, ichthyosis, pityriasis, rubra, pilaris, serborrhoea, keloids, keratosis, neoplasias, scleroderma, warts, benign growths, and a cancer of the skin.
48. The method of claim 45, wherein the nucleic acid molecule or modified nucleic acid molecule reduces the level of an IGF, IGF receptor, or IGF-binding protein in the mammal.
49. The method of claim 45, wherein the nucleic acid molecule or modified nucleic acid molecule is an antisense molecule that reduces the expression of a gene encoding IGF, IGF receptor, or IGF-binding protein.
50. A method for ameliorating a disease in a mammal, the method comprising contacting a cell in the diseased tissue with an effective amount of a nucleic acid molecule, or a chemical modification thereof, wherein the modification produces a modified nucleic acid molecule that has the same length and nucleotide sequence as the nucleic acid molecule prior to modification, and wherein the nucleic acid molecule or modified nucleic acid molecule is capable of inhibiting growth-factor-mediated cell proliferation or inflammation.
51. The method of claim 50, wherein the disease is selected from the group consisting of a hyperneovascular condition, neovascular condition of the retina, neovascular condition of the retina, neovascular condition of the skin, growth-factor-mediated disease, a sclerotic disease, kidney disease, hyper-proliferation of vascular endothelial cells, and hyperplasia.
52. The method of claim 50, wherein the nucleic acid molecule or modified nucleic acid molecule reduces the level of an IGF, IGF receptor, or IGF-binding protein in the mammal.
53. The method of claim 50, wherein the nucleic acid molecule or modified nucleic acid molecule is an antisense molecule that reduces the expression of a gene encoding IGF, IGF receptor, or IGF-binding protein.
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US11163560B1 (en) 2020-04-09 2021-11-02 Capital One Services, Llc Methods and arrangements to process comments

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