Methods of selecting therapeutic molecules

Abstract

The present invention relates to methods of selecting therapeutic molecules. The present disclosure provides methods of identifying molecules that are safe for dosing using a calcium fluctuation assay and / or a sequence score calculation. The present disclosure also includes methods of selecting or identifying molecules with tolerable in vivo neurotoxicity using a calcium fluctuation assay, a sequence score method, an in vivo tolerance assay, or any combination thereof.

Description

[0001] This application is a divisional application of Chinese patent application 201680019958.2, "Method for Selecting Therapeutic Molecules," filed on February 4, 2016.

[0002] Citations of early applications

[0003] This application is a PCT application claiming the benefits of U.S. Provisional Application No. 62 / 112,058, filed February 4, 2015; U.S. Provisional Application No. 62 / 156,684, filed May 4, 2015; and U.S. Provisional Application No. 62 / 279,610, filed January 15, 2016, all of which are incorporated herein by reference in their entirety.

[0004] References to sequence lists submitted electronically via EFS-WEB

[0005] The contents of the electronic submission sequence list (name: 3338.035PC03 SL.txt, size: 339,610 bytes; creation date: February 4, 2016) submitted in this application are incorporated herein by reference in their entirety. Technical Field

[0006] This invention relates to a method for selecting therapeutic molecules with reduced toxicity. The method can be used in vitro or on a computer to screen molecules before administration to laboratory animals, or the method can be used in vivo on laboratory animals. Background Technology

[0007] In the field of targeted therapy, some therapeutic molecules can cause toxic side effects, for example, through non-specific interactions with proteins, stimulation of unwanted immune responses, or accumulation in tissues. A major concern when administering therapeutic molecules to subjects is their potential neurotoxicity. Exposure to neurotoxic molecules can lead to damage to the brain and peripheral nervous system, causing long-term physiological problems. Therefore, it is important that therapeutic molecules are not only effective in treating the desired disease or condition, but also have acceptable toxicity (e.g., neurotoxicity) when administered.

[0008] The identification of the most effective therapeutic molecules typically involves synthesizing a large number of molecules designed to target a specific factor in cells and testing that large number of molecules for activity and toxicity. While animal studies can be conducted to determine toxicity, these studies are ethically and economically undesirable due to the potential for large-scale animal deaths caused by the toxicity of the tested molecules. Therefore, there is a need for improved methods to determine the toxicity (e.g., neurotoxicity) of therapeutic molecules without requiring animal testing. Summary of the Invention

[0009] This disclosure provides a method for testing or determining the acute neurotoxicity of a molecule in vivo, the method comprising measuring, in vitro, fluctuations in intracellular free calcium concentration (“calcium fluctuations”) in neuronal cells in contact with the molecule.

[0010] This disclosure also provides a method for selecting molecules with tolerable acute neurotoxicity in vivo, the method comprising measuring calcium fluctuations in in vitro neuronal cells in contact with the molecule, wherein the molecule exhibits calcium fluctuation levels comparable to controls.

[0011] The methods described above in this disclosure relating to the measurement of calcium fluctuations may further include calculating a sequence score of a molecule (e.g., a polynucleotide containing a nucleotide sequence), wherein the sequence score is calculated by the following formula (I):

[0012] (Number of C nucleotides or their analogues in the nucleotide sequence - Number of G nucleotides or their analogues in the nucleotide sequence) (Number of nucleotides) / Total nucleotide length of the nucleotide sequence (I).

[0013] The present invention also provides a method for determining the acute neurotoxicity in vivo of a molecule containing a nucleotide sequence, the method comprising calculating a sequence score, wherein the sequence score is calculated by the following formula (I):

[0014] (Number of C nucleotides or their analogues in the nucleotide sequence - Number of G nucleotides or their analogues in the nucleotide sequence) (Number of nucleotides) / Total nucleotide length of the nucleotide sequence (I).

[0015] The present invention also provides a method for selecting molecules comprising nucleotide sequences having tolerable acute neurotoxicity in vivo, the method comprising calculating a sequence score using the following formula (I):

[0016] (Number of C nucleotides or their analogues in the nucleotide sequence - Number of G nucleotides or their analogues in the nucleotide sequence) (Number of nucleotides) / Total nucleotide length of the nucleotide sequence (I),

[0017] The nucleotide sequence has a sequence score of 0.2 or higher.

[0018] In other embodiments, this disclosure provides a method for selecting molecules with tolerable acute neurotoxicity in vivo, the method comprising measuring in vivo tolerance. The invention provides a method wherein in vivo tolerance is categorized into one of five tolerance categories. The invention also provides tolerance categories that may be: 1) hyperactivity; 2) decreased activity and arousal; 3) motor dysfunction and / or ataxia; 4) postural and respiratory abnormalities; and 5) tremor and / or seizures. In one embodiment, in vivo tolerance can be measured by injecting the molecule into the mammalian brain and grading the mammalian tolerance in the tolerance categories on a scale of 0-20.

[0019] The method described above in this invention may further include measuring the intensity of microtubules, the expression of target proteins, or the behavioral performance of molecules in neuronal cell cultures.

[0020] The present invention also provides a method for administering molecules tested according to the above method to subjects who require treatment for a disease or condition.

[0021] In some embodiments, the molecule includes proteins, peptides, small molecules, polynucleotides (e.g., antisense oligonucleotides), or any combination thereof.

[0022] Implementation Plan

[0023] E1. A method for testing or determining the acute neurotoxicity of a molecule in vivo, the method comprising measuring, in vitro, calcium fluctuations in neuronal cells in contact with the molecule.

[0024] E2. The method of embodiment 1, wherein the calcium fluctuation of the molecule is compared with the calcium fluctuation of neuronal cells (“control cells”) that are not exposed to the molecule.

[0025] E3. The method of implementation scheme 2, wherein the control cells are solvent control cells.

[0026] E4. The method of embodiment 3, wherein the calcium fluctuation in the neuronal cells in contact with the molecule is about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 99% or higher, about 100% or higher, about 120% or higher, about 140% or higher, about 160% or higher, about 180% or higher, about 200% or higher, about 220% or higher, about 240% or higher, or about 250% or higher, compared to the calcium fluctuation in the solvent control cells.

[0027] E5. A method for selecting or identifying molecules with tolerable acute neurotoxicity in vivo, the method comprising measuring calcium fluctuations in neuronal cells in contact with the molecule in vitro, wherein the neuronal cells in contact with the molecule show calcium fluctuations at levels equivalent to or higher than those of solvent control cells.

[0028] E6. The method of embodiment 5, wherein the calcium fluctuation in the neuronal cells in contact with the molecules is approximately 70% or higher, approximately 75% or higher, approximately 80% or higher, approximately 85% or higher, approximately 90% or higher, approximately 95% or higher, approximately 96% or higher, approximately 97% or higher, approximately 98% or higher, approximately 99% or higher, approximately 100% or higher, approximately 120% or higher, approximately 140% or higher, approximately 160% or higher, approximately 180% or higher, approximately 200% or higher, approximately 220% or higher, approximately 240% or higher, or approximately 250% or higher, compared to the calcium fluctuation in the solvent control cells.

[0029] E7. The method of any one of Implementation Schemes 1-6, wherein the neuronal cells are prepared from primary cortical neurons of mammals.

[0030] E8. The method of any one of the embodiments, wherein the molecule includes small molecules, polynucleotides, proteins, peptides or any combination thereof.

[0031] E9. The method of embodiment 8, wherein the protein comprises an antibody or an antigen-binding fragment thereof, a fusion protein, a cytokine, a cell surface receptor, a hormone, a growth factor, or any combination thereof.

[0032] E10. The method of any one of implementation schemes 1-9, wherein the calcium fluctuation is AMPA receptor-dependent calcium fluctuation.

[0033] E11. The method of any one of embodiments 1-10, wherein calcium fluctuations in Mg 2+ Measurement in the presence of ions.

[0034] E12. The method of implementation scheme 11, wherein Mg 2+ The concentration of the ions is at least about 0.5 mM, at least about 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at least about 0.9 mM, at least about 1 mM, at least about 1.5 mM, at least about 2.0 mM, at least about 2.5 mM, at least about 3.0 mM, at least about 4 mM, at least about 5 mM, or at least about 10 mM.

[0035] E13. A method according to one of embodiments 1-12, wherein calcium fluctuations are determined by measuring the fluorescence of a calcium dye.

[0036] E14. A method according to one of embodiments 1-13, the method further comprising administering the molecule to a subject who requires treatment for a disease or condition.

[0037] E15. The method of implementation scheme 14, wherein the disease or condition is selected from viral infection, neurological disorders (e.g., Alzheimer's disease, progressive supranuclear palsy, Down syndrome, boxer's dementia (chronic traumatic encephalopathy and other traumatic brain injury), frontotemporal dementia linked to chromosome 17 with Parkinson's syndrome (FTDP-17), Lytico-Bodig disease (Guam-type Parkinson's syndrome-dementia complex), tangle-predominant dementia, ganglioglioma, gangliocytoma, meningioma, subacute sclerosing panencephalitis, lead encephalopathy, hemimegalencephaly, tuberous sclerosis, Hallervorden-Spatz disease, Pick's disease). Diseases including: corticobasal ganglia degeneration, argyrophilic granulation disease, corticobasal ganglia degeneration, lipofuscinosis, frontotemporal dementia, supranuclear palsy and frontotemporal lobe degeneration, brain network dysfunction disorders (e.g., all forms of epilepsy and depression), spinal cord disorders, peripheral neuropathy, cranial nerve disorders (e.g., trigeminal neuralgia), autonomic nervous system disorders (e.g., familial autonomic dysfunction or multiple system atrophy), central and peripheral nervous system motor disorders (e.g., Parkinson's disease, essential tremor, amyotrophic lateral sclerosis, Tourette's syndrome, multiple sclerosis or different types of peripheral neuropathy), sleep disorders (e.g., narcolepsy), migraines or other types of headaches (e.g., cluster headaches and tension headaches), lower back and neck pain, central nervous system disorders, neuropsychiatric disorders, attention deficit hyperactivity disorder, autism, Huntington's disease, Rett syndrome, Angelman syndrome. Syndrome, organic mental illness, brain or bone marrow infection (including meningitis) or prion disease, anemia, cancer, leukemia, inflammatory conditions or autoimmune diseases (e.g., arthritis, psoriasis, lupus, multiple sclerosis), bacterial infection, frontotemporal dementia-τ (FTD-τ), frontotemporal dementia linked to chromosome 17 and Parkinson's syndrome (FTDP-17), corticobasal degeneration (CBD), traumatic brain injury, chronic traumatic encephalopathy, HIV-related neurocognitive impairment, auricolic granuloma, Down syndrome-Alzheimer's dementia, amnesic mild cognitive impairment-Alzheimer's dementia, Parkinson's dementia, Hallewarden-Scpatz disease (pantothenic acid kinase-related neurodegeneration), Niemann-Pick disease type C.C) Myotonic dystrophy, amyotrophic lateral sclerosis, hemimegaencephalopathy, tuberous sclerosis relapse, type 2b focal cortical dysplasia, ganglion cell tumor, Dravet syndrome (infantile severe clonic epilepsy), temporal lobe epilepsy, Ohtahara syndrome (early infantile epileptic encephalopathy with repressive outbursts), Lafora body disease, generalized epilepsy with febrile seizures, infantile spasms (West syndrome), Lennox Gastaut syndrome, Angelman syndrome, Rett syndrome, Landau-Kleffner syndrome, focal epilepsy, simple focal epilepsy (without loss of consciousness), focal cognitive impairment epilepsy (disorder of consciousness), focal epilepsy that develops into generalized tonic-clonic (GTC) seizures, generalized seizures (convulsive or nonconvulsive, with bilateral discharges involving subcortical structures), absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures and tonic seizures, autism, autism spectrum disorder, Asperger's disorder, pervasive developmental disorders and any combination thereof.

[0038] E16. The method of any one of embodiments 1-15, wherein the molecule comprises a polynucleotide.

[0039] E17. The method of embodiment 16, the method further comprising calculating a sequence score, wherein the sequence score is calculated by the following formula (I):

[0040] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total nucleotide length (number) of polynucleotides. (I).

[0041] E18. A method for determining acute neurotoxicity in vivo of a molecule containing a polynucleotide, the method comprising calculating a sequence score, wherein the sequence score is calculated by the following formula (I):

[0042] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total nucleotide length (number) of polynucleotides. (I).

[0043] E19. A method for selecting molecules comprising polynucleotides with tolerable acute neurotoxicity in vivo, said method comprising calculating a sequence score using the following formula (I):

[0044] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total nucleotide length (number) of polynucleotides. (I),

[0045] The polynucleotides have a sequence score of 0.2 or higher.

[0046] E20. The method of any one of Implementation Schemes 17-19, wherein the sequence score is greater than or equal to 0.2, greater than or equal to 0.25, greater than or equal to 0.3, greater than or equal to 0.35, greater than or equal to 0.4, greater than or equal to 0.45, greater than or equal to 0.5, greater than or equal to 0.55, greater than or equal to 0.6, greater than or equal to 0.65, greater than or equal to 0.7, greater than or equal to 0.75, greater than or equal to 0.8, greater than or equal to 0.85, greater than or equal to 0.9, greater than or equal to 0.95, greater than or equal to 1.0, greater than or equal to 1.5, greater than or equal to 2.0, greater than or equal to 3.0, or greater than or equal to 4.0.

[0047] E21. The method of any one of embodiments 16-20, wherein the polynucleotide includes DNA or RNA.

[0048] E22. The method of any one of Implementation Schemes 16-21, wherein the polynucleotide is single-stranded.

[0049] E23. The method of any one of embodiments 16-22, wherein the polynucleotide is an antisense oligonucleotide (i.e., an oligomer) of about 10 to about 50 nucleotides in length.

[0050] E24. The method of implementation scheme 23, wherein antisense oligonucleotides regulate the expression of target proteins.

[0051] E25. The method of implementation scheme 23, wherein the antisense oligonucleotide targets the mRNA of the target protein.

[0052] E26. The method of implementation scheme 25, wherein the mRNA is pre-mRNA or mature mRNA.

[0053] E27. The method of any one of embodiments 25-26, wherein the mRNA is expressed in the cell.

[0054] E28. The method of implementation scheme 27, wherein mRNA is expressed in neuronal cells.

[0055] E29. The method of any one of embodiments 23-28, wherein antisense oligonucleotides regulate the mRNA expression of target genes in neuronal cell cultures.

[0056] E30. The method of any one of embodiments 23-29, wherein antisense oligonucleotides regulate the expression of proteins encoded by target proteins in neuronal cell cultures.

[0057] E31. The method of any one of embodiments 23-30, wherein the antisense oligonucleotide is complementary to the mRNA or pre-mRNA of the target gene.

[0058] E32. The method of any one of embodiments 1-31, wherein the method further measures the reduction in in vitro expression of the target protein of the molecule.

[0059] E33. The method of any one of embodiments 1-32, the method further comprising measuring the in vivo tolerance of the molecule.

[0060] E34. The method of embodiment 33, wherein in vivo tolerance is measured by administering the molecule to a mammal and classifying the mammal’s tolerance by tolerance category.

[0061] E35. The method of embodiment 33, wherein the molecule is administered to the brain of a mammal.

[0062] E36. A method for testing or determining the in vivo tolerance of a molecule, the method comprising administering the molecule to a mammal and classifying the mammal’s tolerance by tolerance category.

[0063] E37. The method of any one of embodiments 34-36, wherein the tolerance categories include at least one, at least two, at least three, at least four, or at least five tolerance categories.

[0064] E38. The method of implementation scheme 37, wherein the tolerance category is selected from: 1) hyperactivity; 2) decreased activity and arousal; 3) motor dysfunction and / or ataxia; 4) postural and respiratory abnormalities; 5) tremor and / or seizures and combinations of two or more thereof.

[0065] E39. The method of embodiment 38, wherein the molecule displays an in vivo tolerance score of 0-4 in each tolerance category.

[0066] E40. The method of embodiment 39, wherein the molecules display an in vivo tolerability score with a total value between 0 and 8.

[0067] E41. The method of embodiment 39 or 40, wherein the molecule displays an in vivo tolerability score with a total value between 0 and 6, between 0 and 5, between 0 and 4, between 0 and 3, between 0 and 2, or between 0 and 1.

[0068] E42. A method of any one of embodiments 1-41, said method further comprising measuring a behavioral test score of said molecule.

[0069] E43. The method of implementation scheme 42, wherein behavioral test scores are measured by giving the molecule to a mammal and grading the mammal’s behavioral performance.

[0070] E44. The method of any one of implementation schemes 41-43, wherein the behavioral test is a short-term memory test, a spatial learning and memory test, a gait analysis test, or any combination thereof.

[0071] E45. The method of any one of embodiments 1-44, the method further comprising measuring the microtubule strength of the molecule in a neuronal cell culture.

[0072] E46. The method of embodiment 45, wherein the microtubule strength of the molecule is compared with the microtubule strength of neurons not exposed to the molecule.

[0073] E47. The method of embodiment 46, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%.

[0074] E48. A method of any one of embodiments 1-47, the method further comprising administering the molecule to a subject who requires treatment for a disease or condition.

[0075] E49. The method of implementation scheme 48, wherein the disease or condition is selected from viral infection, neurological disorders (e.g., Alzheimer's dementia, progressive supranuclear palsy, Down syndrome, boxer's dementia (chronic traumatic encephalopathy and other traumatic brain injury), frontotemporal dementia linked to chromosome 17 with Parkinson's syndrome (FTDP-17), Huntington's disease, Rett syndrome, Angelman syndrome, Lytico-Bodig disease (Guam Parkinson's syndrome-dementia complex), tangles-dominant dementia, ganglioglioma, gangliocytoma, meningioma, subacute sclerosing panencephalitis, lead poisoning encephalopathy, hemimegaencephaly, tuberous sclerosis, Hallewarden-Schpattz disease, Pick's disease, corticobasal ganglia degeneration, argyrophilic granuloma, corticobasal ganglia degeneration, lipofuscinosis, frontotemporal dementia, supranuclear palsy and frontotemporal lobe degeneration, and brain network dysfunction disorders (e.g., sore throat). The following conditions are considered: epilepsy and depression; spinal cord disorders; peripheral neuropathy; cranial nerve disorders (e.g., trigeminal neuralgia); autonomic nervous system disorders (e.g., familial autonomic dysfunction or multiple system atrophy); central and peripheral nervous system motor disorders (e.g., Parkinson's disease, essential tremor, amyotrophic lateral sclerosis, Tourette syndrome, multiple sclerosis, or different types of peripheral neuropathy); sleep disorders (e.g., narcolepsy); migraines or other types of headaches (e.g., cluster headaches and tension headaches); lower back and neck pain; central nervous system disorders; neuropsychiatric disorders; attention deficit hyperactivity disorder; autism; Huntington's disease; organic psychosis; brain or bone marrow infections (including meningitis) or prions; anemia; cancer; leukemia; inflammatory conditions or autoimmune diseases (e.g., arthritis, psoriasis, lupus, multiple sclerosis); bacterial infections; frontotemporal dementia. (FTD-τ), frontotemporal dementia and Parkinson's syndrome linked to chromosome 17 (FTDP-17), corticobasal degeneration (CBD), traumatic brain injury, chronic traumatic encephalopathy, HIV-related neurocognitive impairment, auricolic granuloma, Down syndrome-Alzheimer's dementia, amnesic mild cognitive impairment-Alzheimer's dementia, Parkinson's dementia, Hallewarden-Scholes disease (pantothenic acid kinase-related neurodegeneration), Niemann-Pick disease type C, myotonic dystrophy, amyotrophic lateral sclerosis, hemimegaencephaly, tuberous sclerosis relapse, focal cortical dysplasia type 2b, ganglion cell tumor, Dravet syndrome (infantile severe clonic epilepsy), temporal lobe epilepsy, Ohtahara syndrome (early infantile epileptic encephalopathy with repressed bursts), Lafora body disease, generalized epilepsy with febrile seizures, infantile spasms (Wester syndrome), Lennox-Gastaut syndrome, Angelman syndrome, Rett syndrome, LandauKleffner syndrome, focal epilepsy, simple focal epilepsy (without loss of consciousness), focal cognitive impairment epilepsy (disorder of consciousness), focal epilepsy that develops into generalized tonic-clonic (GTC) seizures, generalized seizures (convulsive or nonconvulsive, with bilateral discharges involving subcortical structures), absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures and tonic seizures, autism, autism spectrum disorders, Asperger's disease, pervasive developmental disorders and any combination thereof.

[0076] E50. The method of implementation scheme 49, wherein the molecular treatment or prevention of disease or condition is described.

[0077] E51. The method of any one of embodiments 16-50, wherein the polynucleotide is an antisense oligonucleotide.

[0078] E52. A method according to any one of embodiments 16-51, wherein the polynucleotide comprises at least one nucleotide analog.

[0079] E53. A method according to any one of embodiments 16-52, wherein the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotide analogs.

[0080] E54. The method of embodiment 52 or 53, wherein one or more nucleotide analogs are locked nucleic acids (LNA), 2'-O-alkyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleotide (ANA), 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalated nucleic acid (INA), restricted ethyl nucleoside (cEt), 2'-O-methyl nucleic acid (2'-OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), or any combination thereof.

[0081] E55. The method of any one of embodiments 23-54, wherein the antisense oligonucleotide comprises a nucleoside bond selected from the following: phosphodiester bond, phosphotriester bond, methylphosphonate bond, aminophosphate bond, thiophosphate bond, and combinations thereof.

[0082] E56. The method of any one of Implementation Schemes 23-55, wherein the antisense oligonucleotide is a gapmer, blockmer, mixer, or wingmer.

[0083] E57. The method of any one of embodiments 1-56, wherein when the molecule is administered to laboratory animals, more than 20% of the animals survive.

[0084] E58. A method according to any one of embodiments 1-57, wherein when said molecule is given to laboratory animals, more than 50% of the animals survive.

[0085] E59. A method according to any one of embodiments 1-58, wherein when the molecule is administered to laboratory animals, more than 85% of the animals survive.

[0086] E60. A molecule selected from any one of the methods described in embodiments 1-59.

[0087] E61. A method for treating a disease or condition, said method comprising administering the molecule of embodiment 60.

[0088] E62. The method of implementation scheme 61, wherein the disease or condition is related to neuronal cells.

[0089] E63. A method for testing or determining the acute neurotoxicity of a molecule in vivo, the method comprising the steps of: 1) adding the molecule to a neuronal cell culture, and 2) measuring calcium fluctuations in the neuronal cells in vitro.

[0090] E64. The method of embodiment 63, the method further comprising comparing calcium fluctuations in neuronal cells with calcium fluctuations in neuronal cells not exposed to the molecule (“control cells”).

[0091] E65. The method of implementation scheme 63 or 64, wherein the neuronal cells are prepared from mammalian cortical neurons.

[0092] E66. The method of any one of implementation schemes 63-65, wherein calcium fluctuations are AMPA receptor-dependent.

