WO2000078341A1 - A method for the prophylaxis and/or treatment of medical disorders - Google Patents

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

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WO2000078341A1
WO2000078341A1 PCT/AU2000/000693 AU0000693W WO0078341A1 WO 2000078341 A1 WO2000078341 A1 WO 2000078341A1 AU 0000693 W AU0000693 W AU 0000693W WO 0078341 A1 WO0078341 A1 WO 0078341A1
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igf
receptor
nucleic acid
igfbp
acid molecule
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PCT/AU2000/000693
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French (fr)
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WO2000078341A8 (en
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Christopher John Wraight
George Arthur Werther
Stephanie Ruth Edmondson
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Murdoch Childrens Research Institute
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Priority to JP2001504403A priority Critical patent/JP2003502383A/en
Priority to AU52020/00A priority patent/AU768904B2/en
Priority to NZ515964A priority patent/NZ515964A/en
Priority to EP00936560A priority patent/EP1191941A4/en
Priority to CA002376284A priority patent/CA2376284A1/en
Publication of WO2000078341A1 publication Critical patent/WO2000078341A1/en
Publication of WO2000078341A8 publication Critical patent/WO2000078341A8/en

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    • 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
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • 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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    • C12N15/11—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
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    • 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.
  • 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.
  • 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.
  • 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.
  • a nucleic acid molecule capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation such as psoriasis and one or
  • 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.
  • Figure 1 is a representation of the nucleotide sequence of IGFBP-2.
  • IGFBP-2 insulin-like growth factor binding protein
  • KEYWORDS insulin-like growth factor binding protein insulin-like growth factor binding protein
  • 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)
  • Figure 2 is a representation of the nucleotide sequence of IGFBP-3.
  • ACCESSION M31159 KEYWORDS insulin- like growth factor binding protein SOURCE Human plasma, cDNA to mRNA, clone BP-53.
  • Figure 3 is a representation of the nucleotide sequence of IGF- 1 -receptor.
  • Theria Eutheria; Primates; Haplorhini; Catarrhini; Hominidae.
  • REFERENCE 1 bases 1 to 4989
  • AUTHORS Ullrich, A., Gray, A., Tam,A.W., Yang-Feng,T. , Tsubokawa, M . ,
  • Figure 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
  • Figure 4B is a graphical representation of a scanning imaging desitometry of Western ligand blot (Figure 4A), showing relative band intensities of IGFBP-3 and the 24kDa IGFBP-4 after treatment with phosphorothioate oligonucleotides; * no oligonucleotide added.
  • Figure 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.
  • Figure 5B is a graphical representation of a scanning imaging densitometry of Western ligand blot (Figure 5A), showing relative band intensities of IGFBP-3 after treatment with phosphorothioate oligonucleotides as in Figure 5A, showing IGFBP-3 band intensities expressed as a percentage of the average band intensity from conditioned medium of cells not treated with oligonucleotide.
  • Figure 6 is a graphical representation showing inhibition of IGF-I binding by antisense oligonucleotides to IGF-I receptor.
  • IGFR.AS antisense
  • IGFR.S sense.
  • Figure 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.
  • Figure 8 is a graphical representation showing optimization of IGFBP-3 antisense oligonucleotide concentration as determined by relative IGFBP-3 concentration in culture medium.
  • Figure 9 is a diagramatic representation of a map of IGF-1 Receptor mRNA and position of target ODNs.
  • Figure 10 is a photographical representation showing Lipid-mediated uptake of oligonucleotide in keratinocytes.
  • HaCaT keratinocytes were incubated for 24 hours in medium (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.
  • Figure 12 is a graphical representation of an IGF-1 Receptor mRNA in ODN treated (30nM) HaCaT cells (2 ⁇ g/ml GSV). HaCaT keratinocytes were treated for 96 hours with C-5 propynyl, dU, dC ODNs complexed with cytofectin GSV.
  • 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 (I) are seen at 276 bases.
  • FIG. 13 is a graphical representation of an IGF-1 receptor mRNA in ODN treated (30nM) 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 phosphorlmager 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.
  • Figure 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
  • Figure 15 is a graphical representation of the effect of antisense oligonucleotides on IGF-1 receptor levels on the surface of keratinocytes.
  • Figure 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.
  • Figure 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.
  • Figure 18(a) is a photographical representation of Pregraft, Donor JH, Donor JH, PBS treated, 50 ⁇ l, Donor JH, #50 treated, 50 ⁇ l, 10 ⁇ M.
  • Figure 18(b) is a photographical representation of Donor LB, pregraft, Donor LB, PBS treated (50 ⁇ l), Donor LB, #74 treated (50 ⁇ l, 10 ⁇ M).
  • Figure 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).
  • Figure 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).
  • Figure 19(a) is a photographical representation showing Donor JH pregraft, Donor JH PBS treated 50 ⁇ l, Donor JH #50 treated 50 ⁇ l, 10 ⁇ M.
  • Figure 19(b) is a photographical representation Donor LB pregraft, Donor LB PBS treated 50 ⁇ l, Donor LB #74 treated 50 ⁇ l, 10 ⁇ M.
  • Figure 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.
  • Figure 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.
  • Figure 20 is a graphical representation showing suppression of psoriasis after treatment with oligonucleotide (quantification).
  • Oligonucleotide 50 ⁇ l, lO ⁇ M
  • 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
  • Figure 21 is a photographic representation of ⁇ hKi-67 imunobiological binding.
  • Figure 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.
  • Figure 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.
  • Figure 24 is a graphical representation showing IGFBP-3 mRNA.
  • Figure 25(a) is a graphical representation showing IGFBP-3 mRNA in AON treated (lOOnM) HaCaT cells (2 ⁇ g/ml GSV).
  • Figure 25(b) is a graphical representation showing IGFBP-3 mRNA levels of AON treated (lOOnm) HaCaT cells (2 ⁇ g/ml GSV).
  • Figure 25(c) is a graphical representation showing IGFBP-3 mRNA in AON treated (30nM) HaCaT cells (2 ⁇ g/ml GSV).
  • Figure 25(d) is a graphical representation showing IGFBP-3 mRNA in AON treated (30nM) HaCaT cells (2 ⁇ g/ml GSV).
  • Figure 26(a) is a graphical representation showing IGFBP-3 mRNA in ODN treated (30nM) 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.
  • Figure 26(b) is a graphical representation showing IGFBP-3 mRNA in ODN treated (lOOnM) 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.
  • Figure 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 #56) 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; LT, untreated).
  • Figure 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, (a) Duplicate treated cellular extracts showing the IGF-I receptor at the predicted size of 110 kD
  • Figure 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 (—A—), #50 (— x— ), #64 (— ⁇ —), a random sequence oligonucleotide R451 (— o— ), or treated with GSV lipid alone ( — Q — ) every 24 h for four days (untreated cells, --*--).
  • Competition binding assays using 125 I-IGF-I and the receptor-specific analogue, des(l-3)IGF-I were performed (inset); plotted values are means ⁇ standard error. The mean values were then subjected to Scatchard analysis.
  • Figure 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. A indicates a significant difference between cells treated with AON #64 and all of the control treatments (p ⁇ 0.001).
  • Figure 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.
  • E epidermis; Scale bar, 400 mm; all pictures are at the same magnification
  • 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.
  • Figure 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).
  • Figure 33 is a representation showing a reduction in IGF-I receptor mRNA in HaCaT keratinocytes following treatment with oligonucleotides.
  • Figure 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 0 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, Massachusetts).
  • FIG. 35 is a representation showing a reduction in HaCaT keratinocyte cell number following treatment with oligonucleotides.
  • 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- ⁇ (TGFoc), 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
  • TGFoc transforming growth factor- ⁇
  • TGF ⁇ tumour necrosis factor- ⁇
  • TNF ⁇ tumour necrosis factor- ⁇
  • 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:
  • Acute epidermal hyperplasia following UV has been suggested to increase the risk of keratinocyte carcinogenic transformation [22].
  • IGF-I receptor expression 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.
  • 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.
  • 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 admimstered 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
  • the instant invention is particularly directed to proliferative and/or inflammatory skin disorders such
  • 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.
  • nucleotide sequences of IGFBP-2 and IGFBP-3 are set forth in Figures 1 ( ⁇ 400> 1) and 2 ( ⁇ 400>2), respectively.
  • 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.
  • 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.
  • Alternatively or in addition to the pyrimidine bases are modified by inclusion of a
  • 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.
  • Particularly useful antisense molecules are:
  • 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 conesponding 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 model 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.
  • 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 earners 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.
  • 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.
  • various 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].
  • EXAMPLE 2 Cells were grown to 4 days post confluence in 2cm 2 wells with daily medium changes of Keratinocyte- SFM, then the medium was changed to DMEM (Cytosystems, Australia), with the following additions: 25mM Hepes, 0.19% w/v, sodium bicarbonate, 0.03% w/v glutamine (Sigma Chemical Co, USA), 50IU/ml penicillin and 50 ⁇ g/ml streptomycin (Flow Laboratories).
  • IGF-I or tIGF-I was added to triplicate wells, at the concentrations indicated, in 0.5ml fresh DMEM containing 0.02% v/v bovine serum albumin (Sigma molecular biology grade) and incubated for a further 21 hours.
  • [ 3 H]-Thymidine O.l ⁇ 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.
  • 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 16,000g 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 transfened to nitrocellulose membrane (0.45mm, Schleicher and Schuell, Dassel, Germany) in a buffer containing 25mM Tris, 192mM glycine and 20% v/v methanol.
  • IGFBPs were then visualised by the procedure of Hossenlopp et al [11], using [ l25 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.
  • EXAMPLE 4 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 25mer 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 24mer complementary to the exon 1/intron 1 splice site; BP3AS4, 5'- AGG CGG CTG ACG GCA CTA -3'( ⁇ 400>6), an 18mer 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 18mer sense control sequence equivalent to the start codon region; BP3AS2NS, 5'- CGG AGA TGC CGC ATG CCA GCG CAG G -3' ( ⁇ 400>8), a 25mer randomised sequence with the same GC content as BP3AS2; BP3AS4NS, 5'- GAC AGC GTC GGA GCG ATC -3' ( ⁇ 400>9), an 18mer 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].
  • a sense oligonucleotide conesponding to the start site was used.
  • 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:
  • GCGCTGCTATCCCCA GGGCGAGGGCACTTG GGAGCAGGTTGCAGA CGCTGCTATCCCCAC GGCGAGGGCACTTGT GAGCAGGTTGCAGAC
  • GGCTCCGAGCTGCCC AAGCGCCGGGACGCC GGCGATGACCACTCA
  • AGGAGCCCAAGAAGC AACTGGACCAGGTCC GGGGCCCTCTGGAGC GGAGCCCAAGAAGCT ACTGGACCAGGTCCT GGGCCCTCTGGAGCA
  • GAAGCTGCGACCACC GGTCCTGGAGCGGAT GGAGCACCTCTACTC AAGCTGCGACCACCC GTCCTGGAGCGGATC GAGCACCTCTACTCC
  • GAGCAGCAGGAGGCT GCCAGCCGGTGCCTG GAAAACGGAGAGTGC AGCAGCAGGAGGCTT CCAGCCGGTGCCTGG AAAACGGAGAGTGCT
  • GGTGCACACCCAGCG CCTGCCCCCCGCCCC TGGTGGGTGCTGGAG GTGCACACCCAGCGG CTGCCCCCCCT GGTGGGTGCTGGAGG
  • CACGTATTTATATTT CCTCTCTCTTCCCAG GGGGGAGGAAGGGGG
  • AAGAGACCAGCACCG CAGATGCCACACCTG TGGTCGGGGAGCTGG AGAGACCAGCACCGA AGATGCCACACCTGC GGTCGGGGAGCTGGG
  • GAGCTCGGCACCTCC GCTCCTTCTTGCTTT GGGTACAGGTTTGGG
  • GGCACCTCCCCGGCC TCTTGCTTTCCCCGG AGGTTTGGGGAGGGG GCACCTCCCCGGCCT CTTGCTTTCCCCGGG GGTTTGGGGAGGGGG
  • CTCCCCGGCCTCTCT CTTTCCCCGGGGGAG TGGGGAGGGGGAAGA TCCCCGGCCTCTCTC TTTCCCCGGGGGAGG GGGGAGGGGGAAGAG
  • Antisense oligonucleotides to IGFBP3 may be selected from molecules capable of interacting with one or more of the following sense oligonucleotides:
  • CTGCGGCTGCTGCCT CTACACCGAGCGCTG GGCGCGACCGCTGCA
  • AAAGATAATCATCAT AGTTGACTACGAGTC CAAGCGGGAGACAGA AAGATAATCATCATC GTTGACTACGAGTCT AAGCGGGAGACAGAA
  • ATTCAGAGACTCGAG ACCACATGTTGGTCG GTGTTGCCTATGTAG TTCAGAGACTCGAGC CCACATGTTGGTCGA TGTTGCCTATGTAGA
  • CAGAGACTCGAGCAC ACATGTTGGTCGAAG TTGCCTATGTAGAGA
  • GAGACTCGAGCACAG ATGTTGGTCGAAGCG GCCTATGTAGAGAAC AGACTCGAGCACAGC TGTTGGTCGAAGCGG CCTATGTAGAGAACA
  • GACTCGAGCACAGCA GTTGGTCGAAGCGGC CTATGTAGAGAACAC TATGTAGAGAACACG TATCGAGAATAGGAA ATGCTCCTGGAGCTC
  • GAACACGCTTCACCC ATAGGAAAACCTTTA GGAGCTCACAGCCTT AACACGCTTCACCCC TAGGAAAACCTTTAA GAGCTCACAGCCTTC
  • CTCCCCGTACAGTGC CCCGGTCATCCGGAC TGGTGTCATTTCTGA
  • Antisense oligonucleotides to IGF-I may be selected from molecules capable of interacting with one or more of the following sense oligonucleotides:
  • AGCAGCTGAAGCGCC TCCTGCTCATCTCCA TCACGGTCATTACCG GCAGCTGAAGCGCCT CCTGCTCATCTCCAA CACGGTCATTACCGA
  • GGTGATCGAGGGCTA CCGCAGCTACCGCTT GTTCCGAGTGGCTGG
  • GAGGGCTACCTCCAC TACCGCTTCCCCAAG GTGGCTGGCCTCGAG AGGGCTACCTCCACA ACCGCTTCCCCAAGC TGGCTGGCCTCGAGA
  • AACCTCACGGTCATC ATCTTCGAGATGACC ATTACTCGGGGGGCC ACCTCACGGTCATCC TCTTCGAGATGACCA TTACTCGGGGGGCCA
  • CACGGTCATCCGCGG CGAGATGACCAATCT TCGGGGGGCCATCAG ACGGTCATCCGCGGC GAGATGACCAATCTC CGGGGGGCCATCAGG
  • CTCTCCACTGTGGAC ATTGTGGGGAATAAG ATGGAGGAGAAGCCG
  • TCTCCTTGTCCTTCC TAGAAGGGAATTACT TGTGGGACTGGGACC
  • ACCTTCGCCTCATCC ACGTCCTCGACAACC ACCTGACCATCAAAG

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

A METHOD FOR THE PROPHYLAXIS AND/OR TREATMENT OF
MEDICAL DISORDERS
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
Figure 1 is a representation of the nucleotide sequence of IGFBP-2.
LOCUS HSIGFBP2 1433 bp RNA PRI 31-JAN-1990
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=l
/translation "MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFR
CPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACG
VΥTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGG
LVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEP
KKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCK
MSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEACGVHTQRMQ"
(<400>21)
CDS 118. .234
/note="signal peptide; (AA -39 to -1); NCBI gi : 33011."
/codon_start=l
/translation="MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARA"
(<400>22)
CDS 235. .1101
/note="mature IGFBP-2; (AA 1 to 289); NCBI gi : 33012."
/codon_start=l
/translation="EVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVR
EPGCGCCSVCARLEGEACGVΥTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAE
YGASPEQVADNGDDHSEGGLVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVT
EQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLY
SLHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNE
QQEACGVHTQRMQ" (<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
Figure 2 is a representation of the nucleotide sequence of IGFBP-3.
LOCUS HUMGFIBPA 2474 bp ss- RNA PRI 15-JUN-1990 DEFINITION Human growth hormone-dependent insulin-like growth factor-binding protein mRNA, complete eds.
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 COMMENT NCBI gi: 183115 FEATURES Location/Qualifiers mRNA <1. .2474
/note="GFIBP mRNA"
CDS 110. .985
/gene="IGFBPl"
/note="insulin-like growth factor-binding protein; NCBI gi: 183116."
/codon_start=l
/translation"MQRARPTLWAAALTLLVLLRGPPVARAGASSGGLGPVVRCEPCD
ARALAQCAPPPAVCAELVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEAR
PLQALLDGRGLCVNASAVSRLRAYLLPAPPAPGNASESEEDRSAGSVESPSVSSTHRV
SDPKFHPLHSKIIIIKKGHAKDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREME
DTLNHLKFLNVLSPRGVHIPNCDKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYT
TKGKEDVHCYSMQSK" (<400>24>)
1. .2474
/organism="Homo sapiens"
BASE COUNT 597 a 646 c 651 g 580 t ORIGIN
HUMGFIBPA Length: 2474 May 11, 1994 10:00 Type: N Check: 9946
Figure 3 is a representation of the nucleotide sequence of IGF- 1 -receptor.
LOCUS HSIGFIRR 4989 bp RNA PRI 28-MAR-1991
DEFINITION Human mRNA for insulin-like growth factor I receptor ACCESSION X04434 M24599 KEYWORDS glycoprotein; insulin receptor; insulin-like growth factor I receptor; membrane glycoprotein; receptor; tyrosine kinase. SOURCE human ORGANISM Homo sapiens
Eukaryota; Animalia; Metazoa; Chordata; Vertebrata; Mammalia;
Theria; Eutheria; Primates; Haplorhini; Catarrhini; Hominidae.
REFERENCE 1 (bases 1 to 4989) AUTHORS Ullrich, A., Gray, A., Tam,A.W., Yang-Feng,T. , Tsubokawa, M . ,
Collins, C, Henzel,W., Bon,T.L., Kathuria,S., Chen,E., Jakobs,S.,
Francke,U., Ramachandran, J . and Fuj ita-Yamaguchi, Y.
TITLE Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural dererminants that define functional specificity
JOURNAL EMBO J. 5, 2503-2512 (1986) STANDARD full automatic COMMENT NCBI gi: 33058
FEATURES Location/Qualifiers source 1. .4989
/organism="Homo sapiens"
/tissue_type= "placenta"
/clone_lib=" (lamda)gtlθ"
/clone=" (lambda) IGF-l-R.85, (lambda) IGF-1-R.76 sig_peptide 32. .121 mat_peptide 122. .4132
/note="IGF-I receptor" misc_feature 122. .2251
/note="alpha-subunit (AA 1 - 710)" misc_feature 182. .190
/note= "pot .N-linked glycosylation site (AA 21 - 23) " misc_feature 335. .343
/note= "pot .N-linked glycostlation site (AA 72 - 74) " misc_feature 434. .442
/note="pot .N-linked glycostlation site (AA 105 - 107) " misc_feature 761. .769
/note="pot .N-linked glycostlation site (AA 214 - 216) " misc_feature 971. .979
/note="pot .N-linked glycostlation site (AA 284 - 286) " misc_feature 1280. .1288
/note= "pot .N-linked glycostlation site (AA 387 - 389) " 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
Figure 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 5μM; * no oligonucleotide added.
Figure 4B is a graphical representation of a scanning imaging desitometry of Western ligand blot (Figure 4A), showing relative band intensities of IGFBP-3 and the 24kDa IGFBP-4 after treatment with phosphorothioate oligonucleotides; * no oligonucleotide added.
Figure 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 0.5μM compared with several control oligonucleotides at 0.5μM. (a) oligonucleotide BP3AS2NS; (b) oligonucleotide BP3AS4; (c) oligonucleotide BP3AS4NS; and (untreated), no oligonucleotide added. Figure 5B is a graphical representation of a scanning imaging densitometry of Western ligand blot (Figure 5A), showing relative band intensities of IGFBP-3 after treatment with phosphorothioate oligonucleotides as in Figure 5A, showing IGFBP-3 band intensities expressed as a percentage of the average band intensity from conditioned medium of cells not treated with oligonucleotide.
Figure 6 is a graphical representation showing inhibition of IGF-I binding by antisense oligonucleotides to IGF-I receptor. IGFR.AS: antisense; IGFR.S: sense.
Figure 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.
Figure 8 is a graphical representation showing optimization of IGFBP-3 antisense oligonucleotide concentration as determined by relative IGFBP-3 concentration in culture medium.
Figure 9 is a diagramatic representation of a map of IGF-1 Receptor mRNA and position of target ODNs.
Figure 10 is a photographical representation showing Lipid-mediated uptake of oligonucleotide in keratinocytes. HaCaT keratinocytes were incubated for 24 hours in medium (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.
Figure 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. Figure 12 is a graphical representation of an IGF-1 Receptor mRNA in ODN treated (30nM) HaCaT cells (2μg/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 (I) are seen at 276 bases.
(B) Relative level of IGF-I receptor mRNA following each treatment is shown.
Figure 13 is a graphical representation of an IGF-1 receptor mRNA in ODN treated (30nM) HaCaT cells (2μg/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 phosphorlmager 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; Rl, R4, R7 and R9 = randomised ODNs (Rl =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).
Figure 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 1010M 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.
Figure 15 is a graphical representation of the effect of antisense oligonucleotides on IGF-1 receptor levels on the surface of keratinocytes.
Figure 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.
Figure 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.
Figure 18(a) is a photographical representation of Pregraft, Donor JH, Donor JH, PBS treated, 50μl, Donor JH, #50 treated, 50μl, 10μM.
Figure 18(b) is a photographical representation of Donor LB, pregraft, Donor LB, PBS treated (50μl), Donor LB, #74 treated (50μl, 10μM). Figure 18(c) is a photographical representation of Donor PW, pregraft, Donor PW, R451 treated (50μl, 10μM), Donor LB, #74 treated (50μl, 10μM).
Figure 18(d) is a photographical representation of Donor GM, pregraft, Donor GB, R451 treated (50μl, 10μM), Donor GM, #27 treated (50μl, 10μM).
Figure 19(a) is a photographical representation showing Donor JH pregraft, Donor JH PBS treated 50μl, Donor JH #50 treated 50μl, 10μM.
Figure 19(b) is a photographical representation Donor LB pregraft, Donor LB PBS treated 50μl, Donor LB #74 treated 50μl, 10μM.
Figure 19(c) is a photographical representational showing Donor PW pregraft, Donor PW r451 treated 50μl, 10μM, Donor PW #74 treated 50μl, 10μM.
Figure 19(d) is a photographical representation showing Donor GM pregraft, Donor GM R451 treated 50μl, 10μM, Donor #27 treated 50μl, 10μM.