[0093] E67. The method of any one of embodiments 63-66, wherein calcium fluctuations in Mg 2+ Measurement in the presence of ions.

[0094] E68. The method of implementation scheme 67, wherein Mg 2+ The concentration of the ions is at least about 0.5 mM, at least about 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at least about 0.9 mM, at least about 1 mM, at least about 1.5 mM, at least about 2.0 mM, at least about 2.5 mM, at least about 3.0 mM, at least about 4 mM, at least about 5 mM, or at least about 10 mM.

[0095] E69. The method of any one of embodiments 63-68, wherein the molecule comprises a small molecule, a polynucleotide, a protein, a peptide, or any combination thereof.

[0096] E70. The method of embodiment 69, the method further comprising the step of calculating a sequence score, wherein the sequence score is calculated by the following formula (I):

[0097] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) / Total number of nucleotides in a polynucleotide (I).

[0098] E71. A method for determining acute neurotoxicity in vivo of a molecule containing a nucleotide sequence, the method comprising calculating a sequence score, wherein the sequence score is calculated by the following formula (I):

[0099] (Number of C nucleotides or their analogues in the nucleotide sequence - Number of G nucleotides or their analogues in the nucleotide sequence) (Number of nucleotides) / Total number of nucleotides in polynucleotides (I),

[0100] The nucleotide sequence has a sequence score of 0.2 or higher.

[0101] E72. The method of any one of embodiments 63-71, the method further comprising measuring the in vivo tolerance of the molecule.

[0102] E73. The method of any one of embodiments 63-72, the method further comprising measuring the microtubule strength in a neuronal cell culture.

[0103] E74. The method of any one of embodiments 63-73, wherein the molecule is an antisense oligonucleotide.

[0104] E75. The method of any one of embodiments 63-74, wherein when said molecule is given to laboratory animals, more than 50% of the animals survive.

[0105] E76. The method of any one of embodiments 63-74, wherein when said molecule is given to laboratory animals, more than 85% of the animals survive.

[0106] E77. A molecule for treating a disease or condition, wherein the molecule is determined or identified as having tolerable acute neurotoxicity in vivo by means of the methods of embodiments 63-76.

[0107] E78. The method of implementation scheme 77, wherein the disease or condition is related to neuronal cells.

[0108] E79. An antisense oligonucleotide for treating a neurological disease or condition in a subject requiring treatment, wherein the calcium fluctuation of neurons contacted with the antisense oligonucleotide is about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 99% or higher, about 100% or higher, about 120% or higher, about 140% or higher, about 160% or higher, about 180% or higher, about 200% or higher, about 220% or higher, about 240% or higher, or about 250% or higher.

[0109] E80. An antisense oligonucleotide for treating neurological diseases or conditions, having a sequence score greater than or equal to 0.2, greater than or equal to 0.25, greater than or equal to 0.3, greater than or equal to 0.35, greater than or equal to 0.4, greater than or equal to 0.45, greater than or equal to 0.5, greater than or equal to 0.55, greater than or equal to 0.6, greater than or equal to 0.65, greater than or equal to 0.7, greater than or equal to 0.75, greater than or equal to 0.8, greater than or equal to 0.85, greater than or equal to 0.9, greater than or equal to 0.95, greater than or equal to 1.0, greater than or equal to 1.5, greater than or equal to 2.0, greater than or equal to 3.0, or greater than or equal to 4.0.

[0110] E81. In methods for testing or determining the in vivo neurotoxicity of a molecule, improvements include in vitro measurement of calcium fluctuations in neuronal cells in contact with the molecule.

[0111] E82. In a method for selecting or identifying molecules with tolerable in vivo neurotoxicity, an improvement includes measuring calcium fluctuations in neuronal cells in contact with the molecule in vitro, wherein the neuronal cells in contact with the molecule show calcium fluctuations at levels equivalent to or higher than those of solvent control cells.

[0112] E83. In the method of embodiment 81 or 82, the improvement further includes calculating a sequence score, wherein the molecule comprises a polynucleotide and wherein the sequence score of the polynucleotide is calculated by the following formula (I):

[0113] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total number of nucleotides in polynucleotides (e.g., eye) (I).

[0114] E84. An improvement to the method for determining acute neurotoxicity in vivo of molecules containing polynucleotides includes calculating a sequence score of the polynucleotide, wherein the sequence score is calculated by the following formula (I):

[0115] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total number of nucleotides in polynucleotides (e.g., eye) (I).

[0116] E85. In methods for selecting molecules containing polynucleotides with tolerable acute neurotoxicity in vivo, improvements include calculating sequence scores using the following formula (I):

[0117] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total number of nucleotides in polynucleotides (e.g., eye) (I),

[0118] The polynucleotides have a sequence score of 0.2 or higher.

[0119] E86. In the method of embodiments 81-85, the improvement further includes measuring the reduction in in vitro expression of the target protein of the molecule.

[0120] E87. In any of the methods in embodiments 81-85, the improvement further includes measuring the in vivo tolerance of the molecule.

[0121] E88. In the method of embodiment 87, in vivo tolerance is measured by administering the molecule to a mammal and classifying the mammal’s tolerance by tolerance category. Attached Figure Description

[0122] Figure 1 : A graph showing spontaneous calcium fluctuations in primary neurons. Spontaneous calcium fluctuations in primary neurons were measured as previously described (Murphy et al., 1992, J. Neurosci. 12:4834-4845). The addition of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 3 µM), reduced calcium fluctuations in the total AMPA response by up to 20% during the assay (indicated by the AMPA-labeled lines). Calcium fluctuations were further reduced by approximately 80% when N-methyl-D-aspartate (NMDA) receptor function was blocked by 1 mM MgCl2 (indicated by the NMDA-labeled lines).

[0123] Figure 2 : A graph showing the inhibition of AMPA-mediated calcium fluctuations by antisense oligomers as an indicator of impaired neuronal network activity. The inhibition of NMDA- or AMPA-mediated spontaneous calcium fluctuations by antisense oligomers was evaluated in the presence or absence of 1 mM MgCl2 (representing 100% control in each case). The addition of 25 μM antisense oligomer (TGTgatgcaggaGTT) (SEQ ID NO:304) (ASO-00007) inhibited AMPA receptor-mediated fluctuations, but not NMDA receptor-mediated fluctuations. The negative effects of ASO and other similarly performing oligomers on in vivo central nervous system (CNS) network activity and in vitro electrophysiological spontaneous neuronal activity are shown (data not shown).

[0124] Figure 3 : Correlation analysis of sequence scores relative to in vivo tolerability scores. The sequence score of each oligomer was calculated by substituting appropriate numbers into the following formula: ((number of C nucleotides or analogs - number of G nucleotides) / length (number) of nucleotides in the oligomer)). In a single ventricle ( icv Mice were given 100 µg of oligomer or intrathecal (…) it After administering 900 µg of oligomers or up to 1500 µg to rats, in vivo tolerance scores were calculated based on observations. Rodents were observed according to five categories: 1) hyperactivity; 2) decreased activity and arousal; 3) motor dysfunction and / or ataxia; 4) postural and respiratory abnormalities; and 5) tremors and / or seizures. The total in vivo tolerance score was the sum of the five unit scores; each unit score was measured on a scale of 0-4. Therefore, the total in vivo tolerance score could range from 0 to 20. The sequence score calculated using this formula is located on the X-axis, and the in vivo tolerance score is located on the Y-axis.

[0125] Figure 4 This study demonstrates the effect of τ antisense oligonucleotides on spontaneous calcium fluctuations in primary neurons. Figure 4 List the oligomer name, ASO identifier, ASO sequence, SEQ ID number, MAPT Target start and end positions on the pre-mRNA sequence and calcium fluctuation data as a percentage of control (as described in Example 2 below). Figure 4 Examples of oligomers with mismatched bases are provided in “mm”. Specific mismatched base pairs are shown in bold, underlined, italicized, and highlighted.

[0126] Figure 5 : Shows the in vivo tolerance of exemplary antisense oligonucleotides. Figure 5 List the ASO identifier, ASO sequence, SEQ ID number, MAPT The target start and end positions on the pre-mRNA sequence, the acute tolerance score in vivo (as described in Example 6 below), and the brain residue after administration. MAPT The percentage of mRNA (as described in Example 6 below).

[0127] Figure 6 : This shows a reduction in τ protein due to exemplary antisense oligonucleotides. Figure 6 List the SEQ ID number, oligomer name, ASO identifier, and ASO sequence. MAPT The target start and end positions on the pre-mRNA sequence, the target start point on the mature mRNA sequence, and the normalized Tau / Tuj-1 and Tuj-1 immunocytochemical values ​​(as described in Example 7 below). Figure 6Examples of oligomers with mismatched bases are provided in “mm”. Specific mismatched base pairs are shown in bold, underlined, italicized, and highlighted. Invention Details

[0129] I. Definition

[0130] It should be noted that the term "a" or "an" refers to one or more of the entity; for example, "molecule" should be understood to represent one or more molecules. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this document.

[0131] Furthermore, “and / or” as used herein is to be considered as with or not with each of two specified features or components specifically disclosed herein. Thus, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone) and “B” (alone). Similarly, the term “and / or” as used herein in phrases such as “A, B and / or C” is intended to include any of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0132] It should be understood that wherever the term “comprising” is used to describe aspects, other similar aspects are also provided, expressed as “consisting of” and / or “substantially composed of”.

[0133] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art relating to this disclosure. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0134] Units, prefixes, and symbols are indicated in their internationally recognized form according to the Système International de Unites (SI). Numerical ranges include the numerical values ​​that define the range. Unless otherwise specified, nucleotide sequences are written from left to right in a 5' to 3' direction. Amino acid sequences are written from left to right in an amino to carboxyl direction. The headings provided herein do not limit different aspects of this disclosure and may be used when referring to the specification as a whole. Therefore, terms defined immediately thereafter are defined more fully by reference in their entirety.

[0135] The term “about” as used herein means approximately, roughly, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the listed value. Generally, the term “about” can modify a value to be above or below a specified value by varying, for example, by 10% above or below (higher or lower). For example, about 70% can include 70% - 7% to 70% + 7%, or 63% - 77%.

[0136] The term "therapeutic molecule" refers to any compound that has therapeutic effects in vivo for the treatment of diseases or conditions. Non-limiting examples of therapeutic molecules include naturally occurring, modified, recombinant, or chemically synthesized oligomers, one or more nucleotides, one or more nucleosides, one or more amino acids, polynucleotides, peptides, proteins, polypeptides, or small molecule compounds. Proteins that are therapeutic molecules include, but are not limited to, antibodies or their antigen-binding fragments, fusion proteins, cytokines, cell surface receptors, hormones, growth factors, or any combination thereof.

[0137] In the context of this invention, the term "oligomer" refers to a molecule (i.e., an oligonucleotide) formed by the covalent linkage of two or more nucleotides. An oligomer comprises a continuous nucleotide sequence of about 10 to about 50, for example, 10–20, 16–20, 10–30, 10–35, 10–40, or 10–45 nucleotides in length. The terms "antisense oligomer," "antisense oligonucleotide," and "ASO" as used herein are interchangeable with the term "oligomer." In different embodiments, the oligomers of this invention do not contain RNA (units). In some embodiments, the oligomer comprises one or more DNA units. In one embodiment, the oligomers of this invention are linear molecules or synthesized as linear molecules. In some embodiments, the oligomer is a single-stranded molecule and does not contain, for example, a short region of at least 3, 4, or 5 consecutive nucleotides that is complementary to an equivalent region within the same oligomer (i.e., a double strand)—in this respect, the oligomer is (substantially) not double-stranded. In some embodiments, the oligomer is not substantially double-stranded. In some embodiments, the oligomer is not siRNA. In different embodiments, the oligomers of the present invention consist entirely of continuous nucleotide regions. Therefore, in some embodiments, the oligomers are not substantially self-complementary.

[0138] The terms “nucleic acid,” “nucleotide,” “nucleotide sequence,” or “nucleic acid sequence” are intended to include multiple nucleic acids (e.g., two or more, three or more, etc.). The terms “nucleic acid” or “nucleoside” refer to a single segment of nucleic acid present in a polynucleotide, such as DNA, RNA, or analogs thereof. In some embodiments, the terms “nucleotide,” “unit,” and “monomer” are used interchangeably. It should be understood that when referring to the sequence of a nucleotide or monomer, the sequence of bases (e.g., A, T, G, C, or U) and analogs thereof are referred to. The term “nucleotide sequence” refers to a molecule comprising at least two nucleotides linked together.

[0139] As used herein, the term "nucleotide" refers to a glycoside comprising a sugar moiety, a base moiety, and a covalent linking group (linker) such as a phosphate ester or thiophosphate nucleotide linker, and includes both naturally occurring nucleotides (e.g., DNA or RNA) and non-naturally occurring nucleotides comprising modified sugar and / or base moieties, the latter also referred to herein as "nucleotide analogs." In this document, a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit. In some embodiments, the term "nucleotide analog" refers to a nucleotide having a modified sugar moiety. Other parts of this document disclose non-limiting examples of nucleotides having modified sugar moieties (e.g., LNA), such as 2′-O-methyl, 2′-fluoro(2′-F), 2′-O-methoxyethyl(2′-MOE), and 2′,4′-restricted 2′-O-ethyl(cEt). In other embodiments, the term "nucleotide analog" refers to a nucleotide having a modified base moiety. Nucleotides with modified base moieties include, but are not limited to, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, or 2-chloro-6-aminopurine. In some embodiments, when referring to the sequence scoring formulas disclosed herein, the nucleotide analogue of cytosine is 5-methylcytosine.

[0140] As used herein, the term "polynucleotide" refers to two or more nucleotides linked together in a sequence. Exemplary polynucleotides may include nucleotide sequences having 2, 3, 4, 5, 6, 7, 8, 9, 10, oligonucleotides, 50, 51, or more nucleotides. In some embodiments, polynucleotides include nucleotide sequences longer than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, or 30 nucleotides. In other embodiments, polynucleotides include oligomers (e.g., antisense oligonucleotides). In still other embodiments, polynucleotides include nucleotide sequences encoding proteins or polypeptides. In other embodiments, the polynucleotide includes nucleotide sequences longer than 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, or 5000 nucleotides.

[0141] As used herein, the term "nucleoside" refers to a glycoside comprising a sugar moiety and a base moiety, and therefore can be used when referring to nucleotide units (which are covalently linked by nucleotide bonds between oligomeric nucleotides). In the field of biotechnology, the term "nucleotide" is often used to refer to nucleic acid monomers or units, and therefore can refer to bases in the case of oligonucleotides—for example, "nucleotide sequence" usually refers to a nucleobase sequence (i.e., the presence of a sugar backbone and nucleotide bonds is undisputed). Similarly, particularly in the case of one or more modified oligonucleotides with nucleotide linking groups, the term "nucleotide" can refer to "nucleoside." For example, the term "nucleotide" can be used even when determining the presence or nature of nucleotide bonds.

[0142] As used herein, the term "nucleotide length" refers to the total number of nucleotides (monomers) in a given sequence. For example, the sequence AAAgatgaaatttgctcTTA (SEQ ID NO: 4) has 20 nucleotides; therefore, the nucleotide length of the sequence is 20. The term "nucleotide length" is used interchangeably with "nucleotide number" in this document.

[0143] As used in this article, the term "transcription" can refer to primary transcripts, which are synthesized through DNA transcription and, after processing—namely, pre-mRNA—become messenger RNA (mRNA) and the processed mRNA itself. The term "transcription" is used interchangeably with "pre-mRNA" and "mRNA." After the DNA strand is transcribed into primary transcripts, the newly synthesized primary transcripts are modified in several ways to transform into their mature functional forms, producing different proteins and RNAs, such as mRNA, tRNA, rRNA, lncRNA, miRNA, etc. Therefore, the term "transcription" can include exons, introns, 5' UTRs, and 3' UTRs.

[0144] As used herein, the term "expression" refers to the process by which polynucleotides produce gene products (e.g., RNA or polypeptides). This includes, but is not limited to, the transcription of polynucleotides into messenger RNA (mRNA) and the translation of mRNA into polypeptides. Expression produces "gene products." As used herein, gene products can be nucleic acids (e.g., messenger RNA produced by gene transcription) or polypeptides translated from transcripts. Gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0145] When determining the degree of “complementarity” between the oligomers (or segments thereof) of the present invention and the target segments of nucleic acids encoding mammalian genes (e.g., the segments disclosed herein), the degree of “complementarity” (also “homology” or “identity”) is expressed as the percentage identity (or percentage homology) between the sequence of the oligomer (or its segment) and the best-aligned target segment (or the reverse complementary sequence of the target segment). This percentage is calculated by counting the number of identical aligned bases between the two sequences, dividing by the total number of consecutive monomers in the oligomer, and then multiplying by 100. In such comparisons, if vacancies are present, it is preferable that the vacancies are merely mismatches, rather than regions where the number of monomers within the vacancy differs between the oligomers and the target segments of the present invention.

[0146] The term "complementary sequence" as used in this article refers to a sequence complementary to a reference sequence. Complementarity is a well-known fundamental principle of DNA replication and transcription. Because of this shared property between two DNA or RNA sequences, when they are antiparallel, the nucleotide bases at each position in the sequence will be complementary, much like looking in a mirror and seeing the reverse side of something. Therefore, for example, the complementary sequence of a 5' "ATGC" 3' sequence can be written as 3' "TACG" 5' or 5' "GCAT" 3'. The terms "reverse complementarity," "reverse complementary," and "reverse complementarity" as used in this article are interchangeable with the terms "complementary," "complementary," and "complementarity."

[0147] The term "comparable to" is used in this document to refer to a value that is approximately 30% higher or lower than a reference value to which it is being compared. For example, if a value is approximately 30% lower than a reference value, that value is considered "comparable" to the reference value: for example, 70 is comparable to 100, 80 is comparable to 100, 90 is comparable to 100, 100 is comparable to 100, 110 is comparable to 100, 120 is comparable to 100, and 130 is comparable to 100. The calcium fluctuation level of a molecule comparable to a control calcium fluctuation level means that the calcium fluctuation level of the molecule is ±30%, ±20%, ±10%, or ±5% of the control calcium fluctuation level.

[0148] As used herein, the terms “design” or “oligomeric design” or “ASO sequence” refer to the form of nucleotides (e.g., DNA) and nucleotide analogs (e.g., LNA) in a specified sequence. As used herein, oligomeric designs are indicated by a combination of uppercase and lowercase letters. For example, the oligomeric sequence of tatttccaaattcactttta (SEQ ID NO: 573) can have the following oligomeric designs: ASO-002350 (TAtTTccaaattcactTTTA), ASO-002374 (TAtTTccaaattcacTtTTA), ASO-002386 (TATTtccaaattcaCTttTA), ASO-002227 (TATtTccaaattcactTTTA), ASO-002245 (TAttTCcaaattcactTTTA), ASO-002261 (TATtTccaaattcacTTtTA), ASO-002276... (ATttCcaaattcactTTTA), ASO-002228(TATTtccaaattcaCtTtTA), ASO-002255(TATTtccaaattcactTTTA), ASO-002285(TATTtccaaattcacTTtTA), ASO-002230(TATTtccaaattcacTtTTA), ASO-002256(TATTtccaaattcAcTttTA) or ASO-002279(TATTtccaaattcActTtTA), where uppercase letters indicate nucleotide analogs (e.g., LNA) and lowercase letters indicate nucleotides (e.g., DNA).

[0149] As used herein, the term "chemical structure" of an oligomer refers to a detailed description of the components of an oligomer, such as nucleotides (e.g., DNA), nucleotide analogs (e.g., β-D-oxy-LNA), nucleotide bases (e.g., A, T, G, C, U, or MC), and a backbone structure (e.g., thiophosphates or phosphodiesters). For example, the chemical structure of ASO-002350 could be OxyTsOxyAsDNAtsOxyTsOxyTsOxyTsOxyAs.

[0150] "Efficacy" is typically expressed as IC50 or EC50 values, in nM or pM, unless otherwise specified. IC50 is the median inhibitory concentration of a therapeutic molecule. EC50 is the median effective concentration of a therapeutic molecule relative to a solvent or saline control. In functional assays, IC50 is the concentration that reduces a biological response, such as mRNA transcription or protein expression, to 50% of the level achieved without the therapeutic molecule. In functional assays, EC50 is the concentration of a therapeutic molecule that produces 50% of a biological response, such as mRNA transcription or protein expression. IC50 or EC50 can be calculated using a variety of methods known in the art.

[0151] The term "toxic side effect" refers to an effect that causes weakness in a living subject, including but not limited to death, pain, tremor, seizures, epileptic seizures, motor inhibition, or memory loss. A "toxic" compound can cause toxic side effects when a subject is exposed to it, for example, through injection, ingestion, inhalation, or other routes, and is unsuitable for administration to mammals, such as rodents. In one embodiment, toxic side effects include in vivo neurotoxicity. In another embodiment, toxic side effects are acute in vivo neurotoxicity. A "neurotoxic" compound can alter the normal activity of the nervous system in a way that damages neural tissue, including brain tissue such as neurons and peripheral nerve tissue.

[0152] The term "tolerable" means molecules that are adequately tolerated by living subjects, such as molecules that do not cause visible or detectable adverse effects when administered using general quality of life tests or, as described herein, by measuring in vivo tolerability scores.

[0153] The term "subject," "individual," "animal," "patient," or "mammal" refers to any subject who requires diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, domesticated animals, farm animals, sporting animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, etc.

[0154] The “effective amount” of the therapeutic molecules disclosed herein is an amount sufficient to achieve the clearly specified purpose. The “effective amount” can be determined empirically and in a conventional manner relevant to the specified purpose.

[0155] Terms such as “treatment” or “curing” or “management” or “relief” or “remission” refer to (1) therapeutic measures that cure, slow, alleviate, and / or stop the progression of a diagnosed pathological condition or symptom; and (2) preventive or preventative measures that prevent and / or slow the occurrence of a targeted pathological condition or symptom. Therefore, subjects requiring treatment include subjects with the disease, subjects susceptible to the disease, and subjects whose condition requires prevention. In some embodiments, a subject with a disease or symptom disclosed elsewhere in this document is successfully “treated” if, according to the methods provided herein, the patient exhibits, for example, complete, partial, or temporary relief or elimination of symptoms related to the disease or symptom.

[0156] II. Method for determining in vivo neurotoxicity using calcium fluctuation assay

[0157] This disclosure provides methods for testing or determining the toxicity (e.g., acute neurotoxicity in vivo) of a molecule by measuring certain characteristics of the molecule. This disclosure also provides methods for selecting molecules with reduced toxic side effects. These methods help reduce unnecessary animal killing and / or increase the likelihood that the molecule will be safe for in vivo administration when testing the toxicity of the molecule. The methods of the present invention can also improve the efficiency (i.e., shorten) of the candidate molecule evaluation cycle by shortening the screening period for selecting molecules that do not exhibit acute neurotoxicity in vivo. The methods of the present invention include identifying molecules with reduced or decreased toxicity. For example, molecules can be assayed to determine whether they have low toxicity (e.g., acute neurotoxicity in vivo), and if they are found to have low toxicity, the molecules are selected for further testing or administration to subjects, such as mammals. In some embodiments, if the molecule is found to have low toxicity, it is given to laboratory animals for further testing of the molecule.

[0158] Unbound by any theory, this disclosure identifies a correlation between (i) calcium fluctuations in molecules in in vitro neuronal cells and the sequence score of said molecule (e.g., a polynucleotide containing a certain sequence); (ii) a correlation between calcium fluctuations in molecules and the in vivo neurotoxicity of said molecules; (iii) a correlation between the sequence score of said molecule (e.g., a polynucleotide containing a certain sequence) and the in vivo neurotoxicity of said molecules; or (iv) any combination thereof. In one embodiment, this disclosure indicates that molecules exhibiting calcium fluctuations in neuronal cells comparable to (i.e., less than 30%) calcium fluctuations in neurons not exposed to said molecule, when administered in vivo to mammals, show less in vivo neurotoxicity. In another embodiment, this disclosure indicates that molecules exhibiting calcium fluctuations in neuronal cells comparable to (i.e., less than 30%) calcium fluctuations in neurons not exposed to said molecule, have a sequence score equal to or greater than 0.2. In other embodiments, this disclosure indicates that molecules with a sequence score equal to or greater than 0.2 show less in vivo neurotoxicity when administered in vivo to mammals. Therefore, the identification of the correlation between calcium fluctuation assays, sequence scoring, and in vivo neurotoxicity allows for the prediction of in vivo neurotoxicity based on in vitro calcium fluctuation assays and sequence scoring. In yet another embodiment, as further discussed above, the present invention allows for the prediction of in vivo neurotoxicity based on in vitro calcium fluctuation assays, sequence scoring, and changes in microtubule strength in cells. 。

[0159] On the one hand, this disclosure proposes a calcium fluctuation assay as a method for measuring or predicting molecular toxicity. On the other hand, this disclosure provides a sequence scoring method for measuring or predicting molecular toxicity. In other aspects, this disclosure provides a combined method employing the calcium fluctuation assay and the sequence scoring method. This disclosure also provides an in vivo tolerability assay that can be used alone or in combination with the calcium fluctuation assay and / or the sequence scoring method. The methods disclosed in this application and / or any other methods known in the art can be further combined with the calcium fluctuation assay and / or the sequence scoring method.

[0160] II.A. Calcium Fluctuation Measurement Method

[0161] In one embodiment, the toxicity of the molecule (e.g., acute neurotoxicity in vivo) is tested in vitro by measuring intracellular free calcium fluctuations (calcium fluctuations) in neuronal cells that have been in contact with or have been in contact with the molecule. Examples of methods for measuring calcium fluctuations are discussed in more detail below. In some embodiments, the molecule is considered to have acceptable toxicity (e.g., acute neurotoxicity in vivo) if it does not significantly reduce calcium fluctuations in cells exposed to the molecule compared to calcium fluctuations in control cells. In some embodiments, control cells are cells that are not exposed to the test molecule but otherwise under the same conditions as cells exposed to the test molecule. In some embodiments, the calcium fluctuation assay may include positive control cells (i.e., cells exposed to molecules known to reduce calcium fluctuations to intolerable levels) or negative control cells (i.e., cells exposed to molecules known to not affect calcium fluctuations in cells). In another embodiment, control cells are exposed to a medium that carries the test molecule into a neuronal cell culture, such as water, buffer, or saline, without the test molecule (i.e., a solvent control).

[0162] In one embodiment, this disclosure provides a method for testing, identifying, or determining the acute neurotoxicity of a molecule in vivo, the method comprising measuring, in vitro, calcium fluctuations in neuronal cells that have been in contact with or are in contact with the molecule. In another embodiment, this disclosure includes a method for testing, identifying, or determining the acute neurotoxicity of a molecule in vivo, the method comprising (1) adding the molecule to a neuronal cell culture and (2) measuring calcium fluctuations in the neuronal cells in vitro. In yet another embodiment, this disclosure provides a method for predicting the acute neurotoxicity of a molecule in vivo, comprising the steps of: (1) adding the molecule to a neuronal cell culture and (2) measuring calcium fluctuations in the neuronal cells in vitro.

[0163] In some embodiments, this disclosure provides methods for testing, identifying, or determining the in vivo acute neurotoxicity of a molecule or for selecting or identifying a molecule having tolerable in vivo acute neurotoxicity, the methods comprising (i) measuring calcium fluctuations in neuronal cells in vitro after adding the molecule to a culture of neuronal cells, wherein the calcium fluctuations in the neuronal cells are equivalent to or higher than the calcium fluctuations of a solvent control, and (ii) administering the molecule to a person in need.

[0164] In other embodiments, this disclosure includes methods for selecting or identifying molecules with tolerable acute in vivo neurotoxicity, said methods including in vitro measurement of calcium fluctuations in neuronal cells in contact with the molecule, wherein the contacted neuronal cells show levels of calcium fluctuations equivalent to or higher than those of control cells. In some embodiments, this disclosure provides methods for selecting or identifying molecules with tolerable acute in vivo neurotoxicity, said methods including the steps of: (i) adding the molecule to a neuronal cell culture, and (ii) in vitro measurement of calcium fluctuations in neuronal cells, wherein the neuronal cells containing said molecule show levels of calcium fluctuations equivalent to or higher than those of control cells.

[0165] Calcium fluctuations are important for the normal function of neurons. Cortical neuronal networks exhibit spontaneous calcium fluctuations that lead to the release of the neurotransmitter glutamate. In addition to other related neurons within the network, calcium fluctuations can also modulate the interaction between neurons and related glial cells to release neurotransmitters other than glutamate. Regulated calcium fluctuations are essential for the in vivo homeostasis of neuronal networks for normal brain function (see Shashank et al.). Brain Research , 1006(1): 8–17 (2004); Rose et al, Nature Neurosci. , 4:773 – 774 (2001); Zonta et al., J Physiol Paris. , 96(3-4):193-8 (2002); Pasti et al, J. Neurosci. , 21(2): 477-484 (2001)). Glutamate also activates two distinct channels, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the N-methyl-D-aspartate (NMDA) receptor.

[0166] In some embodiments, the calcium fluctuation measured by the method of the present invention is AMPA-dependent calcium fluctuation. In some embodiments, the calcium fluctuation is NMDA-dependent calcium fluctuation. In some embodiments, the calcium fluctuation is γ-aminobutyric acid (GABA)-dependent calcium fluctuation. In some embodiments, the calcium fluctuation may be a combination of two or more of AMPA-dependent, NMDA-dependent, or GABA-dependent calcium fluctuations.

[0167] In some embodiments, the calcium fluctuations measured by the method of the present invention are AMPA-dependent calcium fluctuations. To measure AMPA-dependent calcium fluctuations, calcium fluctuations can be measured in Mg... 2+ The measurement is performed in the presence of ions (e.g., MgCl2). In some embodiments, the method further includes adding Mg in an amount that allows for the detection of AMPA-dependent calcium fluctuations. 2+Ions (e.g., MgCl2). In some embodiments, the effective ion concentration for AMPA-dependent calcium fluctuations is at least about 0.5 mM. In other embodiments, MgCl2 is used to induce AMPA-dependent calcium fluctuations. 2+ The effective ion concentration of ions (e.g., MgCl2) is at least about 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at least about 0.9 mM, at least about 1 mM, at least about 1.5 mM, at least about 2.0 mM, at least about 2.5 mM, at least about 3.0 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In one specific embodiment, MgCl2 can be used in this method. 2+ The ion concentration is 1 mM. In some embodiments, Mg can be used in the method of the present invention. 2+ The concentration of ions (e.g., MgCl2) is approximately 1 mM to approximately 10 mM, approximately 1 mM to approximately 15 mM, approximately 1 mM to approximately 20 mM, or approximately 1 mM to approximately 25 mM. Mg can be added by adding magnesium salts such as magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, and magnesium sulfate heptahydrate. 2+ ion.

[0168] In some embodiments, calcium fluctuations in the method of the present invention are measured by using fluorescent probes that detect fluctuations in intracellular calcium levels. For example, the detection of intracellular calcium flux can be achieved by staining cells with a fluorescent dye that binds to calcium ions (called a fluorescent calcium indicator), thus producing a detectable change in fluorescence (e.g., Fluo-4 AM and FuraRed AM dyes available from Molecular Probes. Eugene, OR, United States of America).

[0169] Fluorescent dyes used in calcium fluctuation assays often provide proportional detection of intracellular calcium flux by adjusting the fluorescence intensity at the measurement wavelength. In some embodiments, the fluorescence of stained cells (including stained single cells) can be optionally measured at multiple time points or continuously (e.g., in real time) using a confocal or standard fluorescence microscope to provide, for example, time-lag measurements. Those skilled in the art will recognize that other suitable methods may also be available for measuring intracellular calcium flux, such as viral transduction via genetically encoded calcium indicators.

[0170] In one embodiment, the calcium fluctuations measured by the method of the present invention are the cumulative increase in calcium fluctuations within a neuronal cell culture, wherein the time to reach a maximum fluorescence signal constitutes the amplitude of the calcium response. The fluorescence measurements can be analyzed to identify fluctuations in intracellular calcium flux and / or a “threshold” representing the point at which intracellular calcium flux reaches its maximum or minimum within a specified fluctuation. In another embodiment, the calcium fluctuations measured in the method of the present invention are the frequency of calcium fluctuations. The term “fluctuation frequency” refers to the time between fluctuations. In one embodiment, the fluctuation frequency can be determined by the time interval between the start of a first fluctuation in intracellular calcium flux and the start of a second fluctuation in intracellular calcium flux. In other embodiments, the calcium fluctuations measured in the method of the present invention are a combination of fluctuation frequency and amplitude.

[0171] In some embodiments, calcium fluctuations are measured using any method known herein. In some embodiments, calcium fluctuations can be measured using a fluorescence plate reader, such as the Flexstation 2 and 3 plate readers or the FLIPR™ (Fluorescence Imaging Plate Reader). In other embodiments, they can be measured according to Murphy et al. J. Neurosci. The measurement of calcium fluctuations is shown in 12, 4834-4845 (1992).

[0172] Neuronal cells suitable for use in this invention can be isolated from mammalian neuronal cells, such as mouse neurons, rat neurons, human neurons, or other neuronal cells. In some embodiments, the neuronal cells do not express endogenous transcripts encoding proteins; for example, if a human protein is targeted in mouse cells, the endogenous form of the transcript from the mouse cell is absent from its genome. In some embodiments, primary neurons are generated by papain digestion according to the manufacturer's protocol (Worthington Biochemical Corporation, LK0031050). In one embodiment, the forebrain is prepared according to the following example. The forebrain can be excised from hTau mouse E18 BAC-Tg embryos expressing the complete target gene in a mouse MAPT-ineffective background and incubated in a papain / DNase / Earle balanced salt solution (EBSS) at 37°C for 30-45 minutes. After cell pelleting, grinding, and centrifugation, the reaction is terminated by incubation with EBSS containing a protease inhibitor, bovine serum albumin (BSA), and DNase. Cells were homogenized and washed with Neurobasal (NB, Invitrogen) supplemented with 2% B-27, 100 μg / ml penicillin, 85 μg / ml streptomycin, and 0.5 mM glutamine. Cells were seeded at 15,000 cells / well in 96-well optical imaging plates (BD Biosciences) coated with poly-D-lysine on supplemented NB medium.

[0173] In some implementations, calcium fluctuations of molecules with tolerable acute neurotoxicity in vivo are compared with calcium fluctuations in cells not exposed to said molecules. In some embodiments, the calcium fluctuation of the molecule with tolerable acute in vivo toxicity is greater than or equal to about 250%, greater than or equal to about 240%, greater than or equal to about 230%, greater than or equal to about 220%, greater than or equal to about 210%, greater than or equal to about 200%, greater than or equal to about 190%, greater than or equal to about 180%, greater than or equal to about 170%, greater than or equal to about 160%, greater than or equal to about 150%, greater than or equal to about 140%, greater than or equal to about 130%, greater than or equal to about 120%, greater than or equal to about 110%, greater than or equal to about 100%, greater than or equal to about 99%, greater than or equal to about 98%, greater than or equal to about 97%, greater than or equal to about 96%, greater than or equal to about 95%, greater than or equal to about 90%, greater than or equal to about 85%, greater than or equal to about 80%, greater than or equal to about 75%, or greater than or equal to about 70%. The term "greater than or equal to" as used herein may be used interchangeably with "at least". In other embodiments, the calcium fluctuation with tolerable acute in vivo toxicity is greater than or equal to 100% of the calcium fluctuation in solvent control cells. In some embodiments, the calcium fluctuation with tolerable acute in vivo toxicity is greater than or equal to about 70% of the calcium fluctuation in solvent control cells. In some embodiments, the calcium fluctuation with tolerable acute in vivo toxicity is greater than or equal to about 75% of the calcium fluctuation in solvent control cells. In other embodiments, the calcium fluctuation with tolerable acute in vivo toxicity is about 70% to about 250%, about 70% to about 200%, about 75% to about 200%, about 70% to about 180%, about 75% to about 150%, about 80% to about 200%, about 90% to about 200%, about 100% to about 200%, or about 80% to about 250% of the calcium fluctuation in solvent control cells.

[0174] In some embodiments, calcium fluctuations in cells exposed to molecules with tolerable in vivo acute neurotoxicity showed a reduction of less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% compared to calcium fluctuations in solvent control cells. In other embodiments, calcium fluctuations in cells exposed to molecules with tolerable in vivo acute neurotoxicity were reduced by less than about 30%, less than about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% compared to calcium fluctuations in solvent control cells.

[0175] In some embodiments, molecules that cause a reduction in calcium fluctuation greater than the desired reduction are considered molecules with unacceptable neurotoxicity. In these embodiments, molecules that cause a reduction in calcium fluctuation greater than the desired reduction, if administered to a subject, are considered to pose a risk of toxic side effects. In some embodiments, this disclosure provides a method for identifying or determining molecules with intolerable in vivo neurotoxicity, the method comprising measuring calcium fluctuation in neuronal cells in vitro after contact with said molecule. In some embodiments, the calcium fluctuation of the molecule with intolerable in vivo neurotoxicity is less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the calcium fluctuation in solvent control cells. In some embodiments, this disclosure includes a method for identifying or determining molecules with intolerable in vivo neurotoxicity, the method comprising measuring calcium fluctuation in neuronal cells in vitro after contact with said molecule, wherein the calcium fluctuation of said molecule is equal to or less than 50% of the calcium fluctuation in solvent control cells.

[0176] In some embodiments, the molecule is a therapeutic molecule. In other embodiments, the molecule includes small molecules, polynucleotides, proteins, peptides, or any combination thereof. Non-limiting examples of the molecules are described in other parts of this document.

[0177] II.B . Sequence scoring method

[0178] This disclosure also relates to methods for testing or determining the in vivo neurotoxicity of molecules (e.g., polynucleotides) containing nucleotide sequences. In some embodiments, the method includes measuring a sequence score calculated using the following formula (I):

[0179] .

[0180] In other embodiments, the oligomers of the present invention have a sequence score greater than or equal to 0.2.

[0181] In some implementations, the method includes measuring a sequence calculated by the following formula (IA):

[0182] .

[0183] In other embodiments, the oligomers of the present invention have a sequence score greater than or equal to 0.2.

[0184] In these implementations, a sequence score greater than or equal to the cutoff value corresponds to a reduction in the neurotoxicity of the oligomer.

[0185] For example, the nucleotide sequence of ATGCATGCATGCATGC (SEQ ID NO: 3) has a sequence score of 0 ((4C – 4G) / 16). The sequence of GTGCGTGCGTGCGTGC (SEQ ID NO: 732) has a sequence score of -0.25 ((4C – 8G) / 16). The sequence of CTGCCTGCCTGCCTGC (SEQ ID NO: 733) has a sequence score of 0.25 ((8C – 4G) / 16). In some embodiments, a polynucleotide (e.g., an oligomer) containing a nucleotide sequence is considered to have acceptable neurotoxicity if it has a sequence score greater than or equal to about 0.2, greater than or equal to about 0.25, greater than or equal to about 0.3, greater than or equal to about 0.35, greater than or equal to about 0.4, greater than or equal to about 0.45, greater than or equal to about 0.55, greater than or equal to about 0.6, greater than or equal to about 0.65, greater than or equal to about 0.7, greater than or equal to about 0.75, greater than or equal to about 0.8, greater than or equal to about 0.85, greater than or equal to about 0.9, greater than or equal to about 0.95, greater than or equal to about 1.0, greater than or equal to about 1.5, greater than or equal to about 2.0, greater than or equal to about 3.0, or greater than or equal to about 4.0. In some embodiments, a polynucleotide is considered to have acceptable neurotoxicity if it has a sequence score greater than or equal to 0.2, greater than or equal to 0.25, greater than or equal to 0.3, greater than or equal to 0.35, greater than or equal to 0.4, greater than or equal to 0.45, greater than or equal to 0.5, greater than or equal to 0.55, greater than or equal to 0.6, greater than or equal to 0.65, greater than or equal to 0.7, greater than or equal to 0.75, greater than or equal to 0.8, greater than or equal to 0.85, greater than or equal to 0.9, greater than or equal to 0.95, greater than or equal to 1.0, greater than or equal to 1.5, greater than or equal to 2.0, greater than or equal to 3.0, or greater than or equal to 4.0. In some embodiments, a polynucleotide with acceptable neurotoxicity has a sequence score equal to or greater than 0.2.

[0186] In some embodiments, molecules containing nucleotide sequences with sequence scores below a set threshold are considered molecules with unacceptable neurotoxicity. In these embodiments, molecules with sequence scores below a set threshold are considered to pose a risk of toxic side effects if administered to a subject.

[0187] In some embodiments, any of the above-described methods for selecting molecules may be used in combination. When used in combination, if the molecule is selected as having acceptable neurotoxicity for use with more than one method, then the molecule is considered to have a greater chance of having acceptable neurotoxicity when administered to a test subject or patient.

[0188] In some embodiments, this disclosure includes a method for selecting polynucleotides with tolerable acute neurotoxicity in vivo, said method comprising (i) performing the calcium fluctuation assay disclosed herein, and (ii) calculating the sequence score disclosed herein, wherein the calcium fluctuation of the polynucleotide is equal to or greater than 75% of the calcium fluctuation in solvent cells, and the sequence score of the polynucleotide is greater than or equal to 0.25. In some embodiments, the calcium fluctuation assay and / or sequence scoring method is sufficient to predict, identify, or measure the acute neurotoxicity of a molecule in vivo without requiring additional in vivo tolerability studies. The calcium fluctuation assay and / or sequence scoring method is particularly useful for screening multiple candidate molecules for determining their neurotoxicity in vivo.

[0189] II.C. In vivo tolerance assay

[0190] In other embodiments, the invention also relates to methods for selecting or identifying molecules with tolerable in vivo neurotoxicity by performing in vivo tolerability studies. When the number of candidate molecules is small, in vivo tolerability studies can provide a direct indication of in vivo neurotoxicity. In other embodiments, in vivo tolerability studies may be used in conjunction with calcium fluctuation assays and / or sequence scoring methods. In vivo tolerability studies may also be employed after a small number of candidate molecules have been selected through calcium fluctuation assays and / or sequence scoring calculations. In some embodiments, in vivo tolerability scores are measured, for example, by administering the molecules to a mammal (e.g., to the brain of a mammal) via intraventricular (ICV) or intrathecal (IT) administration.

[0191] For example, the molecule can be injected into laboratory animals via ICV or IT. Laboratory animals can be rodents, such as mice, rats, guinea pigs, or hamsters, but can also be another animal commonly used in laboratory experiments. In some embodiments, animals are observed at 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours after molecule injection. Animals are observed for behavioral side effects, and the severity of the side effects is scored on a scale from zero (no side effects) to 20 (convulsions resulting in euthanasia). The tolerance scale can be divided into at least one of the following neurobehavioral categories: 1) hyperactivity, 2) decreased activity and arousal, 3) motor dysfunction / ataxia, 4) postural and respiratory abnormalities, and 5) tremor / convulsions. In some embodiments, the tolerance scale includes at least two, at least three, at least four, or at least five neurobehavioral categories. Each category is scored on a scale of 0-4, with the worst possible total score being 20 and the best possible total score being 0. Behavioral changes in the animal are observed, for example, in a rearing cage, but the animal can be observed in other environments. In some implementations, the animal is removed from the rearing cage for more detailed observation, including measurements of grip strength and righting reflex.

[0192] In some implementations, the cumulative tolerance threshold in vivo after molecular injection is set to 4. For example, Figure 3 Related analyses show that molecules with in vivo tolerance below 4 tend to have sequence scores equal to or higher than 0.2.

[0193] In other embodiments, this disclosure includes methods for identifying or selecting molecules with tolerable in vivo neurotoxicity, the methods comprising (i) performing a calcium fluctuation assay, (ii) calculating a sequence score, (iii) conducting in vivo tolerance studies, and (iv) administering the molecule to a mammal requiring treatment for a disease or condition.

[0194] II.D. Microtubule Strength Assay

[0195] In some embodiments, the methods of this disclosure further include measuring the long-term in vivo toxicity of the molecule. For example, long-term toxicity is determined by measuring the change in tubulin strength in cells caused by the molecule when cells are exposed to it. In some embodiments, the change in tubulin strength is measured in conjunction with one or more of the following: changes in calcium fluctuations, sequence scoring, and / or in vivo tolerance assays. Examples of assays for measuring changes in tubulin strength in cells are provided below. In some embodiments, the molecule shows a tubulin strength in cells greater than or equal to 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% of the tubulin strength in cells not exposed to the molecule (i.e., control cells as defined above). In some embodiments, the tubulin strength in cells not exposed to the test molecule is referred to as the tubulin strength of control cells. In some embodiments, the molecule reduces tubulin strength by less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the tubulin strength in solvent control cells.

[0196] II.E . Behavioral tests

[0197] The method of the present invention may further include measuring the behavioral performance of an animal induced by the molecule. In one embodiment, the method includes a behavioral test score, which can be measured by administering the molecule to a mammal and rating the mammal’s behavioral performance. In some embodiments, the behavioral test is a short-term memory test, a spatial learning and memory test, a gait analysis test, or any combination thereof. In one embodiment, behavioral performance is measured by injecting the molecule into a mammal, such as the mammal’s brain, for example, intraventricular (ICV) or intrathecal (IT) administration, and rating the mammal’s behavioral performance on a scale of 0-4. In some embodiments, the behavioral score is a total score less than or equal to 3, 2, 1, or 0. In some embodiments, the behavioral score is determined as described in Example 5 below.

[0198] In some embodiments, behavioral scores are measured using methods including novel object rejection tests, water maze tests, gait analysis tests, and / or any combination thereof. The therapeutic molecule is administered to laboratory animals via ICV or IT. Laboratory animals can be mammals, such as rodents like mice, rats, guinea pigs, or hamsters, but can also be animals commonly used for laboratory testing. In some embodiments, animals are observed at approximately 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours after molecule injection.