Figure 20 is a graphical representation showing suppression of psoriasis after treatment with oligonucleotide (quantification). Oligonucleotide (50 μl, lOμM) 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)
Figure 21 is a photographic representation of αhKi-67 imunobiological binding.
Figure 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.
Figure 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.
Figure 24 is a graphical representation showing IGFBP-3 mRNA.
Figure 25(a) is a graphical representation showing IGFBP-3 mRNA in AON treated (lOOnM) HaCaT cells (2μg/ml GSV).
Figure 25(b) is a graphical representation showing IGFBP-3 mRNA levels of AON treated (lOOnm) HaCaT cells (2μg/ml GSV).
Figure 25(c) is a graphical representation showing IGFBP-3 mRNA in AON treated (30nM) HaCaT cells (2μg/ml GSV).
Figure 25(d) is a graphical representation showing IGFBP-3 mRNA in AON treated (30nM) HaCaT cells (2μg/ml GSV).
Figure 26(a) is a graphical representation showing IGFBP-3 mRNA in ODN treated (30nM) HaCaT cells (2μg/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. (l-24=IGFBP-3 ODNs; Rl, 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).
Figure 26(b) is a graphical representation showing IGFBP-3 mRNA in ODN treated (lOOnM) HaCaT cells (2μg/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. (l-24=IGFBP-3 ODNs; Rl, 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).
Figure 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 #56) 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; LT, 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 (Uϊ, 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 (A) test was performed; A 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).
Figure 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; C/T, untreated; R451, random sequence oligonucleotide; 64, 50, 27, IGF-I receptor-specific AONs.
Figure 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 (—A—), #50 (— x— ), #64 (—■—), a random sequence oligonucleotide R451 (— o— ), or treated with GSV lipid alone ( — Q — ) every 24 h for four days (untreated cells, --*--). Competition binding assays using 125I-IGF-I and the receptor-specific analogue, des(l-3)IGF-I, were performed (inset); plotted values are means ± standard error. The mean values were then subjected to Scatchard analysis.
Figure 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. A indicates a significant difference between cells treated with AON #64 and all of the control treatments (p< 0.001).
Figure 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: R4 1, 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.
Figure 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 5 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.
Figure 33 is a representation showing a reduction in IGF-I receptor mRNA in HaCaT keratinocytes following treatment with oligonucleotides. HaCaT cell monolayers grown to
10 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, Texas). Band intensity was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale,
15 California).
Figure 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 0 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, Massachusetts). Membranes were incubated with the anti- 5 IGF-I receptor antibody C20 (Sanra Cruz Biotechnology Inc., Santa Cruz, California, 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, California). 0 Figure 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 (TGFoc), 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 admimstered 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 Figures 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 prefened 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 conesponding 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 earners 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 2cm2 wells with daily medium changes of Keratinocyte- SFM, then the medium was changed to DMEM (Cytosystems, Australia), with the following additions: 25mM Hepes, 0.19% w/v, sodium bicarbonate, 0.03% w/v glutamine (Sigma Chemical Co, USA), 50IU/ml penicillin and 50μg/ml streptomycin (Flow Laboratories). After 24 hours, IGF-I or tIGF-I was added to triplicate wells, at the concentrations indicated, in 0.5ml fresh DMEM containing 0.02% v/v bovine serum albumin (Sigma molecular biology grade) and incubated for a further 21 hours. [3H]-Thymidine (O.lμCi/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 (200μl/well), transferred to scintillation vials and radioactivity determined by liquid scintillation counting (Pharmacia Wallac 1410 liquid scintillation counter).
EXAMPLE 3
HaCaT conditioned medium (250μl) was concentrated by adding 750μl cold ethanol, incubating at -20°C for 2 hours and centrifuging at 16,000g 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 transfened to nitrocellulose membrane (0.45mm, Schleicher and Schuell, Dassel, Germany) in a buffer containing 25mM Tris, 192mM glycine and 20% v/v methanol. IGFBPs were then visualised by the procedure of Hossenlopp et al [11], using [l25I]-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 25mer 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 24mer complementary to the exon 1/intron 1 splice site; BP3AS4, 5'- AGG CGG CTG ACG GCA CTA -3'(<400>6), an 18mer 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 18mer sense control sequence equivalent to the start codon region; BP3AS2NS, 5'- CGG AGA TGC CGC ATG CCA GCG CAG G -3' (<400>8), a 25mer randomised sequence with the same GC content as BP3AS2; BP3AS4NS, 5'- GAC AGC GTC GGA GCG ATC -3' (<400>9), an 18mer 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 2cm2 wells with daily medium changes of 0.5ml Keratinocyte-SFM, then subjected to daily medium changes of Keratinocyte-SFM for a further 4 days. Daily additions of 0.5ml fresh Keratinocyte-SFM were then continued for a further 7 days, except that at the time of medium addition, 5μl 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, 5μM and 0.5μM, 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 conesponding to the start site was used. As shown in Figures 4A and 4B, all oligonucleotides at 5μM 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 (Figure 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 0.5μM. 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 0.5μM was compared with a number of control oligonucleotides, including one antisense oligonucleotide to IGFBP-3 that had proved to be ineffective at 0.5μM. As shown in Figures 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 0.5μM, 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 CGCAGGGCCGTGCAC CCGCGCCGCGCTGCC TTCGGGGCGAGGGAG GCAGGGCCGTGCACC CGCGCCGCGCTGCCG
TCGGGGCGAGGGAGG CAGGGCCGTGCACCT GCGCCGCGCTGCCGA
CGGGGCGAGGGAGGA AGGGCCGTGCACCTG CGCCGCGCTGCCGAC
GGGGCGAGGGAGGAG GGGCCGTGCACCTGC GCCGCGCTGCCGACC
GGGCGAGGGAGGAGG GGCCGTGCACCTGCC CCGCGCTGCCGACCG GGCGAGGGAGGAGGA GCCGTGCACCTGCCC CGCGCTGCCGACCGC
GCGAGGGAGGAGGAA. CCGTGCACCTGCCCG GCGCTGCCGACCGCC
CGAGGGAGGAGGAAG CGTGCACCTGCCCGC CGCTGCCGACCGCCA
GAGGGAGGAGGAAGA GTGCACCTGCCCGCC GCTGCCGACCGCCAG
AGGGAGGAGGAAGAA TGCACCTGCCCGCCC CTGCCGACCGCCAGC GGGAGGAGGAAGAAG GCACCTGCCCGCCCG TGCCGACCGCCAGCA
GGAGGAGGAAGAAGC CACCTGCCCGCCCGC GCCGACCGCCAGCAT
GAGGAGGAAGAAGCG ACCTGCCCGCCCGCC CCGACCGCCAGCATG
AGGAGGAAGAAGCGG CCTGCCCGCCCGCCC CGACCGCCAGCATGC
GGAGGAAGAAGCGGA CTGCCCGCCCGCCCG GACCGCCAGCATGCT GAGGAAGAAGCGGAG TGCCCGCCCGCCCGC ACCGCCAGCATGCTG
AGGAAGAAGCGGAGG GCCCGCCCGCCCGCT CCGCCAGCATGCTGC
GGAAGAAGCGGAGGA CCCGCCCGCCCGCTC CGCCAGCATGCTGCC
GAAGAAGCGGAGGAG CCGCCCGCCCGCTCG GCCAGCATGCTGCCG
AAGAAGCGGAGGAGG CGCCCGCCCGCTCGC CCAGCATGCTGCCGA AGAAGCGGAGGAGGC GCCCGCCCGCTCGCT CAGCATGCTGCCGAG
GAAGCGGAGGAGGCG CCCGCCCGCTCGCTC AGCATGCTGCCGAGA
AAGCGGAGGAGGCGG CCGCCCGCTCGCTCG GCATGCTGCCGAGAG
AGCGGAGGAGGCGGC CGCCCGCTCGCTCGC CATGCTGCCGAGAGT
GCGGAGGAGGCGGCT GCCCGCTCGCTCGCT ATGCTGCCGAGAGTG CGGAGGAGGCGGCTC CCCGCTCGCTCGCTC TGCTGCCGAGAGTGG
GGAGGAGGCGGCTCC CCGCTCGCTCGCTCG GCTGCCGAGAGTGGG
GAGGAGGCGGCTCCC CGCTCGCTCGCTCGC CTGCCGAGAGTGGGC
AGGAGGCGGCTCCCG GCTCGCTCGCTCGCC TGCCGAGAGTGGGCT
GGAGGCGGCTCCCGC CTCGCTCGCTCGCCC GCCGAGAGTGGGCTG GAGGCGGCTCCCGCT TCGCTCGCTCGCCCG CCGAGAGTGGGCTGC
AGGCGGCTCCCGCTC CGCTCGCTCGCCCGC CGAGAGTGGGCTGCC
GGCGGCTCCCGCTCG GCTCGCTCGCCCGCC GAGAGTGGGCTGCCC
GCGGCTCCCGCTCGC CTCGCTCGCCCGCCG AGAGTGGGCTGCCCC
CGGCTCCCGCTCGCA TCGCTCGCCCGCCGC GAGTGGGCTGCCCCG GGCTCCCGCTCGCAG CGCTCGCCCGCCGCG AGTGGGCTGCCCCGC
GCTCCCGCTCGCAGG GCTCGCCCGCCGCGC GTGGGCTGCCCCGCG
CTCCCGCTCGCAGGG CTCGCCCGCCGCGCC TGGGCTGCCCCGCGC
TCCCGCTCGCAGGGC TCGCCCGCCGCGCCG GGGCTGCCCCGCGCT
CCCGCTCGCAGGGCC CGCCCGCCGCGCCGC GGCTGCCCCGCGCTG CCGCTCGCAGGGCCG GCCCGCCGCGCCGCG GCTGCCCCGCGCTGC
CGCTCGCAGGGCCGT CCCGCCGCGCCGCGC CTGCCCCGCGCTGCC
GCTCGCAGGGCCGTG CCGCCGCGCCGCGCT TGCCCCGCGCTGCCG
CTCGCAGGGCCGTGC CGCCGCGCCGCGCTG GCCCCGCGCTGCCGC
TCGCAGGGCCGTGCA GCCGCGCCGCGCTGC CCCCGCGCTGCCGCT CCCGCGCTGCCGCTG CTGCTGCTACTGGGC CTGTTCCGCTGCCCG CCGCGCTGCCGCTGC TGCTGCTACTGGGCG TGTTCCGCTGCCCGC CGCGCTGCCGCTGCC GCTGCTACTGGGCGC GTTCCGCTGCCCGCC GCGCTGCCGCTGCCG CTGCTACTGGGCGCG TTCCGCTGCCCGCCC CGCTGCCGCTGCCGC TGCTACTGGGCGCGA TCCGCTGCCCGCCCT GCTGCCGCTGCCGCC GCTACTGGGCGCGAG CCGCTGCCCGCCCTG CTGCCGCTGCCGCCG CTACTGGGCGCGAGT CGCTGCCCGCCCTGC TGCCGCTGCCGCCGC TACTGGGCGCGAGTG GCTGCCCGCCCTGCA GCCGCTGCCGCCGCC ACTGGGCGCGAGTGG CTGCCCGCCCTGCAC CCGCTGCCGCCGCCG CTGGGCGCGAGTGGC TGCCCGCCCTGCACA CGCTGCCGCCGCCGC TGGGCGCGAGTGGCG GCCCGCCCTGCACAC GCTGCCGCCGCCGCC GGGCGCGAGTGGCGG CCCGCCCTGCACACC CTGCCGCCGCCGCCG GGCGCGAGTGGCGGC CCGCCCTGCACACCC TGCCGCCGCCGCCGC GCGCGAGTGGCGGCG CGCCCTGCACACCCG GCCGCCGCCGCCGCT CGCGAGTGGCGGCGG GCCCTGCACACCCGA CCGCCGCCGCCGCTG GCGAGTGGCGGCGGC CCCTGCACACCCGAG CGCCGCCGCCGCTGC CGAGTGGCGGCGGCG CCTGCACACCCGAGC GCCGCCGCCGCTGCT GAGTGGCGGCGGCGG CTGCACACCCGAGCG CCGCCGCCGCTGCTG AGTGGCGGCGGCGGC TGCACACCCGAGCGC CGCCGCCGCTGCTGC GTGGCGGCGGCGGCG GCACACCCGAGCGCC GCCGCCGCTGCTGCC TGGCGGCGGCGGCGG CACACCCGAGCGCCT CCGCCGCTGCTGCCG GGCGGCGGCGGCGGG ACACCCGAGCGCCTG CGCCGCTGCTGCCGC GCGGCGGCGGCGGGG CACCCGAGCGCCTGG GCCGCTGCTGCCGCT CGGCGGCGGCGGGGC ACCCGAGCGCCTGGC CCGCTGCTGCCGCTG GGCGGCGGCGGGGCG CCCGAGCGCCTGGCC CGCTGCTGCCGCTGC GCGGCGGCGGGGCGC CCGAGCGCCTGGCCG GCTGCTGCCGCTGCT CGGCGGCGGGGCGCG CGAGCGCCTGGCCGC CTGCTGCCGCTGCTG GGCGGCGGGGCGCGC GAGCGCCTGGCCGCC TGCTGCCGCTGCTGC GCGGCGGGGCGCGCG AGCGCCTGGCCGCCT GCTGCCGCTGCTGCC CGGCGGGGCGCGCGC GCGCCTGGCCGCCTG CTGCCGCTGCTGCCG GGCGGGGCGCGCGCG CGCCTGGCCGCCTGC TGCCGCTGCTGCCGC GCGGGGCGCGCGCGG GCCTGGCCGCCTGCG GCCGCTGCTGCCGCT CGGGGCGCGCGCGGA CCTGGCCGCCTGCGG CCGCTGCTGCCGCTG GGGGCGCGCGCGGAG CTGGCCGCCTGCGGG CGCTGCTGCCGCTGC GGGCGCGCGCGGAGG TGGCCGCCTGCGGGC GCTGCTGCCGCTGCT GGCGCGCGCGGAGGT GGCCGCCTGCGGGCC CTGCTGCCGCTGCTG GCGCGCGCGGAGGTG GCCGCCTGCGGGCCC TGCTGCCGCTGCTGC CGCGCGCGGAGGTGC CCGCCTGCGGGCCCC GCTGCCGCTGCTGCT GCGCGCGGAGGTGCT CGCCTGCGGGCCCCC CTGCCGCTGCTGCTG CGCGCGGAGGTGCTG GCCTGCGGGCCCCCG TGCCGCTGCTGCTGC GCGCGGAGGTGCTGT CCTGCGGGCCCCCGC GCCGCTGCTGCTGCT CGCGGAGGTGCTGTT CTGCGGGCCCCCGCC CCGCTGCTGCTGCTG GCGGAGGTGCTGTTC TGCGGGCCCCCGCCG CGCTGCTGCTGCTGC CGGAGGTGCTGTTCC GCGGGCCCCCGCCGG GCTGCTGCTGCTGCT GGAGGTGCTGTTCCG CGGGCCCCCGCCGGT CTGCTGCTGCTGCTA GAGGTGCTGTTCCGC GGGCCCCCGCCGGTT TGCTGCTGCTGCTAC AGGTGCTGTTCCGCT GGCCCCCGCCGGTTG GCTGCTGCTGCTACT GGTGCTGTTCCGCTG GCCCCCGCCGGTTGC CTGCTGCTGCTACTG GTGCTGTTCCGCTGC CCCCCGCCGGTTGCG TGCTGCTGCTACTGG TGCTGTTCCGCTGCC CCCCGCCGGTTGCGC GCTGCTGCTACTGGG GCTGTTCCGCTGCCC CCCGCCGGTTGCGCC CCGCCGGTTGCGCCG ATGCCATGCGCGGAG TGCGCCCGGCTGGAG CGCCGGTTGCGCCGC TGCCATGCGCGGAGC GCGCCCGGCTGGAGG GCCGGTTGCGCCGCC GCCATGCGCGGAGCT CGCCCGGCTGGAGGG CCGGTTGCGCCGCCC CCATGCGCGGAGCTC GCCCGGCTGGAGGGC CGGTTGCGCCGCCCG CATGCGCGGAGCTCG CCCGGCTGGAGGGCG GGTTGCGCCGCCCGC ATGCGCGGAGCTCGT CCGGCTGGAGGGCGA GTTGCGCCGCCCGCC TGCGCGGAGCTCGTC CGGCTGGAGGGCGAG TTGCGCCGCCCGCCG GCGCGGAGCTCGTCC GGCTGGAGGGCGAGG TGCGCCGCCCGCCGC CGCGGAGCTCGTCCG GCTGGAGGGCGAGGC GCGCCGCCCGCCGCG GCGGAGCTCGTCCGG CTGGAGGGCGAGGCG CGCCGCCCGCCGCGG CGGAGCTCGTCCGGG TGGAGGGCGAGGCGT GCCGCCCGCCGCGGT GGAGCTCGTCCGGGA GGAGGGCGAGGCGTG CCGCCCGCCGCGGTG GAGCTCGTCCGGGAG GAGGGCGAGGCGTGC CGCCCGCCGCGGTGG AGCTCGTCCGGGAGC AGGGCGAGGCGTGCG GCCCGCCGCGGTGGC GCTCGTCCGGGAGCC GGGCGAGGCGTGCGG CCCGCCGCGGTGGCC CTCGTCCGGGAGCCG GGCGAGGCGTGCGGC CCGCCGCGGTGGCCG TCGTCCGGGAGCCGG GCGAGGCGTGCGGCG CGCCGCGGTGGCCGC CGTCCGGGAGCCGGG CGAGGCGTGCGGCGT GCCGCGGTGGCCGCA GTCCGGGAGCCGGGC GAGGCGTGCGGCGTC CCGCGGTGGCCGCAG TCCGGGAGCCGGGCT AGGCGTGCGGCGTCT CGCGGTGGCCGCAGT CCGGGAGCCGGGCTG GGCGTGCGGCGTCTA GCGGTGGCCGCAGTG CGGGAGCCGGGCTGC GCGTGCGGCGTCTAC CGGTGGCCGCAGTGG GGGAGCCGGGCTGCG CGTGCGGCGTCTACA GGTGGCCGCAGTGGC GGAGCCGGGCTGCGG GTGCGGCGTCTACAC GTGGCCGCAGTGGCC GAGCCGGGCTGCGGC TGCGGCGTCTACACC TGGCCGCAGTGGCCG AGCCGGGCTGCGGCT GCGGCGTCTACACCC GGCCGCAGTGGCCGG GCCGGGCTGCGGCTG CGGCGTCTACACCCC GCCGCAGTGGCCGGA CCGGGCTGCGGCTGC GGCGTCTACACCCCG CCGCAGTGGCCGGAG CGGGCTGCGGCTGCT GCGTCTACACCCCGC CGCAGTGGCCGGAGG GGGCTGCGGCTGCTG CGTCTACACCCCGCG GCAGTGGCCGGAGGC GGCTGCGGCTGCTGC GTCTACACCCCGCGC CAGTGGCCGGAGGCG GCTGCGGCTGCTGCT TCTACACCCCGCGCT AGTGGCCGGAGGCGC CTGCGGCTGCTGCTC CTACACCCCGCGCTG GTGGCCGGAGGCGCC TGCGGCTGCTGCTCG TACACCCCGCGCTGC TGGCCGGAGGCGCCC GCGGCTGCTGCTCGG ACACCCCGCGCTGCG GGCCGGAGGCGCCCG CGGCTGCTGCTCGGT CACCCCGCGCTGCGG GCCGGAGGCGCCCGC GGCTGCTGCTCGGTG ACCCCGCGCTGCGGC CCGGAGGCGCCCGCA GCTGCTGCTCGGTGT CCCCGCGCTGCGGCC CGGAGGCGCCCGCAT CTGCTGCTCGGTGTG CCCGCGCTGCGGCCA GGAGGCGCCCGCATG TGCTGCTCGGTGTGC CCGCGCTGCGGCCAG GAGGCGCCCGCATGC GCTGCTCGGTGTGCG CGCGCTGCGGCCAGG AGGCGCCCGCATGCC CTGCTCGGTGTGCGC GCGCTGCGGCCAGGG GGCGCCCGCATGCCA TGCTCGGTGTGCGCC CGCTGCGGCCAGGGG GCGCCCGCATGCCAT GCTCGGTGTGCGCCC GCTGCGGCCAGGGGC CGCCCGCATGCCATG CTCGGTGTGCGCCCG CTGCGGCCAGGGGCT GCCCGCATGCCATGC TCGGTGTGCGCCCGG TGCGGCCAGGGGCTG CCCGCATGCCATGCG CGGTGTGCGCCCGGC GCGGCCAGGGGCTGC CCGCATGCCATGCGC GGTGTGCGCCCGGCT CGGCCAGGGGCTGCG CGCATGCCATGCGCG GTGTGCGCCCGGCTG GGCCAGGGGCTGCGC GCATGCCATGCGCGG TGTGCGCCCGGCTGG GCCAGGGGCTGCGCT CATGCCATGCGCGGA GTGCGCCCGGCTGGA CCAGGGGCTGCGCTG CAGGGGCTGCGCTGC CTGGTCATGGGCGAG GCCAGCCCGGAGCAG
AGGGGCTGCGCTGCT TGGTCATGGGCGAGG CCAGCCCGGAGCAGG
GGGGCTGCGCTGCTA GGTCATGGGCGAGGG CAGCCCGGAGCAGGT
GGGCTGCGCTGCTAT GTCATGGGCGAGGGC AGCCCGGAGCAGGTT GGCTGCGCTGCTATC TCATGGGCGAGGGCA GCCCGGAGCAGGTTG
GCTGCGCTGCTATCC CATGGGCGAGGGCAC CCCGGAGCAGGTTGC
CTGCGCTGCTATCCC ATGGGCGAGGGCACT CCGGAGCAGGTTGCA
TGCGCTGCTATCCCC TGGGCGAGGGCACTT CGGAGCAGGTTGCAG
GCGCTGCTATCCCCA GGGCGAGGGCACTTG GGAGCAGGTTGCAGA CGCTGCTATCCCCAC GGCGAGGGCACTTGT GAGCAGGTTGCAGAC
GCTGCTATCCCCACC GCGAGGGCACTTGTG AGCAGGTTGCAGACA
CTGCTATCCCCACCC CGAGGGCACTTGTGA GCAGGTTGCAGACAA
TGCTATCCCCACCCG GAGGGCACTTGTGAG CAGGTTGCAGACAAT
GCTATCCCCACCCGG AGGGCACTTGTGAGA AGGTTGCAGACAATG CTATCCCCACCCGGG GGGCACTTGTGAGAA GGTTGCAGACAATGG
TATCCCCACCCGGGC GGCACTTGTGAGAAG GTTGCAGACAATGGC
ATCCCCACCCGGGCT GCACTTGTGAGAAGC TTGCAGACAATGGCG
TCCCCACCCGGGCTC CACTTGTGAGAAGCG TGCAGACAATGGCGA
CCCCACCCGGGCTCC ACTTGTGAGAAGCGC GCAGACAATGGCGAT CCCACCCGGGCTCCG CTTGTGAGAAGCGCC CAGACAATGGCGATG
CCACCCGGGCTCCGA TTGTGAGAAGCGCCG AGACAATGGCGATGA
CACCCGGGCTCCGAG TGTGAGAAGCGCCGG GACAATGGCGATGAC
ACCCGGGCTCCGAGC GTGAGAAGCGCCGGG ACAATGGCGATGACC
CCCGGGCTCCGAGCT TGAGAAGCGCCGGGA CAATGGCGATGACCA CCGGGCTCCGAGCTG GAGAAGCGCCGGGAC AATGGCGATGACCAC
CGGGCTCCGAGCTGC AGAAGCGCCGGGACG ATGGCGATGACCACT
GGGCTCCGAGCTGCC GAAGCGCCGGGACGC TGGCGATGACCACTC
GGCTCCGAGCTGCCC AAGCGCCGGGACGCC GGCGATGACCACTCA
GCTCCGAGCTGCCCC AGCGCCGGGACGCCG GCGATGACCACTCAG CTCCGAGCTGCCCCT GCGCCGGGACGCCGA CGATGACCACTCAGA
TCCGAGCTGCCCCTG CGCCGGGACGCCGAG GATGACCACTCAGAA
CCGAGCTGCCCCTGC GCCGGGACGCCGAGT ATGACCACTCAGAAG
CGAGCTGCCCCTGCA CCGGGACGCCGAGTA TGACCACTCAGAAGG
GAGCTGCCCCTGCAG CGGGACGCCGAGTAT GACCACTCAGAAGGA AGCTGCCCCTGCAGG GGGACGCCGAGTATG ACCACTCAGAAGGAG
GCTGCCCCTGCAGGC GGACGCCGAGTATGG CCACTCAGAAGGAGG
CTGCCCCTGCAGGCG GACGCCGAGTATGGC CACTCAGAAGGAGGC
TGCCCCTGCAGGCGC ACGCCGAGTATGGCG ACTCAGAAGGAGGCC
GCCCCTGCAGGCGCT CGCCGAGTATGGCGC CTCAGAAGGAGGCCT CCCCTGCAGGCGCTG GCCGAGTATGGCGCC TCAGAAGGAGGCCTG
CCCTGCAGGCGCTGG CCGAGTATGGCGCCA CAGAAGGAGGCCTGG
CCTGCAGGCGCTGGT CGAGTATGGCGCCAG AGAAGGAGGCCTGGT
CTGCAGGCGCTGGTC GAGTATGGCGCCAGC GAAGGAGGCCTGGTG
TGCAGGCGCTGGTCA AGTATGGCGCCAGCC AAGGAGGCCTGGTGG GCAGGCGCTGGTCAT GTATGGCGCCAGCCC AGGAGGCCTGGTGGA
CAGGCGCTGGTCATG TATGGCGCCAGCCCG GGAGGCCTGGTGGAG
AGGCGCTGGTCATGG ATGGCGCCAGCCCGG GAGGCCTGGTGGAGA
GGCGCTGGTCATGGG TGGCGCCAGCCCGGA AGGCCTGGTGGAGAA
GCGCTGGTCATGGGC GGCGCCAGCCCGGAG GGCCTGGTGGAGAAC CGCTGGTCATGGGCG GCGCCAGCCCGGAGC GCCTGGTGGAGAACC
GCTGGTCATGGGCGA CGCCAGCCCGGAGCA CCTGGTGGAGAACCA CTGGTGGAGAACCAC AGTGCTGGCCGGAAG CGGGAGAAGGTCACT TGGTGGAGAACCACG GTGCTGGCCGGAAGC GGGAGAAGGTCACTG GGTGGAGAACCACGT TGCTGGCCGGAAGCC GGAGAAGGTCACTGA GTGGAGAACCACGTG GCTGGCCGGAAGCCC GAGAAGGTCACTGAG TGGAGAACCACGTGG CTGGCCGGAAGCCCC AGAAGGTCACTGAGC GGAGAACCACGTGGA TGGCCGGAAGCCCCT GAAGGTCACTGAGCA GAGAACCACGTGGAC GGCCGGAAGCCCCTC AAGGTCACTGAGCAG AGAACCACGTGGACA GCCGGAAGCCCCTCA AGGTCACTGAGCAGC GAACCACGTGGACAG CCGGAAGCCCCTCAA GGTCACTGAGCAGCA AACCACGTGGACAGC CGGAAGCCCCTCAAG GTCACTGAGCAGCAC ACCACGTGGACAGCA GGAAGCCCCTCAAGT TCACTGAGCAGCACC CCACGTGGACAGCAC GAAGCCCCTCAAGTC CACTGAGCAGCACCG CACGTGGACAGCACC AAGCCCCTCAAGTCG ACTGAGCAGCACCGG ACGTGGACAGCACCA AGCCCCTCAAGTCGG CTGAGCAGCACCGGC CGTGGACAGCACCAT GCCCCTCAAGTCGGG TGAGCAGCACCGGCA GTGGACAGCACCATG CCCCTCAAGTCGGGT GAGCAGCACCGGCAG TGGACAGCACCATGA CCCTCAAGTCGGGTA AGCAGCACCGGCAGA GGACAGCACCATGAA CCTCAAGTCGGGTAT GCAGCACCGGCAGAT GACAGCACCATGAAC CTCAAGTCGGGTATG CAGCACCGGCAGATG ACAGCACCATGAACA TCAAGTCGGGTATGA AGCACCGGCAGATGG CAGCACCATGAACAT CAAGTCGGGTATGAA GCACCGGCAGATGGG AGCACCATGAACATG AAGTCGGGTATGAAG CACCGGCAGATGGGC GCACCATGAACATGT AGTCGGGTATGAAGG ACCGGCAGATGGGCA CACCATGAACATGTT GTCGGGTATGAAGGA CCGGCAGATGGGCAA ACCATGAACATGTTG TCGGGTATGAAGGAG CGGCAGATGGGCAAG CCATGAACATGTTGG CGGGTATGAAGGAGC GGCAGATGGGCAAGG CATGAACATGTTGGG GGGTATGAAGGAGCT GCAGATGGGCAAGGG ATGAACATGTTGGGC GGTATGAAGGAGCTG CAGATGGGCAAGGGT TGAACATGTTGGGCG GTATGAAGGAGCTGG AGATGGGCAAGGGTG GAACATGTTGGGCGG TATGAAGGAGCTGGC GATGGGCAAGGGTGG AACATGTTGGGCGGG ATGAAGGAGCTGGCC ATGGGCAAGGGTGGC ACATGTTGGGCGGGG TGAAGGAGCTGGCCG TGGGCAAGGGTGGCA CATGTTGGGCGGGGG GAAGGAGCTGGCCGT GGGCAAGGGTGGCAA ATGTTGGGCGGGGGA AAGGAGCTGGCCGTG GGCAAGGGTGGCAAG TGTTGGGCGGGGGAG AGGAGCTGGCCGTGT GCAAGGGTGGCAAGC GTTGGGCGGGGGAGG GGAGCTGGCCGTGTT CAAGGGTGGCAAGCA TTGGGCGGGGGAGGC GAGCTGGCCGTGTTC AAGGGTGGCAAGCAT TGGGCGGGGGAGGCA AGCTGGCCGTGTTCC AGGGTGGCAAGCATC GGGCGGGGGAGGCAG GCTGGCCGTGTTCCG GGGTGGCAAGCATCA GGCGGGGGAGGCAGT CTGGCCGTGTTCCGG GGTGGCAAGCATCAC GCGGGGGAGGCAGTG TGGCCGTGTTCCGGG GTGGCAAGCATCACC CGGGGGAGGCAGTGC GGCCGTGTTCCGGGA TGGCAAGCATCACCT GGGGGAGGCAGTGCT GCCGTGTTCCGGGAG GGCAAGCATCACCTT GGGGAGGCAGTGCTG CCGTGTTCCGGGAGA GCAAGCATCACCTTG GGGAGGCAGTGCTGG CGTGTTCCGGGAGAA CAAGCATCACCTTGG GGAGGCAGTGCTGGC GTGTTCCGGGAGAAG AAGCATCACCTTGGC GAGGCAGTGCTGGCC TGTTCCGGGAGAAGG AGCATCACCTTGGCC AGGCAGTGCTGGCCG GTTCCGGGAGAAGGT GCATCACCTTGGCCT GGCAGTGCTGGCCGG TTCCGGGAGAAGGTC CATCACCTTGGCCTG GCAGTGCTGGCCGGA TCCGGGAGAAGGTCA ATCACCTTGGCCTGG CAGTGCTGGCCGGAA CCGGGAGAAGGTCAC TCACCTTGGCCTGGA CACCTTGGCCTGGAG CCCTGCCAACAGGAA CTTCCGGATGAGCGG
ACCTTGGCCTGGAGG CCTGCCAACAGGAAC TTCCGGATGAGCGGG
CCTTGGCCTGGAGGA CTGCCAACAGGAACT TCCGGATGAGCGGGG
CTTGGCCTGGAGGAG TGCCAACAGGAACTG CCGGATGAGCGGGGC TTGGCCTGGAGGAGC GCCAACAGGAACTGG CGGATGAGCGGGGCC
TGGCCTGGAGGAGCC CCAACAGGAACTGGA GGATGAGCGGGGCCC
GGCCTGGAGGAGCCC CAACAGGAACTGGAC GATGAGCGGGGCCCT
GCCTGGAGGAGCCCA AACAGGAACTGGACC ATGAGCGGGGCCCTC
CCTGGAGGAGCCCAA ACAGGAACTGGACCA TGAGCGGGGCCCTCT CTGGAGGAGCCCAAG CAGGAACTGGACCAG GAGCGGGGCCCTCTG
TGGAGGAGCCCAAGA AGGAACTGGACCAGG AGCGGGGCCCTCTGG
GGAGGAGCCCAAGAA GGAACTGGACCAGGT GCGGGGCCCTCTGGA
GAGGAGCCCAAGAAG GAACTGGACCAGGTC CGGGGCCCTCTGGAG
AGGAGCCCAAGAAGC AACTGGACCAGGTCC GGGGCCCTCTGGAGC GGAGCCCAAGAAGCT ACTGGACCAGGTCCT GGGCCCTCTGGAGCA
GAGCCCAAGAAGCTG CTGGACCAGGTCCTG GGCCCTCTGGAGCAC
AGCCCAAGAAGCTGC TGGACCAGGTCCTGG GCCCTCTGGAGCACC
GCCCAAGAAGCTGCG GGACCAGGTCCTGGA CCCTCTGGAGCACCT
CCCAAGAAGCTGCGA GACCAGGTCCTGGAG CCTCTGGAGCACCTC CCAAGAAGCTGCGAC ACCAGGTCCTGGAGC CTCTGGAGCACCTCT
CAAGAAGCTGCGACC CCAGGTCCTGGAGCG TCTGGAGCACCTCTA
AAGAAGCTGCGACCA CAGGTCCTGGAGCGG CTGGAGCACCTCTAC
AGAAGCTGCGACCAC AGGTCCTGGAGCGGA TGGAGCACCTCTACT
GAAGCTGCGACCACC GGTCCTGGAGCGGAT GGAGCACCTCTACTC AAGCTGCGACCACCC GTCCTGGAGCGGATC GAGCACCTCTACTCC
AGCTGCGACCACCCC TCCTGGAGCGGATCT AGCACCTCTACTCCC
GCTGCGACCACCCCC CCTGGAGCGGATCTC GCACCTCTACTCCCT
CTGCGACCACCCCCT CTGGAGCGGATCTCC CACCTCTACTCCCTG
TGCGACCACCCCCTG TGGAGCGGATCTCCA ACCTCTACTCCCTGC GCGACCACCCCCTGC GGAGCGGATCTCCAC CCTCTACTCCCTGCA
CGACCACCCCCTGCC GAGCGGATCTCCACC CTCTACTCCCTGCAC
GACCACCCCCTGCCA AGCGGATCTCCACCA TCTACTCCCTGCACA
ACCACCCCCTGCCAG GCGGATCTCCACCAT CTACTCCCTGCACAT
CCACCCCCTGCCAGG CGGATCTCCACCATG TACTCCCTGCACATC CACCCCCTGCCAGGA GGATCTCCACCATGC ACTCCCTGCACATCC
ACCCCCTGCCAGGAC GATCTCCACCATGCG CTCCCTGCACATCCC
CCCCCTGCCAGGACT ATCTCCACCATGCGC TCCCTGCACATCCCC
CCCCTGCCAGGACTC TCTCCACCATGCGCC CCCTGCACATCCCCA
CCCTGCCAGGACTCC CTCCACCATGCGCCT CCTGCACATCCCCAA CCTGCCAGGACTCCC TCCACCATGCGCCTT CTGCACATCCCCAAC
CTGCCAGGACTCCCT CCACCATGCGCCTTC TGCACATCCCCAACT
TGCCAGGACTCCCTG CACCATGCGCCTTCC GCACATCCCCAACTG
GCCAGGACTCCCTGC ACCATGCGCCTTCCG CACATCCCCAACTGT
CCAGGACTCCCTGCC CCATGCGCCTTCCGG ACATCCCCAACTGTG CAGGACTCCCTGCCA CATGCGCCTTCCGGA CATCCCCAACTGTGA
AGGACTCCCTGCCAA ATGCGCCTTCCGGAT ATCCCCAACTGTGAC
GGACTCCCTGCCAAC TGCGCCTTCCGGATG TCCCCAACTGTGACA
GACTCCCTGCCAACA GCGCCTTCCGGATGA CCCCAACTGTGACAA
ACTCCCTGCCAACAG CGCCTTCCGGATGAG CCCAACTGTGACAAG CTCCCTGCCAACAGG GCCTTCCGGATGAGC CCAACTGTGACAAGC
TCCCTGCCAACAGGA CCTTCCGGATGAGCG CAACTGTGACAAGCA AACTGTGACAAGCAT AACGGGCAGCGTGGG ATCCAGGGAGCCCCC ACTGTGACAAGCATG ACGGGCAGCGTGGGG TCCAGGGAGCCCCCA CTGTGACAAGCATGG CGGGCAGCGTGGGGA CCAGGGAGCCCCCAC TGTGACAAGCATGGC GGGCAGCGTGGGGAG CAGGGAGCCCCCACC GTGACAAGCATGGCC GGCAGCGTGGGGAGT AGGGAGCCCCCACCA TGACAAGCATGGCCT GCAGCGTGGGGAGTG GGGAGCCCCCACCAT GACAAGCATGGCCTG CAGCGTGGGGAGTGC GGAGCCCCCACCATC ACAAGCATGGCCTGT AGCGTGGGGAGTGCT GAGCCCCCACCATCC CAAGCATGGCCTGTA GCGTGGGGAGTGCTG AGCCCCCACCATCCG AAGCATGGCCTGTAC CGTGGGGAGTGCTGG GCCCCCACCATCCGG AGCATGGCCTGTACA GTGGGGAGTGCTGGT CCCCCACCATCCGGG GCATGGCCTGTACAA TGGGGAGTGCTGGTG CCCCACCATCCGGGG CATGGCCTGTACAAC GGGGAGTGCTGGTGT CCCACCATCCGGGGG ATGGCCTGTACAACC GGGAGTGCTGGTGTG CCACCATCCGGGGGG TGGCCTGTACAACCT GGAGTGCTGGTGTGT CACCATCCGGGGGGA GGCCTGTACAACCTC GAGTGCTGGTGTGTG ACCATCCGGGGGGAC GCCTGTACAACCTCA AGTGCTGGTGTGTGA CCATCCGGGGGGACC CCTGTACAACCTCAA GTGCTGGTGTGTGAA CATCCGGGGGGACCC CTGTACAACCTCAAA TGCTGGTGTGTGAAC ATCCGGGGGGACCCC TGTACAACCTCAAAC GCTGGTGTGTGAACC TCCGGGGGGACCCCG GTACAACCTCAAACA CTGGTGTGTGAACCC CCGGGGGGACCCCGA TACAACCTCAAACAG TGGTGTGTGAACCCC CGGGGGGACCCCGAG ACAACCTCAAACAGT GGTGTGTGAACCCCA GGGGGGACCCCGAGT CAACCTCAAACAGTG GTGTGTGAACCCCAA GGGGGACCCCGAGTG AACCTCAAACAGTGC TGTGTGAACCCCAAC GGGGACCCCGAGTGT ACCTCAAACAGTGCA GTGTGAACCCCAACA GGGACCCCGAGTGTC CCTCAAACAGTGCAA TGTGAACCCCAACAC GGACCCCGAGTGTCA CTCAAACAGTGCAAG GTGAACCCCAACACC GACCCCGAGTGTCAT TCAAACAGTGCAAGA TGAACCCCAACACCG ACCCCGAGTGTCATC CAAACAGTGCAAGAT GAACCCCAACACCGG CCCCGAGTGTCATCT AAACAGTGCAAGATG AACCCCAACACCGGG CCCGAGTGTCATCTC AACAGTGCAAGATGT ACCCCAACACCGGGA CCGAGTGTCATCTCT ACAGTGCAAGATGTC CCCCAACACCGGGAA CGAGTGTCATCTCTT CAGTGCAAGATGTCT CCCAACACCGGGAAG GAGTGTCATCTCTTC AGTGCAAGATGTCTC CCAACACCGGGAAGC AGTGTCATCTCTTCT GTGCAAGATGTCTCT CAACACCGGGAAGCT GTGTCATCTCTTCTA TGCAAGATGTCTCTG AACACCGGGAAGCTG TGTCATCTCTTCTAC GCAAGATGTCTCTGA ACACCGGGAAGCTGA GTCATCTCTTCTACA CAAGATGTCTCTGAA CACCGGGAAGCTGAT TCATCTCTTCTACAA AAGATGTCTCTGAAC ACCGGGAAGCTGATC CATCTCTTCTACAAT AGATGTCTCTGAACG CCGGGAAGCTGATCC ATCTCTTCTACAATG GATGTCTCTGAACGG CGGGAAGCTGATCCA TCTCTTCTACAATGA ATGTCTCTGAACGGG GGGAAGCTGATCCAG CTCTTCTACAATGAG TGTCTCTGAACGGGC GGAAGCTGATCCAGG TCTTCTACAATGAGC GTCTCTGAACGGGCA GAAGCTGATCCAGGG CTTCTACAATGAGCA TCTCTGAACGGGCAG AAGCTGATCCAGGGA TTCTACAATGAGCAG CTCTGAACGGGCAGC AGCTGATCCAGGGAG TCTACAATGAGCAGC TCTGAACGGGCAGCG GCTGATCCAGGGAGC CTACAATGAGCAGCA CTGAACGGGCAGCGT CTGATCCAGGGAGCC TACAATGAGCAGCAG TGAACGGGCAGCGTG TGATCCAGGGAGCCC ACAATGAGCAGCAGG GAACGGGCAGCGTGG GATCCAGGGAGCCCC CAATGAGCAGCAGGA AATGAGCAGCAGGAG GCAGCCAGCCGGTGC GCAGAAAACGGAGAG
ATGAGCAGCAGGAGG CAGCCAGCCGGTGCC CAGAAAACGGAGAGT
TGAGCAGCAGGAGGC AGCCAGCCGGTGCCT AGAAAACGGAGAGTG
GAGCAGCAGGAGGCT GCCAGCCGGTGCCTG GAAAACGGAGAGTGC AGCAGCAGGAGGCTT CCAGCCGGTGCCTGG AAAACGGAGAGTGCT
GCAGCAGGAGGCTTG CAGCCGGTGCCTGGC AAACGGAGAGTGCTT
CAGCAGGAGGCTTGC AGCCGGTGCCTGGCG AACGGAGAGTGCTTG
AGCAGGAGGCTTGCG GCCGGTGCCTGGCGC ACGGAGAGTGCTTGG
GCAGGAGGCTTGCGG CCGGTGCCTGGCGCC CGGAGAGTGCTTGGG CAGGAGGCTTGCGGG CGGTGCCTGGCGCCC GGAGAGTGCTTGGGT
AGGAGGCTTGCGGGG GGTGCCTGGCGCCCC GAGAGTGCTTGGGTG
GGAGGCTTGCGGGGT GTGCCTGGCGCCCCT AGAGTGCTTGGGTGG
GAGGCTTGCGGGGTG TGCCTGGCGCCCCTG GAGTGCTTGGGTGGT
AGGCTTGCGGGGTGC GCCTGGCGCCCCTGC AGTGCTTGGGTGGTG GGCTTGCGGGGTGCA CCTGGCGCCCCTGCC GTGCTTGGGTGGTGG
GCTTGCGGGGTGCAC CTGGCGCCCCTGCCC TGCTTGGGTGGTGGG
CTTGCGGGGTGCACA TGGCGCCCCTGCCCC GCTTGGGTGGTGGGT
TTGCGGGGTGCACAC GGCGCCCCTGCCCCC CTTGGGTGGTGGGTG
TGCGGGGTGCACACC GCGCCCCTGCCCCCC TTGGGTGGTGGGTGC GCGGGGTGCACACCC CGCCCCTGCCCCCCG TGGGTGGTGGGTGCT
CGGGGTGCACACCCA GCCCCTGCCCCCCGC GGGTGGTGGGTGCTG
GGGGTGCACACCCAG CCCCTGCCCCCCGCC GGTGGTGGGTGCTGG
GGGTGCACACCCAGC CCCTGCCCCCCGCCC GTGGTGGGTGCTGGA
GGTGCACACCCAGCG CCTGCCCCCCGCCCC TGGTGGGTGCTGGAG GTGCACACCCAGCGG CTGCCCCCCGCCCCT GGTGGGTGCTGGAGG
TGCACACCCAGCGGA TGCCCCCCGCCCCTC GTGGGTGCTGGAGGA
GCACACCCAGCGGAT GCCCCCCGCCCCTCT TGGGTGCTGGAGGAT
CACACCCAGCGGATG CCCCCCGCCCCTCTC GGGTGCTGGAGGATT
ACACCCAGCGGATGC CCCCCGCCCCTCTCC GGTGCTGGAGGATTT CACCCAGCGGATGCA CCCCGCCCCTCTCCA GTGCTGGAGGATTTT
ACCCAGCGGATGCAG CCCGCCCCTCTCCAA TGCTGGAGGATTTTC
CCCAGCGGATGCAGT CCGCCCCTCTCCAAA GCTGGAGGATTTTCC
CCAGCGGATGCAGTA CGCCCCTCTCCAAAC CTGGAGGATTTTCCA
CAGCGGATGCAGTAG GCCCCTCTCCAAACA TGGAGGATTTTCCAG AGCGGATGCAGTAGA CCCCTCTCCAAACAC GGAGGATTTTCCAGT
GCGGATGCAGTAGAC CCCTCTCCAAACACC GAGGATTTTCCAGTT
CGGATGCAGTAGACC CCTCTCCAAACACCG AGGATTTTCCAGTTC
GGATGCAGTAGACCG CTCTCCAAACACCGG GGATTTTCCAGTTCT
GATGCAGTAGACCGC TCTCCAAACACCGGC GATTTTCCAGTTCTG ATGCAGTAGACCGCA CTCCAAACACCGGCA ATTTTCCAGTTCTGA
TGCAGTAGACCGCAG TCCAAACACCGGCAG TTTTCCAGTTCTGAC
GCAGTAGACCGCAGC CCAAACACCGGCAGA TTTCCAGTTCTGACA
CAGTAGACCGCAGCC CAAACACCGGCAGAA TTCCAGTTCTGACAC
AGTAGACCGCAGCCA AAACACCGGCAGAAA TCCAGTTCTGACACA GTAGACCGCAGCCAG AACACCGGCAGAAAA CCAGTTCTGACACAC
TAGACCGCAGCCAGC ACACCGGCAGAAAAC CAGTTCTGACACACG
AGACCGCAGCCAGCC CACCGGCAGAAAACG AGTTCTGACACACGT
GACCGCAGCCAGCCG ACCGGCAGAAAACGG GTTCTGACACACGTA
ACCGCAGCCAGCCGG CCGGCAGAAAACGGA TTCTGACACACGTAT CCGCAGCCAGCCGGT CGGCAGAAAACGGAG TCTGACACACGTATT
CGCAGCCAGCCGGTG GGCAGAAAACGGAGA CTGACACACGTATTT TGACACACGTATTTA CCCGGCCTCTCTCTT TCCCCGGGGGAGGAA
GACACACGTATTTAT CCGGCCTCTCTCTTC CCCCGGGGGAGGAAG
ACACACGTATTTATA CGGCCTCTCTCTTCC CCCGGGGGAGGAAGG
CACACGTATTTATAT GGCCTCTCTCTTCCC CCGGGGGAGGAAGGG ACACGTATTTATATT GCCTCTCTCTTCCCA CGGGGGAGGAAGGGG
CACGTATTTATATTT CCTCTCTCTTCCCAG GGGGGAGGAAGGGGG
ACGTATTTATATTTG CTCTCTCTTCCCAGC GGGGAGGAAGGGGGT
CGTATTTATATTTGG TCTCTCTTCCCAGCT GGGAGGAAGGGGGTT
GTATTTATATTTGGA CTCTCTTCCCAGCTG GGAGGAAGGGGGTTG TATTTATATTTGGAA TCTCTTCCCAGCTGC GAGGAAGGGGGTTGT
ATTTATATTTGGAAA CTCTTCCCAGCTGCA AGGAAGGGGGTTGTG
TTTATATTTGGAAAG TCTTCCCAGCTGCAG GGAAGGGGGTTGTGG
TTATATTTGGAAAGA CTTCCCAGCTGCAGA GAAGGGGGTTGTGGT
TATATTTGGAAAGAG TTCCCAGCTGCAGAT AAGGGGGTTGTGGTC ATATTTGGAAAGAGA TCCCAGCTGCAGATG AGGGGGTTGTGGTCG
TATTTGGAAAGAGAC CCCAGCTGCAGATGC GGGGGTTGTGGTCGG
ATTTGGAAAGAGACC CCAGCTGCAGATGCC GGGGTTGTGGTCGGG
TTTGGAAAGAGACCA CAGCTGCAGATGCCA GGGTTGTGGTCGGGG
TTGGAAAGAGACCAG AGCTGCAGATGCCAC GGTTGTGGTCGGGGA TGGAAAGAGACCAGC GCTGCAGATGCCACA GTTGTGGTCGGGGAG
GGAAAGAGACCAGCA CTGCAGATGCCACAC TTGTGGTCGGGGAGC
GAAAGAGACCAGCAC TGCAGATGCCACACC TGTGGTCGGGGAGCT
AAAGAGACCAGCACC GCAGATGCCACACCT GTGGTCGGGGAGCTG
AAGAGACCAGCACCG CAGATGCCACACCTG TGGTCGGGGAGCTGG AGAGACCAGCACCGA AGATGCCACACCTGC GGTCGGGGAGCTGGG
GAGACCAGCACCGAG GATGCCACACCTGCT GTCGGGGAGCTGGGG
AGACCAGCACCGAGC ATGCCACACCTGCTC TCGGGGAGCTGGGGT
GACCAGCACCGAGCT TGCCACACCTGCTCC CGGGGAGCTGGGGTA
ACCAGCACCGAGCTC GCCACACCTGCTCCT GGGGAGCTGGGGTAC CCAGCACCGAGCTCG CCACACCTGCTCCTT GGGAGCTGGGGTACA
CAGCACCGAGCTCGG CACACCTGCTCCTTC GGAGCTGGGGTACAG
AGCACCGAGCTCGGC ACACCTGCTCCTTCT GAGCTGGGGTACAGG
GCACCGAGCTCGGCA CACCTGCTCCTTCTT AGCTGGGGTACAGGT
CACCGAGCTCGGCAC ACCTGCTCCTTCTTG GCTGGGGTACAGGTT ACCGAGCTCGGCACC CCTGCTCCTTCTTGC CTGGGGTACAGGTTT
CCGAGCTCGGCACCT CTGCTCCTTCTTGCT TGGGGTACAGGTTTG
CGAGCTCGGCACCTC TGCTCCTTCTTGCTT GGGGTACAGGTTTGG
GAGCTCGGCACCTCC GCTCCTTCTTGCTTT GGGTACAGGTTTGGG
AGCTCGGCACCTCCC CTCCTTCTTGCTTTC GGTACAGGTTTGGGG GCTCGGCACCTCCCC TCCTTCTTGCTTTCC GTACAGGTTTGGGGA
CTCGGCACCTCCCCG CCTTCTTGCTTTCCC TACAGGTTTGGGGAG
TCGGCACCTCCCCGG CTTCTTGCTTTCCCC ACAGGTTTGGGGAGG
CGGCACCTCCCCGGC TTCTTGCTTTCCCCG CAGGTTTGGGGAGGG
GGCACCTCCCCGGCC TCTTGCTTTCCCCGG AGGTTTGGGGAGGGG GCACCTCCCCGGCCT CTTGCTTTCCCCGGG GGTTTGGGGAGGGGG
CACCTCCCCGGCCTC TTGCTTTCCCCGGGG GTTTGGGGAGGGGGA
ACCTCCCCGGCCTCT TGCTTTCCCCGGGGG TTTGGGGAGGGGGAA
CCTCCCCGGCCTCTC GCTTTCCCCGGGGGA TTGGGGAGGGGGAAG
CTCCCCGGCCTCTCT CTTTCCCCGGGGGAG TGGGGAGGGGGAAGA TCCCCGGCCTCTCTC TTTCCCCGGGGGAGG GGGGAGGGGGAAGAG
CCCCGGCCTCTCTCT TTCCCCGGGGGAGGA GGGAGGGGGAAGAGA GGAGGGGGAAGAGAA AGATTAAAGGAAGGA
GAGGGGGAAGAGAAA GATTAAAGGAAGGAA
AGGGGGAAGAGAAAT ATTAAAGGAAGGAAA
GGGGGAAGAGAAATT TTAAAGGAAGGAAAA GGGGAAGAGAAATTT TAAAGGAAGGAAAAG
GGGAAGAGAAATTTT AAAGGAAGGAAAAGT
GGAAGAGAAATTTTT
GAAGAGAAATTTTTA
AAGAGAAATTTTTAT AGAGAAATTTTTATT
GAGAAATTTTTATTT
AGAAATTTTTATTTT
GAAATTTTTATTTTT
AAATTTTTATTTTTG AATTTTTATTTTTGA
ATTTTTATTTTTGAA
TTTTTATTTTTGAAC
TTTTATTTTTGAACC
TTTATTTTTGAACCC TTATTTTTGAACCCC
TATTTTTGAACCCCT
ATTTTTGAACCCCTG
TTTTTGAACCCCTGT
TTTTGAACCCCTGTG TTTGAACCCCTGTGT
TTGAACCCCTGTGTC
TGAACCCCTGTGTCC
GAACCCCTGTGTCCC
AACCCCTGTGTCCCT ACCCCTGTGTCCCTT
CCCCTGTGTCCCTTT
CCCTGTGTCCCTTTT
CCTGTGTCCCTTTTG
CTGTGTCCCTTTTGC TGTGTCCCTTTTGCA
GTGTCCCTTTTGCAT
TGTCCCTTTTGCATA
GTCCCTTTTGCATAA
TCCCTTTTGCATAAG CCCTTTTGCATAAGA
CCTTTTGCATAAGAT
CTTTTGCATAAGATT
TTTTGCATAAGATTA
TTTGCATAAGATTAA TTGCATAAGATTAAA
TGCATAAGATTAAAG
GCATAAGATTAAAGG
CATAAGATTAAAGGA
ATAAGATTAAAGGAA TAAGATTAAAGGAAG
AAGATTAAAGGAAGG EXAMPLE 7
Antisense oligonucleotides to IGFBP3 may be selected from molecules capable of interacting with one or more of the following sense oligonucleotides:
CTCAGCGCCCAGCCG GCCGTGTACTGTCGC GCAGCGTGCCCCGGT
TCAGCGCCCAGCCGC CCGTGTACTGTCGCC CAGCGTGCCCCGGTT
CAGCGCCCAGCCGCT CGTGTACTGTCGCCC AGCGTGCCCCGGTTG AGCGCCCAGCCGCTT GTGTACTGTCGCCCC GCGTGCCCCGGTTGC
GCGCCCAGCCGCTTC TGTACTGTCGCCCCA CGTGCCCCGGTTGCA
CGCCCAGCCGCTTCC GTACTGTCGCCCCAT GTGCCCCGGTTGCAG
GCCCAGCCGCTTCCT TACTGTCGCCCCATC TGCCCCGGTTGCAGG
CCCAGCCGCTTCCTG ACTGTCGCCCCATCC GCCCCGGTTGCAGGC CCAGCCGCTTCCTGC CTGTCGCCCCATCCC CCCCGGTTGCAGGCG
CAGCCGCTTCCTGCC TGTCGCCCCATCCCT CCCGGTTGCAGGCGT
AGCCGCTTCCTGCCT GTCGCCCCATCCCTG CCGGTTGCAGGCGTC
GCCGCTTCCTGCCTG TCGCCCCATCCCTGC CGGTTGCAGGCGTCA
CCGCTTCCTGCCTGG CGCCCCATCCCTGCG GGTTGCAGGCGTCAT CGCTTCCTGCCTGGA GCCCCATCCCTGCGC GTTGCAGGCGTCATG
GCTTCCTGCCTGGAT CCCCATCCCTGCGCG TTGCAGGCGTCATGC
CTTCCTGCCTGGATT CCCATCCCTGCGCGC TGCAGGCGTCATGCA
TTCCTGCCTGGATTC CCATCCCTGCGCGCC GCAGGCGTCATGCAG
TCCTGCCTGGATTCC CATCCCTGCGCGCCC CAGGCGTCATGCAGC CCTGCCTGGATTCCA ATCCCTGCGCGCCCA AGGCGTCATGCAGCG
CTGCCTGGATTCCAC TCCCTGCGCGCCCAG GGCGTCATGCAGCGG
TGCCTGGATTCCACA CCCTGCGCGCCCAGC GCGTCATGCAGCGGG
GCCTGGATTCCACAG CCTGCGCGCCCAGCC CGTCATGCAGCGGGC
CCTGGATTCCACAGC CTGCGCGCCCAGCCT GTCATGCAGCGGGCG CTGGATTCCACAGCT TGCGCGCCCAGCCTG TCATGCAGCGGGCGC
TGGATTCCACAGCTT GCGCGCCCAGCCTGC CATGCAGCGGGCGCG
GGATTCCACAGCTTC CGCGCCCAGCCTGCC ATGCAGCGGGCGCGA
GATTCCACAGCTTCG GCGCCCAGCCTGCCA TGCAGCGGGCGCGAC
ATTCCACAGCTTCGC CGCCCAGCCTGCCAA GCAGCGGGCGCGACC TTCCACAGCTTCGCG GCCCAGCCTGCCAAG CAGCGGGCGCGACCC
TCCACAGCTTCGCGC CCCAGCCTGCCAAGC AGCGGGCGCGACCCA
CCACAGCTTCGCGCC CCAGCCTGCCAAGCA GCGGGCGCGACCCAC
CACAGCTTCGCGCCG CAGCCTGCCAAGCAG CGGGCGCGACCCACG
ACAGCTTCGCGCCGT AGCCTGCCAAGCAGC GGGCGCGACCCACGC CAGCTTCGCGCCGTG GCCTGCCAAGCAGCG GGCGCGACCCACGCT
AGCTTCGCGCCGTGT CCTGCCAAGCAGCGT GCGCGACCCACGCTC
GCTTCGCGCCGTGTA CTGCCAAGCAGCGTG CGCGACCCACGCTCT
CTTCGCGCCGTGTAC TGCCAAGCAGCGTGC GCGACCCACGCTCTG
TTCGCGCCGTGTACT GCCAAGCAGCGTGCC CGACCCACGCTCTGG TCGCGCCGTGTACTG CCAAGCAGCGTGCCC GACCCACGCTCTGGG
CGCGCCGTGTACTGT CAAGCAGCGTGCCCC ACCCACGCTCTGGGC
GCGCCGTGTACTGTC AAGCAGCGTGCCCCG CCCACGCTCTGGGCC
CGCCGTGTACTGTCG AGCAGCGTGCCCCGG CCACGCTCTGGGCCG CACGCTCTGGGCCGC GGTGGCGCGGGCTGG CGAGCCGTGCGACGC
ACGCTCTGGGCCGCT GTGGCGCGGGCTGGC GAGCCGTGCGACGCG
CGCTCTGGGCCGCTG TGGCGCGGGCTGGCG AGCCGTGCGACGCGC
GCTCTGGGCCGCTGC GGCGCGGGCTGGCGC GCCGTGCGACGCGCG CTCTGGGCCGCTGCG GCGCGGGCTGGCGCG CCGTGCGACGCGCGT
TCTGGGCCGCTGCGC CGCGGGCTGGCGCGA CGTGCGACGCGCGTG
CTGGGCCGCTGCGCT GCGGGCTGGCGCGAG GTGCGACGCGCGTGC
TGGGCCGCTGCGCTG CGGGCTGGCGCGAGC TGCGACGCGCGTGCA
GGGCCGCTGCGCTGA GGGCTGGCGCGAGCT GCGACGCGCGTGCAC GGCCGCTGCGCTGAC GGCTGGCGCGAGCTC CGACGCGCGTGCACT
GCCGCTGCGCTGACT GCTGGCGCGAGCTCG GACGCGCGTGCACTG
CCGCTGCGCTGACTC CTGGCGCGAGCTCGG ACGCGCGTGCACTGG
CGCTGCGCTGACTCT TGGCGCGAGCTCGGG CGCGCGTGCACTGGC
GCTGCGCTGACTCTG GGCGCGAGCTCGGGG GCGCGTGCACTGGCC CTGCGCTGACTCTGC GCGCGAGCTCGGGGG CGCGTGCACTGGCCC
TGCGCTGACTCTGCT CGCGAGCTCGGGGGG GCGTGCACTGGCCCA
GCGCTGACTCTGCTG GCGAGCTCGGGGGGC CGTGCACTGGCCCAG
CGCTGACTCTGCTGG CGAGCTCGGGGGGCT GTGCACTGGCCCAGT
GCTGACTCTGCTGGT GAGCTCGGGGGGCTT TGCACTGGCCCAGTG CTGACTCTGCTGGTG AGCTCGGGGGGCTTG GCACTGGCCCAGTGC
TGACTCTGCTGGTGC GCTCGGGGGGCTTGG CACTGGCCCAGTGCG
GACTCTGCTGGTGCT CTCGGGGGGCTTGGG ACTGGCCCAGTGCGC
ACTCTGCTGGTGCTG TCGGGGGGCTTGGGT CTGGCCCAGTGCGCG
CTCTGCTGGTGCTGC CGGGGGGCTTGGGTC TGGCCCAGTGCGCGC TCTGCTGGTGCTGCT GGGGGGCTTGGGTCC GGCCCAGTGCGCGCC
CTGCTGGTGCTGCTC GGGGGCTTGGGTCCC GCCCAGTGCGCGCCT
TGCTGGTGCTGCTCC GGGGCTTGGGTCCCG CCCAGTGCGCGCCTC
GCTGGTGCTGCTCCG GGGCTTGGGTCCCGT CCAGTGCGCGCCTCC
CTGGTGCTGCTCCGC GGCTTGGGTCCCGTG CAGTGCGCGCCTCCG TGGTGCTGCTCCGCG GCTTGGGTCCCGTGG AGTGCGCGCCTCCGC
GGTGCTGCTCCGCGG CTTGGGTCCCGTGGT GTGCGCGCCTCCGCC
GTGCTGCTCCGCGGG TTGGGTCCCGTGGTG TGCGCGCCTCCGCCC
TGCTGCTCCGCGGGC TGGGTCCCGTGGTGC GCGCGCCTCCGCCCG
GCTGCTCCGCGGGCC GGGTCCCGTGGTGCG CGCGCCTCCGCCCGC CTGCTCCGCGGGCCG GGTCCCGTGGTGCGC GCGCCTCCGCCCGCC
TGCTCCGCGGGCCGC GTCCCGTGGTGCGCT CGCCTCCGCCCGCCG
GCTCCGCGGGCCGCC TCCCGTGGTGCGCTG GCCTCCGCCCGCCGT
CTCCGCGGGCCGCCG CCCGTGGTGCGCTGC CCTCCGCCCGCCGTG
TCCGCGGGCCGCCGG CCGTGGTGCGCTGCG CTCCGCCCGCCGTGT CCGCGGGCCGCCGGT CGTGGTGCGCTGCGA TCCGCCCGCCGTGTG
CGCGGGCCGCCGGTG GTGGTGCGCTGCGAG CCGCCCGCCGTGTGC
GCGGGCCGCCGGTGG TGGTGCGCTGCGAGC CGCCCGCCGTGTGCG
CGGGCCGCCGGTGGC GGTGCGCTGCGAGCC GCCCGCCGTGTGCGC
GGGCCGCCGGTGGCG GTGCGCTGCGAGCCG CCCGCCGTGTGCGCG GGCCGCCGGTGGCGC TGCGCTGCGAGCCGT CCGCCGTGTGCGCGG
GCCGCCGGTGGCGCG GCGCTGCGAGCCGTG CGCCGTGTGCGCGGA
CCGCCGGTGGCGCGG CGCTGCGAGCCGTGC GCCGTGTGCGCGGAG
CGCCGGTGGCGCGGG GCTGCGAGCCGTGCG CCGTGTGCGCGGAGC
GCCGGTGGCGCGGGC CTGCGAGCCGTGCGA CGTGTGCGCGGAGCT CCGGTGGCGCGGGCT TGCGAGCCGTGCGAC GTGTGCGCGGAGCTG
CGGTGGCGCGGGCTG GCGAGCCGTGCGACG TGTGCGCGGAGCTGG GTGCGCGGAGCTGGT ACTGAGCGAGGGCCA CCTTCGCTGCCAGCC
TGCGCGGAGCTGGTG CTGAGCGAGGGCCAG CTTCGCTGCCAGCCG
GCGCGGAGCTGGTGC TGAGCGAGGGCCAGC TTCGCTGCCAGCCGT
CGCGGAGCTGGTGCG GAGCGAGGGCCAGCC TCGCTGCCAGCCGTC GCGGAGCTGGTGCGC AGCGAGGGCCAGCCG CGCTGCCAGCCGTCG
CGGAGCTGGTGCGCG GCGAGGGCCAGCCGT GCTGCCAGCCGTCGC
GGAGCTGGTGCGCGA CGAGGGCCAGCCGTG CTGCCAGCCGTCGCC
GAGCTGGTGCGCGAG GAGGGCCAGCCGTGC TGCCAGCCGTCGCCC
AGCTGGTGCGCGAGC AGGGCCAGCCGTGCG GCCAGCCGTCGCCCG GCTGGTGCGCGAGCC GGGCCAGCCGTGCGG CCAGCCGTCGCCCGA
CTGGTGCGCGAGCCG GGCCAGCCGTGCGGC CAGCCGTCGCCCGAC
TGGTGCGCGAGCCGG GCCAGCCGTGCGGCA AGCCGTCGCCCGACG
GGTGCGCGAGCCGGG CCAGCCGTGCGGCAT GCCGTCGCCCGACGA
GTGCGCGAGCCGGGC CAGCCGTGCGGCATC CCGTCGCCCGACGAG TGCGCGAGCCGGGCT AGCCGTGCGGCATCT CGTCGCCCGACGAGG
GCGCGAGCCGGGCTG GCCGTGCGGCATCTA GTCGCCCGACGAGGC
CGCGAGCCGGGCTGC CCGTGCGGCATCTAC TCGCCCGACGAGGCG
GCGAGCCGGGCTGCG CGTGCGGCATCTACA CGCCCGACGAGGCGC
CGAGCCGGGCTGCGG GTGCGGCATCTACAC GCCCGACGAGGCGCG GAGCCGGGCTGCGGC TGCGGCATCTACACC CCCGACGAGGCGCGA
AGCCGGGCTGCGGCT GCGGCATCTACACCG CCGACGAGGCGCGAC
GCCGGGCTGCGGCTG CGGCATCTACACCGA CGACGAGGCGCGACC
CCGGGCTGCGGCTGC GGCATCTACACCGAG GACGAGGCGCGACCG
CGGGCTGCGGCTGCT GCATCTACACCGAGC ACGAGGCGCGACCGC GGGCTGCGGCTGCTG CATCTACACCGAGCG CGAGGCGCGACCGCT
GGCTGCGGCTGCTGC ATCTACACCGAGCGC GAGGCGCGACCGCTG
GCTGCGGCTGCTGCC TCTACACCGAGCGCT AGGCGCGACCGCTGC
CTGCGGCTGCTGCCT CTACACCGAGCGCTG GGCGCGACCGCTGCA
TGCGGCTGCTGCCTG TACACCGAGCGCTGT GCGCGACCGCTGCAG GCGGCTGCTGCCTGA ACACCGAGCGCTGTG CGCGACCGCTGCAGG
CGGCTGCTGCCTGAC CACCGAGCGCTGTGG GCGACCGCTGCAGGC
GGCTGCTGCCTGACG ACCGAGCGCTGTGGC CGACCGCTGCAGGCG
GCTGCTGCCTGACGT CCGAGCGCTGTGGCT GACCGCTGCAGGCGC
CTGCTGCCTGACGTG CGAGCGCTGTGGCTC ACCGCTGCAGGCGCT TGCTGCCTGACGTGC GAGCGCTGTGGCTCC CCGCTGCAGGCGCTG
GCTGCCTGACGTGCG AGCGCTGTGGCTCCG CGCTGCAGGCGCTGC
CTGCCTGACGTGCGC GCGCTGTGGCTCCGG GCTGCAGGCGCTGCT
TGCCTGACGTGCGCA CGCTGTGGCTCCGGC CTGCAGGCGCTGCTG
GCCTGACGTGCGCAC GCTGTGGCTCCGGCC TGCAGGCGCTGCTGG CCTGACGTGCGCACT CTGTGGCTCCGGCCT GCAGGCGCTGCTGGA
CTGACGTGCGCACTG TGTGGCTCCGGCCTT CAGGCGCTGCTGGAC
TGACGTGCGCACTGA GTGGCTCCGGCCTTC AGGCGCTGCTGGACG
GACGTGCGCACTGAG TGGCTCCGGCCTTCG GGCGCTGCTGGACGG
ACGTGCGCACTGAGC GGCTCCGGCCTTCGC GCGCTGCTGGACGGC CGTGCGCACTGAGCG GCTCCGGCCTTCGCT CGCTGCTGGACGGCC
GTGCGCACTGAGCGA CTCCGGCCTTCGCTG GCTGCTGGACGGCCG
TGCGCACTGAGCGAG TCCGGCCTTCGCTGC CTGCTGGACGGCCGC
GCGCACTGAGCGAGG CCGGCCTTCGCTGCC TGCTGGACGGCCGCG
CGCACTGAGCGAGGG CGGCCTTCGCTGCCA GCTGGACGGCCGCGG GCACTGAGCGAGGGC GGCCTTCGCTGCCAG CTGGACGGCCGCGGG
CACTGAGCGAGGGCC GCCTTCGCTGCCAGC TGGACGGCCGCGGGC GGACGGCCGCGGGCT CTACCTGCTGCCAGC AGACCGCAGCGCCGG GACGGCCGCGGGCTC TACCTGCTGCCAGCG GACCGCAGCGCCGGC ACGGCCGCGGGCTCT ACCTGCTGCCAGCGC ACCGCAGCGCCGGCA CGGCCGCGGGCTCTG CCTGCTGCCAGCGCC CCGCAGCGCCGGCAG GGCCGCGGGCTCTGC CTGCTGCCAGCGCCG CGCAGCGCCGGCAGT GCCGCGGGCTCTGCG TGCTGCCAGCGCCGC GCAGCGCCGGCAGTG CCGCGGGCTCTGCGT GCTGCCAGCGCCGCC CAGCGCCGGCAGTGT CGCGGGCTCTGCGTC CTGCCAGCGCCGCCA AGCGCCGGCAGTGTG GCGGGCTCTGCGTCA TGCCAGCGCCGCCAG GCGCCGGCAGTGTGG CGGGCTCTGCGTCAA GCCAGCGCCGCCAGC CGCCGGCAGTGTGGA GGGCTCTGCGTCAAC CCAGCGCCGCCAGCT GCCGGCAGTGTGGAG GGCTCTGCGTCAACG CAGCGCCGCCAGCTC CCGGCAGTGTGGAGA GCTCTGCGTCAACGC AGCGCCGCCAGCTCC CGGCAGTGTGGAGAG CTCTGCGTCAACGCT GCGCCGCCAGCTCCA GGCAGTGTGGAGAGC TCTGCGTCAACGCTA CGCCGCCAGCTCCAG GCAGTGTGGAGAGCC CTGCGTCAACGCTAG GCCGCCAGCTCCAGG CAGTGTGGAGAGCCC TGCGTCAACGCTAGT CCGCCAGCTCCAGGA AGTGTGGAGAGCCCG GCGTCAACGCTAGTG CGCCAGCTCCAGGAA GTGTGGAGAGCCCGT CGTCAACGCTAGTGC GCCAGCTCCAGGAAA TGTGGAGAGCCCGTC GTCAACGCTAGTGCC CCAGCTCCAGGAAAT GTGGAGAGCCCGTCC TCAACGCTAGTGCCG CAGCTCCAGGAAATG TGGAGAGCCCGTCCG CAACGCTAGTGCCGT AGCTCCAGGAAATGC GGAGAGCCCGTCCGT AACGCTAGTGCCGTC GCTCCAGGAAATGCT GAGAGCCCGTCCGTC ACGCTAGTGCCGTCA CTCCAGGAAATGCTA AGAGCCCGTCCGTCT CGCTAGTGCCGTCAG TCCAGGAAATGCTAG GAGCCCGTCCGTCTC GCTAGTGCCGTCAGC CCAGGAAATGCTAGT AGCCCGTCCGTCTCC CTAGTGCCGTCAGCC CAGGAAATGCTAGTG GCCCGTCCGTCTCCA TAGTGCCGTCAGCCG AGGAAATGCTAGTGA CCCGTCCGTCTCCAG AGTGCCGTCAGCCGC GGAAATGCTAGTGAG CCGTCCGTCTCCAGC GTGCCGTCAGCCGCC GAAATGCTAGTGAGT CGTCCGTCTCCAGCA TGCCGTCAGCCGCCT AAATGCTAGTGAGTC GTCCGTCTCCAGCAC GCCGTCAGCCGCCTG AATGCTAGTGAGTCG TCCGTCTCCAGCACG CCGTCAGCCGCCTGC ATGCTAGTGAGTCGG CCGTCTCCAGCACGC CGTCAGCCGCCTGCG TGCTAGTGAGTCGGA CGTCTCCAGCACGCA GTCAGCCGCCTGCGC GCTAGTGAGTCGGAG GTCTCCAGCACGCAC TCAGCCGCCTGCGCG CTAGTGAGTCGGAGG TCTCCAGCACGCACC CAGCCGCCTGCGCGC TAGTGAGTCGGAGGA CTCCAGCACGCACCG AGCCGCCTGCGCGCC AGTGAGTCGGAGGAA TCCAGCACGCACCGG GCCGCCTGCGCGCCT GTGAGTCGGAGGAAG CCAGCACGCACCGGG CCGCCTGCGCGCCTA TGAGTCGGAGGAAGA CAGCACGCACCGGGT CGCCTGCGCGCCTAC GAGTCGGAGGAAGAC AGCACGCACCGGGTG GCCTGCGCGCCTACC AGTCGGAGGAAGACC GCACGCACCGGGTGT CCTGCGCGCCTACCT GTCGGAGGAAGACCG CACGCACCGGGTGTC CTGCGCGCCTACCTG TCGGAGGAAGACCGC ACGCACCGGGTGTCT TGCGCGCCTACCTGC CGGAGGAAGACCGCA CGCACCGGGTGTCTG GCGCGCCTACCTGCT GGAGGAAGACCGCAG GCACCGGGTGTCTGA CGCGCCTACCTGCTG GAGGAAGACCGCAGC CACCGGGTGTCTGAT GCGCCTACCTGCTGC AGGAAGACCGCAGCG ACCGGGTGTCTGATC CGCCTACCTGCTGCC GGAAGACCGCAGCGC CCGGGTGTCTGATCC GCCTACCTGCTGCCA GAAGACCGCAGCGCC CGGGTGTCTGATCCC CCTACCTGCTGCCAG AAGACCGCAGCGCCG GGGTGTCTGATCCCA GGTGTCTGATCCCAA GAAAGGGCATGCTAA GAGCACAGATACCCA
GTGTCTGATCCCAAG AAAGGGCATGCTAAA AGCACAGATACCCAG
TGTCTGATCCCAAGT AAGGGCATGCTAAAG GCACAGATACCCAGA
GTCTGATCCCAAGTT AGGGCATGCTAAAGA CACAGATACCCAGAA TCTGATCCCAAGTTC GGGCATGCTAAAGAC ACAGATACCCAGAAC
CTGATCCCAAGTTCC GGCATGCTAAAGACA CAGATACCCAGAACT
TGATCCCAAGTTCCA GCATGCTAAAGACAG AGATACCCAGAACTT
GATCCCAAGTTCCAC CATGCTAAAGACAGC GATACCCAGAACTTC
ATCCCAAGTTCCACC ATGCTAAAGACAGCC ATACCCAGAACTTCT TCCCAAGTTCCACCC TGCTAAAGACAGCCA TACCCAGAACTTCTC
CCCAAGTTCCACCCC GCTAAAGACAGCCAG ACCCAGAACTTCTCC
CCAAGTTCCACCCCC CTAAAGACAGCCAGC CCCAGAACTTCTCCT
CAAGTTCCACCCCCT TAAAGACAGCCAGCG CCAGAACTTCTCCTC
AAGTTCCACCCCCTC AAAGACAGCCAGCGC CAGAACTTCTCCTCC AGTTCCACCCCCTCC AAGACAGCCAGCGCT AGAACTTCTCCTCCG
GTTCCACCCCCTCCA AGACAGCCAGCGCTA GAACTTCTCCTCCGA
TTCCACCCCCTCCAT GACAGCCAGCGCTAC AACTTCTCCTCCGAG
TCCACCCCCTCCATT ACAGCCAGCGCTACA ACTTCTCCTCCGAGT
CCACCCCCTCCATTC CAGCCAGCGCTACAA CTTCTCCTCCGAGTC CACCCCCTCCATTCA AGCCAGCGCTACAAA TTCTCCTCCGAGTCC
ACCCCCTCCATTCAA GCCAGCGCTACAAAG TCTCCTCCGAGTCCA
CCCCCTCCATTCAAA CCAGCGCTACAAAGT CTCCTCCGAGTCCAA
CCCCTCCATTCAAAG CAGCGCTACAAAGTT TCCTCCGAGTCCAAG
CCCTCCATTCAAAGA AGCGCTACAAAGTTG CCTCCGAGTCCAAGC CCTCCATTCAAAGAT GCGCTACAAAGTTGA CTCCGAGTCCAAGCG
CTCCATTCAAAGATA CGCTACAAAGTTGAC TCCGAGTCCAAGCGG
TCCATTCAAAGATAA GCTACAAAGTTGACT CCGAGTCCAAGCGGG
CCATTCAAAGATAAT CTACAAAGTTGACTA CGAGTCCAAGCGGGA
CATTCAAAGATAATC TACAAAGTTGACTAC GAGTCCAAGCGGGAG ATTCAAAGATAATCA ACAAAGTTGACTACG AGTCCAAGCGGGAGA
TTCAAAGATAATCAT CAAAGTTGACTACGA GTCCAAGCGGGAGAC
TCAAAGATAATCATC AAAGTTGACTACGAG TCCAAGCGGGAGACA
CAAAGATAATCATCA AAGTTGACTACGAGT CCAAGCGGGAGACAG
AAAGATAATCATCAT AGTTGACTACGAGTC CAAGCGGGAGACAGA AAGATAATCATCATC GTTGACTACGAGTCT AAGCGGGAGACAGAA
AGATAATCATCATCA TTGACTACGAGTCTC AGCGGGAGACAGAAT
GATAATCATCATCAA TGACTACGAGTCTCA GCGGGAGACAGAATA
ATAATCATCATCAAG GACTACGAGTCTCAG CGGGAGACAGAATAT
TAATCATCATCAAGA ACTACGAGTCTCAGA GGGAGACAGAATATG AATCATCATCAAGAA CTACGAGTCTCAGAG GGAGACAGAATATGG
ATCATCATCAAGAAA TACGAGTCTCAGAGC GAGACAGAATATGGT
TCATCATCAAGAAAG ACGAGTCTCAGAGCA AGACAGAATATGGTC
CATCATCAAGAAAGG CGAGTCTCAGAGCAC GACAGAATATGGTCC
ATCATCAAGAAAGGG GAGTCTCAGAGCACA ACAGAATATGGTCCC TCATCAAGAAAGGGC AGTCTCAGAGCACAG CAGAATATGGTCCCT
CATCAAGAAAGGGCA GTCTCAGAGCACAGA AGAATATGGTCCCTG
ATCAAGAAAGGGCAT TCTCAGAGCACAGAT GAATATGGTCCCTGC
TCAAGAAAGGGCATG CTCAGAGCACAGATA AATATGGTCCCTGCC
CAAGAAAGGGCATGC TCAGAGCACAGATAC ATATGGTCCCTGCCG AAGAAAGGGCATGCT CAGAGCACAGATACC TATGGTCCCTGCCGT
AGAAAGGGCATGCTA AGAGCACAGATACCC ATGGTCCCTGCCGTA TGGTCCCTGCCGTAG CAATGTGCTGAGTCC ATTTTATAAGAAAAA GGTCCCTGCCGTAGA AATGTGCTGAGTCCC TTTTATAAGAAAAAG GTCCCTGCCGTAGAG ATGTGCTGAGTCCCA TTTATAAGAAAAAGC TCCCTGCCGTAGAGA TGTGCTGAGTCCCAG TTATAAGAAAAAGCA CCCTGCCGTAGAGAA GTGCTGAGTCCCAGG TATAAGAAAAAGCAG CCTGCCGTAGAGAAA TGCTGAGTCCCAGGG ATAAGAAAAAGCAGT CTGCCGTAGAGAAAT GCTGAGTCCCAGGGG TAAGAAAAAGCAGTG TGCCGTAGAGAAATG CTGAGTCCCAGGGGT AAGAAAAAGCAGTGT GCCGTAGAGAAATGG TGAGTCCCAGGGGTG AGAAAAAGCAGTGTC CCGTAGAGAAATGGA GAGTCCCAGGGGTGT GAAAAAGCAGTGTCG CGTAGAGAAATGGAA AGTCCCAGGGGTGTA AAAAAGCAGTGTCGC GTAGAGAAATGGAAG GTCCCAGGGGTGTAC AAAAGCAGTGTCGCC TAGAGAAATGGAAGA TCCCAGGGGTGTACA AAAGCAGTGTCGCCC AGAGAAATGGAAGAC CCCAGGGGTGTACAC AAGCAGTGTCGCCCT GAGAAATGGAAGACA CCAGGGGTGTACACA AGCAGTGTCGCCCTT AGAAATGGAAGACAC CAGGGGTGTACACAT GCAGTGTCGCCCTTC GAAATGGAAGACACA AGGGGTGTACACATT CAGTGTCGCCCTTCC AAATGGAAGACACAC GGGGTGTACACATTC AGTGTCGCCCTTCCA AATGGAAGACACACT GGGTGTACACATTCC GTGTCGCCCTTCCAA ATGGAAGACACACTG GGTGTACACATTCCC TGTCGCCCTTCCAAA TGGAAGACACACTGA GTGTACACATTCCCA GTCGCCCTTCCAAAG GGAAGACACACTGAA TGTACACATTCCCAA TCGCCCTTCCAAAGG GAAGACACACTGAAT GTACACATTCCCAAC CGCCCTTCCAAAGGC AAGACACACTGAATC TACACATTCCCAACT GCCCTTCCAAAGGCA AGACACACTGAATCA ACACATTCCCAACTG CCCTTCCAAAGGCAG GACACACTGAATCAC CACATTCCCAACTGT CCTTCCAAAGGCAGG ACACACTGAATCACC ACATTCCCAACTGTG CTTCCAAAGGCAGGA CACACTGAATCACCT CATTCCCAACTGTGA TTCCAAAGGCAGGAA ACACTGAATCACCTG ATTCCCAACTGTGAC TCCAAAGGCAGGAAG CACTGAATCACCTGA TTCCCAACTGTGACA CCAAAGGCAGGAAGC ACTGAATCACCTGAA TCCCAACTGTGACAA CAAAGGCAGGAAGCG CTGAATCACCTGAAG CCCAACTGTGACAAG AAAGGCAGGAAGCGG TGAATCACCTGAAGT CCAACTGTGACAAGA AAGGCAGGAAGCGGG GAATCACCTGAAGTT CAACTGTGACAAGAA AGGCAGGAAGCGGGG AATCACCTGAAGTTC AACTGTGACAAGAAG GGCAGGAAGCGGGGC ATCACCTGAAGTTCC ACTGTGACAAGAAGG GCAGGAAGCGGGGCT TCACCTGAAGTTCCT CTGTGACAAGAAGGG CAGGAAGCGGGGCTT CACCTGAAGTTCCTC TGTGACAAGAAGGGA AGGAAGCGGGGCTTC ACCTGAAGTTCCTCA GTGACAAGAAGGGAT GGAAGCGGGGCTTCT CCTGAAGTTCCTCAA TGACAAGAAGGGATT GAAGCGGGGCTTCTG CTGAAGTTCCTCAAT GACAAGAAGGGATTT AAGCGGGGCTTCTGC TGAAGTTCCTCAATG ACAAGAAGGGATTTT AGCGGGGCTTCTGCT GAAGTTCCTCAATGT CAAGAAGGGATTTTA GCGGGGCTTCTGCTG AAGTTCCTCAATGTG AAGAAGGGATTTTAT CGGGGCTTCTGCTGG AGTTCCTCAATGTGC AGAAGGGATTTTATA GGGGCTTCTGCTGGT GTTCCTCAATGTGCT GAAGGGATTTTATAA GGGCTTCTGCTGGTG TTCCTCAATGTGCTG AAGGGATTTTATAAG GGCTTCTGCTGGTGT TCCTCAATGTGCTGA AGGGATTTTATAAGA GCTTCTGCTGGTGTG CCTCAATGTGCTGAG GGGATTTTATAAGAA CTTCTGCTGGTGTGT CTCAATGTGCTGAGT GGATTTTATAAGAAA TTCTGCTGGTGTGTG TCAATGTGCTGAGTC GATTTTATAAGAAAA TCTGCTGGTGTGTGG CTGCTGGTGTGTGGA GGGGAAGGAGGACGT CGCAAGTTAATGTGG TGCTGGTGTGTGGAT GGGAAGGAGGACGTG GCAAGTTAATGTGGA GCTGGTGTGTGGATA GGAAGGAGGACGTGC CAAGTTAATGTGGAG CTGGTGTGTGGATAA GAAGGAGGACGTGCA AAGTTAATGTGGAGC TGGTGTGTGGATAAG AAGGAGGACGTGCAC AGTTAATGTGGAGCT GGTGTGTGGATAAGT AGGAGGACGTGCACT GTTAATGTGGAGCTC GTGTGTGGATAAGTA GGAGGACGTGCACTG TTAATGTGGAGCTCA TGTGTGGATAAGTAT GAGGACGTGCACTGC TAATGTGGAGCTCAA GTGTGGATAAGTATG AGGACGTGCACTGCT AATGTGGAGCTCAAA TGTGGATAAGTATGG GGACGTGCACTGCTA ATGTGGAGCTCAAAT GTGGATAAGTATGGG GACGTGCACTGCTAC TGTGGAGCTCAAATA TGGATAAGTATGGGC ACGTGCACTGCTACA GTGGAGCTCAAATAT GGATAAGTATGGGCA CGTGCACTGCTACAG TGGAGCTCAAATATG GATAAGTATGGGCAG GTGCACTGCTACAGC GGAGCTCAAATATGC ATAAGTATGGGCAGC TGCACTGCTACAGCA GAGCTCAAATATGCC TAAGTATGGGCAGCC GCACTGCTACAGCAT AGCTCAAATATGCCT AAGTATGGGCAGCCT CACTGCTACAGCATG GCTCAAATATGCCTT AGTATGGGCAGCCTC ACTGCTACAGCATGC CTCAAATATGCCTTA GTATGGGCAGCCTCT CTGCTACAGCATGCA TCAAATATGCCTTAT TATGGGCAGCCTCTC TGCTACAGCATGCAG CAAATATGCCTTATT ATGGGCAGCCTCTCC GCTACAGCATGCAGA AAATATGCCTTATTT TGGGCAGCCTCTCCC CTACAGCATGCAGAG AATATGCCTTATTTT GGGCAGCCTCTCCCA TACAGCATGCAGAGC ATATGCCTTATTTTG GGCAGCCTCTCCCAG ACAGCATGCAGAGCA TATGCCTTATTTTGC GCAGCCTCTCCCAGG CAGCATGCAGAGCAA ATGCCTTATTTTGCA CAGCCTCTCCCAGGC AGCATGCAGAGCAAG TGCCTTATTTTGCAC AGCCTCTCCCAGGCT GCATGCAGAGCAAGT GCCTTATTTTGCACA GCCTCTCCCAGGCTA CATGCAGAGCAAGTA CCTTATTTTGCACAA CCTCTCCCAGGCTAC ATGCAGAGCAAGTAG CTTATTTTGCACAAA CTCTCCCAGGCTACA TGCAGAGCAAGTAGA TTATTTTGCACAAAA TCTCCCAGGCTACAC GCAGAGCAAGTAGAC TATTTTGCACAAAAG CTCCCAGGCTACACC CAGAGCAAGTAGACG ATTTTGCACAAAAGA TCCCAGGCTACACCA AGAGCAAGTAGACGC TTTTGCACAAAAGAC CCCAGGCTACACCAC GAGCAAGTAGACGCC TTTGCACAAAAGACT CCAGGCTACACCACC AGCAAGTAGACGCCT TTGCACAAAAGACTG CAGGCTACACCACCA GCAAGTAGACGCCTG TGCACAAAAGACTGC AGGCTACACCACCAA CAAGTAGACGCCTGC GCACAAAAGACTGCC GGCTACACCACCAAG AAGTAGACGCCTGCC CACAAAAGACTGCCA GCTACACCACCAAGG AGTAGACGCCTGCCG ACAAAAGACTGCCAA CTACACCACCAAGGG GTAGACGCCTGCCGC CAAAAGACTGCCAAG TACACCACCAAGGGG TAGACGCCTGCCGCA AAAAGACTGCCAAGG ACACCACCAAGGGGA AGACGCCTGCCGCAA AAAGACTGCCAAGGA CACCACCAAGGGGAA GACGCCTGCCGCAAG AAGACTGCCAAGGAC ACCACCAAGGGGAAG ACGCCTGCCGCAAGT AGACTGCCAAGGACA CCACCAAGGGGAAGG CGCCTGCCGCAAGTT GACTGCCAAGGACAT CACCAAGGGGAAGGA GCCTGCCGCAAGTTA ACTGCCAAGGACATG ACCAAGGGGAAGGAG CCTGCCGCAAGTTAA CTGCCAAGGACATGA CCAAGGGGAAGGAGG CTGCCGCAAGTTAAT TGCCAAGGACATGAC CAAGGGGAAGGAGGA TGCCGCAAGTTAATG GCCAAGGACATGACC AAGGGGAAGGAGGAC GCCGCAAGTTAATGT CCAAGGACATGACCA AGGGGAAGGAGGACG CCGCAAGTTAATGTG CAAGGACATGACCAG AAGGACATGACCAGC GTGAACTGATTTTTT TATGGTTTCTTTGAA AGGACATGACCAGCA TGAACTGATTTTTTT ATGGTTTCTTTGAAT GGACATGACCAGCAG GAACTGATTTTTTTT TGGTTTCTTTGAATG GACATGACCAGCAGC AACTGATTTTTTTTA GGTTTCTTTGAATGG ACATGACCAGCAGCT ACTGATTTTTTTTAA GTTTCTTTGAATGGT CATGACCAGCAGCTG CTGATTTTTTTTAAA TTTCTTTGAATGGTA ATGACCAGCAGCTGG TGATTTTTTTTAAAC TTCTTTGAATGGTAA TGACCAGCAGCTGGC GATTTTTTTTAAACC TCTTTGAATGGTAAA GACCAGCAGCTGGCT ATTTTTTTTAAACCA CTTTGAATGGTAAAC ACCAGCAGCTGGCTA TTTTTTTTAAACCAA TTTGAATGGTAAACT CCAGCAGCTGGCTAC TTTTTTTAAACCAAA TTGAATGGTAAACTT CAGCAGCTGGCTACA TTTTTTAAACCAAAG TGAATGGTAAACTTG AGCAGCTGGCTACAG TTTTTAAACCAAAGT GAATGGTAAACTTGA GCAGCTGGCTACAGC TTTTAAACCAAAGTT AATGGTAAACTTGAG CAGCTGGCTACAGCC TTTAAACCAAAGTTT ATGGTAAACTTGAGC AGCTGGCTACAGCCT TTAAACCAAAGTTTA TGGTAAACTTGAGCA GCTGGCTACAGCCTC TAAACCAAAGTTTAG GGTAAACTTGAGCAT CTGGCTACAGCCTCG AAACCAAAGTTTAGA GTAAACTTGAGCATC TGGCTACAGCCTCGA AACCAAAGTTTAGAA TAAACTTGAGCATCT GGCTACAGCCTCGAT ACCAAAGTTTAGAAA AAACTTGAGCATCTT GCTACAGCCTCGATT CCAAAGTTTAGAAAG AACTTGAGCATCTTT CTACAGCCTCGATTT CAAAGTTTAGAAAGA ACTTGAGCATCTTTT TACAGCCTCGATTTA AAAGTTTAGAAAGAG CTTGAGCATCTTTTC ACAGCCTCGATTTAT AAGTTTAGAAAGAGG TTGAGCATCTTTTCA CAGCCTCGATTTATA AGTTTAGAAAGAGGT TGAGCATCTTTTCAC AGCCTCGATTTATAT GTTTAGAAAGAGGTT GAGCATCTTTTCACT GCCTCGATTTATATT TTTAGAAAGAGGTTT AGCATCTTTTCACTT CCTCGATTTATATTT TTAGAAAGAGGTTTT GCATCTTTTCACTTT CTCGATTTATATTTC TAGAAAGAGGTTTTT CATCTTTTCACTTTC TCGATTTATATTTCT AGAAAGAGGTTTTTG ATCTTTTCACTTTCC CGATTTATATTTCTG GAAAGAGGTTTTTGA TCTTTTCACTTTCCA GATTTATATTTCTGT AAAGAGGTTTTTGAA CTTTTCACTTTCCAG ATTTATATTTCTGTT AAGAGGTTTTTGAAA TTTTCACTTTCCAGT TTTATATTTCTGTTT AGAGGTTTTTGAAAT TTTCACTTTCCAGTA TTATATTTCTGTTTG GAGGTTTTTGAAATG TTCACTTTCCAGTAG TATATTTCTGTTTGT AGGTTTTTGAAATGC TCACTTTCCAGTAGT ATATTTCTGTTTGTG GGTTTTTGAAATGCC CACTTTCCAGTAGTC TATTTCTGTTTGTGG GTTTTTGAAATGCCT ACTTTCCAGTAGTCA ATTTCTGTTTGTGGT TTTTTGAAATGCCTA CTTTCCAGTAGTCAG TTTCTGTTTGTGGTG TTTTGAAATGCCTAT TTTCCAGTAGTCAGC TTCTGTTTGTGGTGA TTTGAAATGCCTATG TTCCAGTAGTCAGCA TCTGTTTGTGGTGAA TTGAAATGCCTATGG TCCAGTAGTCAGCAA CTGTTTGTGGTGAAC TGAAATGCCTATGGT CCAGTAGTCAGCAAA TGTTTGTGGTGAACT GAAATGCCTATGGTT CAGTAGTCAGCAAAG GTTTGTGGTGAACTG AAATGCCTATGGTTT AGTAGTCAGCAAAGA TTTGTGGTGAACTGA AATGCCTATGGTTTC GTAGTCAGCAAAGAG TTGTGGTGAACTGAT ATGCCTATGGTTTCT TAGTCAGCAAAGAGC TGTGGTGAACTGATT TGCCTATGGTTTCTT AGTCAGCAAAGAGCA GTGGTGAACTGATTT GCCTATGGTTTCTTT GTCAGCAAAGAGCAG TGGTGAACTGATTTT CCTATGGTTTCTTTG TCAGCAAAGAGCAGT GGTGAACTGATTTTT CTATGGTTTCTTTGA CAGCAAAGAGCAGTT AGCAAAGAGCAGTTT ACTCGAGCACAGCAC TTGGTCGAAGCGGCC