[0199] In one implementation, behavioral scores are obtained using a novel object recognition (NOR) test. Short-term recognition memory is measured using a novel object recognition (NOR) task. The NOR test is based on the spontaneous behavior of rodents exploring new objects more than familiar ones (Dodart et al., Neuroreport (1997) 8(5): 1173-8; Ennaceur and Delacour, Behav. Brain Res. (1988) 31(1):47-59). The NOR test can be applied similarly to the test shown in Example 5, or can be modified as needed.

[0200] In one implementation, behavioral scores are obtained using a water maze test (e.g., the Morris Water Maze). Spatial learning and memory can be evaluated using the Morris Water Maze test as described in Morris J. Neurosci. (1984) 11(1):47-60) or the test described in Example 5 of this document. In another implementation, spatial learning and memory tests can be evaluated as needed using a modified Morris Water Maze test.

[0201] In one implementation, behavioral scores can be obtained using a gait analysis test (e.g., Catwalk). Catwalk (Noldus, Netherlands) is an automated and computerized gait analysis technique that allows for the objective quantitative determination of multiple static and dynamic gait parameters. Gait analysis tests can be performed using the Catwalk assay shown in Example 5 or, if necessary, by a modified Catwalk test.

[0202] Statistical analysis of behavioral test data can be performed using statistical analysis methods known to those skilled in the art. In some implementations, statistical analysis of behavioral tests is performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). For NOR, data are analyzed using paired t-tests for within-group analysis or by ANOVA followed by Dunnett's post-hoc test for between-group analysis. For Morris Water Maze (MWM), repeated MWM ANOVA is applied to analyze the data collection phase, and one-way ANOVA followed by Dunnett's post-hoc test is applied for probe analysis.

[0203] Unbound by any theory, molecules exhibiting a smaller reduction (70% or more) in calcium fluctuations compared to controls (e.g., saline) have higher sequence scores (e.g., above 0.2). Also unbound by any theory, molecules exhibiting a smaller reduction (70% or more) in calcium fluctuations and higher sequence scores (above 0.2) compared to controls have lower in vivo behavioral scores (e.g., less than 4). In other embodiments, molecules exhibiting a smaller reduction (70% or more) in calcium fluctuations and higher sequence scores (above 0.2) compared to controls demonstrate tolerable acute neurotoxicity in vivo.

[0204] II.F . Diagnosis or treatment

[0205] Molecules selected according to the method of the present invention can be used as research reagents for, for example, diagnostic, therapeutic, and preventative methods. In some embodiments, the present invention provides a method for both selecting and using molecules.

[0206] In other embodiments, the molecule selected according to the method of the present invention is a therapeutic molecule. In other additional embodiments, the method may further include calcium fluctuation assays, sequence scoring methods, and / or in vivo tolerability tests, which may include administering the selected molecule to a subject in need of treatment.

[0207] Therefore, treatment of animals or humans suspected of having a disease or condition can be achieved by administering the molecular therapy of this disclosure. Further, methods are provided for treating mammals (e.g., treating humans) suspected of having or susceptible to a disease or condition by administering a therapeutic or preventative amount of one or more molecules of this disclosure. In some embodiments, this disclosure provides a method of treating mammals (e.g., humans) comprising (1) selecting a molecule with tolerable acute in vivo neurotoxicity (e.g., calcium fluctuation assay, sequence scoring, and / or in vivo tolerance studies) as described elsewhere herein, and (2) administering said molecule to the mammal. Typically, the molecules, conjugates, or pharmaceutical compositions of the present invention are administered in an effective amount. In some embodiments, the molecules or conjugates of the present invention are used in a therapy.

[0208] This disclosure also provides methods for administering molecules to a subject for the treatment of a neurological disease or condition. In some embodiments, the neurological disease is a neurodegenerative disease, epilepsy, idiopathic adult epilepsy, or any combination thereof. In other embodiments, the disease or condition is a neurodegenerative disease with tauopathy (i.e., a neurodegenerative disease involving the accumulation of tau protein in the brain), epilepsy with tauopathy (epilepsy involving the accumulation of tau protein in the brain), epilepsy without tauopathy (epilepsy without the accumulation of tau protein in the brain), idiopathic adult epilepsy without tauopathy (idiopathic adult epilepsy without the accumulation of tau protein in the brain), or any combination thereof. In some embodiments, the disease or condition being treated or prevented is a neurodegenerative disease with tauopathy.

[0209] In some implementations, the disease or condition is progressive supranuclear palsy, Down syndrome, boxer's dementia (chronic traumatic encephalopathy and other traumatic brain injuries), frontotemporal dementia linked to chromosome 17 with Parkinson's syndrome (FTDP-17), Lytico-Bodig disease (Guam-type Parkinson's syndrome-dementia complex), tangles-dominant dementia, ganglioglioma, gangliocytoma, meningioma, subacute sclerosing panencephalitis, lead encephalopathy, hemimegaencephalopathy, tuberous sclerosis, Hallewarden-Schpattz disease, Pick's disease, corticobasal ganglia degeneration, argyrophilic granuloma, corticobasal ganglia degeneration, lipofuscinosis, frontotemporal dementia, supranuclear palsy and frontotemporal lobe degeneration, brain network dysfunction (e.g., all forms of epilepsy and depression), Dravet syndrome, spinal cord disorders, peripheral neuropathy, cranial nerve disorders. Disorders of the nervous system include: trigeminal neuralgia, autonomic nervous system disorders (such as familial autonomic dysfunction or multiple system atrophy), central and peripheral nervous system motor disorders (such as Parkinson's disease, essential tremor, amyotrophic lateral sclerosis, Tourette syndrome, multiple sclerosis, or different types of peripheral neuropathy), sleep disorders (such as narcolepsy), migraines or other types of headaches (such as cluster headaches and tension headaches), lower back and neck pain, central nervous system disorders, neuropsychiatric disorders, attention deficit hyperactivity disorder, autism, Huntington's disease, Tourette syndrome, Angelman syndrome, organic psychosis, brain or bone marrow infection (including meningitis) or prions, anemia, cancer, leukemia, inflammatory conditions or autoimmune diseases (such as arthritis, psoriasis, lupus, multiple sclerosis), bacterial infections, and any combination thereof.

[0210] In some implementations, the disease or condition is a neurodegenerative disease with tau proteinosis, such as progressive supranuclear palsy, frontotemporal dementia-tau (FTD-tau), chromosome 17-linked frontotemporal dementia and Parkinson's syndrome (FTDP-17), corticobasal degeneration (CBD), traumatic brain injury, chronic traumatic encephalopathy, HIV-related neurocognitive impairment, auricolic granuloma, Down syndrome-Alzheimer's dementia, amnesic mild cognitive impairment-Alzheimer's dementia, Parkinson's disease dementia, Hallewarden-Scholes disease (pantothenic acid kinase-related neurodegeneration), Niemann-Pick disease type C, myotonic dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease. In some implementations, the disease or condition is an epileptic condition with tau proteinosis, such as hemimegalencephaly, tuberous sclerosis relapse, type 2b focal cortical dysplasia, or ganglion cell tumor. In some implementations, the disease or condition is an epilepsy without tau protein disease, such as Dravet syndrome (infantile severe clonic epilepsy), temporal lobe epilepsy, Ohtahara syndrome (early infantile epileptic encephalopathy with repressive bursts), Lafora body disease, generalized epilepsy with febrile seizures, infantile spasms (West syndrome), Lennox-Gastaut syndrome, Angelman syndrome, Rett syndrome, and Landau-Kleffner syndrome. In some implementations, the disease or condition is an idiopathic adult epilepsy without tau protein disease, such as focal epilepsy, simple focal epilepsy (without loss of consciousness), focal cognitive impairment epilepsy (disorder of consciousness), focal epilepsy that develops into generalized tonic-clonic (GTC) seizures, generalized seizures (convulsive or nonconvulsive, with bilateral discharges involving subcortical structures), absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures, or tonic seizures. In some implementations, the disease or condition is autism, autism spectrum disorder (e.g., as defined in the Diagnostic and Statistical Manual of Mental Disorders V (DSM-V)), Asperger's syndrome, or pervasive developmental disorder.

[0211] This invention further provides molecules of the invention for treating one or more diseases related to neurons or diseases mentioned herein, such as those selected from the following: Alzheimer's dementia, progressive supranuclear palsy, Down syndrome, boxer's dementia (chronic traumatic encephalopathy and other traumatic brain injuries), frontotemporal dementia linked to chromosome 17 with Parkinson's syndrome (FTDP-17), Lytico-Bodig disease (Guam-type Parkinson's syndrome-dementia complex), tangles-dominant dementia, ganglioglioma, gangliocytoma, meningioma, subacute sclerosing panencephalitis, lead poisoning encephalopathy, hemimegaencephalopathy, tuberous sclerosis, Hallewarden-Spats disease, Pick's disease, corticobasal ganglia degeneration, argyrophilic granuloma, corticobasal ganglia degeneration, lipofuscinosis, frontotemporal dementia, supranuclear palsy, and frontotemporal lobe degeneration (see reviews by Frost et al., Trends Cell Biol (2015) 25: 216-53; Dyment et al., Neurobiol. Aging (2014) Sept 6: S0197-4580; Moussaud et al., Mol. Neurodeg (2014) 29:43; Ross et al., South Med.J. (2014) 107: 715-21, Huntington's disease, Rett syndrome, and Angleman syndrome. Additionally, this invention provides therapeutic molecules for treating diseases including all forms of epilepsy and depression with dysfunction of the brain network (Inoue et al., Epilepsy (2012) 102: 8-12; Xi et al., Med Hypotheses (2011) 76: 897-900; Hou et al., Can. J. Psychiatry (2004) 3: 164-71).

[0212] This disclosure also provides the use of the molecules or conjugates of the present invention in the preparation of medicaments for treating the diseases or conditions mentioned herein, or methods for treating the conditions mentioned herein. Compositions for treating the diseases or conditions mentioned herein are also provided.

[0213] III. Molecules (e.g., therapeutic molecules)

[0214] Molecules to be screened or selected according to the present invention include therapeutic molecules. In one embodiment, the therapeutic molecules include proteins, peptides, polynucleotides (e.g., oligomers), sugars, lipids, liposomes and particles, biomaterials, pharmaceuticals, vitamins, nucleic acids, amino acids, polypeptides, enzyme cofactors, steroids, carbohydrates, heparin, metal-containing agents, receptor antagonists, receptor agonists, receptors or portions of receptors, extracellular matrix proteins, cell surface molecules, antigens, haptens, small molecules, or any combination thereof.

[0215] In some implementations, the therapeutic molecule is a protein, including cytokines, enzymes, growth factors, monoclonal antibodies, antibody fragments, single-chain antibodies, albumins, immunoglobulins, coagulation factors, growth promoters, amylases, lipases, proteases, cellulose, urokinases, galactosidases, staphylococcal kinases, hyaluronidases, tissue plasminogen activators, or any combination thereof.

[0216] In one embodiment, the molecules of the present invention comprise at least one of a therapeutic molecule that is an antigen-binding site (e.g., an antigen-binding site of an antibody, antibody variant, or antibody fragment), a receptor-binding portion of a ligand, or a receptor-binding portion of a ligand.

[0217] In another embodiment, the molecules of the present invention target one or more endogenously produced proteins or peptides, one or more mRNAs or pre-mRNAs encoding proteins or peptides, or one or more genes encoding proteins or peptides in vivo. In some embodiments, the molecules comprise polynucleotides (e.g., oligomers), nucleotides, or small molecules.

[0218] The molecule may also include any therapeutic small molecule or drug as a therapeutic molecule that can be used in the methods disclosed herein. Small molecules may include any therapeutic molecule that is not a peptide, polypeptide, protein, or polynucleotide. Small molecules may include a single nucleotide or nucleoside, such as RNA or DNA.

[0219] In one embodiment, the therapeutic molecule modulates cell activation or inhibition (e.g., by binding to cell surface receptors and resulting in the transmission of activation or inhibition signals). In one embodiment, the therapeutic molecule is capable of initiating signal transduction that leads to cell death (e.g., through cell signaling-induced pathways, complement binding, or exposure to a payload present in the binding molecule (e.g., a toxic payload)), or modulates a disease or condition in a subject (e.g., by mediating or promoting cell killing, or by modulating the amount of bioavailable substances (e.g., by increasing or decreasing the amount of ligands such as TNFα in the subject)). In another embodiment, the molecule of the invention has at least one binding site specific to an antigen, targeting the reduction or elimination of, for example, cell surface antigens or soluble antigens.

[0220] In another embodiment, the binding of the therapeutic molecule of the present invention to a target molecule (e.g., an antigen) results in a reduction or elimination, for example, of the target molecule or cells expressing the target molecule from tissues or circulation. In another embodiment, the therapeutic molecule has at least one binding site specific to a target molecule that can be used to detect the presence of the target molecule (e.g., to detect contaminants or to diagnose a condition or symptom). Exemplary therapeutic molecules are further discussed below.

[0221] III.A . antigen-binding portion

[0222] In some embodiments, the molecules that can be used in this disclosure include at least one therapeutic molecule that is a binding site (e.g., the antigen-binding portion of an antibody). In one embodiment, the molecule used in the methods disclosed herein is a polypeptide.

[0223] In other embodiments, the binding site of the molecule of the present invention includes the antigen-binding portion of an antibody. The term "antigen-binding portion" refers to a polypeptide fragment of an immunoglobulin, antibody, or antibody variant that binds to an antigen or competes for antigen binding (i.e., specifically binds) with an intact antibody (i.e., with the intact antibody from which they are derived). For example, the antigen-binding portion may be derived from any antibody or antibody variant known in the art. The antigen-binding portion may be generated by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include VH and VL regions, Fv, Fab, Fab', and (Fab')2.

[0224] In other embodiments, the therapeutic molecule of the present invention comprises a binding site (e.g., a single-chain variable region or scFv) from a single-chain binding molecule. The techniques described above for generating single-chain antibodies (US Patent No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)) can be adapted to generate single-chain molecules. Single-chain antibodies are formed by linking heavy and light chain fragments of the Fv region via amino acid bridges, resulting in single-chain antibodies. Techniques for assembling functional Fv fragments in *E. coli* can also be employed (Skerra et al., Science 242:1038-1041 (1988)).

[0225] The polypeptides that can be used in this disclosure may include those obtained from the variable region or a portion thereof (e.g., VL and / or VH domains) of an antibody using methods recognized in the art, or those obtained from antibodies recognized in the art using standard molecular biology techniques.

[0226] In one embodiment, the molecules used in this invention are combined with molecules used to treat cancer.

[0227] In other embodiments, the molecules used in this invention may be combined with molecules that can be used to treat autoimmune or inflammatory diseases or conditions.

[0228] For example, molecules (e.g., peptides) can bind to antigens present on immune cells (e.g., B or T cells) or to autoantigens that cause autoimmune diseases or conditions. Examples of autoimmune diseases that can be diagnosed, prevented, or treated by the methods and compositions of the present invention include, but are not limited to, Crohn's disease; inflammatory bowel disease (IBD); systemic lupus erythematosus; ulcerative colitis; rheumatoid arthritis; Goodpasture's syndrome; Graves' disease; Hashimoto's thyroiditis; pemphigus vulgaris; myasthenia gravis; scleroderma; autoimmune hemolytic anemia; autoimmune thrombocytopenic purpura; polymyositis and dermatomyositis; pernicious anemia; Sjögren's syndrome; ankylosing spondylitis; vasculitis; type I diabetes; neurological disorders, multiple sclerosis, and secondary diseases caused by autoimmune diseases.

[0229] In other embodiments, the therapeutic molecules of the present invention bind to target molecules associated with inflammatory diseases or conditions. The term "inflammatory disease or condition" as used herein includes diseases or conditions at least partially caused by or exacerbated by inflammation, such as increased blood flow, edema, or activation of immune cells (e.g., proliferation, increased cytokine production, or phagocytosis). For example, the molecules of the present invention may bind in abnormal amounts (e.g., in amounts that may be beneficially altered, for example, to the subject) to inflammatory factors (e.g., matrix metalloproteinases (MMPs), TNFα, interleukins, plasma proteins, cytokines, lipid metabolites, proteases, toxic groups, mitochondrial proteins, apoptotic proteins, adhesion molecules, etc.) that are included in or present in a region. The inflammatory process is a response of living tissue to damage. Inflammation can be caused by bodily damage, chemicals, microorganisms, tissue necrosis, cancer, or other components. Acute inflammation is transient, for example, lasting only a few days. However, if it is prolonged, it can refer to chronic inflammation.

[0230] Inflammatory conditions include acute, chronic, and relapsing inflammatory conditions. Acute inflammatory conditions are typically short-lived, lasting from a few minutes to 1-2 days, although they can last for several weeks. Key features of acute inflammatory conditions include increased blood flow, fluid and plasma protein exudation (edema), and migration of white blood cells (e.g., neutrophils). Chronic inflammatory conditions generally have a longer duration, ranging from weeks to months to years or even longer, and are histologically associated with the proliferation of lymphocytes and macrophages and with blood vessels and connective tissue. Relapsing inflammatory conditions include those that recur after a period of time or have periodic flare-ups. Examples of relapsing inflammatory conditions include asthma and multiple sclerosis. Some conditions may fall into one or more categories. Inflammatory conditions are generally characterized by fever, redness, swelling, pain, and loss of function. Examples of causes of inflammatory conditions include, but are not limited to, microbial infections (e.g., bacterial, viral, and fungal infections), physical factors (e.g., burns, radiation, and trauma), chemical agents (e.g., toxins and corrosive substances), tissue necrosis, and different types of immune responses. Examples of inflammatory conditions include, but are not limited to, osteoarthritis, rheumatoid arthritis, acute and chronic infections (bacterial, viral, and fungal); acute and chronic bronchitis, sinusitis, and other respiratory infections, including the common cold; acute and chronic gastroenteritis and colitis; acute and chronic cystitis and urethritis; acute respiratory distress syndrome; cystic fibrosis; acute and chronic dermatitis; acute and chronic conjunctivitis; acute and chronic serositis (pericarditis, peritonitis, synovitis, pleurisy, and tendinitis); uremic pericarditis; acute and chronic cholecystitis; acute and chronic vaginitis; acute and chronic uveitis; drug reactions; and burns (thermal, chemical, and electrical).

[0231] In other embodiments, the therapeutic molecules of the present invention bind to molecules that can be used to treat neurological diseases or conditions. For example, peptides may bind to antigens present on nerve cells (e.g., neurons or glial cells). In some embodiments, the antigens associated with neurological conditions may be the aforementioned autoimmune or inflammatory conditions. The term "neurological disease or condition" as used herein includes conditions or illnesses in which the nervous system degenerates (e.g., neurodegenerative diseases) and in subjects in which the nervous system fails to develop normally or fails to regenerate after injury (e.g., bone marrow injury). Examples of neurological disorders that can be diagnosed, prevented, or treated by the methods and compositions of the present invention include, but are not limited to, multiple sclerosis, Huntington's disease, Rett syndrome, Angelman syndrome, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, epilepsy, Dravet syndrome, neuropathic pain, traumatic brain injury, Guillain-Barré syndrome, and chronic inflammatory demyelinating polyneuropathy (CIDP).

[0232] In other respects, the therapeutic molecules of the present invention comprise antigen-binding sites or portions thereof derived from modified forms of antibodies. Exemplary forms of this type include, for example, minibody, diabody, tribody, nanobody, camelids, dabs, tetravalent antibodies, intradiabodies (e.g., Jendreyko et al., 2003. J. Biol. Chem. 278:47813), fusion proteins (e.g., antibody-cytokine fusion proteins, proteins fused to at least a portion of an Fc receptor), and bispecific antibodies.

[0233] III.B. Non-immunoglobulin-binding molecules

[0234] In some embodiments, the therapeutic molecules of the present invention include one or more binding sites derived from non-immunoglobulin-binding molecules. As used herein, a "non-immunoglobulin-binding molecule" is a binding molecule whose binding site comprises a polypeptide derived from sources other than immunoglobulins, but which may be engineered (e.g., mutagenesis) to impart desired binding specificity (e.g., a scaffold or framework).

[0235] Other examples of therapeutic molecules not derived from antibody molecules include receptor-binding sites and ligand-binding sites, which are discussed in more detail below.

[0236] The non-immunoglobulin therapeutic portion may include a binding site portion derived from an immunoglobulin superfamily member that is not an immunoglobulin (e.g., T-cell receptors or cell adhesion proteins (e.g., CTLA-4, N-CAM, telogen effluvium)). Such binding molecules include binding site portions that retain the conformation of immunoglobulin folds and are capable of specifically binding to IGF1-R epitopes. In other embodiments, the non-immunoglobulin binding the molecules of the invention also includes a binding site having a protein topology that is not based on immunoglobulin folds (e.g., ankylosing spondylamine or fibronectin) but is nonetheless capable of specifically binding to a target (e.g., IGF-1R epitope).

[0237] In one embodiment, the therapeutic portion is derived from a fibronectin-binding molecule. Fibronectin-binding molecules (e.g., molecules containing fibronectin type I, II, or III domains) exhibit CDR-like loops, which, unlike immunoglobulins, are independent of intrachain disulfide bonds. In one exemplary embodiment, the fibronectin polypeptide is AdNectin® (Adnexus Therpaeutics, Waltham, MA).

[0238] In another embodiment, the therapeutic molecule of the present invention comprises a binding site derived from Affibody® (Abcam, Cambridge, MA). In another embodiment, the therapeutic molecule of the present invention comprises a binding site derived from Anticalin® (Pieris AG, Friesing, Germany). In another embodiment, the therapeutic molecule of the present invention comprises a binding site derived from a cysteine-rich polypeptide. In other embodiments, the therapeutic molecule of the present invention comprises a binding site derived from a repetitive protein. Other non-immunoglobulin binding sites that can be used in the molecules of the present invention include binding sites derived from Src homology domains (e.g., SH2 or SH3 domains), PDZ domains, β-lactamases, high-affinity protease inhibitors, or small disulfide-bonded protein scaffolds (e.g., scorpion venom).

[0239] III.C . The binding site of receptors or ligands

[0240] In other respects, the molecules of the present invention comprise a ligand-binding site of a receptor and / or a receptor-binding portion of a ligand. Exemplary binding portions of receptors or ligands that may be present in the molecules of the present invention are listed below:

[0241] III.C.1. Cytokines and Cytokine Receptors

[0242] Cytokines have pleiotropic effects on the proliferation, differentiation, and functional activation of lymphocytes. Different cytokines or their receptor-binding moieties can be used in the fusion protein of the present invention as therapeutic molecules, binding sites, and / or domains. Exemplary cytokines include interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, and IL-18), colony-stimulating factors (CSFs) (e.g., granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), and monocyte-macrophage CSF (M-CSF)), tumor necrosis factor (TNF) α and β, cytotoxic T-lymphocyte antigen 4 (CTLA-4), and interferons such as interferon-α, β, or γ (US Patent Nos. 4,925,793 and 4,929,554).