GCAAAGAGCAGTTTG CTCGAGCACAGCACC TGGTCGAAGCGGCCG
CAAAGAGCAGTTTGA TCGAGCACAGCACCC GGTCGAAGCGGCCGA
AAAGAGCAGTTTGAA CGAGCACAGCACCCA GTCGAAGCGGCCGAC AAGAGCAGTTTGAAT GAGCACAGCACCCAG TCGAAGCGGCCGACC
AGAGCAGTTTGAATT AGCACAGCACCCAGA CGAAGCGGCCGACCA
GAGCAGTTTGAATTT GCACAGCACCCAGAC GAAGCGGCCGACCAC
AGCAGTTTGAATTTT CACAGCACCCAGACT AAGCGGCCGACCACT
GCAGTTTGAATTTTC ACAGCACCCAGACTT AGCGGCCGACCACTG CAGTTTGAATTTTCT CAGCACCCAGACTTC GCGGCCGACCACTGA
AGTTTGAATTTTCTT AGCACCCAGACTTCA CGGCCGACCACTGAC
GTTTGAATTTTCTTG GCACCCAGACTTCAT GGCCGACCACTGACT
TTTGAATTTTCTTGT CACCCAGACTTCATG GCCGACCACTGACTT
TTGAATTTTCTTGTC ACCCAGACTTCATGC CCGACCACTGACTTT TGAATTTTCTTGTCG CCCAGACTTCATGCG CGACCACTGACTTTG
GAATTTTCTTGTCGC CCAGACTTCATGCGC GACCACTGACTTTGT
AATTTTCTTGTCGCT CAGACTTCATGCGCC ACCACTGACTTTGTG
ATTTTCTTGTCGCTT AGACTTCATGCGCCC CCACTGACTTTGTGA
TTTTCTTGTCGCTTC GACTTCATGCGCCCG CACTGACTTTGTGAC TTTCTTGTCGCTTCC ACTTCATGCGCCCGT ACTGACTTTGTGACT
TTCTTGTCGCTTCCT CTTCATGCGCCCGTG CTGACTTTGTGACTT
TCTTGTCGCTTCCTA TTCATGCGCCCGTGG TGACTTTGTGACTTA
CTTGTCGCTTCCTAT TCATGCGCCCGTGGA GACTTTGTGACTTAG
TTGTCGCTTCCTATC CATGCGCCCGTGGAA ACTTTGTGACTTAGG TGTCGCTTCCTATCA ATGCGCCCGTGGAAT CTTTGTGACTTAGGC
GTCGCTTCCTATCAA TGCGCCCGTGGAATG TTTGTGACTTAGGCG
TCGCTTCCTATCAAA GCGCCCGTGGAATGC TTGTGACTTAGGCGG
CGCTTCCTATCAAAA CGCCCGTGGAATGCT TGTGACTTAGGCGGC
GCTTCCTATCAAAAT GCCCGTGGAATGCTC GTGACTTAGGCGGCT CTTCCTATCAAAATA CCCGTGGAATGCTCA TGACTTAGGCGGCTG
TTCCTATCAAAATAT CCGTGGAATGCTCAC GACTTAGGCGGCTGT
TCCTATCAAAATATT CGTGGAATGCTCACC ACTTAGGCGGCTGTG
CCTATCAAAATATTC GTGGAATGCTCACCA CTTAGGCGGCTGTGT
CTATCAAAATATTCA TGGAATGCTCACCAC TTAGGCGGCTGTGTT TATCAAAATATTCAG GGAATGCTCACCACA TAGGCGGCTGTGTTG
ATCAAAATATTCAGA GAATGCTCACCACAT AGGCGGCTGTGTTGC
TCAAAATATTCAGAG AATGCTCACCACATG GGCGGCTGTGTTGCC
CAAAATATTCAGAGA ATGCTCACCACATGT GCGGCTGTGTTGCCT
AAAATATTCAGAGAC TGCTCACCACATGTT CGGCTGTGTTGCCTA AAATATTCAGAGACT GCTCACCACATGTTG GGCTGTGTTGCCTAT
AATATTCAGAGACTC CTCACCACATGTTGG GCTGTGTTGCCTATG
ATATTCAGAGACTCG TCACCACATGTTGGT CTGTGTTGCCTATGT
TATTCAGAGACTCGA CACCACATGTTGGTC TGTGTTGCCTATGTA
ATTCAGAGACTCGAG ACCACATGTTGGTCG GTGTTGCCTATGTAG TTCAGAGACTCGAGC CCACATGTTGGTCGA TGTTGCCTATGTAGA
TCAGAGACTCGAGCA CACATGTTGGTCGAA GTTGCCTATGTAGAG
CAGAGACTCGAGCAC ACATGTTGGTCGAAG TTGCCTATGTAGAGA
AGAGACTCGAGCACA CATGTTGGTCGAAGC TGCCTATGTAGAGAA
GAGACTCGAGCACAG ATGTTGGTCGAAGCG GCCTATGTAGAGAAC AGACTCGAGCACAGC TGTTGGTCGAAGCGG CCTATGTAGAGAACA
GACTCGAGCACAGCA GTTGGTCGAAGCGGC CTATGTAGAGAACAC TATGTAGAGAACACG TATCGAGAATAGGAA ATGCTCCTGGAGCTC
ATGTAGAGAACACGC ATCGAGAATAGGAAA TGCTCCTGGAGCTCA
TGTAGAGAACACGCT TCGAGAATAGGAAAA GCTCCTGGAGCTCAC
GTAGAGAACACGCTT CGAGAATAGGAAAAC CTCCTGGAGCTCACA TAGAGAACACGCTTC GAGAATAGGAAAACC TCCTGGAGCTCACAG
AGAGAACACGCTTCA AGAATAGGAAAACCT CCTGGAGCTCACAGC
GAGAACACGCTTCAC GAATAGGAAAACCTT CTGGAGCTCACAGCC
AGAACACGCTTCACC AATAGGAAAACCTTT TGGAGCTCACAGCCT
GAACACGCTTCACCC ATAGGAAAACCTTTA GGAGCTCACAGCCTT AACACGCTTCACCCC TAGGAAAACCTTTAA GAGCTCACAGCCTTC
ACACGCTTCACCCCC AGGAAAACCTTTAAA AGCTCACAGCCTTCT
CACGCTTCACCCCCA GGAAAACCTTTAAAC GCTCACAGCCTTCTG
ACGCTTCACCCCCAC GAAAACCTTTAAACC CTCACAGCCTTCTGT
CGCTTCACCCCCACT AAAACCTTTAAACCC TCACAGCCTTCTGTG GCTTCACCCCCACTC AAACCTTTAAACCCC CACAGCCTTCTGTGG
CTTCACCCCCACTCC AACCTTTAAACCCCG ACAGCCTTCTGTGGT
TTCACCCCCACTCCC ACCTTTAAACCCCGG CAGCCTTCTGTGGTG
TCACCCCCACTCCCC CCTTTAAACCCCGGT AGCCTTCTGTGGTGT
CACCCCCACTCCCCG CTTTAAACCCCGGTC GCCTTCTGTGGTGTC ACCCCCACTCCCCGT TTTAAACCCCGGTCA CCTTCTGTGGTGTCA
CCCCCACTCCCCGTA TTAAACCCCGGTCAT CTTCTGTGGTGTCAT
CCCCACTCCCCGTAC TAAACCCCGGTCATC TTCTGTGGTGTCATT
CCCACTCCCCGTACA AAACCCCGGTCATCC TCTGTGGTGTCATTT
CCACTCCCCGTACAG AACCCCGGTCATCCG CTGTGGTGTCATTTC CACTCCCCGTACAGT ACCCCGGTCATCCGG TGTGGTGTCATTTCT
ACTCCCCGTACAGTG CCCCGGTCATCCGGA GTGGTGTCATTTCTG
CTCCCCGTACAGTGC CCCGGTCATCCGGAC TGGTGTCATTTCTGA
TCCCCGTACAGTGCG CCGGTCATCCGGACA GGTGTCATTTCTGAA
CCCCGTACAGTGCGC CGGTCATCCGGACAT GTGTCATTTCTGAAA CCCGTACAGTGCGCA GGTCATCCGGACATC TGTCATTTCTGAAAC
CCGTACAGTGCGCAC GTCATCCGGACATCC GTCATTTCTGAAACA
CGTACAGTGCGCACA TCATCCGGACATCCC TCATTTCTGAAACAA
GTACAGTGCGCACAG CATCCGGACATCCCA CATTTCTGAAACAAG
TACAGTGCGCACAGG ATCCGGACATCCCAA ATTTCTGAAACAAGG ACAGTGCGCACAGGC TCCGGACATCCCAAC TTTCTGAAACAAGGG
CAGTGCGCACAGGCT CCGGACATCCCAACG TTCTGAAACAAGGGC
AGTGCGCACAGGCTT CGGACATCCCAACGC TCTGAAACAAGGGCG
GTGCGCACAGGCTTT GGACATCCCAACGCA CTGAAACAAGGGCGT
TGCGCACAGGCTTTA GACATCCCAACGCAT TGAAACAAGGGCGTG GCGCACAGGCTTTAT ACATCCCAACGCATG GAAACAAGGGCGTGG
CGCACAGGCTTTATC CATCCCAACGCATGC AAACAAGGGCGTGGA
GCACAGGCTTTATCG ATCCCAACGCATGCT AACAAGGGCGTGGAT
CACAGGCTTTATCGA TCCCAACGCATGCTC ACAAGGGCGTGGATC
ACAGGCTTTATCGAG CCCAACGCATGCTCC CAAGGGCGTGGATCC CAGGCTTTATCGAGA CCAACGCATGCTCCT AAGGGCGTGGATCCC
AGGCTTTATCGAGAA CAACGCATGCTCCTG AGGGCGTGGATCCCT
GGCTTTATCGAGAAT AACGCATGCTCCTGG GGGCGTGGATCCCTC
GCTTTATCGAGAATA ACGCATGCTCCTGGA GGCGTGGATCCCTCA
CTTTATCGAGAATAG CGCATGCTCCTGGAG GCGTGGATCCCTCAA TTTATCGAGAATAGG GCATGCTCCTGGAGC CGTGGATCCCTCAAC
TTATCGAGAATAGGA CATGCTCCTGGAGCT GTGGATCCCTCAACC TGGATCCCTCAACCA TTGGGGACTATTGGA GTATCTAAGAATGTT GGATCCCTCAACCAA TGGGGACTATTGGAG TATCTAAGAATGTTC GATCCCTCAACCAAG GGGGACTATTGGAGA ATCTAAGAATGTTCT ATCCCTCAACCAAGA GGGACTATTGGAGAA TCTAAGAATGTTCTA TCCCTCAACCAAGAA GGACTATTGGAGAAA CTAAGAATGTTCTAG CCCTCAACCAAGAAG GACTATTGGAGAAAA TAAGAATGTTCTAGG CCTCAACCAAGAAGA ACTATTGGAGAAAAT AAGAATGTTCTAGGG CTCAACCAAGAAGAA CTATTGGAGAAAATA AGAATGTTCTAGGGC TCAACCAAGAAGAAT TATTGGAGAAAATAA GAATGTTCTAGGGCA CAACCAAGAAGAATG ATTGGAGAAAATAAG AATGTTCTAGGGCAC AACCAAGAAGAATGT TTGGAGAAAATAAGG ATGTTCTAGGGCACT ACCAAGAAGAATGTT TGGAGAAAATAAGGT TGTTCTAGGGCACTC CCAAGAAGAATGTTT GGAGAAAATAAGGTG GTTCTAGGGCACTCT CAAGAAGAATGTTTA GAGAAAATAAGGTGG TTCTAGGGCACTCTG AAGAAGAATGTTTAT AGAAAATAAGGTGGA TCTAGGGCACTCTGG AGAAGAATGTTTATG GAAAATAAGGTGGAG CTAGGGCACTCTGGG GAAGAATGTTTATGT AAAATAAGGTGGAGT TAGGGCACTCTGGGA AAGAATGTTTATGTC AAATAAGGTGGAGTC AGGGCACTCTGGGAA AGAATGTTTATGTCT AATAAGGTGGAGTCC GGGCACTCTGGGAAC GAATGTTTATGTCTT ATAAGGTGGAGTCCT GGCACTCTGGGAACC AATGTTTATGTCTTC TAAGGTGGAGTCCTA GCACTCTGGGAACCT ATGTTTATGTCTTCA AAGGTGGAGTCCTAC CACTCTGGGAACCTA TGTTTATGTCTTCAA AGGTGGAGTCCTACT ACTCTGGGAACCTAT GTTTATGTCTTCAAG GGTGGAGTCCTACTT CTCTGGGAACCTATA TTTATGTCTTCAAGT GTGGAGTCCTACTTG TCTGGGAACCTATAA TTATGTCTTCAAGTG TGGAGTCCTACTTGT CTGGGAACCTATAAA TATGTCTTCAAGTGA GGAGTCCTACTTGTT TGGGAACCTATAAAG ATGTCTTCAAGTGAC GAGTCCTACTTGTTT GGGAACCTATAAAGG TGTCTTCAAGTGACC AGTCCTACTTGTTTA GGAACCTATAAAGGC GTCTTCAAGTGACCT GTCCTACTTGTTTAA GAACCTATAAAGGCA TCTTCAAGTGACCTG TCCTACTTGTTTAAA AACCTATAAAGGCAG CTTCAAGTGACCTGT CCTACTTGTTTAAAA ACCTATAAAGGCAGG TTCAAGTGACCTGTA CTACTTGTTTAAAAA CCTATAAAGGCAGGT TCAAGTGACCTGTAC TACTTGTTTAAAAAA CTATAAAGGCAGGTA CAAGTGACCTGTACT ACTTGTTTAAAAAAT TATAAAGGCAGGTAT AAGTGACCTGTACTG CTTGTTTAAAAAATA ATAAAGGCAGGTATT AGTGACCTGTACTGC TTGTTTAAAAAATAT TAAAGGCAGGTATTT GTGACCTGTACTGCT TGTTTAAAAAATATG AAAGGCAGGTATTTC TGACCTGTACTGCTT GTTTAAAAAATATGT AAGGCAGGTATTTCG GACCTGTACTGCTTG TTTAAAAAATATGTA AGGCAGGTATTTCGG ACCTGTACTGCTTGG TTAAAAAATATGTAT GGCAGGTATTTCGGG CCTGTACTGCTTGGG TAAAAAATATGTATC GCAGGTATTTCGGGC CTGTACTGCTTGGGG AAAAAATATGTATCT CAGGTATTTCGGGCC TGTACTGCTTGGGGA AAAAATATGTATCTA AGGTATTTCGGGCCC GTACTGCTTGGGGAC AAAATATGTATCTAA GGTATTTCGGGCCCT TACTGCTTGGGGACT AAATATGTATCTAAG GTATTTCGGGCCCTC ACTGCTTGGGGACTA AATATGTATCTAAGA TATTTCGGGCCCTCC CTGCTTGGGGACTAT ATATGTATCTAAGAA ATTTCGGGCCCTCCT TGCTTGGGGACTATT TATGTATCTAAGAAT TTTCGGGCCCTCCTC GCTTGGGGACTATTG ATGTATCTAAGAATG TTCGGGCCCTCCTCT CTTGGGGACTATTGG TGTATCTAAGAATGT TCGGGCCCTCCTCTT CGGGCCCTCCTCTTC CAGGATGGCTTTTGC AGAGTCAGCCTCCAC
GGGCCCTCCTCTTCA AGGATGGCTTTTGCT GAGTCAGCCTCCACA
GGCCCTCCTCTTCAG GGATGGCTTTTGCTG AGTCAGCCTCCACAT
GCCCTCCTCTTCAGG GATGGCTTTTGCTGC GTCAGCCTCCACATT CCCTCCTCTTCAGGA ATGGCTTTTGCTGCG TCAGCCTCCACATTC
CCTCCTCTTCAGGAA TGGCTTTTGCTGCGG CAGCCTCCACATTCA
CTCCTCTTCAGGAAT GGCTTTTGCTGCGGC AGCCTCCACATTCAG
TCCTCTTCAGGAATC GCTTTTGCTGCGGCC GCCTCCACATTCAGA
CCTCTTCAGGAATCT CTTTTGCTGCGGCCC CCTCCACATTCAGAG CTCTTCAGGAATCTT TTTTGCTGCGGCCCC CTCCACATTCAGAGG
TCTTCAGGAATCTTC TTTGCTGCGGCCCCG TCCACATTCAGAGGC
CTTCAGGAATCTTCC TTGCTGCGGCCCCGT CCACATTCAGAGGCA
TTCAGGAATCTTCCT TGCTGCGGCCCCGTG CACATTCAGAGGCAT
TCAGGAATCTTCCTG GCTGCGGCCCCGTGG ACATTCAGAGGCATC CAGGAATCTTCCTGA CTGCGGCCCCGTGGG CATTCAGAGGCATCA
AGGAATCTTCCTGAA TGCGGCCCCGTGGGG ATTCAGAGGCATCAC
GGAATCTTCCTGAAG GCGGCCCCGTGGGGT TTCAGAGGCATCACA
GAATCTTCCTGAAGA CGGCCCCGTGGGGTA TCAGAGGCATCACAA
AATCTTCCTGAAGAC GGCCCCGTGGGGTAG CAGAGGCATCACAAG ATCTTCCTGAAGACA GCCCCGTGGGGTAGG AGAGGCATCACAAGT
TCTTCCTGAAGACAT CCCCGTGGGGTAGGA GAGGCATCACAAGTA
CTTCCTGAAGACATG CCCGTGGGGTAGGAG AGGCATCACAAGTAA
TTCCTGAAGACATGG CCGTGGGGTAGGAGG GGCATCACAAGTAAT
TCCTGAAGACATGGC CGTGGGGTAGGAGGG GCATCACAAGTAATG CCTGAAGACATGGCC GTGGGGTAGGAGGGA CATCACAAGTAATGG
CTGAAGACATGGCCC TGGGGTAGGAGGGAC ATCACAAGTAATGGC
TGAAGACATGGCCCA GGGGTAGGAGGGACA TCACAAGTAATGGCA
GAAGACATGGCCCAG GGGTAGGAGGGACAG CACAAGTAATGGCAC
AAGACATGGCCCAGT GGTAGGAGGGACAGA ACAAGTAATGGCACA AGACATGGCCCAGTC GTAGGAGGGACAGAG CAAGTAATGGCACAA
GACATGGCCCAGTCG TAGGAGGGACAGAGA AAGTAATGGCACAAT
ACATGGCCCAGTCGA AGGAGGGACAGAGAG AGTAATGGCACAATT
CATGGCCCAGTCGAA GGAGGGACAGAGAGA GTAATGGCACAATTC
ATGGCCCAGTCGAAG GAGGGACAGAGAGAC TAATGGCACAATTCT TGGCCCAGTCGAAGG AGGGACAGAGAGACG AATGGCACAATTCTT
GGCCCAGTCGAAGGC GGGACAGAGAGACGG ATGGCACAATTCTTC
GCCCAGTCGAAGGCC GGACAGAGAGACGGG TGGCACAATTCTTCG
CCCAGTCGAAGGCCC GACAGAGAGACGGGA GGCACAATTCTTCGG
CCAGTCGAAGGCCCA ACAGAGAGACGGGAG GCACAATTCTTCGGA CAGTCGAAGGCCCAG CAGAGAGACGGGAGA CACAATTCTTCGGAT
AGTCGAAGGCCCAGG AGAGAGACGGGAGAG ACAATTCTTCGGATG
GTCGAAGGCCCAGGA GAGAGACGGGAGAGT CAATTCTTCGGATGA
TCGAAGGCCCAGGAT AGAGACGGGAGAGTC AATTCTTCGGATGAC
CGAAGGCCCAGGATG GAGACGGGAGAGTCA ATTCTTCGGATGACT GAAGGCCCAGGATGG AGACGGGAGAGTCAG TTCTTCGGATGACTG
AAGGCCCAGGATGGC GACGGGAGAGTCAGC TCTTCGGATGACTGC
AGGCCCAGGATGGCT ACGGGAGAGTCAGCC CTTCGGATGACTGCA
GGCCCAGGATGGCTT CGGGAGAGTCAGCCT TTCGGATGACTGCAG
GCCCAGGATGGCTTT GGGAGAGTCAGCCTC TCGGATGACTGCAGA CCCAGGATGGCTTTT GGAGAGTCAGCCTCC CGGATGACTGCAGAA
CCAGGATGGCTTTTG GAGAGTCAGCCTCCA GGATGACTGCAGAAA GATGACTGCAGAAAA ATTTCTGAGGATAAG TTTTGTCCTCCTTAG ATGACTGCAGAAAAT TTTCTGAGGATAAGC TTTGTCCTCCTTAGC TGACTGCAGAAAATA TTCTGAGGATAAGCT TTGTCCTCCTTAGCA GACTGCAGAAAATAG TCTGAGGATAAGCTC TGTCCTCCTTAGCAC ACTGCAGAAAATAGT CTGAGGATAAGCTCT GTCCTCCTTAGCACA CTGCAGAAAATAGTG TGAGGATAAGCTCTT TCCTCCTTAGCACAA TGCAGAAAATAGTGT GAGGATAAGCTCTTT CCTCCTTAGCACAAT GCAGAAAATAGTGTT AGGATAAGCTCTTTA CTCCTTAGCACAATG CAGAAAATAGTGTTT GGATAAGCTCTTTAA TCCTTAGCACAATGT AGAAAATAGTGTTTT GATAAGCTCTTTAAA CCTTAGCACAATGTA GAAAATAGTGTTTTG ATAAGCTCTTTAAAG CTTAGCACAATGTAA AAAATAGTGTTTTGT TAAGCTCTTTAAAGG TTAGCACAATGTAAA AAATAGTGTTTTGTA AAGCTCTTTAAAGGC TAGCACAATGTAAAA AATAGTGTTTTGTAG AGCTCTTTAAAGGCA AGCACAATGTAAAAA ATAGTGTTTTGTAGT GCTCTTTAAAGGCAA GCACAATGTAAAAAA TAGTGTTTTGTAGTT CTCTTTAAAGGCAAA CACAATGTAAAAAAG AGTGTTTTGTAGTTC TCTTTAAAGGCAAAG ACAATGTAAAAAAGA GTGTTTTGTAGTTCA CTTTAAAGGCAAAGC CAATGTAAAAAAGAA TGTTTTGTAGTTCAA TTTAAAGGCAAAGCT AATGTAAAAAAGAAT GTTTTGTAGTTCAAC TTAAAGGCAAAGCTT ATGTAAAAAAGAATA TTTTGTAGTTCAACA TAAAGGCAAAGCTTT TGTAAAAAAGAATAG TTTGTAGTTCAACAA AAAGGCAAAGCTTTA GTAAAAAAGAATAGT TTGTAGTTCAACAAC AAGGCAAAGCTTTAT TAAAAAAGAATAGTA TGTAGTTCAACAACT AGGCAAAGCTTTATT AAAAAAGAATAGTAA GTAGTTCAACAACTC GGCAAAGCTTTATTT AAAAAGAATAGTAAT TAGTTCAACAACTCA GCAAAGCTTTATTTT AAAAGAATAGTAATA AGTTCAACAACTCAA CAAAGCTTTATTTTC AAAGAATAGTAATAT GTTCAACAACTCAAG AAAGCTTTATTTTCA AAGAATAGTAATATC TTCAACAACTCAAGA AAGCTTTATTTTCAT AGAATAGTAATATCA TCAACAACTCAAGAC AGCTTTATTTTCATC GAATAGTAATATCAG CAACAACTCAAGACG GCTTTATTTTCATCT AATAGTAATATCAGA AACAACTCAAGACGA CTTTATTTTCATCTC ATAGTAATATCAGAA ACAACTCAAGACGAA TTTATTTTCATCTCT TAGTAATATCAGAAC CAACTCAAGACGAAG TTATTTTCATCTCTC AGTAATATCAGAACA AACTCAAGACGAAGC TATTTTCATCTCTCA GTAATATCAGAACAG ACTCAAGACGAAGCT ATTTTCATCTCTCAT TAATATCAGAACAGG CTCAAGACGAAGCTT TTTTCATCTCTCATC AATATCAGAACAGGA TCAAGACGAAGCTTA TTTCATCTCTCATCT ATATCAGAACAGGAA CAAGACGAAGCTTAT TTCATCTCTCATCTT TATCAGAACAGGAAG AAGACGAAGCTTATT TCATCTCTCATCTTT ATCAGAACAGGAAGG AGACGAAGCTTATTT CATCTCTCATCTTTT TCAGAACAGGAAGGA GACGAAGCTTATTTC ATCTCTCATCTTTTG CAGAACAGGAAGGAG ACGAAGCTTATTTCT TCTCTCATCTTTTGT AGAACAGGAAGGAGG CGAAGCTTATTTCTG CTCTCATCTTTTGTC GAACAGGAAGGAGGA GAAGCTTATTTCTGA TCTCATCTTTTGTCC AACAGGAAGGAGGAA AAGCTTATTTCTGAG CTCATCTTTTGTCCT ACAGGAAGGAGGAAT AGCTTATTTCTGAGG TCATCTTTTGTCCTC CAGGAAGGAGGAATG GCTTATTTCTGAGGA CATCTTTTGTCCTCC AGGAAGGAGGAATGG CTTATTTCTGAGGAT ATCTTTTGTCCTCCT GGAAGGAGGAATGGC TTATTTCTGAGGATA TCTTTTGTCCTCCTT GAAGGAGGAATGGCT TATTTCTGAGGATAA CTTTTGTCCTCCTTA AAGGAGGAATGGCTT AGGAGGAATGGCTTG GATTCACCCATGTTT ATTCACACATATATG GGAGGAATGGCTTGC ATTCACCCATGTTTG TTCACACATATATGC GAGGAATGGCTTGCT TTCACCCATGTTTGT TCACACATATATGCA AGGAATGGCTTGCTG TCACCCATGTTTGTT CACACATATATGCAG GGAATGGCTTGCTGG CACCCATGTTTGTTG ACACATATATGCAGA GAATGGCTTGCTGGG ACCCATGTTTGTTGA CACATATATGCAGAG AATGGCTTGCTGGGG CCCATGTTTGTTGAA ACATATATGCAGAGA ATGGCTTGCTGGGGA CCATGTTTGTTGAAC CATATATGCAGAGAA TGGCTTGCTGGGGAG CATGTTTGTTGAACT ATATATGCAGAGAAG GGCTTGCTGGGGAGC ATGTTTGTTGAACTT TATATGCAGAGAAGA GCTTGCTGGGGAGCC TGTTTGTTGAACTTA ATATGCAGAGAAGAT CTTGCTGGGGAGCCC GTTTGTTGAACTTAG TATGCAGAGAAGATA TTGCTGGGGAGCCCA TTTGTTGAACTTAGA ATGCAGAGAAGATAT TGCTGGGGAGCCCAT TTGTTGAACTTAGAG TGCAGAGAAGATATG GCTGGGGAGCCCATC TGTTGAACTTAGAGT GCAGAGAAGATATGT CTGGGGAGCCCATCC GTTGAACTTAGAGTC CAGAGAAGATATGTT TGGGGAGCCCATCCA TTGAACTTAGAGTCA AGAGAAGATATGTTC GGGGAGCCCATCCAG TGAACTTAGAGTCAT GAGAAGATATGTTCT GGGAGCCCATCCAGG GAACTTAGAGTCATT AGAAGATATGTTCTT GGAGCCCATCCAGGA AACTTAGAGTCATTC GAAGATATGTTCTTG GAGCCCATCCAGGAC ACTTAGAGTCATTCT AAGATATGTTCTTGT AGCCCATCCAGGACA CTTAGAGTCATTCTC AGATATGTTCTTGTT GCCCATCCAGGACAC TTAGAGTCATTCTCA GATATGTTCTTGTTA CCCATCCAGGACACT TAGAGTCATTCTCAT ATATGTTCTTGTTAA CCATCCAGGACACTG AGAGTCATTCTCATG TATGTTCTTGTTAAC CATCCAGGACACTGG GAGTCATTCTCATGC ATGTTCTTGTTAACA ATCCAGGACACTGGG AGTCATTCTCATGCT TGTTCTTGTTAACAT TCCAGGACACTGGGA GTCATTCTCATGCTT GTTCTTGTTAACATT CCAGGACACTGGGAG TCATTCTCATGCTTT TTCTTGTTAACATTG CAGGACACTGGGAGC CATTCTCATGCTTTT TCTTGTTAACATTGT AGGACACTGGGAGCA ATTCTCATGCTTTTC CTTGTTAACATTGTA GGACACTGGGAGCAC TTCTCATGCTTTTCT TTGTTAACATTGTAT GACACTGGGAGCACA TCTCATGCTTTTCTT TGTTAACATTGTATA ACACTGGGAGCACAT CTCATGCTTTTCTTT GTTAACATTGTATAC CACTGGGAGCACATA TCATGCTTTTCTTTA TTAACATTGTATACA ACTGGGAGCACATAG CATGCTTTTCTTTAT TAACATTGTATACAA CTGGGAGCACATAGA ATGCTTTTCTTTATA AACATTGTATACAAC TGGGAGCACATAGAG TGCTTTTCTTTATAA ACATTGTATACAACA GGGAGCACATAGAGA GCTTTTCTTTATAAT CATTGTATACAACAT GGAGCACATAGAGAT CTTTTCTTTATAATT ATTGTATACAACATA GAGCACATAGAGATT TTTTCTTTATAATTC TTGTATACAACATAG AGCACATAGAGATTC TTTCTTTATAATTCA TGTATACAACATAGC GCACATAGAGATTCA TTCTTTATAATTCAC GTATACAACATAGCC CACATAGAGATTCAC TCTTTATAATTCACA TATACAACATAGCCC ACATAGAGATTCACC CTTTATAATTCACAC ATACAACATAGCCCC CATAGAGATTCACCC TTTATAATTCACACA TACAACATAGCCCCA ATAGAGATTCACCCA TTATAATTCACACAT ACAACATAGCCCCAA TAGAGATTCACCCAT TATAATTCACACATA CAACATAGCCCCAAA AGAGATTCACCCATG ATAATTCACACATAT AACATAGCCCCAAAT GAGATTCACCCATGT TAATTCACACATATA ACATAGCCCCAAATA AGATTCACCCATGTT AATTCACACATATAT CATAGCCCCAAATAT ATAGCCCCAAATATA AGAGATGCTATATGA CCCAGAGACTGGGCT TAGCCCCAAATATAG GAGATGCTATATGAT CCAGAGACTGGGCTG AGCCCCAAATATAGT AGATGCTATATGATA CAGAGACTGGGCTGC GCCCCAAATATAGTA GATGCTATATGATAC AGAGACTGGGCTGCT CCCCAAATATAGTAA ATGCTATATGATACA GAGACTGGGCTGCTC CCCAAATATAGTAAG TGCTATATGATACAA AGACTGGGCTGCTCT CCAAATATAGTAAGA GCTATATGATACAAC GACTGGGCTGCTCTC CAAATATAGTAAGAT CTATATGATACAACT ACTGGGCTGCTCTCC AAATATAGTAAGATC TATATGATACAACTG CTGGGCTGCTCTCCC AATATAGTAAGATCT ATATGATACAACTGT TGGGCTGCTCTCCCG ATATAGTAAGATCTA TATGATACAACTGTG GGGCTGCTCTCCCGG TATAGTAAGATCTAT ATGATACAACTGTGG GGCTGCTCTCCCGGA ATAGTAAGATCTATA TGATACAACTGTGGC GCTGCTCTCCCGGAG TAGTAAGATCTATAC GATACAACTGTGGCC CTGCTCTCCCGGAGG AGTAAGATCTATACT ATACAACTGTGGCCA TGCTCTCCCGGAGGC GTAAGATCTATACTA TACAACTGTGGCCAT GCTCTCCCGGAGGCC TAAGATCTATACTAG ACAACTGTGGCCATG CTCTCCCGGAGGCCA AAGATCTATACTAGA CAACTGTGGCCATGA TCTCCCGGAGGCCAA AGATCTATACTAGAT AACTGTGGCCATGAC CTCCCGGAGGCCAAA GATCTATACTAGATA ACTGTGGCCATGACT TCCCGGAGGCCAAAC ATCTATACTAGATAA CTGTGGCCATGACTG CCCGGAGGCCAAACC TCTATACTAGATAAT TGTGGCCATGACTGA CCGGAGGCCAAACCC CTATACTAGATAATC GTGGCCATGACTGAG CGGAGGCCAAACCCA TATACTAGATAATCC TGGCCATGACTGAGG GGAGGCCAAACCCAA ATACTAGATAATCCT GGCCATGACTGAGGA GAGGCCAAACCCAAG TACTAGATAATCCTA GCCATGACTGAGGAA AGGCCAAACCCAAGA ACTAGATAATCCTAG CCATGACTGAGGAAA GGCCAAACCCAAGAA CTAGATAATCCTAGA CATGACTGAGGAAAG GCCAAACCCAAGAAG TAGATAATCCTAGAT ATGACTGAGGAAAGG CCAAACCCAAGAAGG AGATAATCCTAGATG TGACTGAGGAAAGGA CAAACCCAAGAAGGT GATAATCCTAGATGA GACTGAGGAAAGGAG AAACCCAAGAAGGTC ATAATCCTAGATGAA ACTGAGGAAAGGAGC AACCCAAGAAGGTCT TAATCCTAGATGAAA CTGAGGAAAGGAGCT ACCCAAGAAGGTCTG AATCCTAGATGAAAT TGAGGAAAGGAGCTC CCCAAGAAGGTCTGG ATCCTAGATGAAATG GAGGAAAGGAGCTCA CCAAGAAGGTCTGGC TCCTAGATGAAATGT AGGAAAGGAGCTCAC CAAGAAGGTCTGGCA CCTAGATGAAATGTT GGAAAGGAGCTCACG AAGAAGGTCTGGCAA CTAGATGAAATGTTA GAAAGGAGCTCACGC AGAAGGTCTGGCAAA TAGATGAAATGTTAG AAAGGAGCTCACGCC GAAGGTCTGGCAAAG AGATGAAATGTTAGA AAGGAGCTCACGCCC AAGGTCTGGCAAAGT GATGAAATGTTAGAG AGGAGCTCACGCCCA AGGTCTGGCAAAGTC ATGAAATGTTAGAGA GGAGCTCACGCCCAG GGTCTGGCAAAGTCA TGAAATGTTAGAGAT GAGCTCACGCCCAGA GTCTGGCAAAGTCAG GAAATGTTAGAGATG AGCTCACGCCCAGAG TCTGGCAAAGTCAGG AAATGTTAGAGATGC GCTCACGCCCAGAGA CTGGCAAAGTCAGGC AATGTTAGAGATGCT CTCACGCCCAGAGAC TGGCAAAGTCAGGCT ATGTTAGAGATGCTA TCACGCCCAGAGACT GGCAAAGTCAGGCTC TGTTAGAGATGCTAT CACGCCCAGAGACTG GCAAAGTCAGGCTCA GTTAGAGATGCTATA ACGCCCAGAGACTGG CAAAGTCAGGCTCAG TTAGAGATGCTATAT CGCCCAGAGACTGGG AAAGTCAGGCTCAGG TAGAGATGCTATATG GCCCAGAGACTGGGC AAGTCAGGCTCAGGG AGTCAGGCTCAGGGA GCTGCATAGAGCTCT CCTATTAGCTTTTCT GTCAGGCTCAGGGAG CTGCATAGAGCTCTC CTATTAGCTTTTCTT TCAGGCTCAGGGAGA TGCATAGAGCTCTCC TATTAGCTTTTCTTT CAGGCTCAGGGAGAC GCATAGAGCTCTCCT ATTAGCTTTTCTTTA AGGCTCAGGGAGACT CATAGAGCTCTCCTT TTAGCTTTTCTTTAT GGCTCAGGGAGACTC ATAGAGCTCTCCTTG TAGCTTTTCTTTATT GCTCAGGGAGACTCT TAGAGCTCTCCTTGA AGCTTTTCTTTATTT CTCAGGGAGACTCTG AGAGCTCTCCTTGAA GCTTTTCTTTATTTT TCAGGGAGACTCTGC GAGCTCTCCTTGAAA CTTTTCTTTATTTTT CAGGGAGACTCTGCC AGCTCTCCTTGAAAA TTTTCTTTATTTTTT AGGGAGACTCTGCCC GCTCTCCTTGAAAAC TTTCTTTATTTTTTT GGGAGACTCTGCCCT CTCTCCTTGAAAACA TTCTTTATTTTTTTA GGAGACTCTGCCCTG TCTCCTTGAAAACAG TCTTTATTTTTTTAA GAGACTCTGCCCTGC CTCCTTGAAAACAGA CTTTATTTTTTTAAC AGACTCTGCCCTGCT TCCTTGAAAACAGAG TTTATTTTTTTAACT GACTCTGCCCTGCTG CCTTGAAAACAGAGG TTATTTTTTTAACTT ACTCTGCCCTGCTGC CTTGAAAACAGAGGG TATTTTTTTAACTTT CTCTGCCCTGCTGCA TTGAAAACAGAGGGG ATTTTTTTAACTTTT TCTGCCCTGCTGCAG TGAAAACAGAGGGGT TTTTTTTAACTTTTT CTGCCCTGCTGCAGA GAAAACAGAGGGGTC TTTTTTAACTTTTTG TGCCCTGCTGCAGAC AAAACAGAGGGGTCT TTTTTAACTTTTTGG GCCCTGCTGCAGACC AAACAGAGGGGTCTC TTTTAACTTTTTGGG CCCTGCTGCAGACCT AACAGAGGGGTCTCA TTTAACTTTTTGGGG CCTGCTGCAGACCTC ACAGAGGGGTCTCAA TTAACTTTTTGGGGG CTGCTGCAGACCTCG CAGAGGGGTCTCAAG TAACTTTTTGGGGGG TGCTGCAGACCTCGG AGAGGGGTCTCAAGA AACTTTTTGGGGGGA GCTGCAGACCTCGGT GAGGGGTCTCAAGAC ACTTTTTGGGGGGAA CTGCAGACCTCGGTG AGGGGTCTCAAGACA CTTTTTGGGGGGAAA TGCAGACCTCGGTGT GGGGTCTCAAGACAT TTTTTGGGGGGAAAA GCAGACCTCGGTGTG GGGTCTCAAGACATT TTTTGGGGGGAAAAG CAGACCTCGGTGTGG GGTCTCAAGACATTC TTTGGGGGGAAAAGT AGACCTCGGTGTGGA GTCTCAAGACATTCT TTGGGGGGAAAAGTA GACCTCGGTGTGGAC TCTCAAGACATTCTG TGGGGGGAAAAGTAT ACCTCGGTGTGGACA CTCAAGACATTCTGC GGGGGGAAAAGTATT CCTCGGTGTGGACAC TCAAGACATTCTGCC GGGGGAAAAGTATTT CTCGGTGTGGACACA CAAGACATTCTGCCT GGGGAAAAGTATTTT TCGGTGTGGACACAC AAGACATTCTGCCTA GGGAAAAGTATTTTT CGGTGTGGACACACG AGACATTCTGCCTAC GGAAAAGTATTTTTG GGTGTGGACACACGC GACATTCTGCCTACC GAAAAGTATTTTTGA GTGTGGACACACGCT ACATTCTGCCTACCT AAAAGTATTTTTGAG TGTGGACACACGCTG CATTCTGCCTACCTA AAAGTATTTTTGAGA GTGGACACACGCTGC ATTCTGCCTACCTAT AAGTATTTTTGAGAA TGGACACACGCTGCA TTCTGCCTACCTATT AGTATTTTTGAGAAG GGACACACGCTGCAT TCTGCCTACCTATTA GTATTTTTGAGAAGT GACACACGCTGCATA CTGCCTACCTATTAG TATTTTTGAGAAGTT ACACACGCTGCATAG TGCCTACCTATTAGC ATTTTTGAGAAGTTT CACACGCTGCATAGA GCCTACCTATTAGCT TTTTTGAGAAGTTTG ACACGCTGCATAGAG CCTACCTATTAGCTT TTTTGAGAAGTTTGT CACGCTGCATAGAGC CTACCTATTAGCTTT TTTGAGAAGTTTGTC ACGCTGCATAGAGCT TACCTATTAGCTTTT TTGAGAAGTTTGTCT CGCTGCATAGAGCTC ACCTATTAGCTTTTC TGAGAAGTTTGTCTT GAGAAGTTTGTCTTG
AGAAGTTTGTCTTGC
GAAGTTTGTCTTGCA
AAGTTTGTCTTGCAA AGTTTGTCTTGCAAT
GTTTGTCTTGCAATG
TTTGTCTTGCAATGT
TTGTCTTGCAATGTA
TGTCTTGCAATGTAT GTCTTGCAATGTATT
TCTTGCAATGTATTT
CTTGCAATGTATTTA
TTGCAATGTATTTAT
TGCAATGTATTTATA GCAATGTATTTATAA
CAATGTATTTATAAA
AATGTATTTATAAAT
ATGTATTTATAAATA
TGTATTTATAAATAG GTATTTATAAATAGT
TATTTATAAATAGTA
ATTTATAAATAGTAA
TTTATAAATAGTAAA
TTATAAATAGTAAAT TATAAATAGTAAATA
ATAAATAGTAAATAA