[0243] Cytokine receptors typically consist of a ligand-specific α chain and a universal β chain. Exemplary cytokine receptors include GM-CSF, IL-3 (US Patent No. 5,639,605), IL-4 (US Patent No. 5,599,905), IL-5 (US Patent No. 5,453,491), IL-10 receptor, IFNγ (EP0240975), and receptors from the TNF family (such as TNFα receptors, such as TNFR-1 (EP417,563), TNFR-2 (EP 417,014), and lymphotoxin β receptors).

[0244] III.C.2. Adhesion proteins

[0245] Adhesion molecules are membrane-bound proteins that allow cells to interact with each other. Various adhesion proteins, including leukocyte homing receptors and cell adhesion molecules or their receptor-binding portions, can be incorporated into the fusion protein of the present invention as therapeutic molecules, binding sites, and / or domains. Leukocyte homing receptors are expressed on the surface of leukocytes during inflammation, including β-1 integrins (e.g., VLA-1, 2, 3, 4, 5, and 6) that mediate binding to extracellular matrix components and β2-integrins (e.g., LFA-1, LPAM-1, CR3, and CR4) that bind to cell adhesion molecules (CAMs) on vascular endothelium. Exemplary CAMs include ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1. Other CAMs include those from the selector protein family, including E-select, L-select, and P-select proteins.

[0246] III.C.3. Chemokines

[0247] Chemokines, chemokines that stimulate leukocyte migration to the site of infection, can also be incorporated into the fusion protein of this invention. Exemplary chemokines include macrophage inflammatory proteins (MIP-1-α and MIP-1-β), neutrophil chemokines, and RANTES (which regulate the secretion and expression of normal T cells).

[0248] III.C.4 Hormones

[0249] Exemplary growth hormones used as therapeutic components in the fusion protein of the present invention include renin, human growth hormone (HGH; U.S. Patent No. 5,834,598), N-methionyl human growth hormone; bovine growth hormone; growth hormone-releasing factor; parathyroid hormone (PTH); thyroid-stimulating hormone (TSH); thyroxine; proinsulin and insulin (U.S. Patent Nos. 5,157,021 and 6,576,608); follicle-stimulating hormone (FSH); calcitonin, luteinizing hormone (LH), leptin, glucagon; bufotoxin; growth hormone; mullerian inhibitory substance; relaxin and pro-relaxin; gonadotropin-related peptide; prolactin; placental prolactin; OB protein or mullerian inhibitory substance.

[0250] III.C.5. Receptors and Ligands

[0251] In one embodiment, the polypeptide of the present invention binds the binding site of a ligand or receptor (e.g., the extracellular domain (ECD)) to at least one genetically fused Fc region (i.e., the scFc region). In some embodiments, a portion of the ligand-binding receptor is derived from receptors selected from the following: immunoglobulin (Ig) superfamily receptors (e.g., soluble T-cell receptors, such as mTCR® (Medigene AG, Munich, Germany), receptors of the aforementioned TNF receptor superfamily (e.g., soluble TNFα receptors of immunoadhesins), receptors of the glial cell-derived neurotrophic factor (GDNF) receptor family (e.g., GFRα3), receptors of the G protein-coupled receptor (GPCR) superfamily, receptors of the tyrosine kinase (TK) receptor superfamily, receptors of the ligand-gated (LG) superfamily, receptors of the chemokine receptor superfamily, receptors of the IL-1 / Toll-like receptor (TLR) superfamily, and receptors of the cytokine receptor superfamily.

[0252] In other embodiments, the binding site or domain of the receptor-binding portion of the ligand is derived from a ligand bound by the antibody or antibody variant described above. For example, the ligand may bind to receptors selected from the following: receptors of the immunoglobulin (Ig) superfamily, receptors of the TNF receptor superfamily, receptors of the G protein-coupled receptor (GPCR) superfamily, receptors of the tyrosine kinase (TK) receptor superfamily, receptors of the ligand-gated (LG) superfamily, receptors of the chemokine receptor superfamily, receptors of the IL-1 / Toll-like receptor (TLR) superfamily, and receptors of the cytokine receptor superfamily. In one exemplary embodiment, the binding site of the receptor-binding portion of the ligand is derived from a ligand belonging to the aforementioned TNF ligand superfamily (e.g., CD40L).

[0253] Growth factors or their receptors (or their receptor-binding or ligand-binding portions) may be incorporated into the fusion protein of the present invention. Exemplary growth factors include vascular endothelial growth factor (VEGF) and its isoforms (US Patent No. 5,194,596); fibroblast growth factor (FGF), including aFGF and bFGF; atrial natriuretic factor (ANF); liver growth factor (HGF; US Patent Nos. 5,227,158 and 6,099,841); neurotrophic factors such as bone-derived neurotrophic factor (BDNF); glial cell 1-derived neurotrophic factor ligands (e.g., GDNF, neurotrophin, artemin, and peripheral trophic factor); neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF) (US Patent Nos. 4,889,919, 4,845,075, 5,910,574, and 5,877,016); and transforming growth factor (TGF) such as TGF-α and TGF-β. (WO 90 / 14359); bone-inducing factors, including bone morphogenetic protein (BMP); insulin-like growth factor-I and-II (IGF-I and IGF-II; US Patent Nos. 6,403,764 and 6,506,874); erythropoietin (EPO); thrombopoietin (TPO); stem cell factor (SCF); thrombopoietin (TPO, c-Mpl ligand) and Wnt peptide (US Patent No. 6,159,462).

[0254] Exemplary growth factor receptors that can be used as the therapeutic component of the present invention include EGF receptors; VEGF receptors (e.g., Flt1 or Flk1 / KDR); PDGF receptors (WO 90 / 14425); HGF receptors (US Patent Nos. 5,648,273 and 5,686,292); and neurotrophic receptors, including low-affinity receptors (LNGFRs) that bind NGF, BDNF, and NT-3, also known as p75NTR or p75, and high-affinity receptors that are members of the receptor tyrosine kinase trk family (e.g., trkA, trkB (EP 455,460), trkC (EP 522,530)).

[0255] III.C.6 . heterodimer receptor

[0256] In one embodiment, a β-specific determinant component, when combined with a cytokine, binds to a first β-signaling component to form a nonfunctional intermediate. This intermediate, which then binds to a second β-signaling component, induces β-receptor dimerization and subsequent signal transduction. This cytokine antagonist can be prepared using the methods of the present invention. Such molecules are described in the art (see, for example, U.S. Patent 6,927,044). In one example, a heterodimer is formed by binding a soluble, specific determinant component of the receptor to the extracellular domain of a first β-signaling component of the cytokine receptor, which binds to a cytokine to form a nonfunctional complex. Exemplary cytokines that can be inhibited by the heterodimer receptor include: IL-1, IL-2, IL-3, IL-4, IL-5, IL-11, IL-15, GMCSF, LIF, INFα, and TGFβ.

[0257] III.D Molecules containing polynucleotides

[0258] The molecules of this disclosure may also comprise polynucleotides (e.g., oligomers). In some embodiments, the nucleotide sequence encodes any polypeptide disclosed in Sections III.A, III.B, and III.C.1-III.C.5 above. In some embodiments, the nucleotide sequence binds to or hybridizes to a nucleic acid sequence (DNA or RNA, such as pre-mRNA or mRNA) encoding one or more polypeptides disclosed in Sections III.A., III.B., and III.C.1-III.C.5 above. The term "nucleotide sequence" herein means a molecule in which more than two nucleotides are linked together as a sequence. In one embodiment, the nucleotide sequence of this disclosure is DNA. In another embodiment, the nucleotide sequence of this disclosure is RNA. In other embodiments, the nucleotide sequence of this disclosure is a combination of DNA and RNA. In still other embodiments, the nucleotide sequence of this disclosure includes one or more chemically modified nucleotides. In other embodiments, the nucleotide sequence comprises a length of at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or at least 11 nucleotides. In other embodiments, the nucleotide sequence comprises at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 45 nucleotides, at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides, at least 3000 nucleotides, or at least 4000 nucleotides. In this respect, the nucleotide sequences of the present invention can exert an indirect inhibitory effect on proteins by reducing the mRNA levels typically found in mammalian cells (e.g., human cells, such as neuronal cells). The methods of the present invention can be used to analyze any type of nucleotide sequence. In some embodiments, the nucleotide sequences targeting pre-mRNA or mRNA that are primarily secreted as proteins in neuronal cells are analyzed in relation to selected features discussed elsewhere in this document. Examples of genes that can be targeted by the nucleotide sequences selected by the methods of this invention include, but are not limited to, microtubule-associated protein τ (encoded by the MAPT gene), cerebral acid-soluble protein 1 (encoded by the BASP1 gene), or amyloid precursor protein (encoded by the APP gene).In some embodiments, the nucleotide sequence used in the method of the present invention is an oligomer.

[0259] III.D.1. Oligomers (Antisense Oligonucleotides)

[0260] In some embodiments, the therapeutic molecule that can be used in this invention is an oligomer. The oligomer has a nucleotide sequence of 10–50, for example 10–30 nucleotides in length, comprising a continuous nucleotide sequence of a total of 10–30 nucleotides.

[0261] In some implementations, the oligomer targets microtubule-associated protein τ (MAPT). In disease-related pathological states, MAPT is also known as neurofibrillary tangles or paired helical fiber-τ (PHF-τ). The sequence of the MAPT gene can be found under publicly available accession number NC_000017.11, and the sequence of the MAPT pre-mRNA transcript can be found under publicly available accession number NG_007398. The sequence of the τ protein can be found under publicly available accession numbers: P10636, P18518, Q14799, Q15549, Q15550, Q15551, Q1RMF6, Q53YB1, Q5CZI7, Q5XWF0, Q6QT54, Q9UDJ3, Q9UMH0, and Q9UQ96, each of which is incorporated herein by reference in its entirety. Natural variants of the MAPT gene product are known. For example, natural variants of the τ protein may contain one or more amino acid substitutions selected from the following: R5H, R5L, D285N, V289A, K574T, L583V, G589V, N596K, N613H, P618L, P618S, G620V, S622N, K634M, S637F, V654M, E659V, K686I, G706R, R723W, or any combination thereof. Therefore, the oligomers of the present invention can be designed to reduce or inhibit the expression of natural variants of the τ protein. The τ protein sequence is provided in SEQ ID NO: 1, and the nucleotide sequence is provided in SEQ ID NO: 2.

[0262] In some implementations, the oligomer targets the pre-mRNA or mRNA encoding brain acid-soluble protein 1 (BASP1). BASP1 is also known as a 22 kDa neuronal tissue-rich acidic protein, neuronal axon membrane protein NAP-22, NAP22, CAP-23, NAP-22, CAP23, or neuronal tissue-rich acidic protein. The BASP1 gene encodes a membrane-bound protein with several transient phosphorylation sites and a PEST motif. The conservation of PEST sequences among different species supports its functional importance. PEST sequences are typically found in proteins with high permeability. The immunological properties of this protein are species-specific. This protein also undergoes N-terminal myristoylation.

[0263] Another example of the target nucleic acid sequence for oligomers is BASP1 pre-mRNA or BASP1 mRNA. The BASP1 cDNA corresponding to BASP1 mRNA is named GenBank accession number NM_006317.4.

[0264] In some implementations, therapeutic molecules (e.g., oligomers) target the pre-mRNA encoding amyloid precursor protein (APP). APP is an essential membrane protein secreted in many tissues and concentrated in the synapses of neurons. Its functions involve acting as a regulator of synapse formation, neuroplasticity, and iron output. APP is best known as a precursor molecule of β-amyloid (Aβ), which is produced by its proteolytic breakdown. β-amyloid is a 37-49 amino acid peptide, and its fibrillar form is a major component of amyloid plaques found in the brains of Alzheimer's disease patients.

[0265] In humans, the APP gene is located on chromosome 21 and contains 18 exons spanning 290 kilobases. Several alternative splicing isoforms of APP have been found in humans, ranging in length from 365 to 770 amino acids, some of which are preferentially expressed in neurons; variations in the ratio of neurons to these isoforms are associated with Alzheimer's dementia. Mutations in key regions of amyloid precursor proteins, including the region that produces amyloid β (Aβ), contribute to familial susceptibility to Alzheimer's dementia. For example, several mutations outside the Aβ region found to be associated with familial Alzheimer's dementia significantly increase Aβ production.

[0266] Another example of an oligomeric target nucleic acid sequence is APP pre-mRNA or APP mRNA. The APP cDNA corresponding to APP mRNA is named GenBank accession number Y00264.

[0267] In some embodiments, the method of the present invention is used to select any therapeutic molecule comprising a nucleotide sequence (e.g., an oligomer) that hybridizes with a region of a MAPT transcript (e.g., SEQ ID NO: 2 (which may be mRNA if “t” is replaced with “u”), a BASP1 transcript, or an APP transcript).

[0268] In one embodiment, any therapeutic molecule comprising a nucleotide sequence (e.g., an oligomer) targeting certain regions of pre-mRNA or mRNA encoding MAPT, BASP1, or APP is prepared for testing its toxicity. The therapeutic molecule comprising the nucleotide sequence can then be subjected to the methods of the invention described elsewhere herein. In some embodiments, examples of oligomers (i.e., antisense oligonucleotides) include, but are not limited to, those listed below. Figure 4 and 5 Oligomers.

[0269] The oligomer may include any oligomer design, such as a nucleotide sugar modified form. In one embodiment, the oligomer includes at least one modified nucleoside, such as at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen modified nucleosides.

[0270] In one embodiment, the oligomer of the present invention comprises modifications independently selected from the three types of modifications (modified sugars, modified bases, and modified nucleoside bonds) or combinations thereof.

[0271] In yet another embodiment, the oligonucleotide contains at least one modified internucleotide bond. In other embodiments, the internucleotide bonds within the continuous nucleotide sequence are phosphate thioester or boronic phosphate internucleotide bonds.

[0272] In some embodiments, the oligomers of the present invention comprise at least one LNA unit or at least one 2'-substituted modified nucleoside.

[0273] The oligomers of the present invention may comprise a nucleotide sequence containing both nucleotides and nucleotide analogs, and may be in the form of a gapmer, blockmer, mixmer, headmer, tailmer, or totalmer. Examples of configurations of gapmers, blockmers, mixmers, headmers, tailmers, or totalmers that may be used with the oligomers of the present invention are described in U.S. Patent Application Publication No. 2012 / 0322851.

[0274] The nucleotides or their sequential nucleotide sequences of the oligomers of the present invention can be coupled by a linker. Each nucleotide is suitably linked to a 3' adjacent nucleotide by a linker. Suitable internucleotide bonds include those listed in WO2007 / 031091, such as the internucleotide bonds in the first paragraph on page 34 of WO2007 / 031091.

[0275] U.S. Publication No. 2011 / 0130441, published on June 2, 2011, mentions oligomeric compounds having at least one bicyclic nucleoside linked to the 3' or 5' end via a neutral nucleoside internucleotide bond. The oligomers of the present invention may therefore have at least one bicyclic nucleoside linked to the 3' or 5' end via a neutral nucleoside internucleotide bond, such as one or more triphosphates, methylphosphonates, MMI, amide-3, formacetal, or thiomethylacetal. The remaining bonds may be thiophosphates.

[0276] In this document, the term "conjugate" is intended to refer to a heterologous molecule formed by covalently or non-covalently linking ("conjugate") an oligomer described herein with one or more nonnucleotide or nonpolynucleotide moieties. Examples of nonnucleotide or nonpolynucleotide moieties include macromolecules such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically, proteins may be antibodies against target proteins. In some embodiments, a typical polymer is polyethylene glycol.

[0277] Therefore, in various embodiments, the oligomers of the present invention comprise both a polynucleotide region typically consisting of a continuous sequence of nucleotides and an additional non-nucleotide region. When referring to the oligomers of the present invention comprising a continuous nucleotide sequence, the compound may contain a non-nucleotide component, such as a conjugate component.

[0278] The present invention also provides conjugates comprising the oligomers of the present invention described herein and at least one nonnucleotide or nonpolynucleotide moiety covalently linked to said oligomer. Thus, in various embodiments in which the oligomers of the present invention comprise the specified nucleic acid or nucleotide sequences disclosed herein, the compounds may also comprise at least one nonnucleotide or nonpolynucleotide moiety covalently linked to said oligomer (e.g., not comprising one or more nucleotides or nucleotide analogs).

[0279] Conjugation (with the conjugated portion) can enhance the activity, cellular distribution, or cellular uptake of the oligomers of the present invention. The portion includes, but is not limited to, antibodies, peptides, lipid portions such as cholesterol portions, bile acids, and thioethers.

[0280] The oligomers of the present invention can also be conjugated with active pharmaceutical substances, such as aspirin, ibuprofen, sulfonamides, antidiabetic drugs, antibacterial drugs, or antibiotics.

[0281] In some implementations, the conjugated portion is a sterol, such as cholesterol.

[0282] IV. Pharmaceutical Compositions and Routes of Administration

[0283] The therapeutic molecules of the present invention can be used in pharmaceutical formulations and compositions. The compositions preferably contain pharmaceutically acceptable diluents, carriers, salts, or excipients.

[0284] The therapeutic molecules of the present invention may be contained in a unit formulation, such as in a pharmaceutically acceptable carrier or diluent, in an amount sufficient to deliver a therapeutically effective dose to a patient without causing serious side effects in the treated patient. However, in some forms of treatment, serious side effects may be acceptable in relation to the positive outcomes of the treatment.

[0285] Formulated pharmaceutical products may contain pharmaceutically acceptable binders and excipients. Capsules, tablets, or pills may contain, for example, compounds such as: microcrystalline cellulose, gum, or gelatin as binders; starch or lactose as excipients; stearates as lubricants; and various sweeteners or flavoring agents. For capsules, the dosage unit may contain a liquid carrier such as fatty oil. Similarly, sugar coating materials or enteric solvents may be part of the dosage unit. Therapeutic molecular formulations may also be emulsions of active pharmaceutical ingredients and lipids that form micellar emulsions.

[0286] Depending on whether local or systemic treatment is required and depending on the site of treatment, the pharmaceutical compositions of the present invention can be administered in various ways. Administration can be (a) oral, (b) pulmonary, such as by inhalation or blowing in a powder or aerosol, including via a nebulizer; intratracheal, intranasal, (c) local, including epidermal, percutaneous, ocular, and mucosal delivery, including vaginal and rectal delivery; or (d) parenteral, including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. In one embodiment, the therapeutic molecule is administered intravenously, intraperitoneally, orally, locally, or as a bolus injection or directly to the target organ. In another embodiment, the therapeutic molecule is administered as a bolus injection intrathecally or intraventricularly.

[0287] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, liquids, and powders. Conventional pharmaceutical carriers, aqueous solutions, powders or oily bases, thickeners, etc., may be necessary or desirable. Examples of topical formulations include those in which the oligomers of the present invention are mixed with topical delivery substances such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Compositions and formulations for oral administration include, but are not limited to, powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, soft capsules, small capsules, tablets, or small pieces. Compositions and formulations for parenteral, intrathecal, intraventricular, or intracardiac administration may include sterile aqueous solutions that may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0288] The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from various components, including but not limited to pre-formulated liquids, self-emulsifying solids, and self-emulsifying semi-solids. Drug delivery to target tissues can be enhanced by carrier-mediated delivery, including but not limited to cationic liposomes, cyclodextrins, porphyrin derivatives, branched dendrimers, polyethyleneimine polymers, nanoparticles, and microspheres (Dass CR. JPharm Pharmacol 2002; 54(l):3-27).

[0289] The pharmaceutical formulations of the present invention, readily available in unit dosage forms, can be prepared according to conventional techniques well-known in the pharmaceutical industry. These techniques include the step of mixing the active ingredient with a pharmaceutical carrier or excipient. Generally, the formulation is prepared by thoroughly and uniformly mixing the active ingredient with a liquid carrier or a finely chopped solid carrier, or both, and then shaping it into a product if necessary.

[0290] For parenteral, subcutaneous, intradermal, or topical administration, formulations may include sterile diluents, buffers, tension modifiers, and antimicrobial agents. Therapeutic molecules may be prepared using carriers that prevent degradation or immediate clearance from the body, including implants or microcapsules with controlled-release properties. For intravenous administration, the carrier may be physiological saline or phosphate-buffered saline. International Publication No. WO2007 / 031091 (A2), published on March 22, 2007, further provides suitable pharmaceutically acceptable diluents, carriers, and excipients.

[0291] Unless otherwise stated, the practice of this invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, Sambrook et al., eds. (1989) *Molecular Cloning: A Laboratory Manual* (2nd ed.; Cold Springs Harbor Laboratory Press); Sambrook et al., eds. (1992) *Molecular Cloning: A Laboratory Manual* (Cold Springs Harbor Laboratory, NY); DN Glover, ed. (1985) *DNA Cloning*, Volumes I and II; Gait, ed. (1984) *Oligonucleotide Synthesis*; Mullis et al., US Patent No. 4,683,195; Hames and Higgins, eds. (1984) *Nucleic Acid Hybridization*; Hames and Higgins, ed. (1984) *Transcription and Translation*; Freshney (1987) *Culture of Animal Cells* (Alan R. Liss, Inc.); Immobilized Cells and Enzymes (IRLPress) (1986); Perbal (1984) *A Practical Guide to...* MolecularCloning; monograph Methods In Enzymology (Academic Press, Inc., NYMiller and Calos (eds., 1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al. (eds., Methods In Enzymology, Vol. 154 & 155); Mayer and Walker (eds., 1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell (eds., 1986) Handbook Of Experimental Immunology, Vol. I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooks, Antisensedrug Technology: Principles, strategies and applications, 2. nd Ed. CRC Press (2007) and Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0292] All references cited above, as well as all references mentioned therein, are incorporated into this article in their entirety through citation. Detailed Implementation

[0293] 1. A method for testing or determining the acute neurotoxicity of a molecule in vivo, the method comprising measuring, in vitro, calcium fluctuations in neuronal cells in contact with the molecule.

[0294] 2. The method of implementation scheme 1, wherein the calcium fluctuation in the neuronal cells in contact with the molecule is about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, about 99% or higher, about 100% or higher, about 120% or higher, about 140% or higher, about 160% or higher, about 180% or higher, about 200% or higher, about 220% or higher, about 240% or higher, or about 250% or higher, compared to the calcium fluctuation in the solvent control cells.

[0295] 3. A method for selecting or identifying a molecule with tolerable acute neurotoxicity in vivo, the method comprising measuring calcium fluctuations in neuronal cells in contact with the molecule in vitro, wherein the neuronal cells in contact with the molecule show calcium fluctuations at levels equivalent to or higher than those of solvent control cells.

[0296] 4. The method of embodiment 3, wherein the calcium fluctuation in the neuronal cells in contact with the molecule is approximately 70% or higher, approximately 75% or higher, approximately 80% or higher, approximately 85% or higher, approximately 90% or higher, approximately 95% or higher, approximately 96% or higher, approximately 97% or higher, approximately 98% or higher, approximately 99% or higher, approximately 100% or higher, approximately 120% or higher, approximately 140% or higher, approximately 160% or higher, approximately 180% or higher, approximately 200% or higher, approximately 220% or higher, approximately 240% or higher, or approximately 250% or higher, compared to the calcium fluctuation in the solvent control cells.