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 TTTTTTTTTTTGAGA TTTTTTTTGAGAAAG TTTTTTTTTTTTTGA TTTTTTTTTTGAGAA TTTTTTTGAGAAAGG TTTTTTTTTTTTGAG TTTTTTTTTGAGAAA TTTTTTGAGAAAGGG TTTTTGAGAAAGGGA GGAGGAGGGTCCCCG CTCTCGCTCTGGCCG TTTTGAGAAAGGGAA GAGGAGGGTCCCCGA TCTCGCTCTGGCCGA TTTGAGAAAGGGAAT AGGAGGGTCCCCGAC CTCGCTCTGGCCGAC TTGAGAAAGGGAATT GGAGGGTCCCCGACC TCGCTCTGGCCGACG TGAGAAAGGGAATTT GAGGGTCCCCGACCT CGCTCTGGCCGACGA GAGAAAGGGAATTTC AGGGTCCCCGACCTC GCTCTGGCCGACGAG AGAAAGGGAATTTCA GGGTCCCCGACCTCG CTCTGGCCGACGAGT GAAAGGGAATTTCAT GGTCCCCGACCTCGC TCTGGCCGACGAGTG AAAGGGAATTTCATC GTCCCCGACCTCGCT CTGGCCGACGAGTGG AAGGGAATTTCATCC TCCCCGACCTCGCTG TGGCCGACGAGTGGA AGGGAATTTCATCCC CCCCGACCTCGCTGT GGCCGACGAGTGGAG GGGAATTTCATCCCA CCCGACCTCGCTGTG GCCGACGAGTGGAGA GGAATTTCATCCCAA CCGACCTCGCTGTGG CCGACGAGTGGAGAA GAATTTCATCCCAAA CGACCTCGCTGTGGG CGACGAGTGGAGAAA AATTTCATCCCAAAT GACCTCGCTGTGGGG GACGAGTGGAGAAAT ATTTCATCCCAAATA ACCTCGCTGTGGGGG ACGAGTGGAGAAATC TTTCATCCCAAATAA CCTCGCTGTGGGGGC CGAGTGGAGAAATCT TTCATCCCAAATAAA CTCGCTGTGGGGGCT GAGTGGAGAAATCTG TCATCCCAAATAAAA TCGCTGTGGGGGCTC AGTGGAGAAATCTGC CATCCCAAATAAAAG CGCTGTGGGGGCTCC GTGGAGAAATCTGCG ATCCCAAATAAAAGG GCTGTGGGGGCTCCT TGGAGAAATCTGCGG TCCCAAATAAAAGGA CTGTGGGGGCTCCTG GGAGAAATCTGCGGG CCCAAATAAAAGGAA TGTGGGGGCTCCTGT GAGAAATCTGCGGGC CCAAATAAAAGGAAT GTGGGGGCTCCTGTT AGAAATCTGCGGGCC CAAATAAAAGGAATG TGGGGGCTCCTGTTT GAAATCTGCGGGCCA AAATAAAAGGAATGA GGGGGCTCCTGTTTC AAATCTGCGGGCCAG AATAAAAGGAATGAA GGGGCTCCTGTTTCT AATCTGCGGGCCAGG ATAAAAGGAATGAAG GGGCTCCTGTTTCTC ATCTGCGGGCCAGGC TAAAAGGAATGAAGT GGCTCCTGTTTCTCT TCTGCGGGCCAGGCA AAAAGGAATGAAGTC GCTCCTGTTTCTCTC CTGCGGGCCAGGCAT AAAGGAATGAAGTCT CTCCTGTTTCTCTCC TGCGGGCCAGGCATC AAGGAATGAAGTCTG TCCTGTTTCTCTCCG GCGGGCCAGGCATCG AGGAATGAAGTCTGG CCTGTTTCTCTCCGC CGGGCCAGGCATCGA GGAATGAAGTCTGGC CTGTTTCTCTCCGCC GGGCCAGGCATCGAC GAATGAAGTCTGGCT TGTTTCTCTCCGCCG GGCCAGGCATCGACA AATGAAGTCTGGCTC GTTTCTCTCCGCCGC GCCAGGCATCGACAT ATGAAGTCTGGCTCC TTTCTCTCCGCCGCG CCAGGCATCGACATC TGAAGTCTGGCTCCG TTCTCTCCGCCGCGC CAGGCATCGACATCC GAAGTCTGGCTCCGG TCTCTCCGCCGCGCT AGGCATCGACATCCG AAGTCTGGCTCCGGA CTCTCCGCCGCGCTC GGCATCGACATCCGC AGTCTGGCTCCGGAG TCTCCGCCGCGCTCT GCATCGACATCCGCA GTCTGGCTCCGGAGG CTCCGCCGCGCTCTC CATCGACATCCGCAA TCTGGCTCCGGAGGA TCCGCCGCGCTCTCG ATCGACATCCGCAAC CTGGCTCCGGAGGAG CCGCCGCGCTCTCGC TCGACATCCGCAACG TGGCTCCGGAGGAGG CGCCGCGCTCTCGCT CGACATCCGCAACGA GGCTCCGGAGGAGGG GCCGCGCTCTCGCTC GACATCCGCAACGAC GCTCCGGAGGAGGGT CCGCGCTCTCGCTCT ACATCCGCAACGACT CTCCGGAGGAGGGTC CGCGCTCTCGCTCTG CATCCGCAACGACTA TCCGGAGGAGGGTCC GCGCTCTCGCTCTGG ATCCGCAACGACTAT CCGGAGGAGGGTCCC CGCTCTCGCTCTGGC TCCGCAACGACTATC CGGAGGAGGGTCCCC GCTCTCGCTCTGGCC CCGCAACGACTATCA CGCAACGACTATCAG GGCTACCTCCACATC CGCTTCCCCAAGCTC
GCAACGACTATCAGC GCTACCTCCACATCC GCTTCCCCAAGCTCA
CAACGACTATCAGCA CTACCTCCACATCCT CTTCCCCAAGCTCAC
AACGACTATCAGCAG TACCTCCACATCCTG TTCCCCAAGCTCACG ACGACTATCAGCAGC ACCTCCACATCCTGC TCCCCAAGCTCACGG
CGACTATCAGCAGCT CCTCCACATCCTGCT CCCCAAGCTCACGGT
GACTATCAGCAGCTG CTCCACATCCTGCTC CCCAAGCTCACGGTC
ACTATCAGCAGCTGA TCCACATCCTGCTCA CCAAGCTCACGGTCA
CTATCAGCAGCTGAA CCACATCCTGCTCAT CAAGCTCACGGTCAT TATCAGCAGCTGAAG CACATCCTGCTCATC AAGCTCACGGTCATT
ATCAGCAGCTGAAGC ACATCCTGCTCATCT AGCTCACGGTCATTA
TCAGCAGCTGAAGCG CATCCTGCTCATCTC GCTCACGGTCATTAC
CAGCAGCTGAAGCGC ATCCTGCTCATCTCC CTCACGGTCATTACC
AGCAGCTGAAGCGCC TCCTGCTCATCTCCA TCACGGTCATTACCG GCAGCTGAAGCGCCT CCTGCTCATCTCCAA CACGGTCATTACCGA
CAGCTGAAGCGCCTG CTGCTCATCTCCAAG ACGGTCATTACCGAG
AGCTGAAGCGCCTGG TGCTCATCTCCAAGG CGGTCATTACCGAGT
GCTGAAGCGCCTGGA GCTCATCTCCAAGGC GGTCATTACCGAGTA
CTGAAGCGCCTGGAG CTCATCTCCAAGGCC GTCATTACCGAGTAC TGAAGCGCCTGGAGA TCATCTCCAAGGCCG TCATTACCGAGTACT
GAAGCGCCTGGAGAA CATCTCCAAGGCCGA CATTACCGAGTACTT
AAGCGCCTGGAGAAC ATCTCCAAGGCCGAG ATTACCGAGTACTTG
AGCGCCTGGAGAACT TCTCCAAGGCCGAGG TTACCGAGTACTTGC
GCGCCTGGAGAACTG CTCCAAGGCCGAGGA TACCGAGTACTTGCT CGCCTGGAGAACTGC TCCAAGGCCGAGGAC ACCGAGTACTTGCTG
GCCTGGAGAACTGCA CCAAGGCCGAGGACT CCGAGTACTTGCTGC
CCTGGAGAACTGCAC CAAGGCCGAGGACTA CGAGTACTTGCTGCT
CTGGAGAACTGCACG AAGGCCGAGGACTAC GAGTACTTGCTGCTG
TGGAGAACTGCACGG AGGCCGAGGACTACC AGTACTTGCTGCTGT GGAGAACTGCACGGT GGCCGAGGACTACCG GTACTTGCTGCTGTT
GAGAACTGCACGGTG GCCGAGGACTACCGC TACTTGCTGCTGTTC
AGAACTGCACGGTGA CCGAGGACTACCGCA ACTTGCTGCTGTTCC
GAACTGCACGGTGAT CGAGGACTACCGCAG CTTGCTGCTGTTCCG
AACTGCACGGTGATC GAGGACTACCGCAGC TTGCTGCTGTTCCGA ACTGCACGGTGATCG AGGACTACCGCAGCT TGCTGCTGTTCCGAG
CTGCACGGTGATCGA GGACTACCGCAGCTA GCTGCTGTTCCGAGT
TGCACGGTGATCGAG GACTACCGCAGCTAC CTGCTGTTCCGAGTG
GCACGGTGATCGAGG ACTACCGCAGCTACC TGCTGTTCCGAGTGG
CACGGTGATCGAGGG CTACCGCAGCTACCG GCTGTTCCGAGTGGC ACGGTGATCGAGGGC TACCGCAGCTACCGC CTGTTCCGAGTGGCT
CGGTGATCGAGGGCT ACCGCAGCTACCGCT TGTTCCGAGTGGCTG
GGTGATCGAGGGCTA CCGCAGCTACCGCTT GTTCCGAGTGGCTGG
GTGATCGAGGGCTAC CGCAGCTACCGCTTC TTCCGAGTGGCTGGC
TGATCGAGGGCTACC GCAGCTACCGCTTCC TCCGAGTGGCTGGCC GATCGAGGGCTACCT CAGCTACCGCTTCCC CCGAGTGGCTGGCCT
ATCGAGGGCTACCTC AGCTACCGCTTCCCC CGAGTGGCTGGCCTC
TCGAGGGCTACCTCC GCTACCGCTTCCCCA GAGTGGCTGGCCTCG
CGAGGGCTACCTCCA CTACCGCTTCCCCAA AGTGGCTGGCCTCGA
GAGGGCTACCTCCAC TACCGCTTCCCCAAG GTGGCTGGCCTCGAG AGGGCTACCTCCACA ACCGCTTCCCCAAGC TGGCTGGCCTCGAGA
GGGCTACCTCCACAT CCGCTTCCCCAAGCT GGCTGGCCTCGAGAG GCTGGCCTCGAGAGC GGCTGGAAACTCTTC CTCAAGGATATTGGG
CTGGCCTCGAGAGCC GCTGGAAACTCTTCT TCAAGGATATTGGGC
TGGCCTCGAGAGCCT CTGGAAACTCTTCTA CAAGGATATTGGGCT
GGCCTCGAGAGCCTC TGGAAACTCTTCTAC AAGGATATTGGGCTT GCCTCGAGAGCCTCG GGAAACTCTTCTACA AGGATATTGGGCTTT
CCTCGAGAGCCTCGG GAAACTCTTCTACAA GGATATTGGGCTTTA
CTCGAGAGCCTCGGA AAACTCTTCTACAAC GATATTGGGCTTTAC
TCGAGAGCCTCGGAG AACTCTTCTACAACT ATATTGGGCTTTACA
CGAGAGCCTCGGAGA ACTCTTCTACAACTA TATTGGGCTTTACAA GAGAGCCTCGGAGAC CTCTTCTACAACTAC ATTGGGCTTTACAAC
AGAGCCTCGGAGACC TCTTCTACAACTACG TTGGGCTTTACAACC
GAGCCTCGGAGACCT CTTCTACAACTACGC TGGGCTTTACAACCT
AGCCTCGGAGACCTC TTCTACAACTACGCC GGGCTTTACAACCTG
GCCTCGGAGACCTCT TCTACAACTACGCCC GGCTTTACAACCTGA CCTCGGAGACCTCTT CTACAACTACGCCCT GCTTTACAACCTGAG
CTCGGAGACCTCTTC TACAACTACGCCCTG CTTTACAACCTGAGG
TCGGAGACCTCTTCC ACAACTACGCCCTGG TTTACAACCTGAGGA
CGGAGACCTCTTCCC CAACTACGCCCTGGT TTACAACCTGAGGAA
GGAGACCTCTTCCCC AACTACGCCCTGGTC TACAACCTGAGGAAC GAGACCTCTTCCCCA ACTACGCCCTGGTCA ACAACCTGAGGAACA
AGACCTCTTCCCCAA CTACGCCCTGGTCAT CAACCTGAGGAACAT
GACCTCTTCCCCAAC TACGCCCTGGTCATC AACCTGAGGAACATT
ACCTCTTCCCCAACC ACGCCCTGGTCATCT ACCTGAGGAACATTA
CCTCTTCCCCAACCT CGCCCTGGTCATCTT CCTGAGGAACATTAC CTCTTCCCCAACCTC GCCCTGGTCATCTTC CTGAGGAACATTACT
TCTTCCCCAACCTCA CCCTGGTCATCTTCG TGAGGAACATTACTC
CTTCCCCAACCTCAC CCTGGTCATCTTCGA GAGGAACATTACTCG
TTCCCCAACCTCACG CTGGTCATCTTCGAG AGGAACATTACTCGG
TCCCCAACCTCACGG TGGTCATCTTCGAGA GGAACATTACTCGGG CCCCAACCTCACGGT GGTCATCTTCGAGAT GAACATTACTCGGGG
CCCAACCTCACGGTC GTCATCTTCGAGATG AACATTACTCGGGGG
CCAACCTCACGGTCA TCATCTTCGAGATGA ACATTACTCGGGGGG
CAACCTCACGGTCAT CATCTTCGAGATGAC CATTACTCGGGGGGC
AACCTCACGGTCATC ATCTTCGAGATGACC ATTACTCGGGGGGCC ACCTCACGGTCATCC TCTTCGAGATGACCA TTACTCGGGGGGCCA
CCTCACGGTCATCCG CTTCGAGATGACCAA TACTCGGGGGGCCAT
CTCACGGTCATCCGC TTCGAGATGACCAAT ACTCGGGGGGCCATC
TCACGGTCATCCGCG TCGAGATGACCAATC CTCGGGGGGCCATCA
CACGGTCATCCGCGG CGAGATGACCAATCT TCGGGGGGCCATCAG ACGGTCATCCGCGGC GAGATGACCAATCTC CGGGGGGCCATCAGG
CGGTCATCCGCGGCT AGATGACCAATCTCA GGGGGGCCATCAGGA
GGTCATCCGCGGCTG GATGACCAATCTCAA GGGGGCCATCAGGAT
GTCATCCGCGGCTGG ATGACCAATCTCAAG GGGGCCATCAGGATT
TCATCCGCGGCTGGA TGACCAATCTCAAGG GGGCCATCAGGATTG CATCCGCGGCTGGAA GACCAATCTCAAGGA GGCCATCAGGATTGA
ATCCGCGGCTGGAAA ACCAATCTCAAGGAT GCCATCAGGATTGAG
TCCGCGGCTGGAAAC CCAATCTCAAGGATA CCATCAGGATTGAGA
CCGCGGCTGGAAACT CAATCTCAAGGATAT CATCAGGATTGAGAA
CGCGGCTGGAAACTC AATCTCAAGGATATT ATCAGGATTGAGAAA GCGGCTGGAAACTCT ATCTCAAGGATATTG TCAGGATTGAGAAAA
CGGCTGGAAACTCTT TCTCAAGGATATTGG CAGGATTGAGAAAAA AGGATTGAGAAAAAT CTGATCCTGGATGCG AAGGAATGTGGGGAC
GGATTGAGAAAAATG TGATCCTGGATGCGG AGGAATGTGGGGACC
GATTGAGAAAAATGC GATCCTGGATGCGGT GGAATGTGGGGACCT
ATTGAGAAAAATGCT ATCCTGGATGCGGTG GAATGTGGGGACCTG TTGAGAAAAATGCTG TCCTGGATGCGGTGT AATGTGGGGACCTGT
TGAGAAAAATGCTGA CCTGGATGCGGTGTC ATGTGGGGACCTGTG
GAGAAAAATGCTGAC CTGGATGCGGTGTCC TGTGGGGACCTGTGT
AGAAAAATGCTGACC TGGATGCGGTGTCCA GTGGGGACCTGTGTC
GAAAAATGCTGACCT GGATGCGGTGTCCAA TGGGGACCTGTGTCC AAAAATGCTGACCTC GATGCGGTGTCCAAT GGGGACCTGTGTCCA
AAAATGCTGACCTCT ATGCGGTGTCCAATA GGGACCTGTGTCCAG
AAATGCTGACCTCTG TGCGGTGTCCAATAA GGACCTGTGTCCAGG
AATGCTGACCTCTGT GCGGTGTCCAATAAC GACCTGTGTCCAGGG
ATGCTGACCTCTGTT CGGTGTCCAATAACT ACCTGTGTCCAGGGA TGCTGACCTCTGTTA GGTGTCCAATAACTA CCTGTGTCCAGGGAC
GCTGACCTCTGTTAC GTGTCCAATAACTAC CTGTGTCCAGGGACC
CTGACCTCTGTTACC TGTCCAATAACTACA TGTGTCCAGGGACCA
TGACCTCTGTTACCT GTCCAATAACTACAT GTGTCCAGGGACCAT
GACCTCTGTTACCTC TCCAATAACTACATT TGTCCAGGGACCATG ACCTCTGTTACCTCT CCAATAACTACATTG GTCCAGGGACCATGG
CCTCTGTTACCTCTC CAATAACTACATTGT TCCAGGGACCATGGA
CTCTGTTACCTCTCC AATAACTACATTGTG CCAGGGACCATGGAG
TCTGTTACCTCTCCA ATAACTACATTGTGG CAGGGACCATGGAGG
CTGTTACCTCTCCAC TAACTACATTGTGGG AGGGACCATGGAGGA TGTTACCTCTCCACT AACTACATTGTGGGG GGGACCATGGAGGAG
GTTACCTCTCCACTG ACTACATTGTGGGGA GGACCATGGAGGAGA
TTACCTCTCCACTGT CTACATTGTGGGGAA GACCATGGAGGAGAA
TACCTCTCCACTGTG TACATTGTGGGGAAT ACCATGGAGGAGAAG
ACCTCTCCACTGTGG ACATTGTGGGGAATA CCATGGAGGAGAAGC CCTCTCCACTGTGGA CATTGTGGGGAATAA CATGGAGGAGAAGCC
CTCTCCACTGTGGAC ATTGTGGGGAATAAG ATGGAGGAGAAGCCG
TCTCCACTGTGGACT TTGTGGGGAATAAGC TGGAGGAGAAGCCGA
CTCCACTGTGGACTG TGTGGGGAATAAGCC GGAGGAGAAGCCGAT
TCCACTGTGGACTGG GTGGGGAATAAGCCC GAGGAGAAGCCGATG CCACTGTGGACTGGT TGGGGAATAAGCCCC AGGAGAAGCCGATGT
CACTGTGGACTGGTC GGGGAATAAGCCCCC GGAGAAGCCGATGTG
ACTGTGGACTGGTCC GGGAATAAGCCCCCA GAGAAGCCGATGTGT
CTGTGGACTGGTCCC GGAATAAGCCCCCAA AGAAGCCGATGTGTG
TGTGGACTGGTCCCT GAATAAGCCCCCAAA GAAGCCGATGTGTGA GTGGACTGGTCCCTG AATAAGCCCCCAAAG AAGCCGATGTGTGAG
TGGACTGGTCCCTGA ATAAGCCCCCAAAGG AGCCGATGTGTGAGA
GGACTGGTCCCTGAT TAAGCCCCCAAAGGA GCCGATGTGTGAGAA
GACTGGTCCCTGATC AAGCCCCCAAAGGAA CCGATGTGTGAGAAG
ACTGGTCCCTGATCC AGCCCCCAAAGGAAT CGATGTGTGAGAAGA CTGGTCCCTGATCCT GCCCCCAAAGGAATG GATGTGTGAGAAGAC
TGGTCCCTGATCCTG CCCCCAAAGGAATGT ATGTGTGAGAAGACC
GGTCCCTGATCCTGG CCCCAAAGGAATGTG TGTGTGAGAAGACCA
GTCCCTGATCCTGGA CCCAAAGGAATGTGG GTGTGAGAAGACCAC
TCCCTGATCCTGGAT CCAAAGGAATGTGGG TGTGAGAAGACCACC CCCTGATCCTGGATG CAAAGGAATGTGGGG GTGAGAAGACCACCA
CCTGATCCTGGATGC AAAGGAATGTGGGGA TGAGAAGACCACCAT GAGAAGACCACCATC CGCTGCCAGAAAATG AACAATGAGTGCTGC
AGAAGACCACCATCA GCTGCCAGAAAATGT ACAATGAGTGCTGCC
GAAGACCACCATCAA CTGCCAGAAAATGTG CAATGAGTGCTGCCA
AAGACCACCATCAAC TGCCAGAAAATGTGC AATGAGTGCTGCCAC AGACCACCATCAACA GCCAGAAAATGTGCC ATGAGTGCTGCCACC
GACCACCATCAACAA CCAGAAAATGTGCCC TGAGTGCTGCCACCC
ACCACCATCAACAAT CAGAAAATGTGCCCA GAGTGCTGCCACCCC
CCACCATCAACAATG AGAAAATGTGCCCAA AGTGCTGCCACCCCG
CACCATCAACAATGA GAAAATGTGCCCAAG GTGCTGCCACCCCGA ACCATCAACAATGAG AAAATGTGCCCAAGC TGCTGCCACCCCGAG
CCATCAACAATGAGT AAATGTGCCCAAGCA GCTGCCACCCCGAGT
CATCAACAATGAGTA AATGTGCCCAAGCAC CTGCCACCCCGAGTG
ATCAACAATGAGTAC ATGTGCCCAAGCACG TGCCACCCCGAGTGC
TCAACAATGAGTACA TGTGCCCAAGCACGT GCCACCCCGAGTGCC CAACAATGAGTACAA GTGCCCAAGCACGTG CCACCCCGAGTGCCT
AACAATGAGTACAAC TGCCCAAGCACGTGT CACCCCGAGTGCCTG
ACAATGAGTACAACT GCCCAAGCACGTGTG ACCCCGAGTGCCTGG
CAATGAGTACAACTA CCCAAGCACGTGTGG CCCCGAGTGCCTGGG
AATGAGTACAACTAC CCAAGCACGTGTGGG CCCGAGTGCCTGGGC ATGAGTACAACTACC CAAGCACGTGTGGGA CCGAGTGCCTGGGCA
TGAGTACAACTACCG AAGCACGTGTGGGAA CGAGTGCCTGGGCAG
GAGTACAACTACCGC AGCACGTGTGGGAAG GAGTGCCTGGGCAGC
AGTACAACTACCGCT GCACGTGTGGGAAGC AGTGCCTGGGCAGCT
GTACAACTACCGCTG CACGTGTGGGAAGCG GTGCCTGGGCAGCTG TACAACTACCGCTGC ACGTGTGGGAAGCGG TGCCTGGGCAGCTGC
ACAACTACCGCTGCT CGTGTGGGAAGCGGG GCCTGGGCAGCTGCA
CAACTACCGCTGCTG GTGTGGGAAGCGGGC CCTGGGCAGCTGCAG
AACTACCGCTGCTGG TGTGGGAAGCGGGCG CTGGGCAGCTGCAGC
ACTACCGCTGCTGGA GTGGGAAGCGGGCGT TGGGCAGCTGCAGCG CTACCGCTGCTGGAC TGGGAAGCGGGCGTG GGGCAGCTGCAGCGC
TACCGCTGCTGGACC GGGAAGCGGGCGTGC GGCAGCTGCAGCGCG
ACCGCTGCTGGACCA GGAAGCGGGCGTGCA GCAGCTGCAGCGCGC
CCGCTGCTGGACCAC GAAGCGGGCGTGCAC CAGCTGCAGCGCGCC
CGCTGCTGGACCACA AAGCGGGCGTGCACC AGCTGCAGCGCGCCT GCTGCTGGACCACAA AGCGGGCGTGCACCG GCTGCAGCGCGCCTG
CTGCTGGACCACAAA GCGGGCGTGCACCGA CTGCAGCGCGCCTGA
TGCTGGACCACAAAC CGGGCGTGCACCGAG TGCAGCGCGCCTGAC
GCTGGACCACAAACC GGGCGTGCACCGAGA GCAGCGCGCCTGACA
CTGGACCACAAACCG GGCGTGCACCGAGAA CAGCGCGCCTGACAA TGGACCACAAACCGC GCGTGCACCGAGAAC AGCGCGCCTGACAAC
GGACCACAAACCGCT CGTGCACCGAGAACA GCGCGCCTGACAACG
GACCACAAACCGCTG GTGCACCGAGAACAA CGCGCCTGACAACGA
ACCACAAACCGCTGC TGCACCGAGAACAAT GCGCCTGACAACGAC
CCACAAACCGCTGCC GCACCGAGAACAATG CGCCTGACAACGACA CACAAACCGCTGCCA CACCGAGAACAATGA GCCTGACAACGACAC
ACAAACCGCTGCCAG ACCGAGAACAATGAG CCTGACAACGACACG
CAAACCGCTGCCAGA CCGAGAACAATGAGT CTGACAACGACACGG
AAACCGCTGCCAGAA CGAGAACAATGAGTG TGACAACGACACGGC
AACCGCTGCCAGAAA GAGAACAATGAGTGC GACAACGACACGGCC ACCGCTGCCAGAAAA AGAACAATGAGTGCT ACAACGACACGGCCT
CCGCTGCCAGAAAAT GAACAATGAGTGCTG CAACGACACGGCCTG AACGACACGGCCTGT GTGCCTGCCTGCCCG GACCGTGACTTCTGC ACGACACGGCCTGTG TGCCTGCCTGCCCGC ACCGTGACTTCTGCG CGACACGGCCTGTGT GCCTGCCTGCCCGCC CCGTGACTTCTGCGC GACACGGCCTGTGTA CCTGCCTGCCCGCCC CGTGACTTCTGCGCC ACACGGCCTGTGTAG CTGCCTGCCCGCCCA GTGACTTCTGCGCCA CACGGCCTGTGTAGC TGCCTGCCCGCCCAA TGACTTCTGCGCCAA ACGGCCTGTGTAGCT GCCTGCCCGCCCAAC GACTTCTGCGCCAAC CGGCCTGTGTAGCTT CCTGCCCGCCCAACA ACTTCTGCGCCAACA GGCCTGTGTAGCTTG CTGCCCGCCCAACAC CTTCTGCGCCAACAT GCCTGTGTAGCTTGC TGCCCGCCCAACACC TTCTGCGCCAACATC CCTGTGTAGCTTGCC GCCCGCCCAACACCT TCTGCGCCAACATCC CTGTGTAGCTTGCCG CCCGCCCAACACCTA CTGCGCCAACATCCT TGTGTAGCTTGCCGC CCGCCCAACACCTAC TGCGCCAACATCCTC GTGTAGCTTGCCGCC CGCCCAACACCTACA GCGCCAACATCCTCA TGTAGCTTGCCGCCA GCCCAACACCTACAG CGCCAACATCCTCAG GTAGCTTGCCGCCAC CCCAACACCTACAGG GCCAACATCCTCAGC TAGCTTGCCGCCACT CCAACACCTACAGGT CCAACATCCTCAGCG AGCTTGCCGCCACTA CAACACCTACAGGTT CAACATCCTCAGCGC GCTTGCCGCCACTAC AACACCTACAGGTTT AACATCCTCAGCGCC CTTGCCGCCACTACT ACACCTACAGGTTTG ACATCCTCAGCGCCG TTGCCGCCACTACTA CACCTACAGGTTTGA CATCCTCAGCGCCGA TGCCGCCACTACTAC ACCTACAGGTTTGAG ATCCTCAGCGCCGAG GCCGCCACTACTACT CCTACAGGTTTGAGG TCCTCAGCGCCGAGA CCGCCACTACTACTA CTACAGGTTTGAGGG CCTCAGCGCCGAGAG CGCCACTACTACTAT TACAGGTTTGAGGGC CTCAGCGCCGAGAGC GCCACTACTACTATG ACAGGTTTGAGGGCT TCAGCGCCGAGAGCA CCACTACTACTATGC CAGGTTTGAGGGCTG CAGCGCCGAGAGCAG CACTACTACTATGCC AGGTTTGAGGGCTGG AGCGCCGAGAGCAGC ACTACTACTATGCCG GGTTTGAGGGCTGGC GCGCCGAGAGCAGCG CTACTACTATGCCGG GTTTGAGGGCTGGCG CGCCGAGAGCAGCGA TACTACTATGCCGGT TTTGAGGGCTGGCGC GCCGAGAGCAGCGAC ACTACTATGCCGGTG TTGAGGGCTGGCGCT CCGAGAGCAGCGACT CTACTATGCCGGTGT TGAGGGCTGGCGCTG CGAGAGCAGCGACTC TACTATGCCGGTGTC GAGGGCTGGCGCTGT GAGAGCAGCGACTCC ACTATGCCGGTGTCT AGGGCTGGCGCTGTG AGAGCAGCGACTCCG CTATGCCGGTGTCTG GGGCTGGCGCTGTGT GAGCAGCGACTCCGA TATGCCGGTGTCTGT GGCTGGCGCTGTGTG AGCAGCGACTCCGAG ATGCCGGTGTCTGTG GCTGGCGCTGTGTGG GCAGCGACTCCGAGG TGCCGGTGTCTGTGT CTGGCGCTGTGTGGA CAGCGACTCCGAGGG GCCGGTGTCTGTGTG TGGCGCTGTGTGGAC AGCGACTCCGAGGGG CCGGTGTCTGTGTGC GGCGCTGTGTGGACC GCGACTCCGAGGGGT CGGTGTCTGTGTGCC GCGCTGTGTGGACCG CGACTCCGAGGGGTT GGTGTCTGTGTGCCT CGCTGTGTGGACCGT GACTCCGAGGGGTTT GTGTCTGTGTGCCTG GCTGTGTGGACCGTG ACTCCGAGGGGTTTG TGTCTGTGTGCCTGC CTGTGTGGACCGTGA CTCCGAGGGGTTTGT GTCTGTGTGCCTGCC TGTGTGGACCGTGAC TCCGAGGGGTTTGTG TCTGTGTGCCTGCCT GTGTGGACCGTGACT CCGAGGGGTTTGTGA CTGTGTGCCTGCCTG TGTGGACCGTGACTT CGAGGGGTTTGTGAT TGTGTGCCTGCCTGC GTGGACCGTGACTTC GAGGGGTTTGTGATC GTGTGCCTGCCTGCC TGGACCGTGACTTCT AGGGGTTTGTGATCC TGTGCCTGCCTGCCC GGACCGTGACTTCTG GGGGTTTGTGATCCA GGGTTTGTGATCCAC ATCCGCAACGGCAGC CCGAAGGTCTGTGAG GGTTTGTGATCCACG TCCGCAACGGCAGCC CGAAGGTCTGTGAGG GTTTGTGATCCACGA CCGCAACGGCAGCCA GAAGGTCTGTGAGGA TTTGTGATCCACGAC CGCAACGGCAGCCAG AAGGTCTGTGAGGAA TTGTGATCCACGACG GCAACGGCAGCCAGA AGGTCTGTGAGGAAG TGTGATCCACGACGG CAACGGCAGCCAGAG GGTCTGTGAGGAAGA GTGATCCACGACGGC AACGGCAGCCAGAGC GTCTGTGAGGAAGAA TGATCCACGACGGCG ACGGCAGCCAGAGCA TCTGTGAGGAAGAAA GATCCACGACGGCGA CGGCAGCCAGAGCAT CTGTGAGGAAGAAAA ATCCACGACGGCGAG GGCAGCCAGAGCATG TGTGAGGAAGAAAAG TCCACGACGGCGAGT GCAGCCAGAGCATGT GTGAGGAAGAAAAGA CCACGACGGCGAGTG CAGCCAGAGCATGTA TGAGGAAGAAAAGAA CACGACGGCGAGTGC AGCCAGAGCATGTAC GAGGAAGAAAAGAAA ACGACGGCGAGTGCA GCCAGAGCATGTACT AGGAAGAAAAGAAAA CGACGGCGAGTGCAT CCAGAGCATGTACTG GGAAGAAAAGAAAAC GACGGCGAGTGCATG CAGAGCATGTACTGC GAAGAAAAGAAAACA ACGGCGAGTGCATGC AGAGCATGTACTGCA AAGAAAAGAAAACAA CGGCGAGTGCATGCA GAGCATGTACTGCAT AGAAAAGAAAACAAA GGCGAGTGCATGCAG AGCATGTACTGCATC GAAAAGAAAACAAAG GCGAGTGCATGCAGG GCATGTACTGCATCC AAAAGAAAACAAAGA CGAGTGCATGCAGGA CATGTACTGCATCCC AAAGAAAACAAAGAC GAGTGCATGCAGGAG ATGTACTGCATCCCT AAGAAAACAAAGACC AGTGCATGCAGGAGT TGTACTGCATCCCTT AGAAAACAAAGACCA GTGCATGCAGGAGTG GTACTGCATCCCTTG GAAAACAAAGACCAT TGCATGCAGGAGTGC TACTGCATCCCTTGT AAAACAAAGACCATT GCATGCAGGAGTGCC ACTGCATCCCTTGTG AAACAAAGACCATTG CATGCAGGAGTGCCC CTGCATCCCTTGTGA AACAAAGACCATTGA ATGCAGGAGTGCCCC TGCATCCCTTGTGAA ACAAAGACCATTGAT TGCAGGAGTGCCCCT GCATCCCTTGTGAAG CAAAGACCATTGATT GCAGGAGTGCCCCTC CATCCCTTGTGAAGG AAAGACCATTGATTC CAGGAGTGCCCCTCG ATCCCTTGTGAAGGT AAGACCATTGATTCT AGGAGTGCCCCTCGG TCCCTTGTGAAGGTC AGACCATTGATTCTG GGAGTGCCCCTCGGG CCCTTGTGAAGGTCC GACCATTGATTCTGT GAGTGCCCCTCGGGC CCTTGTGAAGGTCCT ACCATTGATTCTGTT AGTGCCCCTCGGGCT CTTGTGAAGGTCCTT CCATTGATTCTGTTA GTGCCCCTCGGGCTT TTGTGAAGGTCCTTG CATTGATTCTGTTAC TGCCCCTCGGGCTTC TGTGAAGGTCCTTGC ATTGATTCTGTTACT GCCCCTCGGGCTTCA GTGAAGGTCCTTGCC TTGATTCTGTTACTT CCCCTCGGGCTTCAT TGAAGGTCCTTGCCC TGATTCTGTTACTTC CCCTCGGGCTTCATC GAAGGTCCTTGCCCG GATTCTGTTACTTCT CCTCGGGCTTCATCC AAGGTCCTTGCCCGA ATTCTGTTACTTCTG CTCGGGCTTCATCCG AGGTCCTTGCCCGAA TTCTGTTACTTCTGC TCGGGCTTCATCCGC GGTCCTTGCCCGAAG TCTGTTACTTCTGCT CGGGCTTCATCCGCA GTCCTTGCCCGAAGG CTGTTACTTCTGCTC GGGCTTCATCCGCAA TCCTTGCCCGAAGGT TGTTACTTCTGCTCA GGCTTCATCCGCAAC CCTTGCCCGAAGGTC GTTACTTCTGCTCAG GCTTCATCCGCAACG CTTGCCCGAAGGTCT TTACTTCTGCTCAGA CTTCATCCGCAACGG TTGCCCGAAGGTCTG TACTTCTGCTCAGAT TTCATCCGCAACGGC TGCCCGAAGGTCTGT ACTTCTGCTCAGATG TCATCCGCAACGGCA GCCCGAAGGTCTGTG CTTCTGCTCAGATGC CATCCGCAACGGCAG CCCGAAGGTCTGTGA TTCTGCTCAGATGCT TCTGCTCAGATGCTC AACATCCGACGGGGG CTCATCGAGGTGGTG
CTGCTCAGATGCTCC ACATCCGACGGGGGA TCATCGAGGTGGTGA
TGCTCAGATGCTCCA CATCCGACGGGGGAA CATCGAGGTGGTGAC
GCTCAGATGCTCCAA ATCCGACGGGGGAAT ATCGAGGTGGTGACG CTCAGATGCTCCAAG TCCGACGGGGGAATA TCGAGGTGGTGACGG
TCAGATGCTCCAAGG CCGACGGGGGAATAA CGAGGTGGTGACGGG
CAGATGCTCCAAGGA CGACGGGGGAATAAC GAGGTGGTGACGGGC
AGATGCTCCAAGGAT GACGGGGGAATAACA AGGTGGTGACGGGCT
GATGCTCCAAGGATG ACGGGGGAATAACAT GGTGGTGACGGGCTA ATGCTCCAAGGATGC CGGGGGAATAACATT GTGGTGACGGGCTAC
TGCTCCAAGGATGCA GGGGGAATAACATTG TGGTGACGGGCTACG
GCTCCAAGGATGCAC GGGGAATAACATTGC GGTGACGGGCTACGT
CTCCAAGGATGCACC GGGAATAACATTGCT GTGACGGGCTACGTG
TCCAAGGATGCACCA GGAATAACATTGCTT TGACGGGCTACGTGA CCAAGGATGCACCAT GAATAACATTGCTTC GACGGGCTACGTGAA
CAAGGATGCACCATC AATAACATTGCTTCA ACGGGCTACGTGAAG
AAGGATGCACCATCT ATAACATTGCTTCAG CGGGCTACGTGAAGA
AGGATGCACCATCTT TAACATTGCTTCAGA GGGCTACGTGAAGAT
GGATGCACCATCTTC AACATTGCTTCAGAG GGCTACGTGAAGATC GATGCACCATCTTCA ACATTGCTTCAGAGC GCTACGTGAAGATCC
ATGCACCATCTTCAA CATTGCTTCAGAGCT CTACGTGAAGATCCG
TGCACCATCTTCAAG ATTGCTTCAGAGCTG TACGTGAAGATCCGC
GCACCATCTTCAAGG TTGCTTCAGAGCTGG ACGTGAAGATCCGCC
CACCATCTTCAAGGG TGCTTCAGAGCTGGA CGTGAAGATCCGCCA ACCATCTTCAAGGGC GCTTCAGAGCTGGAG GTGAAGATCCGCCAT
CCATCTTCAAGGGCA CTTCAGAGCTGGAGA TGAAGATCCGCCATT
CATCTTCAAGGGCAA TTCAGAGCTGGAGAA GAAGATCCGCCATTC
ATCTTCAAGGGCAAT TCAGAGCTGGAGAAC AAGATCCGCCATTCT
TCTTCAAGGGCAATT CAGAGCTGGAGAACT AGATCCGCCATTCTC CTTCAAGGGCAATTT AGAGCTGGAGAACTT GATCCGCCATTCTCA
TTCAAGGGCAATTTG GAGCTGGAGAACTTC ATCCGCCATTCTCAT
TCAAGGGCAATTTGC AGCTGGAGAACTTCA TCCGCCATTCTCATG
CAAGGGCAATTTGCT GCTGGAGAACTTCAT CCGCCATTCTCATGC
AAGGGCAATTTGCTC CTGGAGAACTTCATG CGCCATTCTCATGCC AGGGCAATTTGCTCA TGGAGAACTTCATGG GCCATTCTCATGCCT
GGGCAATTTGCTCAT GGAGAACTTCATGGG CCATTCTCATGCCTT
GGCAATTTGCTCATT GAGAACTTCATGGGG CATTCTCATGCCTTG
GCAATTTGCTCATTA AGAACTTCATGGGGC ATTCTCATGCCTTGG
CAATTTGCTCATTAA GAACTTCATGGGGCT TTCTCATGCCTTGGT AATTTGCTCATTAAC AACTTCATGGGGCTC TCTCATGCCTTGGTC
ATTTGCTCATTAACA ACTTCATGGGGCTCA CTCATGCCTTGGTCT
TTTGCTCATTAACAT CTTCATGGGGCTCAT TCATGCCTTGGTCTC
TTGCTCATTAACATC TTCATGGGGCTCATC CATGCCTTGGTCTCC
TGCTCATTAACATCC TCATGGGGCTCATCG ATGCCTTGGTCTCCT GCTCATTAACATCCG CATGGGGCTCATCGA TGCCTTGGTCTCCTT
CTCATTAACATCCGA ATGGGGCTCATCGAG GCCTTGGTCTCCTTG
TCATTAACATCCGAC TGGGGCTCATCGAGG CCTTGGTCTCCTTGT
CATTAACATCCGACG GGGGCTCATCGAGGT CTTGGTCTCCTTGTC
ATTAACATCCGACGG GGGCTCATCGAGGTG TTGGTCTCCTTGTCC TTAACATCCGACGGG GGCTCATCGAGGTGG TGGTCTCCTTGTCCT
TAACATCCGACGGGG GCTCATCGAGGTGGT GGTCTCCTTGTCCTT GTCTCCTTGTCCTTC CTAGAAGGGAATTAC CTGTGGGACTGGGAC
TCTCCTTGTCCTTCC TAGAAGGGAATTACT TGTGGGACTGGGACC
CTCCTTGTCCTTCCT AGAAGGGAATTACTC GTGGGACTGGGACCA
TCCTTGTCCTTCCTA GAAGGGAATTACTCC TGGGACTGGGACCAC CCTTGTCCTTCCTAA AAGGGAATTACTCCT GGGACTGGGACCACC
CTTGTCCTTCCTAAA AGGGAATTACTCCTT GGACTGGGACCACCG
TTGTCCTTCCTAAAA GGGAATTACTCCTTC GACTGGGACCACCGC
TGTCCTTCCTAAAAA GGAATTACTCCTTCT ACTGGGACCACCGCA
GTCCTTCCTAAAAAA GAATTACTCCTTCTA CTGGGACCACCGCAA TCCTTCCTAAAAAAC AATTACTCCTTCTAC TGGGACCACCGCAAC
CCTTCCTAAAAAACC ATTACTCCTTCTACG GGGACCACCGCAACC
CTTCCTAAAAAACCT TTACTCCTTCTACGT GGACCACCGCAACCT
TTCCTAAAAAACCTT TACTCCTTCTACGTC GACCACCGCAACCTG
TCCTAAAAAACCTTC ACTCCTTCTACGTCC ACCACCGCAACCTGA CCTAAAAAACCTTCG CTCCTTCTACGTCCT CCACCGCAACCTGAC
CTAAAAAACCTTCGC TCCTTCTACGTCCTC CACCGCAACCTGACC
TAAAAAACCTTCGCC CCTTCTACGTCCTCG ACCGCAACCTGACCA
AAAAAACCTTCGCCT CTTCTACGTCCTCGA CCGCAACCTGACCAT
AAAAACCTTCGCCTC TTCTACGTCCTCGAC CGCAACCTGACCATC AAAACCTTCGCCTCA TCTACGTCCTCGACA GCAACCTGACCATCA
AAACCTTCGCCTCAT CTACGTCCTCGACAA CAACCTGACCATCAA
AACCTTCGCCTCATC TACGTCCTCGACAAC AACCTGACCATCAAA
ACCTTCGCCTCATCC ACGTCCTCGACAACC ACCTGACCATCAAAG
CCTTCGCCTCATCCT CGTCCTCGACAACCA CCTGACCATCAAAGC CTTCGCCTCATCCTA GTCCTCGACAACCAG CTGACCATCAAAGCA
TTCGCCTCATCCTAG TCCTCGACAACCAGA TGACCATCAAAGCAG
TCGCCTCATCCTAGG CCTCGACAACCAGAA GACCATCAAAGCAGG
CGCCTCATCCTAGGA CTCGACAACCAGAAC ACCATCAAAGCAGGG
GCCTCATCCTAGGAG TCGACAACCAGAACT CCATCAAAGCAGGGA CCTCATCCTAGGAGA CGACAACCAGAACTT CATCAAAGCAGGGAA
CTCATCCTAGGAGAG GACAACCAGAACTTG ATCAAAGCAGGGAAA
TCATCCTAGGAGAGG ACAACCAGAACTTGC TCAAAGCAGGGAAAA
CATCCTAGGAGAGGA CAACCAGAACTTGCA CAAAGCAGGGAAAAT
ATCCTAGGAGAGGAG AACCAGAACTTGCAG AAAGCAGGGAAAATG TCCTAGGAGAGGAGC ACCAGAACTTGCAGC AAGCAGGGAAAATGT
CCTAGGAGAGGAGCA CCAGAACTTGCAGCA AGCAGGGAAAATGTA
CTAGGAGAGGAGCAG CAGAACTTGCAGCAA GCAGGGAAAATGTAC
TAGGAGAGGAGCAGC AGAACTTGCAGCAAC CAGGGAAAATGTACT
AGGAGAGGAGCAGCT GAACTTGCAGCAACT AGGGAAAATGTACTT GGAGAGGAGCAGCTA AACTTGCAGCAACTG GGGAAAATGTACTTT
GAGAGGAGCAGCTAG ACTTGCAGCAACTGT GGAAAATGTACTTTG
AGAGGAGCAGCTAGA CTTGCAGCAACTGTG GAAAATGTACTTTGC
GAGGAGCAGCTAGAA TTGCAGCAACTGTGG AAAATGTACTTTGCT
AGGAGCAGCTAGAAG TGCAGCAACTGTGGG AAATGTACTTTGCTT GGAGCAGCTAGAAGG GCAGCAACTGTGGGA AATGTACTTTGCTTT
GAGCAGCTAGAAGGG CAGCAACTGTGGGAC ATGTACTTTGCTTTC
AGCAGCTAGAAGGGA AGCAACTGTGGGACT TGTACTTTGCTTTCA
GCAGCTAGAAGGGAA GCAACTGTGGGACTG GTACTTTGCTTTCAA
CAGCTAGAAGGGAAT CAACTGTGGGACTGG TACTTTGCTTTCAAT AGCTAGAAGGGAATT AACTGTGGGACTGGG ACTTTGCTTTCAATC
GCTAGAAGGGAATTA ACTGTGGGACTGGGA CTTTGCTTTCAATCC TTTGCTTTCAATCCC GTGACGGGGACTAAA AACGGGGAGAGAGCC
TTGCTTTCAATCCCA TGACGGGGACTAAAG ACGGGGAGAGAGCCT
TGCTTTCAATCCCAA GACGGGGACTAAAGG CGGGGAGAGAGCCTC
GCTTTCAATCCCAAA ACGGGGACTAAAGGG GGGGAGAGAGCCTCC CTTTCAATCCCAAAT CGGGGACTAAAGGGC GGGAGAGAGCCTCCT
TTTCAATCCCAAATT GGGGACTAAAGGGCG GGAGAGAGCCTCCTG
TTCAATCCCAAATTA GGGACTAAAGGGCGC GAGAGAGCCTCCTGT
TCAATCCCAAATTAT GGACTAAAGGGCGCC AGAGAGCCTCCTGTG
CAATCCCAAATTATG GACTAAAGGGCGCCA GAGAGCCTCCTGTGA AATCCCAAATTATGT ACTAAAGGGCGCCAA AGAGCCTCCTGTGAA
ATCCCAAATTATGTG CTAAAGGGCGCCAAA GAGCCTCCTGTGAAA
TCCCAAATTATGTGT TAAAGGGCGCCAAAG AGCCTCCTGTGAAAG
CCCAAATTATGTGTT AAAGGGCGCCAAAGC GCCTCCTGTGAAAGT
CCAAATTATGTGTTT AAGGGCGCCAAAGCA CCTCCTGTGAAAGTG CAAATTATGTGTTTC AGGGCGCCAAAGCAA CTCCTGTGAAAGTGA
AAATTATGTGTTTCC GGGCGCCAAAGCAAA TCCTGTGAAAGTGAC
AATTATGTGTTTCCG GGCGCCAAAGCAAAG CCTGTGAAAGTGACG
ATTATGTGTTTCCGA GCGCCAAAGCAAAGG CTGTGAAAGTGACGT
TTATGTGTTTCCGAA CGCCAAAGCAAAGGG TGTGAAAGTGACGTC TATGTGTTTCCGAAA GCCAAAGCAAAGGGG GTGAAAGTGACGTCC
ATGTGTTTCCGAAAT CCAAAGCAAAGGGGA TGAAAGTGACGTCCT
TGTGTTTCCGAAATT CAAAGCAAAGGGGAC GAAAGTGACGTCCTG
GTGTTTCCGAAATTT AAAGCAAAGGGGACA AAAGTGACGTCCTGC
TGTTTCCGAAATTTA AAGCAAAGGGGACAT AAGTGACGTCCTGCA GTTTCCGAAATTTAC AGCAAAGGGGACATA AGTGACGTCCTGCAT
TTTCCGAAATTTACC GCAAAGGGGACATAA GTGACGTCCTGCATT
TTCCGAAATTTACCG CAAAGGGGACATAAA TGACGTCCTGCATTT
TCCGAAATTTACCGC AAAGGGGACATAAAC GACGTCCTGCATTTC
CCGAAATTTACCGCA AAGGGGACATAAACA ACGTCCTGCATTTCA CGAAATTTACCGCAT AGGGGACATAAACAC CGTCCTGCATTTCAC
GAAATTTACCGCATG GGGGACATAAACACC GTCCTGCATTTCACC
AAATTTACCGCATGG GGGACATAAACACCA TCCTGCATTTCACCT
AATTTACCGCATGGA GGACATAAACACCAG CCTGCATTTCACCTC
ATTTACCGCATGGAG GACATAAACACCAGG CTGCATTTCACCTCC TTTACCGCATGGAGG ACATAAACACCAGGA TGCATTTCACCTCCA
TTACCGCATGGAGGA CATAAACACCAGGAA GCATTTCACCTCCAC
TACCGCATGGAGGAA ATAAACACCAGGAAC CATTTCACCTCCACC