[0297] 5. The method of any one of embodiments 1-4, wherein the molecule includes small molecules, polynucleotides, proteins, peptides, or any combination thereof.

[0298] 6. The method of any one of embodiments 1-5, wherein the calcium fluctuation is AMPA receptor-dependent calcium fluctuation.

[0299] 7. The method of any one of embodiments 1-6, wherein the calcium fluctuation is within the Mg... 2+ Measurement in the presence of ions.

[0300] 8. The method of embodiment 1 or 3, wherein the molecule comprises a polynucleotide, the method further comprising calculating a sequence score, wherein the sequence score is calculated by the following formula (I):

[0301] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total nucleotide length (number) of polynucleotides. (I).

[0302] 9. A method for determining acute neurotoxicity in vivo of a molecule containing a polynucleotide, the method comprising calculating a sequence score, wherein the sequence score is calculated by the following formula (I):

[0303] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total nucleotide length (number) of polynucleotides. (I).

[0304] 10. A method for selecting molecules comprising polynucleotides with tolerable acute neurotoxicity in vivo, the method comprising calculating a sequence score using the following formula (I):

[0305] (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) Total nucleotide length (number) of polynucleotides. (I),

[0306] The polynucleotides thereon have a sequence score of 0.2 or higher.

[0307] 11. The method of any one of embodiments 1-10, wherein the method further comprises measuring the in vivo tolerance of the molecule.

[0308] 12. The method of implementation scheme 11, wherein the tolerance category is selected from: 1) hyperactivity; 2) decreased activity and arousal; 3) motor dysfunction and / or ataxia; 4) postural and respiratory abnormalities; 5) tremor and / or seizures, and combinations of two or more thereof.

[0309] 13. The method of embodiment 12, wherein the molecules display an in vivo tolerability score with a total value between 0 and 8.

[0310] 14. The method of any one of embodiments 1-13, wherein the method further comprises measuring a behavioral test score of the molecule.

[0311] 15. The method of implementation scheme 14, wherein the behavioral test is a short-term memory test, a spatial learning and memory test, a gait analysis test, or any combination thereof.

[0312] 16. The method of any one of embodiments 1-15, the method further comprising measuring the microtubule strength of the molecule in a neuronal cell culture.

[0313] 17. The method of embodiment 16, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1%.

[0314] 18. The method of any one of embodiments 1-17, wherein when the molecule is administered to laboratory animals, more than 20% of the animals survive.

[0315] 19. A molecule selected by any of the methods in implementation schemes 1-18.

[0316] 20. A method for treating a disease or condition, said method comprising administering the molecule of embodiment 19.

[0317] The following examples are provided by way of illustration rather than limitation. Example

[0318] Example 1: Molecular Construction

[0319] Various molecules (e.g., oligomers) were designed to target the 3' UTR of MAPT pre-mRNA. For example, oligomers were constructed to target nucleotides 134,821-138,940 and 72,802-73,072 of SEQ ID NO: 2. Exemplary sequences of the oligomers are described in [reference needed]. Figure 4 , 5 And 6. In some embodiments, the oligomer is designed as a gapmer or mixmer. The same approach can be applied to any other sequence disclosed herein. The gapmer is constructed to contain an LNA (uppercase letter), such as a β-deoxy LNA located at the 5' and 3' ends, and to contain a phosphate thioester backbone, but the LNA can be replaced by any other nucleotide analogue, and the backbone can be other backbone types (e.g., phosphodiester bonds, phosphotriester bonds, methylphosphonate bonds, aminophosphate bonds, or combinations thereof).

[0320] Oligomers were synthesized using methods well known in the art. Exemplary methods for preparing such oligomers are described in Barciszewski et al., Chapter 10 – "Locked Nucleic Acid Aptamers", in Nucleic Acid and Peptide Aptamers: Methods and Protocols, vol. 535, Gunter Mayer (ed.) (2009).

[0321] Example 2: Spontaneous calcium fluctuation measurement of antisense oligonucleotides

[0322] To measure spontaneous calcium fluctuations in primary cortical neurons, primary rat cortical neurons were prepared from Sprague-Dawley rat embryos (E19). Cells were seeded at 25,000 cells / well in 25 µl / well Neurobasal medium on 384-well poly-D-lysine-coated FLIPR plates (Greiner Bio-One), containing B27 supplementation and 2 mM glutamine (day 1, DIV1). Cells were grown at 37°C in 5% CO2 for 11 days, with an additional 25 µl of medium provided on day 4 (“DIV04”) and day 8 (“DIV08”) for use on day 11 (“DIV11”). On experimental days, the medium was removed from the plates, and cells were washed once with 50 µl / well of 37°C assay buffer (containing 2 mM CaCl2 and 10 mM Hopes pH 7.4 Hank balanced salt solution). Fluctuations were tested with or without 1 mM MgCl2. Figure 1Cells were loaded with the cell-permanent fluorescent calcium dye, fluo-4 AM (Life Technologies). 2.5 mm of Fluo-4 AM was prepared in DMSO containing 20% ​​plutonic F-127 and then diluted 1:1000 in assay buffer. Cells were incubated with 20 µl of 2.5 µM fluo-4 AM at 37°C in 5% CO2 for 1 hour. After 1 hour, 20 µl of room temperature assay buffer was added, and the cells were allowed to equilibrate to room temperature for an additional 10 minutes before being placed in a fluorescence imaging plate reader (FLIPR). The baseline signal (measuring intracellular calcium) was read for 100 seconds (1 read / second) before adding the antisense oligomer. The oligomer was added to the FLIPR containing 20 µl of 75 µM assay buffer to a final concentration of 25 µM using a 384-well head. The FLIPR signal was read for another 200 seconds (1 read / second) after adding the oligomer. At the second 5 minutes following the addition, plate readings on the FLIPR were performed (300 times, 1-second points) to allow for additional data capture. The raw data from the 5-minute readings were output using Excel, and the peak amplitude and frequency were calculated. These were calculated by measuring the average FLIPR signal over 300 seconds of readings from the control (untreated) wells. For the treated wells, a scoring system was developed that assigned a score of 1 for each 1-second reading when the signal enhancement was greater than 50% of the average control value (calculated above). Readings with enhancement less than 50% of the average control value were assigned a score of 0 for each 1-second reading. For each treatment, a total score was calculated and converted to a control % for plotting purposes. If antisense oligomers produced greater calcium fluctuations than untreated cells, the percentage of control was expressed as greater than 100%. Figure 4 ).

[0323] As previously described, the effect of oligomers on spontaneous calcium fluctuations in primary neurons was measured under both the presence and absence of 1 mM MgCl2 (Murphy et al., 1992). J. Neurosci (12:4834-4845). The same procedure was performed for isolated N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated calcium fluctuations. Figure 1 The data provided show that the addition of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 3µM) reduced calcium fluctuations by up to 20% of the total AMPA response in the assay. Figure 1 (As indicated by the AMPA-marked lines). When (NMDA) receptor function was blocked by 1 mM MgCl2, calcium fluctuations were further reduced by approximately 80%. Figure 1 (NMDA marker lines are shown).

[0324] Evaluation of antisense oligomer inhibition of spontaneous calcium fluctuations mediated by NMDA or AMPA in the presence or absence of 1 mM MgCl2 (representing 100% control in each case); Figure 2 Adding 25 μM antisense oligomers (ASO) inhibits AMPA receptor-mediated fluctuations rather than NMDA receptor-mediated fluctuations. Figure 2 ASO and other similar oligomers showed negative effects on in vivo central nervous system (CNS) network activity and in vitro electrophysiological spontaneous neuronal activity (data not shown). The effects of τ-antisense oligonucleotides on spontaneous calcium fluctuations in primary neurons are summarized in... Figure 4 See Murphy et al. J. Neurosci. 12, 4834 – 4845 (1992).

[0325] The oligomers of this invention were used to measure a reduction in calcium fluctuations in neuronal cells, and this was compared with calcium fluctuations in control cells (i.e., neuronal cells not treated with oligomers). The effect of τ-antisense oligonucleotides on spontaneous calcium fluctuations in primary neurons is described in [link to relevant documentation]. Figure 4 Oligomers in neurons that exhibited AMPA-mediated fluctuations equal to or greater than 75% of the calcium fluctuations in untreated control cells were selected for further analysis.

[0326] Example 3: Measurement of calcium fluctuations using small molecules

[0327] The effect of small molecules on calcium fluctuations can be measured using essentially the same method provided in Example 2. To measure spontaneous calcium fluctuations in primary cortical neurons, primary rat cortical neurons can be prepared. Cells can be seeded and allowed to grow until appropriate for use. As described in Example 2, the effect of small molecules on spontaneous calcium fluctuations in primary neurons can be measured, as previously described, under both the presence and absence of 1 mg MgCl2 (Murphy et al., 1992). J. Neurosci (12:4834-4845). Cells can be loaded with a permanently fluorescent calcium dye. Cells can be co-incubated and equilibrated to room temperature to measure fluorescence intensity. Raw data can be output, and peak amplitude and frequency can be calculated.

[0328] This study can evaluate the inhibition of spontaneous calcium fluctuations mediated by NMDA or AMPA. The addition of the small molecule will inhibit AMPA receptor-mediated fluctuations. The negative effects of the small molecule, which reduces calcium fluctuations to below 70% of control levels, are expected to impact in vivo CNS network activity and in vitro electrophysiological spontaneous neuronal activity.

[0329] Example 4: Measurement of calcium fluctuations using therapeutic proteins

[0330] The effects of therapeutic proteins, such as antibodies or their antigen-binding fragments, fusion proteins, cytokines, cell surface receptors, hormones, or growth factors, on calcium fluctuations can be measured using essentially the same methods provided in Example 2. To measure spontaneous calcium fluctuations in primary cortical neurons, primary rat cortical neurons can be prepared. Cells can be seeded and allowed to grow for use at an appropriate date. As described in Example 2, the effects of therapeutic proteins on spontaneous calcium fluctuations in primary neurons can be measured under both the presence and absence of 1 mM MgCl2, as previously described (Murphy et al., 1992, J. Neurosci. 12:4834-4845). Cells can be loaded with a cellular permanent fluorescent calcium dye. Cells can be incubated and equilibrated to room temperature to measure fluorescence intensity. Raw data can be output, and peak amplitude and frequency can be calculated.

[0331] Therapeutic proteins can be used to evaluate the inhibition of spontaneous calcium fluctuations mediated by NMDA or AMPA. The addition of therapeutic proteins will inhibit AMPA receptor-mediated fluctuations. Therapeutic proteins that reduce calcium fluctuations to below 70% of control levels are expected to negatively impact in vivo CNS network activity and in vitro electrophysiological spontaneous neuronal activity.

[0332] Example 5: Sequence Score Calculation

[0333] Sequence scores were calculated for each oligomer to predict its fitness. The sequence score is a mathematical calculation for all oligomer assays and is based on the percentage of G and C nucleotides or their analogues within the specified oligomer sequence. The following formula was applied to all oligomers to calculate the sequence score:

[0334] .

[0335] For the oligomer ASO-000013 (SEQ ID NO: 686; sequence score 0.25), an example calculation is given: ATTtccaaattcaCTT: 4-0 / 16= 0.25 sequence score.

[0336] The sequence score of the selected oligomer was calculated for further research. To determine the cutoff value of the sequence score, an in vivo tolerability study was conducted as shown in Example 6.

[0337] Example 6: In vivo tolerance

[0338] The in vivo tolerance of the oligomers was tested to see how well the animals tolerated them when they were injected with the oligomers.

[0339] Subjects

[0340] In vivo tolerance of the oligomers was tested in mice and rats. Animals used for T qPCR and behavioral studies were 3–4 adult female C57Bl / 6J mice (20–30 g; Jackson Laboratories, Bar Harbor, ME) per cage. Animals were kept in a group chamber maintained at a constant temperature (21 ± 2 °C) and humidity (50 ± 10%) with 12 hours of lighting per day (lights on at 06:00). In some cases, transgenic mice expressing the τ gene from the human PAC,H1 haplotype driven by the τ promoter (Polydoro et al., J. Neurosci. (2009) 29(34): 10741–9), and in which male and female transgenic mice (30–40 g) with natural mouse τ gene deletion were used to evaluate pharmacodynamic endpoints and tissue drug concentrations. For intrathecal infusion studies, female Sprague-Dawley rats (180–225 g at test; Harlan) were housed individually in a group room maintained at a constant temperature (21 ± 2 °C) and humidity (50 ± 10%) with 12 hours of lighting per day (lights on at 06:00). All animals had free access to food and water throughout the study. Behavioral studies were conducted between 07:00 and 15:00. Animals were cared for according to the guidelines of the Bristol-Myers Squibb Animal Management and Use Committee and the "Laboratory Animal Management and Use Guidelines" published by the National Institutes of Health. The study protocol was approved by the Bristol-Myers Squibb Animal Management and Use Committee.

[0341] Administration route—intraventricular or intrathecal injection.

[0342] Through the ventricles ( icvThe oligomer was administered to mice via injection or intrathecal injection. Intraventricular injection was performed using a Hamilton microinjector equipped with a 27 or 30 gauge needle, following the method of Haley and McCormick. The needle was fitted with a polyethylene sheath 2.5 mm from the tip to limit its penetration into the brain. Mice were anesthetized with isoflurane (1.5–4%). The mouse, weighing 20–30 g, was held by loosening the skin at the back of the neck with the thumb and forefinger of one hand. Gentle but firm pressure was applied, and the animal’s head was then secured by pressing against a firm, flat, horizontal surface. The needle tip was then inserted into the scalp and skull, approximately 1 mm lateral to the anterior fontanelle and 1 mm posteriorly. Once the needle was positioned, the antisense oligonucleotide in 5 μL of saline solution was injected into the right (or left) lateral ventricle over 20–30 seconds. The needle was left in place for 10 seconds before being removed. This procedure does not require surgery or incision. The animal was kept warm on a heating pad until it recovered from the procedure. Brain tissue (right anterior cortical region) was collected on dry ice or RNAlater for drug concentration analysis and T qPCR at multiple time points after drug administration (e.g., 1 week to 16 weeks after administration).

[0343] For intrathecal (IT) injection in mice, the animals were held under mild isoflurane anesthesia (1.5–5%). The mouse was held firmly in one hand by a pelvic strap, and a 30G 1 / 2-inch needle attached to a Hamilton syringe was inserted into the tissue between the dorsal surfaces of L5 and L6, perpendicular to the spine. A sudden lateral movement of the tail was observed as the needle entered the subarachnoid space. This reflex was used as an indicator of successful needle placement for IT administration. A volume of 5–10 µL of antisense oligonucleotide was slowly (over approximately 60 seconds) injected into the subarachnoid space.

[0344] For intrathecal injection in rats, the method described by Yaksh and Rudy, Physiol. Behav. (1976) 17(6):1031-6 was used, with surgical implantation of the intrathecal cannula. Rats anesthetized with isoflurane via the cephalic pyramid were mounted in a stereotactic frame. A skin incision was made approximately 3 cm from the line connecting the ear to the midline and extending towards the tail. The muscles connecting to the occipital coronal region of the skull were incised approximately 3 mm laterally on both sides of the midline. Using a retractor or forceps, the muscles were dissected near the tail to expose the cisternaeida at the base of the skull. Fascia and tissue were carefully removed from the cisternaeida. The curved end of a 16-22 gauge needle was used to create a 1-2 mm lateral incision in the cisternaeida. A sterile IT cannula made of polyethylene tubing (PE10 tubing stretched to approximately 1.3 mm outer diameter) is inserted through the incision and carefully advanced through the subarachnoid space near the tail, rotating it between the thumb and forefinger while gently pulling the tail end with the other hand to align it with the bone marrow. If any resistance is encountered, the cannula is slightly pulled back and slowly advanced again. Once the cannula is advanced to the desired area, it is flushed with 20 µL of sterile saline and passed through the cranial tip approximately 1 cm through the skin using a 19-gauge needle. The cannula is then plugged with a pin. Postoperatively, rats are given oral antibiotics for 5 days. At least 5 days postoperatively, a single antisense oligonucleotide injection is diluted in water and delivered in volumes of 10–50 µL at a rate of 10 µL / min using a programmable infusion pump (Knopf). A brief 5 µL saline flush is given immediately before the antisense oligonucleotide delivery, and immediately after the oligonucleotide delivery, a 10 µL saline flush is given at a rate of 10 µL / min to cover the dead volume of the cannula (6–7 µL). Animals were also given 1-2X / week 20ul saline rinses until used in experiments.

[0345] Acute tolerance behavior assessment

[0346] One hour after a single injection of the antisense oligonucleotide ICV or IT, animal behavioral side effects were observed and their severity was scored on a scale of 0 (no side effects) to 20 (convulsions resulting in euthanasia). The tolerance scale was divided into five neurobehavioral categories: 1) hyperactivity, 2) decreased activity and arousal, 3) motor dysfunction / ataxia, 4) postural and respiratory abnormalities, and 5) tremor / convulsions. Each category was scored on a scale of 0-4, with 20 being the worst possible total score. Changes in animal behavior were observed in the rearing cages before being removed for more detailed observation, including measurements of grip strength and righting reflex.

[0347] New Object Recognition

[0348] Short-term recognition memory was measured using a novel object recognition (NOR) task. The NOR test is based on the spontaneous behavior of rodents exploring novel objects over familiar ones (Dodart et al., Neuroreport (1997) 8(5): 1173-8; Ennaceur and Delacour, Behav. Brain Res. (1988) 31(1):47-59). Mice that remembered objects from the training period showed a preference for novel objects from the test period after a 1-hour retention period between the training (T1) and test (T2) periods. For these experiments, animals were treated for 3 days and acclimatized to a test chamber (48 cm x 38 cm x 20 cm) the day before the test period. The test chamber was made of polyethylene and lined with vinyl sheets. On the test day, animals were placed in the rectangular test chamber and allowed to explore two identical objects (7.6 cm high x 5.1 cm wide) for a 15-minute training period. One hour later, the mice were returned to the laboratory for a 10-minute test period, this time with one object they had observed during training and a new object. The objects were thoroughly cleaned with 25% ethanol between training and testing periods and between subjects, and again with a mild detergent at the end of each day. Object exploration was considered complete when the animal's nose pointed at the object. Exploration was recorded using ObjectScan tracking software (Cleversys, Reston, VA). Data was reported as the percentage of time spent exploring the object (i.e., new time / new + familiar time * 100).

[0349] Morris Water Maze

[0350] Spatial learning and memory were evaluated using the Morris water maze test (Morris J. Neurosci. (1984) 11(1):47-60). The water maze consisted of a pool of water with a diameter of 120 cm. White, non-toxic wall paint was used to make the water opaque (20℃±1). The pool was surrounded by different maze markings.

[0351] Prior to hidden platform training, mice were exposed to a water maze pool by allowing all mice to swim beneath a rectangular passage during two pre-training trials. An escape platform was placed in the center of the passage. If a mouse could not find and climb onto the platform during a 60-second trial, it was led to the platform and allowed to sit for a maximum of 10 seconds. After pre-training, mice underwent hidden platform training, during which a 10x10 cm platform was submerged 1.5 cm below the surface. The platform location remained the same throughout the training, while the descent position was randomly changed in four daily trials and between four days of training. Mice received two sessions per day for four consecutive days. Each session consisted of two trials with a 10-minute interval between trials. The maximum allowed time for each trial was 60 seconds. If a mouse could not find or climb onto the platform, it was led to it by the experimenter. After each training trial, all mice were allowed to sit on the platform for 10 seconds.

[0352] For the exploration test, the platform was removed, and each mouse was allowed to swim for 60 seconds. The descent position for the exploration test was 180° from the platform position used during the hidden platform training. After 60 seconds, the mouse was led to the platform position before being retrieved from the pool. For early memory retrieval, the mice were explored 2 hours after the last hidden platform training session; long-term memory recall was evaluated 16 hours after the last hidden platform training session. Two hours after the 16-hour exploration test, all mice underwent visible platform training, in which local markers (using Lego-built poles) were placed on the hidden platform. The mice were given two training sessions. All behavior was recorded using a video tracking system (Cleversys Inc.). Escape latency, distance traveled, swimming path, swimming speed, and platform intersections were automatically recorded for subsequent analysis.

[0353] Catwalk

[0354] Catwalk (Noldus, Netherlands) is an automated and computerized gait analysis technique that allows for the objective and quantitative measurement of multiple static and dynamic gait parameters. Mice are placed at one end of the catwalk and allowed to explore freely for 3 minutes or until they have performed 5 compliance tests, whichever occurs first. Data are reported and categorized using Catwalk software. The mean of the categorized tests is used for data analysis. Target metrics include, but are not limited to: the distance between the imprint location or hind paw position and the anterior position of the ipsilateral forepaw, the start and end of bipedal stance, paw swing speed, and the duration of paw contact with the glass plate during a paw stance or walking cycle.

[0355] Behavioral statistics

[0356] Statistical analysis of all behavioral tests was performed using GraphPad Prism (GraphPad Software, Inc., LaJolla, CA). For NOR, paired t-tests were used for within-group analysis or ANOVA followed by Dunnett's post-hoc test was used for between-group analysis. For MWM, repeated MWM ANOVA analysis was used during the data collection phase, and univariate ANOVA followed by Dunnett's post-hoc test was used for probe analysis.

[0357] result

[0358] The acute in vivo tolerance of the oligomers, as determined by the above assay method, is shown in the figure. Figure 5 The cumulative tolerance threshold in vivo was set to 4 after ICV injection of 100 μg oligomers.

[0359] Furthermore, the correlation between sequence scores of each oligomer and the acute tolerability of the oligomers in vivo was investigated. Correlation analysis showed that oligomers with an in vivo tolerability lower than 4 tended to have sequence scores equal to or higher than 0.2. See also Figure 3 .therefore, Figure 3 Sequence scores representing oligomers can be used to predict in vivo tolerance to oligomers.

[0360] Example 7: In vitro reduction of τ protein

[0361] In response to the expression of a complete person MAPT The gene, in mouse primary neurons, has the ability to reduce τ protein as a transgenic rod granule, targeting... MAPT The oligomers of the 3' UTR of the transcript were tested (C57-bl6 BAC-TghTau; Polydoro et al.). J. Neurosci. (2009) 29 (34): 10747-9). Primary hTau mouse embryonic forebrain neuron cultures do not express endogenous mouse τ because mouse τ is knocked out. Primary neurons were generated by papain digestion according to the manufacturer's protocol (Worthington Biochemical Corporation, LK0031050). In short, from mice with MAPT failure expressing intact human microtubule-associated protein T (T)... MAPTForebrains were excised from hTau mouse E18 BAC-Tg embryos carrying the α gene and incubated in papain / DNase / Earle balanced salt solution (EBSS) at 37°C for 30–45 min. After grinding and centrifugation of the cell pellet, the reaction was terminated by incubation with EBSS containing protease inhibitors, bovine serum albumin (BSA), and DNase. Cells were ground and washed with Neurobasal (NB, Invitrogen) supplemented with 2% B-27, 100 μg / ml penicillin, 85 μg / ml streptomycin, and 0.5 mM glutamine. Cells were seeded at 15,000 cells / well in poly-D-lysine-coated 96-well optical imaging plates (BD Biosciences) supplemented with NB medium.

[0362] In obtaining the expresser MAPTAfter primary hTau mouse embryonic forebrain neuron cultures were obtained, the cultures were treated with oligomers to inhibit τ mRNA and protein expression. Immunocytochemistry and imaging were then performed to measure the inhibition. One day post-inoculation (DIV 1), half of the neurobasal (NB) supplemented medium from the primary hTau mouse embryonic forebrain neuron cultures was removed and replaced with NB supplemented medium containing varying concentrations of LNA oligomers. The primary hTau neuron cultures were cultured with LNA oligomers until day 13 post-inoculation (DIV 13). At DIV 13, the cultures were washed with calcium- and magnesium-deficient Dulbecco phosphate-buffered saline (DPBS, Invitrogen) and fixed for 15 minutes in 4% paraformaldehyde / 4% sucrose / DPBS. The cultures were then washed and permeabilized in DPBS containing 0.1% Triton X-100 (TX-100) and 3% BSA at room temperature for 1 hour. Cultures were washed and then incubated for 2 hours at room temperature with a first antibody (1:500) in DPBS containing 3% BSA and 0.1% TX-100, Tau5 antibody (to measure τ protein, Invitrogen AHB0042), and a β-III tubulin (TuJ-1) antibody (to measure neurite spurs, Abcam ab41489) (1:500). Cultures were then washed and incubated for 1 hour at room temperature with a second antibody (1:500) in DPBS containing 3% BSA and 0.1% TX-100, Hoeschst 33342 nuclear dye (1:800, Invitrogen), and Alexa Fluor fluorescently conjugated (Invitrogen). Cultures were thoroughly washed and stored in DPBS until imaging. Imaging was performed using the Cellomics VTi automated immunofluorescence imaging system. Briefly, untreated wells were used, and the saturation level of each fluorophore channel was set to 70%. Twelve consecutive images were then acquired from each well, and the total fluorescence intensity and total fluorescence area of ​​τ and TuJ-1 proteins were calculated using Cellomics VTi SpotDetector (version 4) image analysis software. To evaluate the reduction in τ protein resulting from oligomer treatment, the ratio of total Tau5 fluorescence intensity to total TuJ-1 fluorescence area (Tau / TuJ-1) for each well was generated, and all data were then normalized to the mean Tau / TuJ-1 ratio of untreated wells. TuJ-1 intensity was used as an internal standard for each sample. To evaluate neurite / neuronal toxicity from oligomer treatment, the total TuJ-1 fluorescence area for each well was normalized to the mean total TuJ-1 fluorescence area of ​​untreated wells. Nucleus counts for each well were also obtained as an alternative measure of toxicity associated with LNA oligomer treatment. Data are presented as mean ± SD. For immunocytochemistry, data points are presented as mean ± SD from triplicate of treated wells.The efficacy values ​​were generated from wells treated with a wide range of LNA oligomers, from which normalized Tau / Tuj-1 and Tuj-1 values ​​were obtained and compared with normalized values ​​of saline control samples. Analysis was performed using nonlinear regression with the highest and lowest values ​​set as fixed values ​​of 100% and 0%, respectively, where 100% suppression indicates a complete reduction in signal compared to the control sample. Figure 3 For qPCR, data were analyzed using one-way ANOVA and Dunnett's multiple comparison test to compare the saline and LNA oligomer treatment groups. Statistical significance was defined as p < 0.05.

[0363] The reduction of τ protein by individual oligomers was compared with that by saline. The results of the τ protein reduction compared to saline are shown in [Figure number missing]. Figure 6 If the tau protein level in neurons treated with antisense oligonucleotides is equal to or higher than that in control cells, then %inhibition indicates zero inhibition. If present, "ND" indicates "not measured" and "TBD" indicates "to be measured".

[0364] Example 8: Oligomer Preferred Selection

[0365] The properties of the selected oligomers can be described as shown in Table 1. Based on these criteria, certain oligomers are selected for additional in vitro and in vivo dose-response testing.

[0366] Determination method Priority Selection Criteria τ protein decrease τ protein decreased by >70% (5μM oligomers). Calcium fluctuations Calcium fluctuations decreased by <25%. Sequence scoring Sequence score ≥ 0.20