ACCGCATGGAGGAAG TAAACACCAGGAACA ATTTCACCTCCACCA
CCGCATGGAGGAAGT AAACACCAGGAACAA TTTCACCTCCACCAC CGCATGGAGGAAGTG AACACCAGGAACAAC TTCACCTCCACCACC
GCATGGAGGAAGTGA ACACCAGGAACAACG TCACCTCCACCACCA
CATGGAGGAAGTGAC CACCAGGAACAACGG CACCTCCACCACCAC
ATGGAGGAAGTGACG ACCAGGAACAACGGG ACCTCCACCACCACG
TGGAGGAAGTGACGG CCAGGAACAACGGGG CCTCCACCACCACGT GGAGGAAGTGACGGG CAGGAACAACGGGGA CTCCACCACCACGTC
GAGGAAGTGACGGGG AGGAACAACGGGGAG TCCACCACCACGTCG
AGGAAGTGACGGGGA GGAACAACGGGGAGA CCACCACCACGTCGA
GGAAGTGACGGGGAC GAACAACGGGGAGAG CACCACCACGTCGAA
GAAGTGACGGGGACT AACAACGGGGAGAGA ACCACCACGTCGAAG AAGTGACGGGGACTA ACAACGGGGAGAGAG CCACCACGTCGAAGA
AGTGACGGGGACTAA CAACGGGGAGAGAGC CACCACGTCGAAGAA ACCACGTCGAAGAAT GACTACAGGGATCTC AAGAATGTCACAGAG CCACGTCGAAGAATC ACTACAGGGATCTCA AGAATGTCACAGAGT CACGTCGAAGAATCG CTACAGGGATCTCAT GAATGTCACAGAGTA ACGTCGAAGAATCGC TACAGGGATCTCATC AATGTCACAGAGTAT CGTCGAAGAATCGCA ACAGGGATCTCATCA ATGTCACAGAGTATG GTCGAAGAATCGCAT CAGGGATCTCATCAG TGTCACAGAGTATGA TCGAAGAATCGCATC AGGGATCTCATCAGC GTCACAGAGTATGAT CGAAGAATCGCATCA GGGATCTCATCAGCT TCACAGAGTATGATG GAAGAATCGCATCAT GGATCTCATCAGCTT CACAGAGTATGATGG AAGAATCGCATCATC GATCTCATCAGCTTC ACAGAGTATGATGGG AGAATCGCATCATCA ATCTCATCAGCTTCA CAGAGTATGATGGGC GAATCGCATCATCAT TCTCATCAGCTTCAC AGAGTATGATGGGCA AATCGCATCATCATA CTCATCAGCTTCACC GAGTATGATGGGCAG ATCGCATCATCATAA TCATCAGCTTCACCG AGTATGATGGGCAGG TCGCATCATCATAAC CATCAGCTTCACCGT GTATGATGGGCAGGA CGCATCATCATAACC ATCAGCTTCACCGTT TATGATGGGCAGGAT GCATCATCATAACCT TCAGCTTCACCGTTT ATGATGGGCAGGATG CATCATCATAACCTG CAGCTTCACCGTTTA TGATGGGCAGGATGC ATCATCATAACCTGG AGCTTCACCGTTTAC GATGGGCAGGATGCC TCATCATAACCTGGC GCTTCACCGTTTACT ATGGGCAGGATGCCT CATCATAACCTGGCA CTTCACCGTTTACTA TGGGCAGGATGCCTG ATCATAACCTGGCAC TTCACCGTTTACTAC GGGCAGGATGCCTGC TCATAACCTGGCACC TCACCGTTTACTACA GGCAGGATGCCTGCG CATAACCTGGCACCG CACCGTTTACTACAA GCAGGATGCCTGCGG ATAACCTGGCACCGG ACCGTTTACTACAAG CAGGATGCCTGCGGC TAACCTGGCACCGGT CCGTTTACTACAAGG AGGATGCCTGCGGCT AACCTGGCACCGGTA CGTTTACTACAAGGA GGATGCCTGCGGCTC ACCTGGCACCGGTAC GTTTACTACAAGGAA GATGCCTGCGGCTCC CCTGGCACCGGTACC TTTACTACAAGGAAG ATGCCTGCGGCTCCA CTGGCACCGGTACCG TTACTACAAGGAAGC TGCCTGCGGCTCCAA TGGCACCGGTACCGG TACTACAAGGAAGCA GCCTGCGGCTCCAAC GGCACCGGTACCGGC ACTACAAGGAAGCAC CCTGCGGCTCCAACA GCACCGGTACCGGCC CTACAAGGAAGCACC CTGCGGCTCCAACAG CACCGGTACCGGCCC TACAAGGAAGCACCC TGCGGCTCCAACAGC ACCGGTACCGGCCCC ACAAGGAAGCACCCT GCGGCTCCAACAGCT CCGGTACCGGCCCCC CAAGGAAGCACCCTT CGGCTCCAACAGCTG CGGTACCGGCCCCCT AAGGAAGCACCCTTT GGCTCCAACAGCTGG GGTACCGGCCCCCTG AGGAAGCACCCTTTA GCTCCAACAGCTGGA GTACCGGCCCCCTGA GGAAGCACCCTTTAA CTCCAACAGCTGGAA TACCGGCCCCCTGAC GAAGCACCCTTTAAG TCCAACAGCTGGAAC ACCGGCCCCCTGACT AAGCACCCTTTAAGA CCAACAGCTGGAACA CCGGCCCCCTGACTA AGCACCCTTTAAGAA CAACAGCTGGAACAT CGGCCCCCTGACTAC GCACCCTTTAAGAAT AACAGCTGGAACATG GGCCCCCTGACTACA CACCCTTTAAGAATG ACAGCTGGAACATGG GCCCCCTGACTACAG ACCCTTTAAGAATGT CAGCTGGAACATGGT CCCCCTGACTACAGG CCCTTTAAGAATGTC AGCTGGAACATGGTG CCCCTGACTACAGGG CCTTTAAGAATGTCA GCTGGAACATGGTGG CCCTGACTACAGGGA CTTTAAGAATGTCAC CTGGAACATGGTGGA CCTGACTACAGGGAT TTTAAGAATGTCACA TGGAACATGGTGGAC CTGACTACAGGGATC TTAAGAATGTCACAG GGAACATGGTGGACG TGACTACAGGGATCT TAAGAATGTCACAGA GAACATGGTGGACGT AACATGGTGGACGTG TTACTACATGGGCTG GTGACCCTCACCATG
ACATGGTGGACGTGG TACTACATGGGCTGA TGACCCTCACCATGG
CATGGTGGACGTGGA ACTACATGGGCTGAA GACCCTCACCATGGT
ATGGTGGACGTGGAC CTACATGGGCTGAAG ACCCTCACCATGGTG TGGTGGACGTGGACC TACATGGGCTGAAGC CCCTCACCATGGTGG
GGTGGACGTGGACCT ACATGGGCTGAAGCC CCTCACCATGGTGGA
GTGGACGTGGACCTC CATGGGCTGAAGCCC CTCACCATGGTGGAG
TGGACGTGGACCTCC ATGGGCTGAAGCCCT TCACCATGGTGGAGA
GGACGTGGACCTCCC TGGGCTGAAGCCCTG CACCATGGTGGAGAA GACGTGGACCTCCCG GGGCTGAAGCCCTGG ACCATGGTGGAGAAC
ACGTGGACCTCCCGC GGCTGAAGCCCTGGA CCATGGTGGAGAACG
CGTGGACCTCCCGCC GCTGAAGCCCTGGAC CATGGTGGAGAACGA
GTGGACCTCCCGCCC CTGAAGCCCTGGACT ATGGTGGAGAACGAC
TGGACCTCCCGCCCA TGAAGCCCTGGACTC TGGTGGAGAACGACC GGACCTCCCGCCCAA GAAGCCCTGGACTCA GGTGGAGAACGACCA
GACCTCCCGCCCAAC AAGCCCTGGACTCAG GTGGAGAACGACCAT
ACCTCCCGCCCAACA AGCCCTGGACTCAGT TGGAGAACGACCATA
CCTCCCGCCCAACAA GCCCTGGACTCAGTA GGAGAACGACCATAT
CTCCCGCCCAACAAG CCCTGGACTCAGTAC GAGAACGACCATATC TCCCGCCCAACAAGG CCTGGACTCAGTACG AGAACGACCATATCC
CCCGCCCAACAAGGA CTGGACTCAGTACGC GAACGACCATATCCG
CCGCCCAACAAGGAC TGGACTCAGTACGCC AACGACCATATCCGT
CGCCCAACAAGGACG GGACTCAGTACGCCG ACGACCATATCCGTG
GCCCAACAAGGACGT GACTCAGTACGCCGT CGACCATATCCGTGG CCCAACAAGGACGTG ACTCAGTACGCCGTT GACCATATCCGTGGG
CCAACAAGGACGTGG CTCAGTACGCCGTTT ACCATATCCGTGGGG
CAACAAGGACGTGGA TCAGTACGCCGTTTA CCATATCCGTGGGGC
AACAAGGACGTGGAG CAGTACGCCGTTTAC CATATCCGTGGGGCC
ACAAGGACGTGGAGC AGTACGCCGTTTACG ATATCCGTGGGGCCA CAAGGACGTGGAGCC GTACGCCGTTTACGT TATCCGTGGGGCCAA
AAGGACGTGGAGCCC TACGCCGTTTACGTC ATCCGTGGGGCCAAG
AGGACGTGGAGCCCG ACGCCGTTTACGTCA TCCGTGGGGCCAAGA
GGACGTGGAGCCCGG CGCCGTTTACGTCAA CCGTGGGGCCAAGAG
GACGTGGAGCCCGGC GCCGTTTACGTCAAG CGTGGGGCCAAGAGT ACGTGGAGCCCGGCA CCGTTTACGTCAAGG GTGGGGCCAAGAGTG
CGTGGAGCCCGGCAT CGTTTACGTCAAGGC TGGGGCCAAGAGTGA
GTGGAGCCCGGCATC GTTTACGTCAAGGCT GGGGCCAAGAGTGAG
TGGAGCCCGGCATCT TTTACGTCAAGGCTG GGGCCAAGAGTGAGA
GGAGCCCGGCATCTT TTACGTCAAGGCTGT GGCCAAGAGTGAGAT GAGCCCGGCATCTTA TACGTCAAGGCTGTG GCCAAGAGTGAGATC
AGCCCGGCATCTTAC ACGTCAAGGCTGTGA CCAAGAGTGAGATCT
GCCCGGCATCTTACT CGTCAAGGCTGTGAC CAAGAGTGAGATCTT
CCCGGCATCTTACTA GTCAAGGCTGTGACC AAGAGTGAGATCTTG
CCGGCATCTTACTAC TCAAGGCTGTGACCC AGAGTGAGATCTTGT CGGCATCTTACTACA CAAGGCTGTGACCCT GAGTGAGATCTTGTA
GGCATCTTACTACAT AAGGCTGTGACCCTC AGTGAGATCTTGTAC
GCATCTTACTACATG AGGCTGTGACCCTCA GTGAGATCTTGTACA
CATCTTACTACATGG GGCTGTGACCCTCAC TGAGATCTTGTACAT
ATCTTACTACATGGG GCTGTGACCCTCACC GAGATCTTGTACATT TCTTACTACATGGGC CTGTGACCCTCACCA AGATCTTGTACATTC
CTTACTACATGGGCT TGTGACCCTCACCAT GATCTTGTACATTCG ATCTTGTACATTCGC CTTTCAGCATCGAAC TCTCTGCCCAACGGC
TCTTGTACATTCGCA TTTCAGCATCGAACT CTCTGCCCAACGGCA
CTTGTACATTCGCAC TTCAGCATCGAACTC TCTGCCCAACGGCAA
TTGTACATTCGCACC TCAGCATCGAACTCC CTGCCCAACGGCAAC TGTACATTCGCACCA CAGCATCGAACTCCT TGCCCAACGGCAACC
GTACATTCGCACCAA AGCATCGAACTCCTC GCCCAACGGCAACCT
TACATTCGCACCAAT GCATCGAACTCCTCT CCCAACGGCAACCTG
ACATTCGCACCAATG CATCGAACTCCTCTT CCAACGGCAACCTGA
CATTCGCACCAATGC ATCGAACTCCTCTTC CAACGGCAACCTGAG ATTCGCACCAATGCT TCGAACTCCTCTTCT AACGGCAACCTGAGT
TTCGCACCAATGCTT CGAACTCCTCTTCTC ACGGCAACCTGAGTT
TCGCACCAATGCTTC GAACTCCTCTTCTCA CGGCAACCTGAGTTA
CGCACCAATGCTTCA AACTCCTCTTCTCAG GGCAACCTGAGTTAC
GCACCAATGCTTCAG ACTCCTCTTCTCAGT GCAACCTGAGTTACT CACCAATGCTTCAGT CTCCTCTTCTCAGTT CAACCTGAGTTACTA
ACCAATGCTTCAGTT TCCTCTTCTCAGTTA AACCTGAGTTACTAC
CCAATGCTTCAGTTC CCTCTTCTCAGTTAA ACCTGAGTTACTACA
CAATGCTTCAGTTCC CTCTTCTCAGTTAAT CCTGAGTTACTACAT
AATGCTTCAGTTCCT TCTTCTCAGTTAATC CTGAGTTACTACATT ATGCTTCAGTTCCTT CTTCTCAGTTAATCG TGAGTTACTACATTG
TGCTTCAGTTCCTTC TTCTCAGTTAATCGT GAGTTACTACATTGT
GCTTCAGTTCCTTCC TCTCAGTTAATCGTG AGTTACTACATTGTG
CTTCAGTTCCTTCCA CTCAGTTAATCGTGA GTTACTACATTGTGC
TTCAGTTCCTTCCAT TCAGTTAATCGTGAA TTACTACATTGTGCG TCAGTTCCTTCCATT CAGTTAATCGTGAAG TACTACATTGTGCGC
CAGTTCCTTCCATTC AGTTAATCGTGAAGT ACTACATTGTGCGCT
AGTTCCTTCCATTCC GTTAATCGTGAAGTG CTACATTGTGCGCTG
GTTCCTTCCATTCCC TTAATCGTGAAGTGG TACATTGTGCGCTGG
TTCCTTCCATTCCCT TAATCGTGAAGTGGA ACATTGTGCGCTGGC TCCTTCCATTCCCTT AATCGTGAAGTGGAA CATTGTGCGCTGGCA
CCTTCCATTCCCTTG ATCGTGAAGTGGAAC ATTGTGCGCTGGCAG
CTTCCATTCCCTTGG TCGTGAAGTGGAACC TTGTGCGCTGGCAGC
TTCCATTCCCTTGGA CGTGAAGTGGAACCC TGTGCGCTGGCAGCG
TCCATTCCCTTGGAC GTGAAGTGGAACCCT GTGCGCTGGCAGCGG CCATTCCCTTGGACG TGAAGTGGAACCCTC TGCGCTGGCAGCGGC
CATTCCCTTGGACGT GAAGTGGAACCCTCC GCGCTGGCAGCGGCA
ATTCCCTTGGACGTT AAGTGGAACCCTCCC CGCTGGCAGCGGCAG
TTCCCTTGGACGTTC AGTGGAACCCTCCCT GCTGGCAGCGGCAGC
TCCCTTGGACGTTCT GTGGAACCCTCCCTC CTGGCAGCGGCAGCC CCCTTGGACGTTCTT TGGAACCCTCCCTCT TGGCAGCGGCAGCCT
CCTTGGACGTTCTTT GGAACCCTCCCTCTC GGCAGCGGCAGCCTC
CTTGGACGTTCTTTC GAACCCTCCCTCTCT GCAGCGGCAGCCTCA
TTGGACGTTCTTTCA AACCCTCCCTCTCTG CAGCGGCAGCCTCAG
TGGACGTTCTTTCAG ACCCTCCCTCTCTGC AGCGGCAGCCTCAGG GGACGTTCTTTCAGC CCCTCCCTCTCTGCC GCGGCAGCCTCAGGA
GACGTTCTTTCAGCA CCTCCCTCTCTGCCC CGGCAGCCTCAGGAC
ACGTTCTTTCAGCAT CTCCCTCTCTGCCCA GGCAGCCTCAGGACG
CGTTCTTTCAGCATC TCCCTCTCTGCCCAA GCAGCCTCAGGACGG
GTTCTTTCAGCATCG CCCTCTCTGCCCAAC CAGCCTCAGGACGGC TTCTTTCAGCATCGA CCTCTCTGCCCAACG AGCCTCAGGACGGCT
TCTTTCAGCATCGAA CTCTCTGCCCAACGG GCCTCAGGACGGCTA CCTCAGGACGGCTAC CCCATCAGGAAGTAT AACCCCAAGACTGAG
CTCAGGACGGCTACC CCATCAGGAAGTATG ACCCCAAGACTGAGG
TCAGGACGGCTACCT CATCAGGAAGTATGC CCCCAAGACTGAGGT
CAGGACGGCTACCTT ATCAGGAAGTATGCC CCCAAGACTGAGGTG AGGACGGCTACCTTT TCAGGAAGTATGCCG CCAAGACTGAGGTGT
GGACGGCTACCTTTA CAGGAAGTATGCCGA CAAGACTGAGGTGTG
GACGGCTACCTTTAC AGGAAGTATGCCGAC AAGACTGAGGTGTGT
ACGGCTACCTTTACC GGAAGTATGCCGACG AGACTGAGGTGTGTG
CGGCTACCTTTACCG GAAGTATGCCGACGG GACTGAGGTGTGTGG GGCTACCTTTACCGG AAGTATGCCGACGGC ACTGAGGTGTGTGGT
GCTACCTTTACCGGC AGTATGCCGACGGCA CTGAGGTGTGTGGTG
CTACCTTTACCGGCA GTATGCCGACGGCAC TGAGGTGTGTGGTGG
TACCTTTACCGGCAC TATGCCGACGGCACC GAGGTGTGTGGTGGG
ACCTTTACCGGCACA ATGCCGACGGCACCA AGGTGTGTGGTGGGG CCTTTACCGGCACAA TGCCGACGGCACCAT GGTGTGTGGTGGGGA
CTTTACCGGCACAAT GCCGACGGCACCATC GTGTGTGGTGGGGAG
TTTACCGGCACAATT CCGACGGCACCATCG TGTGTGGTGGGGAGA
TTACCGGCACAATTA CGACGGCACCATCGA GTGTGGTGGGGAGAA
TACCGGCACAATTAC GACGGCACCATCGAC TGTGGTGGGGAGAAA ACCGGCACAATTACT ACGGCACCATCGACA GTGGTGGGGAGAAAG
CCGGCACAATTACTG CGGCACCATCGACAT TGGTGGGGAGAAAGG
CGGCACAATTACTGC GGCACCATCGACATT GGTGGGGAGAAAGGG
GGCACAATTACTGCT GCACCATCGACATTG GTGGGGAGAAAGGGC
GCACAATTACTGCTC CACCATCGACATTGA TGGGGAGAAAGGGCC CACAATTACTGCTCC ACCATCGACATTGAG GGGGAGAAAGGGCCT
ACAATTACTGCTCCA CCATCGACATTGAGG GGGAGAAAGGGCCTT
CAATTACTGCTCCAA CATCGACATTGAGGA GGAGAAAGGGCCTTG
AATTACTGCTCCAAA ATCGACATTGAGGAG GAGAAAGGGCCTTGC
ATTACTGCTCCAAAG TCGACATTGAGGAGG AGAAAGGGCCTTGCT TTACTGCTCCAAAGA CGACATTGAGGAGGT GAAAGGGCCTTGCTG
TACTGCTCCAAAGAC GACATTGAGGAGGTC AAAGGGCCTTGCTGC
ACTGCTCCAAAGACA ACATTGAGGAGGTCA AAGGGCCTTGCTGCG
CTGCTCCAAAGACAA CATTGAGGAGGTCAC AGGGCCTTGCTGCGC
TGCTCCAAAGACAAA ATTGAGGAGGTCACA GGGCCTTGCTGCGCC GCTCCAAAGACAAAA TTGAGGAGGTCACAG GGCCTTGCTGCGCCT
CTCCAAAGACAAAAT TGAGGAGGTCACAGA GCCTTGCTGCGCCTG
TCCAAAGACAAAATC GAGGAGGTCACAGAG CCTTGCTGCGCCTGC
CCAAAGACAAAATCC AGGAGGTCACAGAGA CTTGCTGCGCCTGCC
CAAAGACAAAATCCC GGAGGTCACAGAGAA TTGCTGCGCCTGCCC AAAGACAAAATCCCC GAGGTCACAGAGAAC TGCTGCGCCTGCCCC
AAGACAAAATCCCCA AGGTCACAGAGAACC GCTGCGCCTGCCCCA
AGACAAAATCCCCAT GGTCACAGAGAACCC CTGCGCCTGCCCCAA
GACAAAATCCCCATC GTCACAGAGAACCCC TGCGCCTGCCCCAAA
ACAAAATCCCCATCA TCACAGAGAACCCCA GCGCCTGCCCCAAAA CAAAATCCCCATCAG CACAGAGAACCCCAA CGCCTGCCCCAAAAC
AAAATCCCCATCAGG ACAGAGAACCCCAAG GCCTGCCCCAAAACT
AAATCCCCATCAGGA CAGAGAACCCCAAGA CCTGCCCCAAAACTG
AATCCCCATCAGGAA AGAGAACCCCAAGAC CTGCCCCAAAACTGA
ATCCCCATCAGGAAG GAGAACCCCAAGACT TGCCCCAAAACTGAA TCCCCATCAGGAAGT AGAACCCCAAGACTG GCCCCAAAACTGAAG
CCCCATCAGGAAGTA GAACCCCAAGACTGA CCCCAAAACTGAAGC CCCAAAACTGAAGCC AAAGTCTTTGAGAAT AGGAAGCGGAGAGAT CCAAAACTGAAGCCG AAGTCTTTGAGAATT GGAAGCGGAGAGATG CAAAACTGAAGCCGA AGTCTTTGAGAATTT GAAGCGGAGAGATGT AAAACTGAAGCCGAG GTCTTTGAGAATTTC AAGCGGAGAGATGTC AAACTGAAGCCGAGA TCTTTGAGAATTTCC AGCGGAGAGATGTCA AACTGAAGCCGAGAA CTTTGAGAATTTCCT GCGGAGAGATGTCAT ACTGAAGCCGAGAAG TTTGAGAATTTCCTG CGGAGAGATGTCATG CTGAAGCCGAGAAGC TTGAGAATTTCCTGC GGAGAGATGTCATGC TGAAGCCGAGAAGCA TGAGAATTTCCTGCA GAGAGATGTCATGCA GAAGCCGAGAAGCAG GAGAATTTCCTGCAC AGAGATGTCATGCAA AAGCCGAGAAGCAGG AGAATTTCCTGCACA GAGATGTCATGCAAG AGCCGAGAAGCAGGC GAATTTCCTGCACAA AGATGTCATGCAAGT GCCGAGAAGCAGGCC AATTTCCTGCACAAC GATGTCATGCAAGTG CCGAGAAGCAGGCCG ATTTCCTGCACAACT ATGTCATGCAAGTGG CGAGAAGCAGGCCGA TTTCCTGCACAACTC TGTCATGCAAGTGGC GAGAAGCAGGCCGAG TTCCTGCACAACTCC GTCATGCAAGTGGCC AGAAGCAGGCCGAGA TCCTGCACAACTCCA TCATGCAAGTGGCCA GAAGCAGGCCGAGAA CCTGCACAACTCCAT CATGCAAGTGGCCAA AAGCAGGCCGAGAAG CTGCACAACTCCATC ATGCAAGTGGCCAAC AGCAGGCCGAGAAGG TGCACAACTCCATCT TGCAAGTGGCCAACA GCAGGCCGAGAAGGA GCACAACTCCATCTT GCAAGTGGCCAACAC CAGGCCGAGAAGGAG CACAACTCCATCTTC CAAGTGGCCAACACC AGGCCGAGAAGGAGG ACAACTCCATCTTCG AAGTGGCCAACACCA GGCCGAGAAGGAGGA CAACTCCATCTTCGT AGTGGCCAACACCAC GCCGAGAAGGAGGAG AACTCCATCTTCGTG GTGGCCAACACCACC CCGAGAAGGAGGAGG ACTCCATCTTCGTGC TGGCCAACACCACCA CGAGAAGGAGGAGGC CTCCATCTTCGTGCC GGCCAACACCACCAT GAGAAGGAGGAGGCT TCCATCTTCGTGCCC GCCAACACCACCATG AGAAGGAGGAGGCTG CCATCTTCGTGCCCA CCAACACCACCATGT GAAGGAGGAGGCTGA CATCTTCGTGCCCAG CAACACCACCATGTC AAGGAGGAGGCTGAA ATCTTCGTGCCCAGA AACACCACCATGTCC AGGAGGAGGCTGAAT TCTTCGTGCCCAGAC ACACCACCATGTCCA GGAGGAGGCTGAATA CTTCGTGCCCAGACC CACCACCATGTCCAG GAGGAGGCTGAATAC TTCGTGCCCAGACCT ACCACCATGTCCAGC AGGAGGCTGAATACC TCGTGCCCAGACCTG CCACCATGTCCAGCC GGAGGCTGAATACCG CGTGCCCAGACCTGA CACCATGTCCAGCCG GAGGCTGAATACCGC GTGCCCAGACCTGAA ACCATGTCCAGCCGA AGGCTGAATACCGCA TGCCCAGACCTGAAA CCATGTCCAGCCGAA GGCTGAATACCGCAA GCCCAGACCTGAAAG CATGTCCAGCCGAAG GCTGAATACCGCAAA CCCAGACCTGAAAGG ATGTCCAGCCGAAGC CTGAATACCGCAAAG CCAGACCTGAAAGGA TGTCCAGCCGAAGCA TGAATACCGCAAAGT CAGACCTGAAAGGAA GTCCAGCCGAAGCAG GAATACCGCAAAGTC AGACCTGAAAGGAAG TCCAGCCGAAGCAGG AATACCGCAAAGTCT GACCTGAAAGGAAGC CCAGCCGAAGCAGGA ATACCGCAAAGTCTT ACCTGAAAGGAAGCG CAGCCGAAGCAGGAA TACCGCAAAGTCTTT CCTGAAAGGAAGCGG AGCCGAAGCAGGAAC ACCGCAAAGTCTTTG CTGAAAGGAAGCGGA GCCGAAGCAGGAACA CCGCAAAGTCTTTGA TGAAAGGAAGCGGAG CCGAAGCAGGAACAC CGCAAAGTCTTTGAG GAAAGGAAGCGGAGA CGAAGCAGGAACACC GCAAAGTCTTTGAGA AAAGGAAGCGGAGAG GAAGCAGGAACACCA CAAAGTCTTTGAGAA AAGGAAGCGGAGAGA AAGCAGGAACACCAC AGCAGGAACACCACG CTGGAGACAGAGTAC ACTGTCATTTCTAAC GCAGGAACACCACGG TGGAGACAGAGTACC CTGTCATTTCTAACC CAGGAACACCACGGC GGAGACAGAGTACCC TGTCATTTCTAACCT AGGAACACCACGGCC GAGACAGAGTACCCT GTCATTTCTAACCTT GGAACACCACGGCCG AGACAGAGTACCCTT TCATTTCTAACCTTC GAACACCACGGCCGC GACAGAGTACCCTTT CATTTCTAACCTTCG AACACCACGGCCGCA ACAGAGTACCCTTTC ATTTCTAACCTTCGG ACACCACGGCCGCAG CAGAGTACCCTTTCT TTTCTAACCTTCGGC CACCACGGCCGCAGA AGAGTACCCTTTCTT TTCTAACCTTCGGCC ACCACGGCCGCAGAC GAGTACCCTTTCTTT TCTAACCTTCGGCCT CCACGGCCGCAGACA AGTACCCTTTCTTTG CTAACCTTCGGCCTT CACGGCCGCAGACAC GTACCCTTTCTTTGA TAACCTTCGGCCTTT ACGGCCGCAGACACC TACCCTTTCTTTGAG AACCTTCGGCCTTTC CGGCCGCAGACACCT ACCCTTTCTTTGAGA ACCTTCGGCCTTTCA GGCCGCAGACACCTA CCCTTTCTTTGAGAG CCTTCGGCCTTTCAC GCCGCAGACACCTAC CCTTTCTTTGAGAGC CTTCGGCCTTTCACA CCGCAGACACCTACA CTTTCTTTGAGAGCA TTCGGCCTTTCACAT CGCAGACACCTACAA TTTCTTTGAGAGCAG TCGGCCTTTCACATT GCAGACACCTACAAC TTCTTTGAGAGCAGA CGGCCTTTCACATTG CAGACACCTACAACA TCTTTGAGAGCAGAG GGCCTTTCACATTGT AGACACCTACAACAT CTTTGAGAGCAGAGT GCCTTTCACATTGTA GACACCTACAACATC TTTGAGAGCAGAGTG CCTTTCACATTGTAC ACACCTACAACATCA TTGAGAGCAGAGTGG CTTTCACATTGTACC CACCTACAACATCAC TGAGAGCAGAGTGGA TTTCACATTGTACCG ACCTACAACATCACC GAGAGCAGAGTGGAT TTCACATTGTACCGC CCTACAACATCACCG AGAGCAGAGTGGATA TCACATTGTACCGCA CTACAACATCACCGA GAGCAGAGTGGATAA CACATTGTACCGCAT TACAACATCACCGAC AGCAGAGTGGATAAC ACATTGTACCGCATC ACAACATCACCGACC GCAGAGTGGATAACA CATTGTACCGCATCG CAACATCACCGACCC CAGAGTGGATAACAA ATTGTACCGCATCGA AACATCACCGACCCG AGAGTGGATAACAAG TTGTACCGCATCGAT ACATCACCGACCCGG GAGTGGATAACAAGG TGTACCGCATCGATA CATCACCGACCCGGA AGTGGATAACAAGGA GTACCGCATCGATAT ATCACCGACCCGGAA GTGGATAACAAGGAG TACCGCATCGATATC TCACCGACCCGGAAG TGGATAACAAGGAGA ACCGCATCGATATCC CACCGACCCGGAAGA GGATAACAAGGAGAG CCGCATCGATATCCA ACCGACCCGGAAGAG GATAACAAGGAGAGA CGCATCGATATCCAC CCGACCCGGAAGAGC ATAACAAGGAGAGAA GCATCGATATCCACA CGACCCGGAAGAGCT TAACAAGGAGAGAAC CATCGATATCCACAG GACCCGGAAGAGCTG AACAAGGAGAGAACT ATCGATATCCACAGC ACCCGGAAGAGCTGG ACAAGGAGAGAACTG TCGATATCCACAGCT CCCGGAAGAGCTGGA CAAGGAGAGAACTGT CGATATCCACAGCTG CCGGAAGAGCTGGAG AAGGAGAGAACTGTC GATATCCACAGCTGC CGGAAGAGCTGGAGA AGGAGAGAACTGTCA ATATCCACAGCTGCA GGAAGAGCTGGAGAC GGAGAGAACTGTCAT TATCCACAGCTGCAA GAAGAGCTGGAGACA GAGAGAACTGTCATT ATCCACAGCTGCAAC AAGAGCTGGAGACAG AGAGAACTGTCATTT TCCACAGCTGCAACC AGAGCTGGAGACAGA GAGAACTGTCATTTC CCACAGCTGCAACCA GAGCTGGAGACAGAG AGAACTGTCATTTCT CACAGCTGCAACCAC AGCTGGAGACAGAGT GAACTGTCATTTCTA ACAGCTGCAACCACG GCTGGAGACAGAGTA AACTGTCATTTCTAA CAGCTGCAACCACGA AGCTGCAACCACGAG TTTGCAAGGACTATG ACCTGGGAGCCAAGG
GCTGCAACCACGAGG TTGCAAGGACTATGC CCTGGGAGCCAAGGC
CTGCAACCACGAGGC TGCAAGGACTATGCC CTGGGAGCCAAGGCC
TGCAACCACGAGGCT GCAAGGACTATGCCC TGGGAGCCAAGGCCT GCAACCACGAGGCTG CAAGGACTATGCCCG GGGAGCCAAGGCCTG
CAACCACGAGGCTGA AAGGACTATGCCCGC GGAGCCAAGGCCTGA
AACCACGAGGCTGAG AGGACTATGCCCGCA GAGCCAAGGCCTGAA
ACCACGAGGCTGAGA GGACTATGCCCGCAG AGCCAAGGCCTGAAA
CCACGAGGCTGAGAA GACTATGCCCGCAGA GCCAAGGCCTGAAAA CACGAGGCTGAGAAG ACTATGCCCGCAGAA CCAAGGCCTGAAAAC
ACGAGGCTGAGAAGC CTATGCCCGCAGAAG CAAGGCCTGAAAACT
CGAGGCTGAGAAGCT TATGCCCGCAGAAGG AAGGCCTGAAAACTC
GAGGCTGAGAAGCTG ATGCCCGCAGAAGGA AGGCCTGAAAACTCC
AGGCTGAGAAGCTGG TGCCCGCAGAAGGAG GGCCTGAAAACTCCA GGCTGAGAAGCTGGG GCCCGCAGAAGGAGC GCCTGAAAACTCCAT
GCTGAGAAGCTGGGC CCCGCAGAAGGAGCA CCTGAAAACTCCATC
CTGAGAAGCTGGGCT CCGCAGAAGGAGCAG CTGAAAACTCCATCT
TGAGAAGCTGGGCTG CGCAGAAGGAGCAGA TGAAAACTCCATCTT
GAGAAGCTGGGCTGC GCAGAAGGAGCAGAT GAAAACTCCATCTTT AGAAGCTGGGCTGCA CAGAAGGAGCAGATG AAAACTCCATCTTTT
GAAGCTGGGCTGCAG AGAAGGAGCAGATGA AAACTCCATCTTTTT
AAGCTGGGCTGCAGC GAAGGAGCAGATGAC AACTCCATCTTTTTA
AGCTGGGCTGCAGCG AAGGAGCAGATGACA ACTCCATCTTTTTAA
GCTGGGCTGCAGCGC AGGAGCAGATGACAT CTCCATCTTTTTAAA CTGGGCTGCAGCGCC GGAGCAGATGACATT TCCATCTTTTTAAAG
TGGGCTGCAGCGCCT GAGCAGATGACATTC CCATCTTTTTAAAGT
GGGCTGCAGCGCCTC AGCAGATGACATTCC CATCTTTTTAAAGTG
GGCTGCAGCGCCTCC GCAGATGACATTCCT ATCTTTTTAAAGTGG
GCTGCAGCGCCTCCA CAGATGACATTCCTG TCTTTTTAAAGTGGC CTGCAGCGCCTCCAA AGATGACATTCCTGG CTTTTTAAAGTGGCC
TGCAGCGCCTCCAAC GATGACATTCCTGGG TTTTTAAAGTGGCCG
GCAGCGCCTCCAACT ATGACATTCCTGGGC TTTTAAAGTGGCCGG
CAGCGCCTCCAACTT TGACATTCCTGGGCC TTTAAAGTGGCCGGA
AGCGCCTCCAACTTC GACATTCCTGGGCCA TTAAAGTGGCCGGAA GCGCCTCCAACTTCG ACATTCCTGGGCCAG TAAAGTGGCCGGAAC
CGCCTCCAACTTCGT CATTCCTGGGCCAGT AAAGTGGCCGGAACC
GCCTCCAACTTCGTC ATTCCTGGGCCAGTG AAGTGGCCGGAACCT
CCTCCAACTTCGTCT TTCCTGGGCCAGTGA AGTGGCCGGAACCTG
CTCCAACTTCGTCTT TCCTGGGCCAGTGAC GTGGCCGGAACCTGA TCCAACTTCGTCTTT CCTGGGCCAGTGACC TGGCCGGAACCTGAG
CCAACTTCGTCTTTG CTGGGCCAGTGACCT GGCCGGAACCTGAGA
CAACTTCGTCTTTGC TGGGCCAGTGACCTG GCCGGAACCTGAGAA
AACTTCGTCTTTGCA GGGCCAGTGACCTGG CCGGAACCTGAGAAT
ACTTCGTCTTTGCAA GGCCAGTGACCTGGG CGGAACCTGAGAATC CTTCGTCTTTGCAAG GCCAGTGACCTGGGA GGAACCTGAGAATCC
TTCGTCTTTGCAAGG CCAGTGACCTGGGAG GAACCTGAGAATCCC
TCGTCTTTGCAAGGA CAGTGACCTGGGAGC AACCTGAGAATCCCA
CGTCTTTGCAAGGAC AGTGACCTGGGAGCC ACCTGAGAATCCCAA
GTCTTTGCAAGGACT GTGACCTGGGAGCCA CCTGAGAATCCCAAT TCTTTGCAAGGACTA TGACCTGGGAGCCAA CTGAGAATCCCAATG
CTTTGCAAGGACTAT GACCTGGGAGCCAAG TGAGAATCCCAATGG GAGAATCCCAATGGA GTTGAGGATCAGCGA GGGGCCAAGCTAAAC AGAATCCCAATGGAT TTGAGGATCAGCGAG GGGCCAAGCTAAACC GAATCCCAATGGATT TGAGGATCAGCGAGA GGCCAAGCTAAACCG AATCCCAATGGATTG GAGGATCAGCGAGAA GCCAAGCTAAACCGG ATCCCAATGGATTGA AGGATCAGCGAGAAT CCAAGCTAAACCGGC TCCCAATGGATTGAT GGATCAGCGAGAATG CAAGCTAAACCGGCT CCCAATGGATTGATT GATCAGCGAGAATGT AAGCTAAACCGGCTA CCAATGGATTGATTC ATCAGCGAGAATGTG AGCTAAACCGGCTAA CAATGGATTGATTCT TCAGCGAGAATGTGT GCTAAACCGGCTAAA AATGGATTGATTCTA CAGCGAGAATGTGTG CTAAACCGGCTAAAC ATGGATTGATTCTAA AGCGAGAATGTGTGT TAAACCGGCTAAACC TGGATTGATTCTAAT GCGAGAATGTGTGTC AAACCGGCTAAACCC GGATTGATTCTAATG CGAGAATGTGTGTCC AACCGGCTAAACCCG GATTGATTCTAATGT GAGAATGTGTGTCCA ACCGGCTAAACCCGG ATTGATTCTAATGTA AGAATGTGTGTCCAG CCGGCTAAACCCGGG TTGATTCTAATGTAT GAATGTGTGTCCAGA CGGCTAAACCCGGGG TGATTCTAATGTATG AATGTGTGTCCAGAC GGCTAAACCCGGGGA GATTCTAATGTATGA ATGTGTGTCCAGACA GCTAAACCCGGGGAA ATTCTAATGTATGAA TGTGTGTCCAGACAG CTAAACCCGGGGAAC TTCTAATGTATGAAA GTGTGTCCAGACAGG TAAACCCGGGGAACT TCTAATGTATGAAAT TGTGTCCAGACAGGA AAACCCGGGGAACTA CTAATGTATGAAATA GTGTCCAGACAGGAA AACCCGGGGAACTAC TAATGTATGAAATAA TGTCCAGACAGGAAT ACCCGGGGAACTACA AATGTATGAAATAAA GTCCAGACAGGAATA CCCGGGGAACTACAC ATGTATGAAATAAAA TCCAGACAGGAATAC CCGGGGAACTACACA TGTATGAAATAAAAT CCAGACAGGAATACA CGGGGAACTACACAG GTATGAAATAAAATA CAGACAGGAATACAG GGGGAACTACACAGC TATGAAATAAAATAC AGACAGGAATACAGG GGGAACTACACAGCC ATGAAATAAAATACG GACAGGAATACAGGA GGAACTACACAGCCC TGAAATAAAATACGG ACAGGAATACAGGAA GAACTACACAGCCCG GAAATAAAATACGGA CAGGAATACAGGAAG AACTACACAGCCCGG AAATAAAATACGGAT AGGAATACAGGAAGT ACTACACAGCCCGGA AATAAAATACGGATC GGAATACAGGAAGTA CTACACAGCCCGGAT ATAAAATACGGATCA GAATACAGGAAGTAT TACACAGCCCGGATT TAAAATACGGATCAC AATACAGGAAGTATG ACACAGCCCGGATTC AAAATACGGATCACA ATACAGGAAGTATGG CACAGCCCGGATTCA AAATACGGATCACAA TACAGGAAGTATGGA ACAGCCCGGATTCAG AATACGGATCACAAG ACAGGAAGTATGGAG CAGCCCGGATTCAGG ATACGGATCACAAGT CAGGAAGTATGGAGG AGCCCGGATTCAGGC TACGGATCACAAGTT AGGAAGTATGGAGGG GCCCGGATTCAGGCC ACGGATCACAAGTTG GGAAGTATGGAGGGG CCCGGATTCAGGCCA CGGATCACAAGTTGA GAAGTATGGAGGGGC CCGGATTCAGGCCAC GGATCACAAGTTGAG AAGTATGGAGGGGCC CGGATTCAGGCCACA GATCACAAGTTGAGG AGTATGGAGGGGCCA GGATTCAGGCCACAT ATCACAAGTTGAGGA GTATGGAGGGGCCAA GATTCAGGCCACATC TCACAAGTTGAGGAT TATGGAGGGGCCAAG ATTCAGGCCACATCT CACAAGTTGAGGATC ATGGAGGGGCCAAGC TTCAGGCCACATCTC ACAAGTTGAGGATCA TGGAGGGGCCAAGCT TCAGGCCACATCTCT CAAGTTGAGGATCAG GGAGGGGCCAAGCTA CAGGCCACATCTCTC AAGTTGAGGATCAGC GAGGGGCCAAGCTAA AGGCCACATCTCTCT AGTTGAGGATCAGCG AGGGGCCAAGCTAAA GGCCACATCTCTCTC GCCACATCTCTCTCT GTCCAGGCCAAAACA CCCGTCGCTGTCCTG CCACATCTCTCTCTG TCCAGGCCAAAACAG CCGTCGCTGTCCTGT CACATCTCTCTCTGG CCAGGCCAAAACAGG CGTCGCTGTCCTGTT ACATCTCTCTCTGGG CAGGCCAAAACAGGA GTCGCTGTCCTGTTG CATCTCTCTCTGGGA AGGCCAAAACAGGAT TCGCTGTCCTGTTGA ATCTCTCTCTGGGAA GGCCAAAACAGGATA CGCTGTCCTGTTGAT TCTCTCTCTGGGAAT GCCAAAACAGGATAT GCTGTCCTGTTGATC CTCTCTCTGGGAATG CCAAAACAGGATATG CTGTCCTGTTGATCG TCTCTCTGGGAATGG CAAAACAGGATATGA TGTCCTGTTGATCGT CTCTCTGGGAATGGG AAAACAGGATATGAA GTCCTGTTGATCGTG TCTCTGGGAATGGGT AAACAGGATATGAAA TCCTGTTGATCGTGG CTCTGGGAATGGGTC AACAGGATATGAAAA CCTGTTGATCGTGGG TCTGGGAATGGGTCG ACAGGATATGAAAAC CTGTTGATCGTGGGA CTGGGAATGGGTCGT CAGGATATGAAAACT TGTTGATCGTGGGAG TGGGAATGGGTCGTG AGGATATGAAAACTT GTTGATCGTGGGAGG GGGAATGGGTCGTGG GGATATGAAAACTTC TTGATCGTGGGAGGG GGAATGGGTCGTGGA GATATGAAAACTTCA TGATCGTGGGAGGGT GAATGGGTCGTGGAC ATATGAAAACTTCAT GATCGTGGGAGGGTT AATGGGTCGTGGACA TATGAAAACTTCATC ATCGTGGGAGGGTTG ATGGGTCGTGGACAG ATGAAAACTTCATCC TCGTGGGAGGGTTGG TGGGTCGTGGACAGA TGAAAACTTCATCCA CGTGGGAGGGTTGGT GGGTCGTGGACAGAT GAAAACTTCATCCAT GTGGGAGGGTTGGTG GGTCGTGGACAGATC AAAACTTCATCCATC TGGGAGGGTTGGTGA GTCGTGGACAGATCC AAACTTCATCCATCT GGGAGGGTTGGTGAT TCGTGGACAGATCCT AACTTCATCCATCTG GGAGGGTTGGTGATT CGTGGACAGATCCTG ACTTCATCCATCTGA GAGGGTTGGTGATTA GTGGACAGATCCTGT CTTCATCCATCTGAT AGGGTTGGTGATTAT TGGACAGATCCTGTG TTCATCCATCTGATC GGGTTGGTGATTATG GGACAGATCCTGTGT TCATCCATCTGATCA GGTTGGTGATTATGC GACAGATCCTGTGTT CATCCATCTGATCAT GTTGGTGATTATGCT ACAGATCCTGTGTTC ATCCATCTGATCATC TTGGTGATTATGCTG CAGATCCTGTGTTCT TCCATCTGATCATCG TGGTGATTATGCTGT AGATCCTGTGTTCTT CCATCTGATCATCGC GGTGATTATGCTGTA GATCCTGTGTTCTTC CATCTGATCATCGCT GTGATTATGCTGTAC ATCCTGTGTTCTTCT ATCTGATCATCGCTC TGATTATGCTGTACG TCCTGTGTTCTTCTA TCTGATCATCGCTCT GATTATGCTGTACGT CCTGTGTTCTTCTAT CTGATCATCGCTCTG ATTATGCTGTACGTC CTGTGTTCTTCTATG TGATCATCGCTCTGC TTATGCTGTACGTCT TGTGTTCTTCTATGT GATCATCGCTCTGCC TATGCTGTACGTCTT GTGTTCTTCTATGTC ATCATCGCTCTGCCC ATGCTGTACGTCTTC TGTTCTTCTATGTCC TCATCGCTCTGCCCG TGCTGTACGTCTTCC GTTCTTCTATGTCCA CATCGCTCTGCCCGT GCTGTACGTCTTCCA TTCTTCTATGTCCAG ATCGCTCTGCCCGTC CTGTACGTCTTCCAT TCTTCTATGTCCAGG TCGCTCTGCCCGTCG TGTACGTCTTCCATA CTTCTATGTCCAGGC CGCTCTGCCCGTCGC GTACGTCTTCCATAG TTCTATGTCCAGGCC GCTCTGCCCGTCGCT TACGTCTTCCATAGA TCTATGTCCAGGCCA CTCTGCCCGTCGCTG ACGTCTTCCATAGAA CTATGTCCAGGCCAA TCTGCCCGTCGCTGT CGTCTTCCATAGAAA TATGTCCAGGCCAAA CTGCCCGTCGCTGTC GTCTTCCATAGAAAG ATGTCCAGGCCAAAA TGCCCGTCGCTGTCC TCTTCCATAGAAAGA TGTCCAGGCCAAAAC GCCCGTCGCTGTCCT CTTCCATAGAAAGAG TTCCATAGAAAGAGA TCTGTGAACCCGGAG TGGGAGGTGGCTCGG
TCCATAGAAAGAGAA CTGTGAACCCGGAGT GGGAGGTGGCTCGGG
CCATAGAAAGAGAAA TGTGAACCCGGAGTA GGAGGTGGCTCGGGA
CATAGAAAGAGAAAT GTGAACCCGGAGTAC GAGGTGGCTCGGGAG ATAGAAAGAGAAATA TGAACCCGGAGTACT AGGTGGCTCGGGAGA
TAGAAAGAGAAATAA GAACCCGGAGTACTT GGTGGCTCGGGAGAA
AGAAAGAGAAATAAC AACCCGGAGTACTTC GTGGCTCGGGAGAAG
GAAAGAGAAATAACA ACCCGGAGTACTTCA TGGCTCGGGAGAAGA
AAAGAGAAATAACAG CCCGGAGTACTTCAG GGCTCGGGAGAAGAT AAGAGAAATAACAGC CCGGAGTACTTCAGC GCTCGGGAGAAGATC
AGAGAAATAACAGCA CGGAGTACTTCAGCG CTCGGGAGAAGATCA
GAGAAATAACAGCAG GGAGTACTTCAGCGC TCGGGAGAAGATCAC
AGAAATAACAGCAGG GAGTACTTCAGCGCT CGGGAGAAGATCACC
GAAATAACAGCAGGC AGTACTTCAGCGCTG GGGAGAAGATCACCA AAATAACAGCAGGCT GTACTTCAGCGCTGC GGAGAAGATCACCAT
AATAACAGCAGGCTG TACTTCAGCGCTGCT GAGAAGATCACCATG
ATAACAGCAGGCTGG ACTTCAGCGCTGCTG AGAAGATCACCATGA
TAACAGCAGGCTGGG CTTCAGCGCTGCTGA GAAGATCACCATGAG
AACAGCAGGCTGGGG TTCAGCGCTGCTGAT AAGATCACCATGAGC ACAGCAGGCTGGGGA TCAGCGCTGCTGATG AGATCACCATGAGCC
CAGCAGGCTGGGGAA CAGCGCTGCTGATGT GATCACCATGAGCCG
AGCAGGCTGGGGAAT AGCGCTGCTGATGTG ATCACCATGAGCCGG
GCAGGCTGGGGAATG GCGCTGCTGATGTGT TCACCATGAGCCGGG
CAGGCTGGGGAATGG CGCTGCTGATGTGTA CACCATGAGCCGGGA AGGCTGGGGAATGGA GCTGCTGATGTGTAC ACCATGAGCCGGGAA
GGCTGGGGAATGGAG CTGCTGATGTGTACG CCATGAGCCGGGAAC
GCTGGGGAATGGAGT TGCTGATGTGTACGT CATGAGCCGGGAACT
CTGGGGAATGGAGTG GCTGATGTGTACGTT ATGAGCCGGGAACTT
TGGGGAATGGAGTGC CTGATGTGTACGTTC TGAGCCGGGAACTTG GGGGAATGGAGTGCT TGATGTGTACGTTCC GAGCCGGGAACTTGG
GGGAATGGAGTGCTG GATGTGTACGTTCCT AGCCGGGAACTTGGG
GGAATGGAGTGCTGT ATGTGTACGTTCCTG GCCGGGAACTTGGGC
GAATGGAGTGCTGTA TGTGTACGTTCCTGA CCGGGAACTTGGGCA
AATGGAGTGCTGTAT GTGTACGTTCCTGAT CGGGAACTTGGGCAG ATGGAGTGCTGTATG TGTACGTTCCTGATG GGGAACTTGGGCAGG
TGGAGTGCTGTATGC GTACGTTCCTGATGA GGAACTTGGGCAGGG
GGAGTGCTGTATGCC TACGTTCCTGATGAG GAACTTGGGCAGGGG
GAGTGCTGTATGCCT ACGTTCCTGATGAGT AACTTGGGCAGGGGT
AGTGCTGTATGCCTC CGTTCCTGATGAGTG ACTTGGGCAGGGGTC GTGCTGTATGCCTCT GTTCCTGATGAGTGG CTTGGGCAGGGGTCG
TGCTGTATGCCTCTG TTCCTGATGAGTGGG TTGGGCAGGGGTCGT
GCTGTATGCCTCTGT TCCTGATGAGTGGGA TGGGCAGGGGTCGTT
CTGTATGCCTCTGTG CCTGATGAGTGGGAG GGGCAGGGGTCGTTT
TGTATGCCTCTGTGA CTGATGAGTGGGAGG GGCAGGGGTCGTTTG GTATGCCTCTGTGAA TGATGAGTGGGAGGT GCAGGGGTCGTTTGG
TATGCCTCTGTGAAC GATGAGTGGGAGGTG CAGGGGTCGTTTGGG
ATGCCTCTGTGAACC ATGAGTGGGAGGTGG AGGGGTCGTTTGGGA
TGCCTCTGTGAACCC TGAGTGGGAGGTGGC GGGGTCGTTTGGGAT
GCCTCTGTGAACCCG GAGTGGGAGGTGGCT GGGTCGTTTGGGATG CCTCTGTGAACCCGG AGTGGGAGGTGGCTC GGTCGTTTGGGATGG
CTCTGTGAACCCGGA GTGGGAGGTGGCTCG GTCGTTTGGGATGGT TCGTTTGGGATGGTC CCTGAAACCAGAGTG GAGAGGATTGAGTTT
CGTTTGGGATGGTCT CTGAAACCAGAGTGG AGAGGATTGAGTTTC
GTTTGGGATGGTCTA TGAAACCAGAGTGGC GAGGATTGAGTTTCT
TTTGGGATGGTCTAT GAAACCAGAGTGGCC AGGATTGAGTTTCTC TTGGGATGGTCTATG AAACCAGAGTGGCCA GGATTGAGTTTCTCA
TGGGATGGTCTATGA AACCAGAGTGGCCAT GATTGAGTTTCTCAA
GGGATGGTCTATGAA ACCAGAGTGGCCATT ATTGAGTTTCTCAAC
GGATGGTCTATGAAG CCAGAGTGGCCATTA TTGAGTTTCTCAACG
GATGGTCTATGAAGG CAGAGTGGCCATTAA TGAGTTTCTCAACGA ATGGTCTATGAAGGA AGAGTGGCCATTAAA GAGTTTCTCAACGAA
TGGTCTATGAAGGAG GAGTGGCCATTAAAA AGTTTCTCAACGAAG
GGTCTATGAAGGAGT AGTGGCCATTAAAAC GTTTCTCAACGAAGC
GTCTATGAAGGAGTT GTGGCCATTAAAACA TTTCTCAACGAAGCT
TCTATGAAGGAGTTG TGGCCATTAAAACAG TTCTCAACGAAGCTT CTATGAAGGAGTTGC GGCCATTAAAACAGT TCTCAACGAAGCTTC
TATGAAGGAGTTGCC GCCATTAAAACAGTG CTCAACGAAGCTTCT
ATGAAGGAGTTGCCA CCATTAAAACAGTGA TCAACGAAGCTTCTG
TGAAGGAGTTGCCAA CATTAAAACAGTGAA CAACGAAGCTTCTGT
GAAGGAGTTGCCAAG ATTAAAACAGTGAAC AACGAAGCTTCTGTG AAGGAGTTGCCAAGG TTAAAACAGTGAACG ACGAAGCTTCTGTGA
AGGAGTTGCCAAGGG TAAAACAGTGAACGA CGAAGCTTCTGTGAT
GGAGTTGCCAAGGGT AAAACAGTGAACGAG GAAGCTTCTGTGATG
GAGTTGCCAAGGGTG AAACAGTGAACGAGG AAGCTTCTGTGATGA
AGTTGCCAAGGGTGT AACAGTGAACGAGGC AGCTTCTGTGATGAA GTTGCCAAGGGTGTG ACAGTGAACGAGGCC GCTTCTGTGATGAAG
TTGCCAAGGGTGTGG CAGTGAACGAGGCCG CTTCTGTGATGAAGG
TGCCAAGGGTGTGGT AGTGAACGAGGCCGC TTCTGTGATGAAGGA
GCCAAGGGTGTGGTG GTGAACGAGGCCGCA TCTGTGATGAAGGAG
CCAAGGGTGTGGTGA TGAACGAGGCCGCAA CTGTGATGAAGGAGT CAAGGGTGTGGTGAA GAACGAGGCCGCAAG TGTGATGAAGGAGTT
AAGGGTGTGGTGAAA AACGAGGCCGCAAGC GTGATGAAGGAGTTC
AGGGTGTGGTGAAAG ACGAGGCCGCAAGCA TGATGAAGGAGTTCA
GGGTGTGGTGAAAGA CGAGGCCGCAAGCAT GATGAAGGAGTTCAA
GGTGTGGTGAAAGAT GAGGCCGCAAGCATG ATGAAGGAGTTCAAT GTGTGGTGAAAGATG AGGCCGCAAGCATGC TGAAGGAGTTCAATT
TGTGGTGAAAGATGA GGCCGCAAGCATGCG GAAGGAGTTCAATTG
GTGGTGAAAGATGAA GCCGCAAGCATGCGT AAGGAGTTCAATTGT
TGGTGAAAGATGAAC CCGCAAGCATGCGTG AGGAGTTCAATTGTC
GGTGAAAGATGAACC CGCAAGCATGCGTGA GGAGTTCAATTGTCA GTGAAAGATGAACCT GCAAGCATGCGTGAG GAGTTCAATTGTCAC
TGAAAGATGAACCTG CAAGCATGCGTGAGA AGTTCAATTGTCACC
GAAAGATGAACCTGA AAGCATGCGTGAGAG GTTCAATTGTCACCA
AAAGATGAACCTGAA AGCATGCGTGAGAGG TTCAATTGTCACCAT
AAGATGAACCTGAAA GCATGCGTGAGAGGA TCAATTGTCACCATG AGATGAACCTGAAAC CATGCGTGAGAGGAT CAATTGTCACCATGT
GATGAACCTGAAACC ATGCGTGAGAGGATT AATTGTCACCATGTG
ATGAACCTGAAACCA TGCGTGAGAGGATTG ATTGTCACCATGTGG
TGAACCTGAAACCAG GCGTGAGAGGATTGA TTGTCACCATGTGGT
GAACCTGAAACCAGA CGTGAGAGGATTGAG TGTCACCATGTGGTG AACCTGAAACCAGAG GTGAGAGGATTGAGT GTCACCATGTGGTGC
ACCTGAAACCAGAGT TGAGAGGATTGAGTT TCACCATGTGGTGCG CACCATGTGGTGCGA GTCATCATGGAACTG CTGAGGCCAGAAATG ACCATGTGGTGCGAT TCATCATGGAACTGA TGAGGCCAGAAATGG CCATGTGGTGCGATT CATCATGGAACTGAT GAGGCCAGAAATGGA CATGTGGTGCGATTG ATCATGGAACTGATG AGGCCAGAAATGGAG ATGTGGTGCGATTGC TCATGGAACTGATGA GGCCAGAAATGGAGA TGTGGTGCGATTGCT CATGGAACTGATGAC GCCAGAAATGGAGAA GTGGTGCGATTGCTG ATGGAACTGATGACA CCAGAAATGGAGAAT TGGTGCGATTGCTGG TGGAACTGATGACAC CAGAAATGGAGAATA GGTGCGATTGCTGGG GGAACTGATGACACG AGAAATGGAGAATAA GTGCGATTGCTGGGT GAACTGATGACACGG GAAATGGAGAATAAT TGCGATTGCTGGGTG AACTGATGACACGGG AAATGGAGAATAATC GCGATTGCTGGGTGT ACTGATGACACGGGG AATGGAGAATAATCC CGATTGCTGGGTGTG CTGATGACACGGGGC ATGGAGAATAATCCA GATTGCTGGGTGTGG TGATGACACGGGGCG TGGAGAATAATCCAG ATTGCTGGGTGTGGT GATGACACGGGGCGA GGAGAATAATCCAGT TTGCTGGGTGTGGTG ATGACACGGGGCGAT GAGAATAATCCAGTC TGCTGGGTGTGGTGT TGACACGGGGCGATC AGAATAATCCAGTCC GCTGGGTGTGGTGTC GACACGGGGCGATCT GAATAATCCAGTCCT CTGGGTGTGGTGTCC ACACGGGGCGATCTC AATAATCCAGTCCTA TGGGTGTGGTGTCCC CACGGGGCGATCTCA ATAATCCAGTCCTAG GGGTGTGGTGTCCCA ACGGGGCGATCTCAA TAATCCAGTCCTAGC GGTGTGGTGTCCCAA CGGGGCGATCTCAAA AATCCAGTCCTAGCA GTGTGGTGTCCCAAG GGGGCGATCTCAAAA ATCCAGTCCTAGCAC TGTGGTGTCCCAAGG GGGCGATCTCAAAAG TCCAGTCCTAGCACC GTGGTGTCCCAAGGC GGCGATCTCAAAAGT CCAGTCCTAGCACCT TGGTGTCCCAAGGCC GCGATCTCAAAAGTT CAGTCCTAGCACCTC GGTGTCCCAAGGCCA CGATCTCAAAAGTTA AGTCCTAGCACCTCC GTGTCCCAAGGCCAG GATCTCAAAAGTTAT GTCCTAGCACCTCCA TGTCCCAAGGCCAGC ATCTCAAAAGTTATC TCCTAGCACCTCCAA GTCCCAAGGCCAGCC TCTCAAAAGTTATCT CCTAGCACCTCCAAG TCCCAAGGCCAGCCA CTCAAAAGTTATCTC CTAGCACCTCCAAGC CCCAAGGCCAGCCAA TCAAAAGTTATCTCC TAGCACCTCCAAGCC CCAAGGCCAGCCAAC CAAAAGTTATCTCCG AGCACCTCCAAGCCT CAAGGCCAGCCAACA AAAAGTTATCTCCGG GCACCTCCAAGCCTG AAGGCCAGCCAACAC AAAGTTATCTCCGGT CACCTCCAAGCCTGA AGGCCAGCCAACACT AAGTTATCTCCGGTC ACCTCCAAGCCTGAG GGCCAGCCAACACTG AGTTATCTCCGGTCT CCTCCAAGCCTGAGC GCCAGCCAACACTGG GTTATCTCCGGTCTC CTCCAAGCCTGAGCA CCAGCCAACACTGGT TTATCTCCGGTCTCT TCCAAGCCTGAGCAA CAGCCAACACTGGTC TATCTCCGGTCTCTG CCAAGCCTGAGCAAG AGCCAACACTGGTCA ATCTCCGGTCTCTGA CAAGCCTGAGCAAGA GCCAACACTGGTCAT TCTCCGGTCTCTGAG AAGCCTGAGCAAGAT CCAACACTGGTCATC CTCCGGTCTCTGAGG AGCCTGAGCAAGATG CAACACTGGTCATCA TCCGGTCTCTGAGGC GCCTGAGCAAGATGA AACACTGGTCATCAT CCGGTCTCTGAGGCC CCTGAGCAAGATGAT ACACTGGTCATCATG CGGTCTCTGAGGCCA CTGAGCAAGATGATT CACTGGTCATCATGG GGTCTCTGAGGCCAG TGAGCAAGATGATTC ACTGGTCATCATGGA GTCTCTGAGGCCAGA GAGCAAGATGATTCA CTGGTCATCATGGAA TCTCTGAGGCCAGAA AGCAAGATGATTCAG TGGTCATCATGGAAC CTCTGAGGCCAGAAA GCAAGATGATTCAGA GGTCATCATGGAACT TCTGAGGCCAGAAAT CAAGATGATTCAGAT AAGATGATTCAGATG GCCAATAAGTTCGTC GAAGATTTCACAGTC AGATGATTCAGATGG CCAATAAGTTCGTCC AAGATTTCACAGTCA GATGATTCAGATGGC CAATAAGTTCGTCCA AGATTTCACAGTCAA ATGATTCAGATGGCC AATAAGTTCGTCCAC GATTTCACAGTCAAA TGATTCAGATGGCCG ATAAGTTCGTCCACA ATTTCACAGTCAAAA GATTCAGATGGCCGG TAAGTTCGTCCACAG TTTCACAGTCAAAAT ATTCAGATGGCCGGA AAGTTCGTCCACAGA TTCACAGTCAAAATC TTCAGATGGCCGGAG AGTTCGTCCACAGAG TCACAGTCAAAATCG TCAGATGGCCGGAGA GTTCGTCCACAGAGA CACAGTCAAAATCGG CAGATGGCCGGAGAG TTCGTCCACAGAGAC ACAGTCAAAATCGGA AGATGGCCGGAGAGA TCGTCCACAGAGACC CAGTCAAAATCGGAG GATGGCCGGAGAGAT CGTCCACAGAGACCT AGTCAAAATCGGAGA ATGGCCGGAGAGATT GTCCACAGAGACCTT GTCAAAATCGGAGAT TGGCCGGAGAGATTG TCCACAGAGACCTTG TCAAAATCGGAGATT GGCCGGAGAGATTGC CCACAGAGACCTTGC CAAAATCGGAGATTT GCCGGAGAGATTGCA CACAGAGACCTTGCT AAAATCGGAGATTTT CCGGAGAGATTGCAG ACAGAGACCTTGCTG AAATCGGAGATTTTG CGGAGAGATTGCAGA CAGAGACCTTGCTGC AATCGGAGATTTTGG GGAGAGATTGCAGAC AGAGACCTTGCTGCC ATCGGAGATTTTGGT GAGAGATTGCAGACG GAGACCTTGCTGCCC TCGGAGATTTTGGTA AGAGATTGCAGACGG AGACCTTGCTGCCCG CGGAGATTTTGGTAT GAGATTGCAGACGGC GACCTTGCTGCCCGG GGAGATTTTGGTATG AGATTGCAGACGGCA ACCTTGCTGCCCGGA GAGATTTTGGTATGA GATTGCAGACGGCAT CCTTGCTGCCCGGAA AGATTTTGGTATGAC ATTGCAGACGGCATG CTTGCTGCCCGGAAT GATTTTGGTATGACG TTGCAGACGGCATGG TTGCTGCCCGGAATT ATTTTGGTATGACGC TGCAGACGGCATGGC TGCTGCCCGGAATTG TTTTGGTATGACGCG GCAGACGGCATGGCA GCTGCCCGGAATTGC TTTGGTATGACGCGA CAGACGGCATGGCAT CTGCCCGGAATTGCA TTGGTATGACGCGAG AGACGGCATGGCATA TGCCCGGAATTGCAT TGGTATGACGCGAGA GACGGCATGGCATAC GCCCGGAATTGCATG GGTATGACGCGAGAT ACGGCATGGCATACC CCCGGAATTGCATGG GTATGACGCGAGATA CGGCATGGCATACCT CCGGAATTGCATGGT TATGACGCGAGATAT GGCATGGCATACCTC CGGAATTGCATGGTA ATGACGCGAGATATC GCATGGCATACCTCA GGAATTGCATGGTAG TGACGCGAGATATCT CATGGCATACCTCAA GAATTGCATGGTAGC GACGCGAGATATCTA ATGGCATACCTCAAC AATTGCATGGTAGCC ACGCGAGATATCTAT TGGCATACCTCAACG ATTGCATGGTAGCCG CGCGAGATATCTATG GGCATACCTCAACGC TTGCATGGTAGCCGA GCGAGATATCTATGA GCATACCTCAACGCC TGCATGGTAGCCGAA CGAGATATCTATGAG CATACCTCAACGCCA GCATGGTAGCCGAAG GAGATATCTATGAGA ATACCTCAACGCCAA CATGGTAGCCGAAGA AGATATCTATGAGAC TACCTCAACGCCAAT ATGGTAGCCGAAGAT GATATCTATGAGACA ACCTCAACGCCAATA TGGTAGCCGAAGATT ATATCTATGAGACAG CCTCAACGCCAATAA GGTAGCCGAAGATTT TATCTATGAGACAGA CTCAACGCCAATAAG GTAGCCGAAGATTTC ATCTATGAGACAGAC TCAACGCCAATAAGT TAGCCGAAGATTTCA TCTATGAGACAGACT CAACGCCAATAAGTT AGCCGAAGATTTCAC CTATGAGACAGACTA AACGCCAATAAGTTC GCCGAAGATTTCACA TATGAGACAGACTAT ACGCCAATAAGTTCG CCGAAGATTTCACAG ATGAGACAGACTATT CGCCAATAAGTTCGT CGAAGATTTCACAGT TGAGACAGACTATTA GAGACAGACTATTAC ATGTCTCCTGAGTCC TGGTCCTTCGGGGTC AGACAGACTATTACC TGTCTCCTGAGTCCC GGTCCTTCGGGGTCG GACAGACTATTACCG GTCTCCTGAGTCCCT GTCCTTCGGGGTCGT ACAGACTATTACCGG TCTCCTGAGTCCCTC TCCTTCGGGGTCGTC CAGACTATTACCGGA CTCCTGAGTCCCTCA CCTTCGGGGTCGTCC AGACTATTACCGGAA TCCTGAGTCCCTCAA CTTCGGGGTCGTCCT GACTATTACCGGAAA CCTGAGTCCCTCAAG TTCGGGGTCGTCCTC ACTATTACCGGAAAG CTGAGTCCCTCAAGG TCGGGGTCGTCCTCT CTATTACCGGAAAGG TGAGTCCCTCAAGGA CGGGGTCGTCCTCTG TATTACCGGAAAGGA GAGTCCCTCAAGGAT GGGGTCGTCCTCTGG ATTACCGGAAAGGAG AGTCCCTCAAGGATG GGGTCGTCCTCTGGG TTACCGGAAAGGAGG GTCCCTCAAGGATGG GGTCGTCCTCTGGGA TACCGGAAAGGAGGC TCCCTCAAGGATGGA GTCGTCCTCTGGGAG ACCGGAAAGGAGGCA CCCTCAAGGATGGAG TCGTCCTCTGGGAGA CCGGAAAGGAGGCAA CCTCAAGGATGGAGT CGTCCTCTGGGAGAT CGGAAAGGAGGCAAA CTCAAGGATGGAGTC GTCCTCTGGGAGATC GGAAAGGAGGCAAAG TCAAGGATGGAGTCT TCCTCTGGGAGATCG GAAAGGAGGCAAAGG CAAGGATGGAGTCTT CCTCTGGGAGATCGC AAAGGAGGCAAAGGG AAGGATGGAGTCTTC CTCTGGGAGATCGCC AAGGAGGCAAAGGGC AGGATGGAGTCTTCA TCTGGGAGATCGCCA AGGAGGCAAAGGGCT GGATGGAGTCTTCAC CTGGGAGATCGCCAC GGAGGCAAAGGGCTG GATGGAGTCTTCACC TGGGAGATCGCCACA GAGGCAAAGGGCTGC ATGGAGTCTTCACCA GGGAGATCGCCACAC AGGCAAAGGGCTGCT TGGAGTCTTCACCAC GGAGATCGCCACACT GGCAAAGGGCTGCTG GGAGTCTTCACCACT GAGATCGCCACACTG GCAAAGGGCTGCTGC GAGTCTTCACCACTT AGATCGCCACACTGG CAAAGGGCTGCTGCC AGTCTTCACCACTTA GATCGCCACACTGGC AAAGGGCTGCTGCCC GTCTTCACCACTTAC ATCGCCACACTGGCC AAGGGCTGCTGCCCG TCTTCACCACTTACT TCGCCACACTGGCCG AGGGCTGCTGCCCGT CTTCACCACTTACTC CGCCACACTGGCCGA GGGCTGCTGCCCGTG TTCACCACTTACTCG GCCACACTGGCCGAG GGCTGCTGCCCGTGC TCACCACTTACTCGG CCACACTGGCCGAGC GCTGCTGCCCGTGCG CACCACTTACTCGGA CACACTGGCCGAGCA CTGCTGCCCGTGCGC ACCACTTACTCGGAC ACACTGGCCGAGCAG TGCTGCCCGTGCGCT CCACTTACTCGGACG CACTGGCCGAGCAGC GCTGCCCGTGCGCTG CACTTACTCGGACGT ACTGGCCGAGCAGCC CTGCCCGTGCGCTGG ACTTACTCGGACGTC CTGGCCGAGCAGCCC TGCCCGTGCGCTGGA CTTACTCGGACGTCT TGGCCGAGCAGCCCT GCCCGTGCGCTGGAT TTACTCGGACGTCTG GGCCGAGCAGCCCTA CCCGTGCGCTGGATG TACTCGGACGTCTGG GCCGAGCAGCCCTAC CCGTGCGCTGGATGT ACTCGGACGTCTGGT CCGAGCAGCCCTACC CGTGCGCTGGATGTC CTCGGACGTCTGGTC CGAGCAGCCCTACCA GTGCGCTGGATGTCT TCGGACGTCTGGTCC GAGCAGCCCTACCAG TGCGCTGGATGTCTC CGGACGTCTGGTCCT AGCAGCCCTACCAGG GCGCTGGATGTCTCC GGACGTCTGGTCCTT GCAGCCCTACCAGGG CGCTGGATGTCTCCT GACGTCTGGTCCTTC CAGCCCTACCAGGGC GCTGGATGTCTCCTG ACGTCTGGTCCTTCG AGCCCTACCAGGGCT CTGGATGTCTCCTGA CGTCTGGTCCTTCGG GCCCTACCAGGGCTT TGGATGTCTCCTGAG GTCTGGTCCTTCGGG CCCTACCAGGGCTTG GGATGTCTCCTGAGT TCTGGTCCTTCGGGG CCTACCAGGGCTTGT GATGTCTCCTGAGTC CTGGTCCTTCGGGGT CTACCAGGGCTTGTC TACCAGGGCTTGTCC CTTCTGGACAAGCCA ATGTGCTGGCAGTAT
ACCAGGGCTTGTCCA TTCTGGACAAGCCAG TGTGCTGGCAGTATA
CCAGGGCTTGTCCAA TCTGGACAAGCCAGA GTGCTGGCAGTATAA
CAGGGCTTGTCCAAC CTGGACAAGCCAGAC TGCTGGCAGTATAAC AGGGCTTGTCCAACG TGGACAAGCCAGACA GCTGGCAGTATAACC
GGGCTTGTCCAACGA GGACAAGCCAGACAA CTGGCAGTATAACCC
GGCTTGTCCAACGAG GACAAGCCAGACAAC TGGCAGTATAACCCC
GCTTGTCCAACGAGC ACAAGCCAGACAACT GGCAGTATAACCCCA
CTTGTCCAACGAGCA CAAGCCAGACAACTG GCAGTATAACCCCAA TTGTCCAACGAGCAA AAGCCAGACAACTGT CAGTATAACCCCAAG
TGTCCAACGAGCAAG AGCCAGACAACTGTC AGTATAACCCCAAGA
GTCCAACGAGCAAGT GCCAGACAACTGTCC GTATAACCCCAAGAT
TCCAACGAGCAAGTC CCAGACAACTGTCCT TATAACCCCAAGATG
CCAACGAGCAAGTCC CAGACAACTGTCCTG ATAACCCCAAGATGA CAACGAGCAAGTCCT AGACAACTGTCCTGA TAACCCCAAGATGAG
AACGAGCAAGTCCTT GACAACTGTCCTGAC AACCCCAAGATGAGG
ACGAGCAAGTCCTTC ACAACTGTCCTGACA ACCCCAAGATGAGGC
CGAGCAAGTCCTTCG CAACTGTCCTGACAT CCCCAAGATGAGGCC
GAGCAAGTCCTTCGC AACTGTCCTGACATG CCCAAGATGAGGCCT AGCAAGTCCTTCGCT ACTGTCCTGACATGC CCAAGATGAGGCCTT
GCAAGTCCTTCGCTT CTGTCCTGACATGCT CAAGATGAGGCCTTC
CAAGTCCTTCGCTTC TGTCCTGACATGCTG AAGATGAGGCCTTCC
AAGTCCTTCGCTTCG GTCCTGACATGCTGT AGATGAGGCCTTCCT
AGTCCTTCGCTTCGT TCCTGACATGCTGTT GATGAGGCCTTCCTT GTCCTTCGCTTCGTC CCTGACATGCTGTTT ATGAGGCCTTCCTTC
TCCTTCGCTTCGTCA CTGACATGCTGTTTG TGAGGCCTTCCTTCC
CCTTCGCTTCGTCAT TGACATGCTGTTTGA GAGGCCTTCCTTCCT
CTTCGCTTCGTCATG GACATGCTGTTTGAA AGGCCTTCCTTCCTG
TTCGCTTCGTCATGG ACATGCTGTTTGAAC GGCCTTCCTTCCTGG TCGCTTCGTCATGGA CATGCTGTTTGAACT GCCTTCCTTCCTGGA
CGCTTCGTCATGGAG ATGCTGTTTGAACTG CCTTCCTTCCTGGAG
GCTTCGTCATGGAGG TGCTGTTTGAACTGA CTTCCTTCCTGGAGA
CTTCGTCATGGAGGG GCTGTTTGAACTGAT TTCCTTCCTGGAGAT
TTCGTCATGGAGGGC CTGTTTGAACTGATG TCCTTCCTGGAGATC TCGTCATGGAGGGCG TGTTTGAACTGATGC CCTTCCTGGAGATCA
CGTCATGGAGGGCGG GTTTGAACTGATGCG CTTCCTGGAGATCAT
GTCATGGAGGGCGGC TTTGAACTGATGCGC TTCCTGGAGATCATC
TCATGGAGGGCGGCC TTGAACTGATGCGCA TCCTGGAGATCATCA
CATGGAGGGCGGCCT TGAACTGATGCGCAT CCTGGAGATCATCAG ATGGAGGGCGGCCTT GAACTGATGCGCATG CTGGAGATCATCAGC
TGGAGGGCGGCCTTC AACTGATGCGCATGT TGGAGATCATCAGCA
GGAGGGCGGCCTTCT ACTGATGCGCATGTG GGAGATCATCAGCAG
GAGGGCGGCCTTCTG CTGATGCGCATGTGC GAGATCATCAGCAGC
AGGGCGGCCTTCTGG TGATGCGCATGTGCT AGATCATCAGCAGCA GGGCGGCCTTCTGGA GATGCGCATGTGCTG GATCATCAGCAGCAT
GGCGGCCTTCTGGAC ATGCGCATGTGCTGG ATCATCAGCAGCATC
GCGGCCTTCTGGACA TGCGCATGTGCTGGC TCATCAGCAGCATCA
CGGCCTTCTGGACAA GCGCATGTGCTGGCA CATCAGCAGCATCAA
GGCCTTCTGGACAAG CGCATGTGCTGGCAG ATCAGCAGCATCAAA GCCTTCTGGACAAGC GCATGTGCTGGCAGT TCAGCAGCATCAAAG
CCTTCTGGACAAGCC CATGTGCTGGCAGTA CAGCAGCATCAAAGA AGCAGCATCAAAGAG TACAGCGAGGAGAAC GAGAACATGGAGAGC
GCAGCATCAAAGAGG ACAGCGAGGAGAACA AGAACATGGAGAGCG
CAGCATCAAAGAGGA CAGCGAGGAGAACAA GAACATGGAGAGCGT
AGCATCAAAGAGGAG AGCGAGGAGAACAAG AACATGGAGAGCGTC GCATCAAAGAGGAGA GCGAGGAGAACAAGC ACATGGAGAGCGTCC
CATCAAAGAGGAGAT CGAGGAGAACAAGCT CATGGAGAGCGTCCC
ATCAAAGAGGAGATG GAGGAGAACAAGCTG ATGGAGAGCGTCCCC
TCAAAGAGGAGATGG AGGAGAACAAGCTGC TGGAGAGCGTCCCCC
CAAAGAGGAGATGGA GGAGAACAAGCTGCC GGAGAGCGTCCCCCT AAAGAGGAGATGGAG GAGAACAAGCTGCCC GAGAGCGTCCCCCTG
AAGAGGAGATGGAGC AGAACAAGCTGCCCG AGAGCGTCCCCCTGG
AGAGGAGATGGAGCC GAACAAGCTGCCCGA GAGCGTCCCCCTGGA
GAGGAGATGGAGCCT AACAAGCTGCCCGAG AGCGTCCCCCTGGAC
AGGAGATGGAGCCTG ACAAGCTGCCCGAGC GCGTCCCCCTGGACC GGAGATGGAGCCTGG CAAGCTGCCCGAGCC CGTCCCCCTGGACCC
GAGATGGAGCCTGGC AAGCTGCCCGAGCCG GTCCCCCTGGACCCC
AGATGGAGCCTGGCT AGCTGCCCGAGCCGG TCCCCCTGGACCCCT
GATGGAGCCTGGCTT GCTGCCCGAGCCGGA CCCCCTGGACCCCTC
ATGGAGCCTGGCTTC CTGCCCGAGCCGGAG CCCCTGGACCCCTCG TGGAGCCTGGCTTCC TGCCCGAGCCGGAGG CCCTGGACCCCTCGG
GGAGCCTGGCTTCCG GCCCGAGCCGGAGGA CCTGGACCCCTCGGC
GAGCCTGGCTTCCGG CCCGAGCCGGAGGAG CTGGACCCCTCGGCC
AGCCTGGCTTCCGGG CCGAGCCGGAGGAGC TGGACCCCTCGGCCT
GCCTGGCTTCCGGGA CGAGCCGGAGGAGCT GGACCCCTCGGCCTC CCTGGCTTCCGGGAG GAGCCGGAGGAGCTG GACCCCTCGGCCTCC
CTGGCTTCCGGGAGG AGCCGGAGGAGCTGG ACCCCTCGGCCTCCT
TGGCTTCCGGGAGGT GCCGGAGGAGCTGGA CCCCTCGGCCTCCTC
GGCTTCCGGGAGGTC CCGGAGGAGCTGGAC CCCTCGGCCTCCTCG
GCTTCCGGGAGGTCT CGGAGGAGCTGGACC CCTCGGCCTCCTCGT CTTCCGGGAGGTCTC GGAGGAGCTGGACCT CTCGGCCTCCTCGTC
TTCCGGGAGGTCTCC GAGGAGCTGGACCTG TCGGCCTCCTCGTCC
TCCGGGAGGTCTCCT AGGAGCTGGACCTGG CGGCCTCCTCGTCCT
CCGGGAGGTCTCCTT GGAGCTGGACCTGGA GGCCTCCTCGTCCTC
CGGGAGGTCTCCTTC GAGCTGGACCTGGAG GCCTCCTCGTCCTCC GGGAGGTCTCCTTCT AGCTGGACCTGGAGC CCTCCTCGTCCTCCC
GGAGGTCTCCTTCTA GCTGGACCTGGAGCC CTCCTCGTCCTCCCT
GAGGTCTCCTTCTAC CTGGACCTGGAGCCA TCCTCGTCCTCCCTG
AGGTCTCCTTCTACT TGGACCTGGAGCCAG CCTCGTCCTCCCTGC
GGTCTCCTTCTACTA GGACCTGGAGCCAGA CTCGTCCTCCCTGCC GTCTCCTTCTACTAC GACCTGGAGCCAGAG TCGTCCTCCCTGCCA
TCTCCTTCTACTACA ACCTGGAGCCAGAGA CGTCCTCCCTGCCAC
CTCCTTCTACTACAG CCTGGAGCCAGAGAA GTCCTCCCTGCCACT
TCCTTCTACTACAGC CTGGAGCCAGAGAAC TCCTCCCTGCCACTG
CCTTCTACTACAGCG TGGAGCCAGAGAACA CCTCCCTGCCACTGC CTTCTACTACAGCGA GGAGCCAGAGAACAT CTCCCTGCCACTGCC
TTCTACTACAGCGAG GAGCCAGAGAACATG TCCCTGCCACTGCCC
TCTACTACAGCGAGG AGCCAGAGAACATGG CCCTGCCACTGCCCG
CTACTACAGCGAGGA GCCAGAGAACATGGA CCTGCCACTGCCCGA
TACTACAGCGAGGAG CCAGAGAACATGGAG CTGCCACTGCCCGAC ACTACAGCGAGGAGA CAGAGAACATGGAGA TGCCACTGCCCGACA
CTACAGCGAGGAGAA AGAGAACATGGAGAG GCCACTGCCCGACAG CCACTGCCCGACAGA GGGGTGCTGGTCCTC ATGAACGGGGGCCGC
CACTGCCCGACAGAC GGGTGCTGGTCCTCC TGAACGGGGGCCGCA
ACTGCCCGACAGACA GGTGCTGGTCCTCCG GAACGGGGGCCGCAA
CTGCCCGACAGACAC GTGCTGGTCCTCCGC AACGGGGGCCGCAAG TGCCCGACAGACACT TGCTGGTCCTCCGCG ACGGGGGCCGCAAGA
GCCCGACAGACACTC GCTGGTCCTCCGCGC CGGGGGCCGCAAGAA
CCCGACAGACACTCA CTGGTCCTCCGCGCC GGGGGCCGCAAGAAC
CCGACAGACACTCAG TGGTCCTCCGCGCCA GGGGCCGCAAGAACG
CGACAGACACTCAGG GGTCCTCCGCGCCAG GGGCCGCAAGAACGA GACAGACACTCAGGA GTCCTCCGCGCCAGC GGCCGCAAGAACGAG
ACAGACACTCAGGAC TCCTCCGCGCCAGCT GCCGCAAGAACGAGC
CAGACACTCAGGACA CCTCCGCGCCAGCTT CCGCAAGAACGAGCG
AGACACTCAGGACAC CTCCGCGCCAGCTTC CGCAAGAACGAGCGG
GACACTCAGGACACA TCCGCGCCAGCTTCG GCAAGAACGAGCGGG ACACTCAGGACACAA CCGCGCCAGCTTCGA CAAGAACGAGCGGGC
CACTCAGGACACAAG CGCGCCAGCTTCGAC AAGAACGAGCGGGCC
ACTCAGGACACAAGG GCGCCAGCTTCGACG AGAACGAGCGGGCCT
CTCAGGACACAAGGC CGCCAGCTTCGACGA GAACGAGCGGGCCTT
TCAGGACACAAGGCC GCCAGCTTCGACGAG AACGAGCGGGCCTTG CAGGACACAAGGCCG CCAGCTTCGACGAGA ACGAGCGGGCCTTGC
AGGACACAAGGCCGA CAGCTTCGACGAGAG CGAGCGGGCCTTGCC
GGACACAAGGCCGAG AGCTTCGACGAGAGA GAGCGGGCCTTGCCG
GACACAAGGCCGAGA GCTTCGACGAGAGAC AGCGGGCCTTGCCGC
ACACAAGGCCGAGAA CTTCGACGAGAGACA GCGGGCCTTGCCGCT CACAAGGCCGAGAAC TTCGACGAGAGACAG CGGGCCTTGCCGCTG
ACAAGGCCGAGAACG TCGACGAGAGACAGC GGGCCTTGCCGCTGC
CAAGGCCGAGAACGG CGACGAGAGACAGCC GGCCTTGCCGCTGCC
AAGGCCGAGAACGGC GACGAGAGACAGCCT GCCTTGCCGCTGCCC
AGGCCGAGAACGGCC ACGAGAGACAGCCTT CCTTGCCGCTGCCCC GGCCGAGAACGGCCC CGAGAGACAGCCTTA CTTGCCGCTGCCCCA
GCCGAGAACGGCCCC GAGAGACAGCCTTAC TTGCCGCTGCCCCAG
CCGAGAACGGCCCCG AGAGACAGCCTTACG TGCCGCTGCCCCAGT
CGAGAACGGCCCCGG GAGACAGCCTTACGC GCCGCTGCCCCAGTC
GAGAACGGCCCCGGC AGACAGCCTTACGCC CCGCTGCCCCAGTCT AGAACGGCCCCGGCC GACAGCCTTACGCCC CGCTGCCCCAGTCTT
GAACGGCCCCGGCCC ACAGCCTTACGCCCA GCTGCCCCAGTCTTC
AACGGCCCCGGCCCT CAGCCTTACGCCCAC CTGCCCCAGTCTTCG
ACGGCCCCGGCCCTG AGCCTTACGCCCACA TGCCCCAGTCTTCGA
CGGCCCCGGCCCTGG GCCTTACGCCCACAT GCCCCAGTCTTCGAC GGCCCCGGCCCTGGG CCTTACGCCCACATG CCCCAGTCTTCGACC
GCCCCGGCCCTGGGG CTTACGCCCACATGA CCCAGTCTTCGACCT
CCCCGGCCCTGGGGT TTACGCCCACATGAA CCAGTCTTCGACCTG
CCCGGCCCTGGGGTG TACGCCCACATGAAC CAGTCTTCGACCTGC
CCGGCCCTGGGGTGC ACGCCCACATGAACG AGTCTTCGACCTGCT CGGCCCTGGGGTGCT CGCCCACATGAACGG GTCTTCGACCTGCTG
GGCCCTGGGGTGCTG GCCCACATGAACGGG TCTTCGACCTGCTGA
GCCCTGGGGTGCTGG CCCACATGAACGGGG CTTCGACCTGCTGAT
CCCTGGGGTGCTGGT CCACATGAACGGGGG TTCGACCTGCTGATC
CCTGGGGTGCTGGTC CACATGAACGGGGGC TCGACCTGCTGATCC CTGGGGTGCTGGTCC ACATGAACGGGGGCC CGACCTGCTGATCCT
TGGGGTGCTGGTCCT CATGAACGGGGGCCG GACCTGCTGATCCTT ACCTGCTGATCCTTG GCGCAGCGGGGTGGG TCCTGTACCTCAGTG CCTGCTGATCCTTGG CGCAGCGGGGTGGGG CCTGTACCTCAGTGG CTGCTGATCCTTGGA GCAGCGGGGTGGGGG CTGTACCTCAGTGGA TGCTGATCCTTGGAT CAGCGGGGTGGGGGG TGTACCTCAGTGGAT GCTGATCCTTGGATC AGCGGGGTGGGGGGG GTACCTCAGTGGATC CTGATCCTTGGATCC GCGGGGTGGGGGGGG TACCTCAGTGGATCT TGATCCTTGGATCCT CGGGGTGGGGGGGGA ACCTCAGTGGATCTT GATCCTTGGATCCTG GGGGTGGGGGGGGAG CCTCAGTGGATCTTC ATCCTTGGATCCTGA GGGTGGGGGGGGAGA CTCAGTGGATCTTCA TCCTTGGATCCTGAA GGTGGGGGGGGAGAG TCAGTGGATCTTCAG CCTTGGATCCTGAAT GTGGGGGGGGAGAGA CAGTGGATCTTCAGT CTTGGATCCTGAATC TGGGGGGGGAGAGAG AGTGGATCTTCAGTT TTGGATCCTGAATCT GGGGGGGGAGAGAGA GTGGATCTTCAGTTC TGGATCCTGAATCTG GGGGGGGAGAGAGAG TGGATCTTCAGTTCT GGATCCTGAATCTGT GGGGGGAGAGAGAGT GGATCTTCAGTTCTG GATCCTGAATCTGTG GGGGGAGAGAGAGTT GATCTTCAGTTCTGC ATCCTGAATCTGTGC GGGGAGAGAGAGTTT ATCTTCAGTTCTGCC TCCTGAATCTGTGCA GGGAGAGAGAGTTTT TCTTCAGTTCTGCCC CCTGAATCTGTGCAA GGAGAGAGAGTTTTA CTTCAGTTCTGCCCT CTGAATCTGTGCAAA GAGAGAGAGTTTTAA TTCAGTTCTGCCCTT TGAATCTGTGCAAAC AGAGAGAGTTTTAAC TCAGTTCTGCCCTTG GAATCTGTGCAAACA GAGAGAGTTTTAACA CAGTTCTGCCCTTGC AATCTGTGCAAACAG AGAGAGTTTTAACAA AGTTCTGCCCTTGCT ATCTGTGCAAACAGT GAGAGTTTTAACAAT GTTCTGCCCTTGCTG TCTGTGCAAACAGTA AGAGTTTTAACAATC TTCTGCCCTTGCTGC CTGTGCAAACAGTAA GAGTTTTAACAATCC TCTGCCCTTGCTGCC TGTGCAAACAGTAAC AGTTTTAACAATCCA CTGCCCTTGCTGCCC GTGCAAACAGTAACG GTTTTAACAATCCAT TGCCCTTGCTGCCCG TGCAAACAGTAACGT TTTTAACAATCCATT GCCCTTGCTGCCCGC GCAAACAGTAACGTG TTTAACAATCCATTC CCCTTGCTGCCCGCG CAAACAGTAACGTGT TTAACAATCCATTCA CCTTGCTGCCCGCGG AAACAGTAACGTGTG TAACAATCCATTCAC CTTGCTGCCCGCGGG AACAGTAACGTGTGC AACAATCCATTCACA TTGCTGCCCGCGGGA ACAGTAACGTGTGCG ACAATCCATTCACAA TGCTGCCCGCGGGAG CAGTAACGTGTGCGC CAATCCATTCACAAG GCTGCCCGCGGGAGA AGTAACGTGTGCGCA AATCCATTCACAAGC CTGCCCGCGGGAGAC GTAACGTGTGCGCAC ATCCATTCACAAGCC TGCCCGCGGGAGACA TAACGTGTGCGCACG TCCATTCACAAGCCT GCCCGCGGGAGACAG AACGTGTGCGCACGC CCATTCACAAGCCTC CCCGCGGGAGACAGC ACGTGTGCGCACGCG CATTCACAAGCCTCC CCGCGGGAGACAGCT CGTGTGCGCACGCGC ATTCACAAGCCTCCT CGCGGGAGACAGCTT GTGTGCGCACGCGCA TTCACAAGCCTCCTG GCGGGAGACAGCTTC TGTGCGCACGCGCAG TCACAAGCCTCCTGT CGGGAGACAGCTTCT GTGCGCACGCGCAGC CACAAGCCTCCTGTA GGGAGACAGCTTCTC TGCGCACGCGCAGCG ACAAGCCTCCTGTAC GGAGACAGCTTCTCT GCGCACGCGCAGCGG CAAGCCTCCTGTACC GAGACAGCTTCTCTG CGCACGCGCAGCGGG AAGCCTCCTGTACCT AGACAGCTTCTCTGC GCACGCGCAGCGGGG AGCCTCCTGTACCTC GACAGCTTCTCTGCA CACGCGCAGCGGGGT GCCTCCTGTACCTCA ACAGCTTCTCTGCAG ACGCGCAGCGGGGTG CCTCCTGTACCTCAG CAGCTTCTCTGCAGT CGCGCAGCGGGGTGG CTCCTGTACCTCAGT AGCTTCTCTGCAGTA GCTTCTCTGCAGTAA CAGCTTTTTATTCCC CTTAATGACAACACT
CTTCTCTGCAGTAAA AGCTTTTTATTCCCT TTAATGACAACACTT
TTCTCTGCAGTAAAA GCTTTTTATTCCCTG TAATGACAACACTTA
TCTCTGCAGTAAAAC CTTTTTATTCCCTGC AATGACAACACTTAA CTCTGCAGTAAAACA TTTTTATTCCCTGCC ATGACAACACTTAAT
TCTGCAGTAAAACAC TTTTATTCCCTGCCC TGACAACACTTAATA
CTGCAGTAAAACACA TTTATTCCCTGCCCA GACAACACTTAATAG
TGCAGTAAAACACAT TTATTCCCTGCCCAA ACAACACTTAATAGC
GCAGTAAAACACATT TATTCCCTGCCCAAA CAACACTTAATAGCA CAGTAAAACACATTT ATTCCCTGCCCAAAC AACACTTAATAGCAA
AGTAAAACACATTTG TTCCCTGCCCAAACC ACACTTAATAGCAAC
GTAAAACACATTTGG TCCCTGCCCAAACCC CACTTAATAGCAACA
TAAAACACATTTGGG CCCTGCCCAAACCCT ACTTAATAGCAACAG
AAAACACATTTGGGA CCTGCCCAAACCCTT CTTAATAGCAACAGA AAACACATTTGGGAT CTGCCCAAACCCTTA TTAATAGCAACAGAG
AACACATTTGGGATG TGCCCAAACCCTTAA TAATAGCAACAGAGC
ACACATTTGGGATGT GCCCAAACCCTTAAC AATAGCAACAGAGCA
CACATTTGGGATGTT CCCAAACCCTTAACT ATAGCAACAGAGCAC
ACATTTGGGATGTTC CCAAACCCTTAACTG TAGCAACAGAGCACT CATTTGGGATGTTCC CAAACCCTTAACTGA AGCAACAGAGCACTT
ATTTGGGATGTTCCT AAACCCTTAACTGAC GCAACAGAGCACTTG
TTTGGGATGTTCCTT AACCCTTAACTGACA CAACAGAGCACTTGA
TTGGGATGTTCCTTT ACCCTTAACTGACAT AACAGAGCACTTGAG
TGGGATGTTCCTTTT CCCTTAACTGACATG ACAGAGCACTTGAGA GGGATGTTCCTTTTT CCTTAACTGACATGG CAGAGCACTTGAGAA
GGATGTTCCTTTTTT CTTAACTGACATGGG AGAGCACTTGAGAAC
GATGTTCCTTTTTTC TTAACTGACATGGGC GAGCACTTGAGAACC
ATGTTCCTTTTTTCA TAACTGACATGGGCC AGCACTTGAGAACCA
TGTTCCTTTTTTCAA AACTGACATGGGCCT GCACTTGAGAACCAG GTTCCTTTTTTCAAT ACTGACATGGGCCTT CACTTGAGAACCAGT
TTCCTTTTTTCAATA CTGACATGGGCCTTT ACTTGAGAACCAGTC
TCCTTTTTTCAATAT TGACATGGGCCTTTA CTTGAGAACCAGTCT
CCTTTTTTCAATATG GACATGGGCCTTTAA TTGAGAACCAGTCTC
CTTTTTTCAATATGC ACATGGGCCTTTAAG TGAGAACCAGTCTCC TTTTTTCAATATGCA CATGGGCCTTTAAGA GAGAACCAGTCTCCT
TTTTTCAATATGCAA ATGGGCCTTTAAGAA AGAACCAGTCTCCTC
TTTTCAATATGCAAG TGGGCCTTTAAGAAC GAACCAGTCTCCTCA
TTTCAATATGCAAGC GGGCCTTTAAGAACC AACCAGTCTCCTCAC
TTCAATATGCAAGCA GGCCTTTAAGAACCT ACCAGTCTCCTCACT TCAATATGCAAGCAG GCCTTTAAGAACCTT CCAGTCTCCTCACTC
CAATATGCAAGCAGC CCTTTAAGAACCTTA CAGTCTCCTCACTCT
AATATGCAAGCAGCT CTTTAAGAACCTTAA AGTCTCCTCACTCTG
ATATGCAAGCAGCTT TTTAAGAACCTTAAT GTCTCCTCACTCTGT
TATGCAAGCAGCTTT TTAAGAACCTTAATG TCTCCTCACTCTGTC ATGCAAGCAGCTTTT TAAGAACCTTAATGA CTCCTCACTCTGTCC
TGCAAGCAGCTTTTT AAGAACCTTAATGAC TCCTCACTCTGTCCC
GCAAGCAGCTTTTTA AGAACCTTAATGACA CCTCACTCTGTCCCT
CAAGCAGCTTTTTAT GAACCTTAATGACAA CTCACTCTGTCCCTG
AAGCAGCTTTTTATT AACCTTAATGACAAC TCACTCTGTCCCTGT AGCAGCTTTTTATTC ACCTTAATGACAACA CACTCTGTCCCTGTC
GCAGCTTTTTATTCC CCTTAATGACAACAC ACTCTGTCCCTGTCC CTCTGTCCCTGTCCT AACGGAAAAATAATT TGAGGAAGTGGCTGT TCTGTCCCTGTCCTT ACGGAAAAATAATTG GAGGAAGTGGCTGTC CTGTCCCTGTCCTTC CGGAAAAATAATTGC AGGAAGTGGCTGTCC TGTCCCTGTCCTTCC GGAAAAATAATTGCC GGAAGTGGCTGTCCC GTCCCTGTCCTTCCC GAAAAATAATTGCCA GAAGTGGCTGTCCCT TCCCTGTCCTTCCCT AAAAATAATTGCCAC AAGTGGCTGTCCCTG CCCTGTCCTTCCCTG AAAATAATTGCCACA AGTGGCTGTCCCTGT CCTGTCCTTCCCTGT AAATAATTGCCACAA GTGGCTGTCCCTGTG CTGTCCTTCCCTGTT AATAATTGCCACAAG TGGCTGTCCCTGTGG TGTCCTTCCCTGTTC ATAATTGCCACAAGT GGCTGTCCCTGTGGC GTCCTTCCCTGTTCT TAATTGCCACAAGTC GCTGTCCCTGTGGCC TCCTTCCCTGTTCTC AATTGCCACAAGTCC CTGTCCCTGTGGCCC CCTTCCCTGTTCTCC ATTGCCACAAGTCCA TGTCCCTGTGGCCCC CTTCCCTGTTCTCCC TTGCCACAAGTCCAG GTCCCTGTGGCCCCA TTCCCTGTTCTCCCT TGCCACAAGTCCAGC TCCCTGTGGCCCCAT TCCCTGTTCTCCCTT GCCACAAGTCCAGCT CCCTGTGGCCCCATC CCCTGTTCTCCCTTT CCACAAGTCCAGCTG CCTGTGGCCCCATCC CCTGTTCTCCCTTTC CACAAGTCCAGCTGG CTGTGGCCCCATCCA CTGTTCTCCCTTTCT ACAAGTCCAGCTGGG TGTGGCCCCATCCAA TGTTCTCCCTTTCTC CAAGTCCAGCTGGGA GTGGCCCCATCCAAC GTTCTCCCTTTCTCT AAGTCCAGCTGGGAA TGGCCCCATCCAACC TTCTCCCTTTCTCTC AGTCCAGCTGGGAAG GGCCCCATCCAACCA TCTCCCTTTCTCTCT GTCCAGCTGGGAAGC GCCCCATCCAACCAC CTCCCTTTCTCTCTC TCCAGCTGGGAAGCC CCCCATCCAACCACT TCCCTTTCTCTCTCC CCAGCTGGGAAGCCC CCCATCCAACCACTG CCCTTTCTCTCTCCT CAGCTGGGAAGCCCT CCATCCAACCACTGT CCTTTCTCTCTCCTC AGCTGGGAAGCCCTT CATCCAACCACTGTA CTTTCTCTCTCCTCT GCTGGGAAGCCCTTT ATCCAACCACTGTAC TTTCTCTCTCCTCTC CTGGGAAGCCCTTTT TCCAACCACTGTACA TTCTCTCTCCTCTCT TGGGAAGCCCTTTTT CCAACCACTGTACAC TCTCTCTCCTCTCTG GGGAAGCCCTTTTTA CAACCACTGTACACA CTCTCTCCTCTCTGC GGAAGCCCTTTTTAT AACCACTGTACACAC TCTCTCCTCTCTGCT GAAGCCCTTTTTATC ACCACTGTACACACC CTCTCCTCTCTGCTT AAGCCCTTTTTATCA CCACTGTACACACCC TCTCCTCTCTGCTTC AGCCCTTTTTATCAG CACTGTACACACCCG CTCCTCTCTGCTTCA GCCCTTTTTATCAGT ACTGTACACACCCGC TCCTCTCTGCTTCAT CCCTTTTTATCAGTT CTGTACACACCCGCC CCTCTCTGCTTCATA CCTTTTTATCAGTTT TGTACACACCCGCCT CTCTCTGCTTCATAA CTTTTTATCAGTTTG GTACACACCCGCCTG TCTCTGCTTCATAAC TTTTTATCAGTTTGA TACACACCCGCCTGA CTCTGCTTCATAACG TTTTATCAGTTTGAG ACACACCCGCCTGAC TCTGCTTCATAACGG TTTATCAGTTTGAGG CACACCCGCCTGACA CTGCTTCATAACGGA TTATCAGTTTGAGGA ACACCCGCCTGACAC TGCTTCATAACGGAA TATCAGTTTGAGGAA CACCCGCCTGACACC GCTTCATAACGGAAA ATCAGTTTGAGGAAG ACCCGCCTGACACCG CTTCATAACGGAAAA TCAGTTTGAGGAAGT CCCGCCTGACACCGT TTCATAACGGAAAAA CAGTTTGAGGAAGTG CCGCCTGACACCGTG TCATAACGGAAAAAT AGTTTGAGGAAGTGG CGCCTGACACCGTGG CATAACGGAAAAATA GTTTGAGGAAGTGGC GCCTGACACCGTGGG ATAACGGAAAAATAA TTTGAGGAAGTGGCT CCTGACACCGTGGGT TAACGGAAAAATAAT TTGAGGAAGTGGCTG CTGACACCGTGGGTC TGACACCGTGGGTCA TTATCTTTCACCTTT CCAAGGCTGTTACCA GACACCGTGGGTCAT TATCTTTCACCTTTC CAAGGCTGTTACCAT ACACCGTGGGTCATT ATCTTTCACCTTTCT AAGGCTGTTACCATT CACCGTGGGTCATTA TCTTTCACCTTTCTA AGGCTGTTACCATTT ACCGTGGGTCATTAC CTTTCACCTTTCTAG GGCTGTTACCATTTT CCGTGGGTCATTACA TTTCACCTTTCTAGG GCTGTTACCATTTTA CGTGGGTCATTACAA TTCACCTTTCTAGGG CTGTTACCATTTTAA GTGGGTCATTACAAA TCACCTTTCTAGGGA TGTTACCATTTTAAC TGGGTCATTACAAAA CACCTTTCTAGGGAC GTTACCATTTTAACG GGGTCATTACAAAAA ACCTTTCTAGGGACA TTACCATTTTAACGC GGTCATTACAAAAAA CCTTTCTAGGGACAT TACCATTTTAACGCT GTCATTACAAAAAAA CTTTCTAGGGACATG ACCATTTTAACGCTG TCATTACAAAAAAAC TTTCTAGGGACATGA CCATTTTAACGCTGC CATTACAAAAAAACA TTCTAGGGACATGAA CATTTTAACGCTGCC ATTACAAAAAAACAC TCTAGGGACATGAAA ATTTTAACGCTGCCT TTACAAAAAAACACG CTAGGGACATGAAAT TTTTAACGCTGCCTA TACAAAAAAACACGT TAGGGACATGAAATT TTTAACGCTGCCTAA ACAAAAAAACACGTG AGGGACATGAAATTT TTAACGCTGCCTAAT CAAAAAAACACGTGG GGGACATGAAATTTA TAACGCTGCCTAATT AAAAAAACACGTGGA GGACATGAAATTTAC AACGCTGCCTAATTT AAAAAACACGTGGAG GACATGAAATTTACA ACGCTGCCTAATTTT AAAAACACGTGGAGA ACATGAAATTTACAA CGCTGCCTAATTTTG AAAACACGTGGAGAT CATGAAATTTACAAA GCTGCCTAATTTTGC AAACACGTGGAGATG ATGAAATTTACAAAG CTGCCTAATTTTGCC AACACGTGGAGATGG TGAAATTTACAAAGG TGCCTAATTTTGCCA ACACGTGGAGATGGA GAAATTTACAAAGGG GCCTAATTTTGCCAA CACGTGGAGATGGAA AAATTTACAAAGGGC CCTAATTTTGCCAAA ACGTGGAGATGGAAA AATTTACAAAGGGCC CTAATTTTGCCAAAA CGTGGAGATGGAAAT ATTTACAAAGGGCCA TAATTTTGCCAAAAT GTGGAGATGGAAATT TTTACAAAGGGCCAT AATTTTGCCAAAATC TGGAGATGGAAATTT TTACAAAGGGCCATC ATTTTGCCAAAATCC GGAGATGGAAATTTT TACAAAGGGCCATCG TTTTGCCAAAATCCT GAGATGGAAATTTTT ACAAAGGGCCATCGT TTTGCCAAAATCCTG AGATGGAAATTTTTA CAAAGGGCCATCGTT TTGCCAAAATCCTGA GATGGAAATTTTTAC AAAGGGCCATCGTTC TGCCAAAATCCTGAA ATGGAAATTTTTACC AAGGGCCATCGTTCA GCCAAAATCCTGAAC TGGAAATTTTTACCT AGGGCCATCGTTCAT CCAAAATCCTGAACT GGAAATTTTTACCTT GGGCCATCGTTCATC CAAAATCCTGAACTT GAAATTTTTACCTTT GGCCATCGTTCATCC AAAATCCTGAACTTT AAATTTTTACCTTTA GCCATCGTTCATCCA AAATCCTGAACTTTC AATTTTTACCTTTAT CCATCGTTCATCCAA AATCCTGAACTTTCT ATTTTTACCTTTATC CATCGTTCATCCAAG ATCCTGAACTTTCTC TTTTTACCTTTATCT ATCGTTCATCCAAGG TCCTGAACTTTCTCC TTTTACCTTTATCTT TCGTTCATCCAAGGC CCTGAACTTTCTCCC TTTACCTTTATCTTT CGTTCATCCAAGGCT CTGAACTTTCTCCCT TTACCTTTATCTTTC GTTCATCCAAGGCTG TGAACTTTCTCCCTC TACCTTTATCTTTCA TTCATCCAAGGCTGT GAACTTTCTCCCTCA ACCTTTATCTTTCAC TCATCCAAGGCTGTT AACTTTCTCCCTCAT CCTTTATCTTTCACC CATCCAAGGCTGTTA ACTTTCTCCCTCATC CTTTATCTTTCACCT ATCCAAGGCTGTTAC CTTTCTCCCTCATCG TTTATCTTTCACCTT TCCAAGGCTGTTACC TTTCTCCCTCATCGG TTCTCCCTCATCGGC GCATGGCAGCTGGTT CCATCCGACTGCCCC TCTCCCTCATCGGCC CATGGCAGCTGGTTG CATCCGACTGCCCCT CTCCCTCATCGGCCC ATGGCAGCTGGTTGC ATCCGACTGCCCCTG TCCCTCATCGGCCCG TGGCAGCTGGTTGCT TCCGACTGCCCCTGC CCCTCATCGGCCCGG GGCAGCTGGTTGCTC CCGACTGCCCCTGCT CCTCATCGGCCCGGC GCAGCTGGTTGCTCC CGACTGCCCCTGCTG CTCATCGGCCCGGCG CAGCTGGTTGCTCCA GACTGCCCCTGCTGT TCATCGGCCCGGCGC AGCTGGTTGCTCCAT ACTGCCCCTGCTGTG CATCGGCCCGGCGCT GCTGGTTGCTCCATT CTGCCCCTGCTGTGC ATCGGCCCGGCGCTG CTGGTTGCTCCATTT TGCCCCTGCTGTGCT TCGGCCCGGCGCTGA TGGTTGCTCCATTTG GCCCCTGCTGTGCTG CGGCCCGGCGCTGAT GGTTGCTCCATTTGA CCCCTGCTGTGCTGC GGCCCGGCGCTGATT GTTGCTCCATTTGAG CCCTGCTGTGCTGCT GCCCGGCGCTGATTC TTGCTCCATTTGAGA CCTGCTGTGCTGCTC CCCGGCGCTGATTCC TGCTCCATTTGAGAG CTGCTGTGCTGCTCA CCGGCGCTGATTCCT GCTCCATTTGAGAGA TGCTGTGCTGCTCAA CGGCGCTGATTCCTC CTCCATTTGAGAGAC GCTGTGCTGCTCAAG GGCGCTGATTCCTCG TCCATTTGAGAGACA CTGTGCTGCTCAAGG GCGCTGATTCCTCGT CCATTTGAGAGACAC TGTGCTGCTCAAGGC CGCTGATTCCTCGTG CATTTGAGAGACACG GTGCTGCTCAAGGCC GCTGATTCCTCGTGT ATTTGAGAGACACGC TGCTGCTCAAGGCCA CTGATTCCTCGTGTC TTTGAGAGACACGCT GCTGCTCAAGGCCAC TGATTCCTCGTGTCC TTGAGAGACACGCTG CTGCTCAAGGCCACA GATTCCTCGTGTCCG TGAGAGACACGCTGG TGCTCAAGGCCACAG ATTCCTCGTGTCCGG GAGAGACACGCTGGC GCTCAAGGCCACAGG TTCCTCGTGTCCGGA AGAGACACGCTGGCG CTCAAGGCCACAGGC TCCTCGTGTCCGGAG GAGACACGCTGGCGA TCAAGGCCACAGGCA CCTCGTGTCCGGAGG AGACACGCTGGCGAC CAAGGCCACAGGCAC CTCGTGTCCGGAGGC GACACGCTGGCGACA AAGGCCACAGGCACA TCGTGTCCGGAGGCA ACACGCTGGCGACAC AGGCCACAGGCACAC CGTGTCCGGAGGCAT CACGCTGGCGACACA GGCCACAGGCACACA GTGTCCGGAGGCATG ACGCTGGCGACACAC GCCACAGGCACACAG TGTCCGGAGGCATGG CGCTGGCGACACACT CCACAGGCACACAGG GTCCGGAGGCATGGG GCTGGCGACACACTC CACAGGCACACAGGT TCCGGAGGCATGGGT CTGGCGACACACTCC ACAGGCACACAGGTC CCGGAGGCATGGGTG TGGCGACACACTCCG CAGGCACACAGGTCT CGGAGGCATGGGTGA GGCGACACACTCCGT AGGCACACAGGTCTC GGAGGCATGGGTGAG GCGACACACTCCGTC GGCACACAGGTCTCA GAGGCATGGGTGAGC CGACACACTCCGTCC GCACACAGGTCTCAT AGGCATGGGTGAGCA GACACACTCCGTCCA CACACAGGTCTCATT GGCATGGGTGAGCAT ACACACTCCGTCCAT ACACAGGTCTCATTG GCATGGGTGAGCATG CACACTCCGTCCATC CACAGGTCTCATTGC CATGGGTGAGCATGG ACACTCCGTCCATCC ACAGGTCTCATTGCT ATGGGTGAGCATGGC CACTCCGTCCATCCG CAGGTCTCATTGCTT TGGGTGAGCATGGCA ACTCCGTCCATCCGA AGGTCTCATTGCTTC GGGTGAGCATGGCAG CTCCGTCCATCCGAC GGTCTCATTGCTTCT GGTGAGCATGGCAGC TCCGTCCATCCGACT GTCTCATTGCTTCTG GTGAGCATGGCAGCT CCGTCCATCCGACTG TCTCATTGCTTCTGA TGAGCATGGCAGCTG CGTCCATCCGACTGC CTCATTGCTTCTGAC GAGCATGGCAGCTGG GTCCATCCGACTGCC TCATTGCTTCTGACT AGCATGGCAGCTGGT TCCATCCGACTGCCC CATTGCTTCTGACTA ATTGCTTCTGACTAG CTCTCAGTGAAGGTG
TTGCTTCTGACTAGA TCTCAGTGAAGGTGG
TGCTTCTGACTAGAT CTCAGTGAAGGTGGG
GCTTCTGACTAGATT TCAGTGAAGGTGGGG CTTCTGACTAGATTA CAGTGAAGGTGGGGA
TTCTGACTAGATTAT AGTGAAGGTGGGGAG
TCTGACTAGATTATT GTGAAGGTGGGGAGA
CTGACTAGATTATTA TGAAGGTGGGGAGAA
TGACTAGATTATTAT GAAGGTGGGGAGAAG GACTAGATTATTATT AAGGTGGGGAGAAGC
ACTAGATTATTATTT AGGTGGGGAGAAGCT
CTAGATTATTATTTG GGTGGGGAGAAGCTG
TAGATTATTATTTGG GTGGGGAGAAGCTGA
AGATTATTATTTGGG TGGGGAGAAGCTGAA GATTATTATTTGGGG GGGGAGAAGCTGAAC
ATTATTATTTGGGGG GGGAGAAGCTGAACC
TTATTATTTGGGGGA GGAGAAGCTGAACCG
TATTATTTGGGGGAA GAGAAGCTGAACCGG
ATTATTTGGGGGAAC AGAAGCTGAACCGGC TTATTTGGGGGAACT
TATTTGGGGGAACTG
ATTTGGGGGAACTGG
TTTGGGGGAACTGGA
TTGGGGGAACTGGAC TGGGGGAACTGGACA
GGGGGAACTGGACAC
GGGGAACTGGACACA
GGGAACTGGACACAA
GGAACTGGACACAAT GAACTGGACACAATA
AACTGGACACAATAG
ACTGGACACAATAGG
CTGGACACAATAGGT
TGGACACAATAGGTC GGACACAATAGGTCT
GACACAATAGGTCTT
ACACAATAGGTCTTT
CACAATAGGTCTTTC
ACAATAGGTCTTTCT CAATAGGTCTTTCTC
AATAGGTCTTTCTCT
ATAGGTCTTTCTCTC
TAGGTCTTTCTCTCA
AGGTCTTTCTCTCAG GGTCTTTCTCTCAGT
GTCTTTCTCTCAGTG
TCTTTCTCTCAGTGA
CTTTCTCTCAGTGAA
TTTCTCTCAGTGAAG TTCTCTCAGTGAAGG
TCTCTCAGTGAAGGT 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 Figures 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'- CGGAGATGCCGC ATGCC AGCGCAGG-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 0.5ml 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 lOOμl 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 Figure 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 (30,000cpm/well) at
4°C.
EXAMPLE 11 Experiments involving ribozymes are generally conducted as described in Intemaitonal 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 15mer 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 Figure 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, 30nM) and cytofectin GSV (2μg/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. Figure 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 (30nM or 100 nM) plus cytofectin GSV (2μg/ml or 5μg/ml) over 120 hours, viewed by fluorescence microscopy, tryptan blue stained, and counted. The graphs in Figure 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 2μg/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 (30nM) were completed with cytofectin GSV (2μg/ml) and added tot he 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, TX 77033) 96 hours following the first 5 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 10 templates were obtained from Ambion, Inc. (2130 Woodward St., Houston, TX 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.
15 Figure 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
20 276 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 Figure 13. The graph depicts the relative level of IGF-I receptor mRNA after treatment with oligonucleotides 25 complementary to the human IGF-I receptor mRNA (26-86), four randomized sequence oligonucleotides (Rl, 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 Figure 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 Figure 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 IGFOI 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 30nM random sequence or antisense oligonucleotide and 2μm/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 1ml cold PBS. Serum-free DMEM containing 10" 10M125I-IGF-I was added with or without the IGF-I analogue, des (1-3) IGF-I, at 10"UM to 10/ 7M. Cells were incubated at 4 °C for 17 hours with gentle shaking, then washed three times with 1ml cold PBS and lysed in 250μl 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 Figure 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 Figure 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 tot he 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 Figure 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, 50μl, 10μM 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. Figure 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:
Figure imgf000102_0001
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 Figures 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. Figures 18(a)-(d) are shown at lOOx magnification; figures 19(a)-(d) are shown at 400x 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 Figure 20.
As shown in Figures 18(a)-(d) and 19(a)-(d), the vehicle-treated (control) grafts were marginally thinner than thepregraft 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 Figure 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 G,, 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 Figure 21 as immunohistochemical sections at lOOx magnification. The top panel of Figure 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 Figure 21 also exhibits evidence of proliferation, including parakeratosis and Ki67-positive cells appearing as brown-staining nuclei. The center panel of Figure 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 foruptake 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. Figure 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 (10μM R451) complexed with cytofectin GSV (lOμg/ml). Figure 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, Del 5 mer phosphorothioate oligodeoxyribonucleotides. Figure 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 (30nM or lOOnM) were complexed with GSV cytofectin (2μg/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 (15μg) 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.
Figures 25(a)-(d) provide graphs which depict results in this screening process. In these graphs, Rl 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 Figure 26. As demonstrated, at a concentration of 30nM, 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 lOOnM, 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). Figure 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. Figure 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. Figure 28(a) shows duplicate treated cellular extracts following the IGF-I receptor at the predicted size of 110 kD. Figure 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(l-3)IGF-I were performed. Results are shown in Figure 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 Figure 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 Figure 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 Figure 31. In Figure 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 Figure 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 Figure 32 shows the reversal of epidermal hyperplasia correlating with reduced IGF-I receptor mRNA in grafted psoriasis lesions treated with antisense oligonucleotides. Figure 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). Figure 32(b) shows the same lesion prior to grafting and after oligonucleotide treatment as in Figure 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 Figure 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
Figure 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 aprotimn 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, California) for 1 hour at room temperature and developed using the Vistra ECF western blotting kit (Amersham). The results shown in Figure 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 Figure 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 referced to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. REFERENCES:
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Claims

CLAIMS:
1. 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 growth factor mediated cell proliferation and/or inflammation and/or other medical disorders.
2. A method according to claim 1 wherein cell proliferation and/or inflammation or other medical disorder is mediated by at least one of insulin-like growth factor I (IGF-I), keratinocyte growth factor (KGF), transforming growth factor-α (TGFα), tumour necrosis factor-α (TNFα), interleukin (IL) -1 (IL-1), IL-4, IL-6, IL-8 and/or basic fibroblast growth factor (bFGF).
3. A method according to claim 2 wherein cell proliferation and/or inflammation or other medical disorder is mediated by IGF-I.
4. A method according to claim 1 wherein the nucleic acid molecule inhibits or otherwise reduces IGF-I mediated cell proliferation and/or inflammation or other medical disorder.
5. A method according to claim 1 wherein the proliferative or inflammatory skin disorder is psoriasis, ichthyosis, pityriasis, rubra, pilaris, serbonhoea, keloids, keratosis, neoplasias, scleroderma, warts, benign growths or cancers of the skin.
6. A method according to claim 5 wherein the skin condition is psoriasis.
7. A method according to claim 1 wherein the other medical disorder is a hyperneovascular condition such as a neovascular condition of the retina, brain or skin, growth factor-mediated malignancies, other sclerotic disease, kidney disease or hyperproliferation of the inside of blood vessels or any other hyperplasia.
8. A method according to claim 1 or 4 or 6 or 7 wherein the mammal is a human.
9. A method according to claim 1 or 4 or 6 or 7 wherein the nucleic acid molecule is capable of inhibiting, reducing or otherwise interfering with IGF-I-interaction with its receptor.
10. A method according to claim 9 wherein the nucleic acid molecule is an antisense molecule capable of reducing expression of a gene encoding IGF-I, IGF-I-receptor or an IGF binding protein (IGFBP).
11. A method according to claim 10 wherein the nucleic acid molecule is an antisense molecule capable of reducing expression of a gene encoding IGFBP-2, -3, -4, -5 or - 6.
12. A method according to claim 11 wherein the nucleic acid molecule is an antisense molecule capable of reducing expression of a gene encoding IGFBP-2 or IGFBP-3.
13. A method according to claim 10 or 12 wherein the antisense molecule is at least about 15 nucleotides in length and is capable of interacting with at least one sequence selected from the list set forth in Example 6 or Example 7 or Example 8.
14. A method according to claim 12 wherein the antisense molecule comprises the nucleotide sequence:
5'-ATCTCTCCGCTTCCTTTC-3' (<400>10)
15. A method according to claim 12 wherein the antisense molecule is selected from the following:
UCCGGAGCCAGACUU (<400>12) CACAGUUGCUGCAAG (<400>13) UCUCCGCUUCCUUUC (<400>14) AGCCCCCACAGCGAG (<400>15) GCCUUGGAGAUGAGC (<400>16) UAACAGAGGUCAGCA (<400>17) GGAUCAGGGACCAGU (<400>18) CGGCAAGCUACACAG (<400>19) GGCAGGCAGGCACAC (<400>20)
16. A method according to claim 15 wherein the antisense molecule in <400>12, <400>13 or <400>14.
17. A method according to claim 15 wherein the antisense molecule in <400>12.
18. A nucleic acid molecule comprising at least about 10 nucleotides capable of hybridising to or forming a heteroduplex or otherwise interacting with a complementary form of <400>12 to <400>20 inclusive.
19. A nucleic acid molecule comprising at least about 15 nucleotides capable of hybridising to or form a heteroduplex or otherwise interacting with a complementary form of <400>12 to <400>14 inclusive.
20. A method of ameliorating the effects of psoriasis or other medical disorder, said method comprising contacting proliferating skin or skin capable of proliferation or cell otherwise 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 other 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.
21. A method according to claim 20 wherein the IGFBP is IGFBP-2 or IGFBP-3.
22. A method according to claim 20 or 21 wherein the mammal is a human.
23. A method according to claim 22 wherein the nucleic acid molecule is capable of interacting with a nucleotide sequence selected from the list set forth in <400>12 to <400>14 inclusive.
24. A method according to claim 23 wherein the nucleic acid molecule comprises the nucleotide sequence selected from <400>12 to <400>14.
25. A composition comprising a nucleic acid molecule capable of inhibiting or otherwise reducing IGF-I mediated cell proliferation or other medical disorder said composition further comprising one or more pharmaceutically acceptable earners and/or diluents.
26. A composition according to claim 25 wherein the nucleic acid molecule is antisense molecule to a gene encoding IGF-I, IGF-I-receptor or an IGFBP.
27. A composition according to claim 26 wherein the nucleic acid molecule is selected from <400>12 to <400>20 inclusive.
28. A composition according to claim 26 selected from <400>12 to <400>14 inclusive.
29. 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.
30. A method according to claim 29 wherein the proliferative or inflammatory skin disorder is psoriasis, ichthyosis, pityriasis, rubra, pilaris, serbonhoea, keloids, keratosis, neoplasias, scleroderma, warts, benign growths or cancers of the skin.
31. A method according to claim 30 wherein the proliferative or inflammatory skin disorder is psoriasis.
32. A method according to claim 29 or 30 or 31 wherein the nucleic acid molecule is capable of inhibiting, reducing or otherwise interfering with IGF-I-interaction with its receptor.
33. A method according to claim 32 wherein the nucleic acid molecule is an antisense molecule capable of reducing expression of a gene encoding IGF-I, IGF-I-receptor or an IGF binding protein (IGFBP).
34. A method according to claim 33 wherein the nucleic acid molecule is an antisense molecule capable of reducing expression of a gene encoding IGFBP-2, -3, -4, -5 or - 6.
35. A method according to claim 34 wherein the nucleic acid molecule is an antisense molecule capable of reducing expression of a gene encoding IGFBP-2 or IGFBP-3.
36. A method according to claim 33 wherein the nucleic acid molecule is an antisense molecule capable of reducing expression of a gene encoding IGF-I receptor.
37. A method according to any one of claims 29 to 36 wherein the antisense molecule is at least about 15 nucleotides in length and is capable of interacting with at least one sequence selected from the list set forth in Example 6 or Example 7 or Example 8.
38. A method according to claim 37 wherein the antisense molecule comprises the nucleotide sequence:
5'-ATCTCTCCGCTTCCTTTC-3' (<400>10)
39. A method according to claim 37 wherein the antisense molecule is selected from the following:
UCCGGAGCCAGACUU (<400>12) CACAGUUGCUGCAAG (<400>13) UCUCCGCUUCCUUUC (<400>14) AGCCCCCACAGCGAG (<400>15) GCCUUGGAGAUGAGC (<400>16) UAACAGAGGUCAGCA (<400>17) GGAUCAGGGACCAGU (<400>18) CGGCAAGCUACACAG (<400>19) GGCAGGCAGGCACAC (<400>20)
40. A method according to claim 39 wherein the antisense molecule in <400>12, <400>13 or <400>14.
41. A method according to claim 40 wherein the antisense molecule in <400>12.
42. A method according to claim 39 wherein the UV treatment occurs simultaneously with or following contact with the nucleic acid molecule or its chemical analogue.
43. Use of an antisense molecule directed to the gene encoding IGF-I receptor or its mRNA as adjunct therapy in combination with UV treatment to reduce proliferation and/or inflammation of keratinocyte cells.
44. Use according to claim 43 in the treatment of psoriasis.
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