[0367] In other embodiments, oligomers may be selected based on the following characteristics: (1) a reduction of >30% in τ protein (5 μM oligomers); (2) a reduction of <25% in calcium fluctuations; and (3) a sequence score equal to or greater than 0.2. sequence list <110> Bristol-Myers Squibb Roche Copenhagen Innovation Center <120> Methods for selecting therapeutic molecules <130> 3338.035PC03 / ELE / C-K / SAS <150> 62 / 279,610 <151> 2016-01-15 <150> 62 / 112,058 <151> 2015-02-04 <150> 62 / 156,684 <151> 2015-05-04 <160> 805 <170> PatentIn version 3.5 <210> 1 <211> 758 <212> PRT <213> Artificial sequence <220> <223> Microtubule-associated protein tau (Tau) protein sequence <400> 1 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Glu Pro Glu Ser 115 120 125 Gly Lys Val Val Gln Glu Gly Phe Leu Arg Glu Pro Gly Pro Pro Gly 130 135 140 Leu Ser His Gln Leu Met Ser Gly Met Pro Gly Ala Pro Leu Leu Pro 145 150 155 160 Glu Gly Pro Arg Glu Ala Thr Arg Gln Pro Ser Gly Thr Gly Pro Glu 165 170 175 Asp Thr Glu Gly Gly Arg His Ala Pro Glu Leu Leu Lys His Gln Leu 180 185 190 Leu Gly Asp Leu His Gln Glu Gly Pro Pro Leu Lys Gly Ala Gly Gly 195 200 205 Lys Glu Arg Pro Gly Ser Lys Glu Glu Val Asp Glu Asp Arg Asp Val 210 215 220 Asp Glu Ser Ser Pro Gln Asp Ser Pro Pro Ser Lys Ala Ser Pro Ala 225 230 235 240 Gln Asp Gly Arg Pro Pro Gln Thr Ala Ala Arg Glu Ala Thr Ser Ile 245 250 255 Pro Gly Phe Pro Ala Glu Gly Ala Ile Pro Leu Pro Val Asp Phe Leu 260 265 270 Ser Lys Val Ser Thr Glu Ile Pro Ala Ser Glu Pro Asp Gly Pro Ser 275 280 285 Val Gly Arg Ala Lys Gly Gln Asp Ala Pro Leu Glu Phe Thr Phe His 290 295 300 Val Glu Ile Thr Pro Asn Val Gln Lys Glu Gln Ala His Ser Glu Glu 305 310 315 320 His Leu Gly Arg Ala Ala Phe Pro Gly Ala Pro Gly Glu Gly Pro Glu 325 330 335 Ala Arg Gly Pro Ser Leu Gly Glu Asp Thr Lys Glu Ala Asp Leu Pro 340 345 350 Glu Pro Ser Glu Lys Gln Pro Ala Ala Ala Pro Arg Gly Lys Pro Val 355 360 365 Ser Arg Val Pro Gln Leu Lys Ala Arg Met Val Ser Lys Ser Lys Asp 370 375 380 Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Thr Ser Thr Arg Ser Ser 385 390 395 400 Ala Lys Thr Leu Lys Asn Arg Pro Cys Leu Ser Pro Lys His Pro Thr 405 410 415 Pro Gly Ser Ser Asp Pro Leu Ile Gln Pro Ser Ser Pro Ala Val Cys 420 425 430 Pro Glu Pro Pro Ser Ser Pro Lys His Val Ser Ser Val Thr Ser Arg 435 440 445 Thr Gly Ser Ser Gly Ala Lys Glu Met Lys Leu Lys Gly Ala Asp Gly 450 455 460 Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys 465 470 475 480 Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro 485 490 495 Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser 500 505 510 Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg 515 520 525 Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala 530 535 540 Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu 545 550 555 560 Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys 565 570 575 Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly Gly Lys Val 580 585 590 Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys 595 600 605 Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val Gln 610 615 620 Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly 625 630 635 640 Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val 645 650 655 Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly 660 665 670 Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile 675 680 685 Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp 690 695 700 His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr 705 710 715 720 Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met 725 730 735 Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val Ser Ala Ser 740 745 750 Leu Ala Lys Gln Gly Leu 755 <210> 2 <211> 140924 <212> DNA <213> Human (Homo Sapiens) <400> 2 gggattacag gcgtgagcca ccacacccag cccagaatgt ttattagaat gcacaattaa 60 taccagaggc agtggggaag gaaggactga gcagaggagg aagttgagtt gtgattcaac 120 ccaacaactg cctggctggc atggggagct ctggagttaa atagggccat cagactttcc 180 cagtgtgggg ccaacatgac tgggtcttta tacccccacc tctgtcagtc actcaacgtg 240 gtctccctgc aacaaggtga ctcttgcagc cgagacaatc cctgaaggga cagaggctga 300 agcctgtctg ccaacagcac tcccagtggc tggaacaagt ccttccctat aggggaatct 360 gggcggcaca cctccatctc catgtccatc acatacgata tcacagacat ttaaatattt 420 tgataactgt acataagagt ttcctttata atcttataga tcttatttta tgcatttgaa 480 aatattcttc tgagacaggg cttttatcat attgccatag ggtgccacga tataaaaaag 540 gttaaatact ctctgattca gaagtatcca atgatgactt ctctctcatg catttaattg 600 aaaatctggt tttctcctt ctctgctagt tctctacctc tctccccacc tcccacatca 660 tagcctattc acatatgtct gaatctcatg atagacaagt tcaggttctt ttcccaggtt 720 cttttacca catcccccca cccccacata aaaagtatat atggcacagc ctaggttcca 780 cccaaatcct ttctcctctt cttcctgggc ccacaactct cctacataca ttggtatacc 840 ttgcgcttag ggatggccat gtgactaagt tctaacagtg gaacatgatc agatgccact 900 tccagcctct aagacagcca gtgtgtttc tccataagct ccttctcttc ctcccaactg 960 gagactctaa atgatgaccc tgcctcaagc aagcaaacaa caagtccctc aggggtggtg 1020 taggctgcaa atggaaggag cttgagtccc aaaccttcca cggagaaggc tggctaccaa 1080 cctggatcac tcacccaaga ctgctcgaag agttggtttg aaccattgtg ttttggggtc 1140 tatttattac aacagtttag cttgctttgt gaatagattt agtggcagag cctccaaatt 1200 ctatagatac attgatctca gtcctaaccg catctggaac accattaaat aaaggaattg 1260 caaacccaga gaaggtaatg aatttgtcta aggtcataca agatggctag gatcaggacc 1320 caactctcca gttttctttc ttctctgcta ttctgccttc tgtgatccta cataagtggg 1380 catgattgta taacatatgc ggccatgaga tttctctttc agcaagagaa agggacagga 1440 agaaagagag ggaatgcatt ttcttggcct gaattagtgt gagccattag ttacctacat 1500 tgactaaatt atctggaatg aacattcaac tctacatcac atatagttaa aatgacagat 1560 ctgcttaaga ttgtttctag catacgttat ttcaatttag gcaaatgtga ccattcagtg 1620 tgaggggacc atactgtcat taggtccctg tcagttctca attatactgt tatcttagag 1680 ggggaaaaat gtgaaatttg aatgtagacg agtgttgatt tgactgctac agtttatttt 1740 acgtatagaa ataaaataat gtgtagcaaa agcattatta caaagatgat aatgaaataa 1800 ctagtattta taatagtata atagtatagt atttataata gtatgatagt ttaatgacta 1860 tttgtcagat gttgtgtaag aaactttata cacacacaca cacacacctc atttaattcc 1920 tgtatcaatc aggatacagg acgctgtggt aacaactcct caaatctcgg tggcttgcac 1980 aacaaatgct tatttctttt ttttttttga caccaagtct tgctctgtaa caggctggag 2040 tgcaatggtg caatctcggc tcactgcagc ctctgcctcc tgggttcaag cgattctcct 2100 gcctcagtct ctcgagtagc tgggaacaca ggcacgcgcc accacatctg gctaattttt 2160 gtgattttag tagagatggg atttcaccat gttgctcagg ctggccttga actcctgacc 2220 tcaagcgatc cacccacctc agcctcccaa agtgctggga ttacaggcat gagccactgc 2280 gcccagcccc aaatgttat ttcttgctca tgtgacatgt acttcctcga gttttcctt 2340 cctgagatct aagctgaagg aacagctctc tggagccacg ccattctggt ggcggaaagg 2400 aagagtaaaa gtggtagaac cttgcaatgc tcttgaagcg cctatttgga atgtctacat 2460 catgtaaatg gtaatggaca agtatgtata atccccacac caaaaaaagg ggacactatt 2520 ggggacaata accacatttc aatgctgcaa gacggatatt gactgcaccc ccttcccact 2580 ttcagaaaga agaagagtaa tttgctgaa ctccttctag agactggaaa tgtcccttcc 2640 agttggggtg attagggaag gctttggtaa aatttgagct agagtttgaa ggttaggtag 2700 actactggtg ggtgaagaaa gaacaaggac ctttgtaggc aaaggaaaac ctcagaatta 2760 cagaggtgga aaagagttc tagtcaagcc acttcagctg gctacagagt aggtgggaaa 2820 gaaaatggga ggacaagggc tcagatg gggggttggg gcattggggggzgacttgaa 2880 agctaaacta aggggttgaa cttaatttag gaggcagtta gaagcttta catattttg 2940 agcaagagag tgacatatt aaaatgatct gggccaggtg tggtggctca cacctgtaat 3000 cccagcactt tgggaggtg aggagcttgg gtcacctgag gtcaggagat cgagaccagc 3060 ctggccaaca tggtgaatc ccgtcctact aaaaatacaa aattagccg ggagtggtgg 3120 catatgcctg taatcccagt agctgggagg ctgagacagg aaatcgctt gaacccggga 3180 aacaggttgc agtgagccga gatcgtgcca ctgcactcca gcctgggca cagagcgaga 3240 ctccatctca aaaaaaaaaaaaacac aaaaaacc aaaaataat aaaaaatg 3300 atcacttctg atactgatc taactagggg ttgcagggtg ggctgatata gggagaaact 3360 ggagagcaag gagatcacta agtccctac atgtccagaa ccagataga ggtcttgaac 3420 taggatggtg gcagttagaa cacaac aaaagtcaa ttccaggctg agtgcagtgg 3480 ctcatgcttg taatcccac gcttgggag gctgaggtgg gagttagaa gcagcctggg 3540 siacactgca agaccctc tctaaaaaaaaaaaaaaa aaagttagcc aggtgtggtg 3600 gtgcccacct gtagtcccag caactcagaa ggctgaggtg ggaagattgc ttgagcccca 3660 ggagttcaag cttgccgtga gctacgatg tgccactgca ctccagcctg agcagacct 3720 tgtctccaaa aaaaggtcaa ttccactgac tttctagg tgtacaccat caggggcag 3780 ctccatctcc aggccattgg ctcatgagac attctgt cagaagctta gggcagattg 3840 ctttgagcaa gccccatgg tggttcac tcctactct ttggtatat gcccctgt 3900 ttaaaaataa agttaatatg catttaaaaaaaaaggg aaaaggtca gttccagaaa 3960 ctgtgtgaat aaagcatttt acttgcttt tctattaatc tataacatat gttgattttt 4020 taaaaagaat atagagcta tgcaattgg agctcaaga caactccca tctccctagg 4080 aggagatggc tgccttaac cccctacat agaatcatc ccactgctg gggttaact 4140 tgatgttggg gaatgaaaa atccaagcta aggccgaagc ctggggcctg ggcgaccagc 4200 agaatgagga caatctgtca gttcaggct gaggtgcgtc tccagggga caatctctag 4260 ctggccctta aacattcaga cttcaagctc tattacagc ataaggtgt ttcaaagac 4320 gtgatacaaa taactgcaaa tgctctgcga tgtgttaagc actgtttgaa attcgtctaa 4380 tttaagattt ttttctga cgtaacggtt agatcacgt ttcttttt ttaagtacag 4440 ttctactgta ttgtactga gttagcttgc tttaagccga ttgttagg aaggattca 4500 ccttggtcag taacaaaaa ggtgggaaaaaagcaggag aaggaagca gcctggggga 4560 aagagacctt agccaggggg gcggttcgg gactacgaag ggtcggggcg gacggactcg 4620 agggccggcc acgtggagg cgctcagga cttctgtagg aggacacc gccccaggct 4680 gactgaaagt aaagggcagc ggacccagcg gcggagccac tggccttgcc ccgaccccgc 4740 atggcccgaa gaggacacc cacccccaca acgacacacaa gactccaact acaggaggtg 4800 gagaaagcgc gtgcgccacg gaacgcgcgt gcgcgctgcg gtcagcgccg cggcctgagg 4860 cgtagcggga gggggaccgc gaagggcag cgccgagagg aacgagccgg gagacgccgg 4920 acggccgagc ggcagggcgc tcgcgcgcgc ccactagtgg ccggaggaga aggctcccgc 4980 ggaggccgcg ctgcccgccc cctcccctgg ggaggctcgc gttcccgctg ctcgcgcctg 5040 cgccgcccgc cggcctcagg aacgcgccct cttcgccggc gcgcgccctc gcagtcaccg 5100 ccacccacca gctccggcac caacagcagc gccgctgcca ccgcccacct tctgccgccg 5160 ccaccacagc caccttctcc tcctccgctg tcctctcccg tcctcgcctc tgtcgactat 5220 caggtaagcg ccgcggctcc gaaatctgcc tcgccgtccg cctctgtgca cccctgcgcc 5280 gccgcccctc gccctccctc tccgcagact ggggcttcgt gcgccgggca tcggtcgggg 5340 ccaccgcagg gcccctccct gcctcccctg ctcgggggct ggggccaggg cggcctggaa 5400 agggacctga gcaagggatg cacgcacgcg tgagtgcgcg cgtgtgtgtg tgctggaggg 5460 tcttcaccac cagattcgcg cagaccccag gtggaggctg tgccggcagg gtggggcgcg 5520 gcggcggtga cttgggggag ggggctgccc ttcactctcg actgcagcct tttgccgcaa 5580 tgggcgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg gaggggtccg 5640 ataacgaccc ccgaaaccga atctgaaatc cgctgtccct gccgctgttc gccatcagct 5700 ctaagaaaga cgtggatcgg gttctagaaa agatgactcc ctgcacgccc ctccctgcac 5760 ctcccgagca gtgattccga cagggccttc actgcccctg attttaggcg ggggccggcc 5820 ccctcccctt ttcctccttc agaaacccgt aggggacatt tgggggctgg gagaaatcga 5880 ggagatgggg aggggtccac gcgctgtcac tttagttgcc cttccccctg cgcacgcctg 5940 gcacagagac gcgagcagcg ccgtgcctga gaacagtgcg cggatcccac tgtgcacgct 6000 cgcaaaggca gggttcacct ggcctggcga tgtggacgga ctcggcggcc gctggtcccc 6060 gttcgcgggc acgcacagcc gcagccacgc acggatgggc gcggggctgc aggtgcatct 6120 cggggcggat ttctttctca gcgctcggag cgcagggcgc ccggcgtgtg cgctccctgc 6180 cggaggcgcg gggctggcgc gcagggctcg cccctcactg cggcagtggg tgtggaccct 6240 ggtgggcgag gaagggggag gataggctgt gcctcctccc actcccgccc ccagcccccc 6300 tttttttccc cctcggaacg cgaggtgcca tcttttttcg gcgtgtcacg tctttacggt 6360 gccatgccaa accgggtggc cgggcttcat aggacagggc ggggcctggc attaaaggga 6420 gggggacaat cagcgctgaa atcttggcgt tttgctgctg cgggcgtgag cactgggggc 6480 gttcgcccag caccttcttc gggggctctt tgctttgtct gtagaggtta cgtgatctgc 6540 gctcccagcc ctggtttctg gcttttattc tgagggtgtt cagtcaacct cccccctacg 6600 cccatgcgcc tctctttcct ttttcgctcc tcatttccga gcccattgtt ggatctcgag 6660 gcttgctggg ttcgatgaac tcgagtcaac cccccgaccc ccggcacgca tggaacgggc 6720 gtgaccgcgc gcagcctcgt ctcggagtct gccggcgccg ggaagcttct gaagggatgg 6780 gattcgagtc tccgtgcgcg ctgcgggcgg cggcagaggg atctcgcccc tccctacacc 6840 ccaagtgtcc tgagggccac gccacaccag gttgcccagc gagggacgct ggctacccat 6900 ccggggatgg gtggggagcc ctggcggggc ctctccggct ttacgccctg ttgcttcgcc 6960 tggccggaga atgtgaggaa ggggcataag gttactggtg cttcggccac acccatcttt 7020 ctgagcccac tggactgggc gcagaggggg gattgccatg gaaaccacag gtgtccggag 7080 aggggatctt ggggctggcc tcaccccttc cctgcggaga ttggggaccc tggggtaggg 7140 ggagccgcgc ccagtcggcc tcctggagga cacgggagga agccccgaac ccccgcgcct 7200 gaggctgttt ctgattggcc cctggaggcc gcagacacgc agataggcgg ccctgggtgt 7260 atttttatta atattatgtc cgtactgatt aatattattt atcttaaata aatttcaccc 7320 gtgtccaagt tcaccgcgcc cccaaaaccg agtctggggc ggcaggggga actcctggcc 7380 aacgaatcca tgcctcgccc tcctgtgatg aacctggtac gcacggtttt ctggttaatt 7440 ctatcgctga aaactggtgc ggggggcgca cttctgagac ggaagagcat ctaggagctg 7500 aatcctccac gcgggtcgcc caggttgatc tgaatttctg gggaatggct tggctgcccg 7560 cccgggacca ggccgaccct ccttgacggt ggcgtagagg gctggagcct gggtactgcg 7620 aggctcctcg catggctggg cccgccgcga ggggttgcag agcggctcag ggatcgattc 7680 aagcatcgtc tctcctccct cgcccccaga cagagctggg cgcggggttc cccttccaga 7740 tggagcgagg gtctcggggt ggccccggaa aaggggagcc cgcggccacg gctacgtatt 7800 gccatctcgc gagcagagat gtcacctcct gccttttggag gaaagggagc ccggtgggga 7860 tgagcgcatt tagcccaatg ctgggaacaa agcgcactcc gcgcttctgc gatttcgctc cattttgaaa tgtgttggcg ctttggtggg gccgctgcgg tgggcaaggc cggggcgct 7980. gttaatggag gaacctcagg gggacggtcc ttcgtaggaa actctatcct ggctctgcgc gcgctttaag gaaatggctt ccctccagga cctcgaggga tgcagctttt gcgcggatga 8100. cggtggggtg ctgaaccagc cggtgcgcct ctggaaatgt ctgggcacgg atcctggggc 8160 catcgacgac tcctccccat tcccagcagg cgggagctct tacattccga gcgagtgacc cctctcaccc tctggcgctc acacacctgt aactccaaac ctccgtctca gaatggtcca ggctggaagg gatgatgggg gctccgacag cgactgccta gctcacccct ctgcgtgctc 8340 aggctccagg ctcagcagg ccaatttgag ttctatctga tccccctcgg ccccttaact 8460. gacccatcct acaggagaca gggaaatgtc tttcctaccg cggttgattc tggggtgtca ttttgtgttt tgtgatggct gcttatattt actgtataag cattgtattt actgtataag cattgtatta taattactgt ataagctgct tatatttact gtataagcat ctccaaatcc 8580 tccctacg taaacaaatt aatggataaa cagataagtg tatcccctgc ccccacccct 8640 gctacgcagg tccggagtga ctcttgaagc tcatacattc cttggccaag ttgcttc 8700 taacagatgt ttatatagca ataacctggc ttggctcttg ggttcacctt tggacgattt 8760 ggggaagggg cttgttggct ttgctgggtt ttggatgagt gacagtccat gactgttcct 8820 gctggaaggg cgtgactttt aagtggttc tatatcagg cattgctcct ccgacaggaa 8880 caaaagaaat ggatactgcc cataaattgt tagaaaactt agaatcgctt tgattgagga 8940 aaggttagat ttattccggt tggaaaaagt ggcctttcta ttaaacgtgc cctttgaccc 9000 tcatgccctt ggaggtcggt gccagcctgg agatgggata agattgtggt tttccttctg 9060 cctttttaac atctgttgtt acagtccatt tgttgaaaat ttaaagaaac tgttttattc 9120 cactttccct cagcatttat gtgtgtggtt tcagtagctc tgtggctata tgtacgaaca 9180 cgtgttattt ttccaattgg acatgtgata attttccaac tggaccttgc cttctattga 9240 tgtatttatt tagcatcttc cttactccct ccttgaaaaa gaatcactca aaaacaaata 9300 aaaacagccg taggggccta atacagtgct agacatacaa gaggtattcg gtccatacca 9360 aatggatttt atccatgaag gataaatggg gaaatacagt gggaagcagg tgggaaactg 9420 cgtttgactc tgctctttcc tccaccacca ctttcctcat caccgtgttc agagaccccc 9480 aaagccccct cacactccca gaaacacccc cctggccact cctaacttgc catgcccagg 9540 agttaggtgc ttccactagt gacatggagc tggcgtttgg ggggcacctc agcaggtgac 9600 gggaagagaa gaccccagcc tcaccagctg gggctcagca gggagaggag tcctcatgtt 9660 ccagcaggga ctctcagctg ttttcctgta aaaccatggt tctcaactgg gggccactga 9720 gatgtctaga gagatgtttt tgttttcaca actcggggagag ggtgctactg acatcttgtg 9780 ggtagaggcc aggaatgctg ttaaacatcc tacaaggaag gcacaggaca gtctcctaca 9840 tcaaaatatg acccagtccc aatgtcacca ctgctggggt tgacactggc actgctatct 9900 taattacatt cattgagtgt cttttaggag gccctattct aagtgcttgc taagattatc 9960 tcatttaatc ctcacaacac ttccgctatg tagcaggtgc tgttattatc tccgtgatgg 10020 ggaaactgaa gcacagagag ggttagtaac ttgctaaagg tcacagagcc agtgggtggt 10080 ggagctggtt gcctgacact agttccctcc cctctcagcc acatgtgggt ttacttggcc 10140 attgtggact agtctgggaa cccagatatg atctataaca ttgacccagt agaatattga 10200 ttccaaaacc actgtctcac aaatgaattt ttacaagagt ctgtaatcgg agcatgaccc 10260 agaataaggt tagggagatg tggagttaaa gctctcaatt tcttatctgg ccccgacaca 10320 gagagcaagg catttcactc tacattggtg ctctgtttat aaaacaaaga gcaaatatct 10380 cttcctaagg tccttaaacc tcttccccca atccagggtt tctggactgc tctgccatat 10440 gacggggcag ctggtttgat tgacccaggg aaggctggaa atcaagactg ggggatcaag 10500 acgtagattc agtgtggcca aggtcaagtc tctgaggttt agggacatca gatccccagc 10560 ttaggttctg tacctcggca aggtgaaagc gttggcgccc actgatgagg cctgctctga 10620 gattgtgggt gtgggttgag ttgggtgggc ataggcaagt cctcttgtaa gaatcttttg 10680 gcaaagatgg gcctgggagg cttttctcac ttcctggggc ccaggctttg caataagtat 10740 tccattatac tgtggtacct tggggctacc tgagaatcct ctgtctcgcc cctgttgcct 10800 tgccaaagag tttgctgtcc aagaattcct ttcctgtctc caggtgccat gctcctgcca 10860 cctctgccag gttccctgcc tgcccagatg gctcccaact gagtgtgagg aggaatttga 10920 gacaggtttt gagctttctg ggttctccag ttaggaaact ttctgtaagc atgcagatag 10980 aatgggcttc agcaaaatac aaactcgaac aacttccatg tatagtccct taattttctt 11040 tgcttttttc atatttcatc aggctccatg ctgagcccaa tcagggaccc gatagaaatc 11100 caaacaccat gtcagcgagt ccccaagaaa tgcattttgt gccaaggcta ttcaaggaag 11160 gtttgggagc agctcaaggg cagacactgt taccctcccc caggtcccca gtgcagggca 11220 gtgttctgca tgtggaggca gtttggccta atggttaagg aggtaggctc tgatcgggcc 11280 tcctgggcac aaatcccagc tccctgctca ctgtgagacc taagccatat tgtttagctg 11340 cttggagagt tttttgtcat ccacaacttg gagtatgatg gtacctgtct cacgggttgc 11400 catggggttc acacaagcta acccggtact cactagggcc aagcacatag taactgctca 11460 gtaaatggca tcatcggcgg tgtcctgtgg atgagtgctt gtgattggct gaatgaccag 11520 aggggtctaa agatcctggt gatggaatca gttgtacaga taaattgtta cactgagtag 11580 ggatcaagat aggaaaagtc ggcaactacc cagctcccct gcaccaaact gggcagaagt 11640 ggatcctctg aaaattgcac acacccatgt ttaaatgtac acacagaact cttgccacag 11700 gcaagcggag atttgtcatc tgctgtccct gcctcatctt cttcctgaaa tccactccat 11760 gccaggaata aactgcatgc tctccaccag cccaaactga cctgccttcc cgccagccat 11820 cccgggcagg gtgacctggc ttagtacatc gggttcagag atctttccag tttatcgtt 11880 gaataaaaag tgagggctga tcgagaaagt aatggcagtc agggaaggcg aaagggtaa 11940 12000 agatttcagg gtggttggtt gagagtagag tagaaag ttaaaag caaacttgtg 12060 gtgtactgaa tcttaggaat tccatgtatc caataagtat agtcatttat gaattaataa 12120 attcggccta agaagccttc ttatcgctta aatcaagact aagtaacaat atatcagttt 12180 taaaaagtca ttatatcaga aaatcattta aatgatacac atagatttcc aagattttac 12240 tttaaccgaa actatataaa tgtgaatttg ttcacccatc ttttgacaca gggctcaggt 12300 cttctcttgg tgtctggatc agccagttga aatttcttgt ctgttttgcc tatgccacat 12360 taataatgca ctgtctgggt cctccgattt cagtttggat tttgggttta cattgtggag 12420 tcatctgaat gcagaatcct tcagggattt tactttttttttttttttc atggtcttta 12480 ccatcccatt tgatagtaaa tattactcac ctttatgaag tctttccaaa acattcaact 12540 aaattttctt aaaatcattg aatgatttga agagcttatt cctcagcact tttactccat 12600 cagcttgcac cttattttt aatctttttt tgagacggag tctcgctcta tcgcccaggc 12660 ttaagtgcaa tggcgcgatc ttggctcact gcgacctcca cctcctgggt tcaagcaatt 12720 ccgcctcagc ctccgccgta gccgggacta caggtacaca ccataatgct cggctgattt 12780 ttgtattttt gtagggatgg ggtatcgcca tgttggccag gctggtcccg aacttctgac 12840 ccaagtgatc cacccaccctc ggccccca agtgctggga ttacaggtgt gagccaccgc 12900 gcccggccag cttgcacctt atttaggata tgtgattatt atagcaagtc tggtgtacat 12960 acaagatttt gatgggcac agatgacctt tagtaagtgc tggctgtga tagaggcag 13020 tcctgactgc agatcaggct gtgtggaccc cagccttgca tgtttacaga ccttcatgtc 13080 ttattcttac agggtatcag agaacacct actggggaa cttataatt agtaaaggt 13140 gggcattctc cccgcccatc ttctgtctgt ctgccaggac tagcacagca cttgaagtc 13200 attcacatag atcccaact tagaggggta aaatcctcct siacacactg aaataagtt 13260 taaattccct ttgctatatt aactcccctg aggaagagt cttagatcaa tgtccacac 13320 taaaaacagt ttaatcag caagtgagaa ttaaatctga agcaatgat ataatgtttt 13380 cattcattcc tctccttgg cccgtccac cctactgcta aatccaggca tcaaagagaa 13440 gagggacata attatctcta gtcccagctg ctggttttcc ttccagccta tggcccagtt 13500 ttctgtttta ctgagaaggc tggtgatgtt atcttgggat ctaagtctgc agtttcacca 13560 caaaaagtcc agggatgcac tttcatgctt gtgtcctcct ccctgggata gcaaggatat 13620 tagagaaccc ctggctctgt aattgcttgt catgtgctct acagacgcca cagaatgcca 13680 agaacgaagt gctgggaagg acaaattcat ggaaccgtgg gacggtgctc ctcccccagc 13740 gtaaaggaca gctcctcctc ctgaattgga gccagcgttc taaatcatgt gtcaacagag 13800 ttgtcctgga tcggatccag ttctgccatt gatttgcagg tcatttcagt ggtacctgtt 13860 tccagttgtt cttaattgaa cagtggcacc aaactattgt cttgcctcat ccccctccca 13920 tggcctgtcc cccaaaaga gacttcttgg gtaattaatc agggcaacat caggcagtct 13980 gggcgcggtg gctcacgcct gtaatcccag cactttggga ggccgaggcg ggcagatcat 14040 gaggttagga gattgagacc atcctggctt tgtgaaaccc cgtctctact aaaaatacaa 14100 aaaattagcc gggcgtggtg gcgggcgcct gtagtcccag ctactcgaga ggctgaggca 14160 ggggaatggc gtgaacccgg gaggtggagg ttgcagtg ccgagatcgc accactgcac 14220 tctagcctgg gcgacagagc tagactctt ctcaaaaaa aaaaaaaaaa ggaatctctt 14280 tggttttatta tatttttttt fatherfather fatherfather fatherfather fatherfather 14340 daddy daddy daddy daddy daddy daddy daddy 14400 father father father father father father father father father 14460 father father father father father father father father 14520 fatherfather fatherfather fatherfather fatherfather fatherfather 14580 daddy daddy daddy daddy daddy daddy daddy 14640 fatherfather fatherfather fatherfather fatherfather gttafather 14700 fatherfather father father father father father father father father grandfather father father father father father father father 14820 gdaddad daddydad aaaaacatat daddy daddy daddy 14880 great-grandfather great-grandfather great-grandfather great-grandfather great-grandfather 14940 fatherfather father father father father father father aatgagg ccaggctcgg 15000 tggctcacac ttgtaatccc agcactgtgg gaggatcact tgaagccagg agtctgagac 15060 tagcctgggc aaaaaaaa gatcctgtct ctacaaagg aactgtaaa aattagctgg 15120 gcatgatggc atgtgtctgt agccctagct acttgggagg ccgaagcagg aggatcgctt 15180 gagcccagga gttcaggct acagtgagct atgattgtcc catagcactc cagcctgggt 15240 aacacagcaa ggccctgtct ctaaactttt ttttttat tctatttata tttacatgta 15300 tttaaatgtg atattcact acctatttgt tgcatgcctg catttttat actggggcttg 15360 ccaaaaaccc gaacagcttt ctactttgac aatgtatcag atttaatc agcaatgt 15420 taataagcca agcaaggtt atatatgca ataaaactgt tgtctataac ctcctgttac 15480 actggggcac agcaaaagtc atggtgtagt cgcatgtgaa cctgtccctt tcatagctgc 15540 tcattgccag gaaacatcag gaatagccat ttggaagagt catcagccct cccaccatcc 15600 gttttctgtc ttgtcttttc cctatgagca ggggaaattc cacgctggcc ccaatcccca 15660 gtgcagcggc tcagcctctg cctctgctgc tggtccccat gaggccagct tagaaacgga 15720 ggattttgca gaacatccct aaatccgctt gaataatgaa gtgatcattc ataaactcac 15780 ctgaacctta ttaaaccta tttaatattt ttcctggata atcctatagg gataacttgc 15840 ctcctgggct tctctccacc gggttcagtt cttcctttag tggtgaagtt cctcccttct 15900 tagcatctca actgtgcctg agaaaaggcc agtggcggct gcactctgtt ccctgtggag 15960 tgttaataaa gactgaataa attgaaataa atccctttca atgtcattaa gtgctataaa 16020 taatcatgaa ccaatgttcg atggctgatg agaaatgcaa gaaaaaattt ttaatcagta 16080 ggattcataa gttgacaatc tgggccaagt taaaaaaaat aaaaataaaaa agacttttaa 16140 aaagatctta tcgtttgtta ccagtaagac tgaattccag aagcaagcta ctccctcatt 16200 tgtgggcccc tgttatcact ggctgcttag ggttgccaag ccctgaattc atttgtcaac 16260 taagagattt ttggccaaga ttaagatttc ccatgcctcc atatttccat ctgagaaatg 16320 gagattatac tgtcttcccc ctcagaatgg atgataatgt ggtctctctt ctgttcgcat 16380 agtcatagaa ctgaaataaa acaacttaag agaattcctt tgagcttctc agaagtgctg 16440 cagggctggg ggatgcctcc caggagccgc agtcaggtgc tgatctgaag tctttggtgg 16500 gctgacttta gcctgacctg aaatagtata gctgctgcca cctggctccc ttagcgtcag 16560 tcagacggtg cagctggttc ctaggggtga gggctgagcc agcagggtcc gtgcccagga 16620 gggatgcatg ggtggccaca gcccagcctg cactgatctt gtctgtcccc ttctttggaa 16680 ggaaggagcc ccaaaccagg gtgcaagaca gtgggtgggg gtgccttgag catgacctca 16740 agtgatttcc agcccctgcc agtgctgact tctctgggga agggctggga cttccttctg 16800 ggctcaagtc acgacccttg gatggaattt cctgggagct tttctgtttt ttctggagtt 16860 ttcagtttt tcctaaccag acagggactt ggtacagaat ctcatattct aattatgcct 16920 aggagcagcc tctccccacc actcacagtg tttagcatgt gacaggaatc gattaaggca 16980 tgagtgatta attaaagcc aggcattgac ttggatggtg tatattctg acatctgttt 17040 ggtgtcaaag gcacggggca ggcgcgttaa ttgaactgct tgcacctggc atttgaattg 17100 agccagagcg gggctaaagt cagtttgcct tcaccctgta aatggagggt ttctccggag 17160 cgtggatggt gggaggtatt tcagggtgta tgcataaccc ccaccctgac aatggcccat 17220 ctcttctcca gcgtggccag gtttgagtgc cagtcctggg tgtccagtgg ccccatagcc 17280 ttgcgtttta gtaaaatgct gcccccatta ccacctggtc tgtgcacttc ggtcactgga 17340 atttgccatc ttccagtccc gaatgtggca agccatggag ccttaagctc ttctccctcc 17400 acatcctgga acagacccgc cagtttcttc caggcattgc ctcagtttgc ccctctgttt 17460 ccagtcacac tctcaccagc gataaaatga ttttagacct tatcatctca ccctcggatc 17520 cttatggaaa caataatgag ttgttccctg tttcaattcc aaaattcata tccaatccgt 17580 tttgcatgcc attgccaaat tcctcccaga gcaaccccgt cacctgccct ggccctctcc 17640 aagtgtggtc ctgccatggg catcgcctgc taagccaagc tggcctcgag ctgcctgccc 17700 gggtccccac accttggctc acctccctgc ccagtcccgc ctcctgccag cctgccctgt 17760 ggctccttca tagatgccgt gctctttctg ccccttgctc acccatggca gccttgcccc 17820 tctctccctg ccccaccccc tatttaaatt gacctgacct tcctcagtgt ccatcttccc 17880 cgaagctttc cccagccttg gcactcaagg tccagaggct acgcgtttcc tctcacctgt 17940 ggcagcgccg tgctccccag tgcctcacag tttccttctt gcccccgctt cctgtgtagg 18000 actcatctgc ccacaggttg cacgtcctgt gagggcaagg actgtgtctt atgtgacttt 18060 ccttctccag tcacagagct gggcacatag atagctcaaa accctcttta ttaacacagt 18120 tggatgttga gaaatcaaac aggccaatgt caaatgagct ctccttattt aaatcaagtc 18180 agttctccac ctcctagcac tcagttccag tactctatat acatggaaat aataaaaaac 18240 acatttcctt tgaaacattc tataatcgtt cctttgccct acttcagacc aacttaacgc 18300 actccccatt ggtccaaatg agttttgcta tacgaagaatg ctgataataa tagcagcagt 18360 ggattattct gctaaaacca ttgcctcgtt aatcctcagt cccgaggtgg ggattattat 18420 cctcattttg cagagaagca aactgagact cagagatttc acagctgggg agggagccag 18480 ctcatccctc tgtccaggcc caagctctct cccgcttgcc ttcctgcctc tgcaacctca 18540 gagcatcccc catctggttc tactgcctgt gctagtcgtg caggagccaa aagacacgtc 18600 tttagtgcta aggactggag aagccatgcc ctccagcctc tgtgaatggg tcatatgtaa 18660 catgagcctg gagaattat ttgaaaccaa aggcaagcct ctaaaccagg ctgctgcttc 18720 atggcgccgg tgacggcaga accaaattta gtgctgtggg caggtccaca cttatcaaat 18780 agagaagctc attttcttc cggctcacat caagcatgaa aaatgttcac acataccccc 18840 cacacaca tgctttccgg aggggtccat gtggctagag gctggaagat gtggatgaga 18900 ggagcctggc aggtaagccc agggaagatg acattcagct tcccagacag catctacagg 18960 gagaaattta attaaaagtg gggcggtttc cctgagcaag gcagacaaag tcagccctct 19020 actgttaaga aaaagggtca cagtgagagg ggaggtgagg agactgagtc tgtattttct 19080 agtctgttgg gctacactac ctgatccccc ttcctcaaaa atccacttta ctttccccat 19140 gtctacacca atgtggttca cactctggga ccaggaaaag ggggagtgat ggggaacaga 19200 gaagggagga gctcacacag ctgaggctgg ggttatgcat atcgaattac ttagaatttg 19260 caacctcaca gggtactttc atggcgttga aatacacttc ccacagccac cctccctcta 19320 actaaaagca agagtcattt ctcagttctg gtcttgcctc ccacgttctc ctccacattt 19380 aagaaaatcc accagctaca aagtgaagat accatatgtg atatcccacc ctagtttctg 19440 ttttatcagg gtttggagca ggtggagcag gcagagggat catttcagcc tataaattgt 19500 attaagggtg agtactgagt cattcttcaa gaaaagtttt agaagcatcc aaaactgaag 19560 ggtggagcca cctggagaca gtatcatcag tcctggcccc gagcatggcc tgcataggcc 19620 cccatggatc ccagcgggag ctgcagagtg cgggcacctt ggcacacagc cctgagtgca 19680 aaattaggag ctgggcagag ggcatctctc tgtcgccatt gggcagccca gggcacactg 19740 gtcatagcct tagaccacga acaccctgtg cccggggac agatgcaacc agtgtgccct 19800 gggctgccca atggcaacag agagatcgac acctggaccc catgtcacgg ggactccact 19860 actaaggctc ctaagactgc caccttccag tgggataagc cctgcctcct actgggccca 19920 caatgtgcag agaacacttg ggactacctg gctttctgga tacacaaata ttgatccaat 19980 ctggactaat tagaaggtca gtccccaataa caaatcgaag tcagctgggc gtgatggctc 20040 actcctataa tcccagcact ttgggaggct gaggtgggca gatcatttga agccagaagt 20100 tcaagaccag cctgggcaac atagcaaaac cctgtctcta ctaaaatac aaataattag 20160 gctgggtgtg gtggctcatg cctgtaatcc caacagtttg ggaggctgag gcaggtggtc 20220 acctgaggtc aggagtttga gaccagcctg gccaacaggg tgaaaccccg tgtctactaa 20280 aaacataaaa attagccaag catgatggca tgtgcctata atcctggcta ctagggaggc 20340 tgagacagga gagaatcgct tgaatccagg aggtggttgc agtgagctga gatggtgcca 20400 ctgcactcca gcctggttga cagagcaaga ctctgtctca aaaaaaaaaa aaaaaaaaa 20460 aaaagccatg cctggtggag cactacgtgt aatctcagct atttgggagg ctgaggcacg 20520 agaatcactt gaacctggga ggcagtggtt gcagtgagct gagatcgcgc cactgcactc 20580 cagcctgggc gacagagtga gtgagactcc atttcaaaaa aataataaat ctgagtcact 20640 ttaatattgt tatttggatg tcaacctcta ggtgtttgag acaggagagt gatatgggggg 20700 cactggaaac acacaggcac ggggtgtcct cacacttggg tagcccacac gatgtgattt 20760 cagggtgctg ggaggtcccc ccactcccca aattactaac aagtggatag tactttacag 20820 tttatatgat ctcatttgat tcttaacatg agcctgtgag tgaaaaattc cttcccctct 20880 tctacagatt aggacgttga gattcaggga ggttcagagg gattcaggga agtcaagtgg 20940 cacctggagt cccgtggcta atttgaggcc ggtaggggat tcgaacccag gatttgtgct 21000 tcttatgcct gggcttctgc tccctggggc atggtctcc ccctagcttt cccattcact 21060 gctttagcct aggggtccta cccttatta aactgccagt gcctcactgc tttctcccc 21120 caaagacaaa aaaaagtgt tttgcttt gttttgttt tcatggggcag agacctggaa 21180 ttcagcttg agaatttgtg ccatatgata aaaatcaa cagatggctt ttccttaaa 21240 aaaaaaaaaa aaaaaaacta agatgtattt gcagtgaggc attattgta ccaaaagtg 21300 ctcaccacac tgtagtcatg ggggcaggag gcagccgcgg gtgaagggag aaatcttgga 21360 gtccaggcag cccccttctg gggctgaactg gggagctggg gtgctgcca gccctgccag 21420 gttctcctag gaggcggcag ctcatatggc tgtgggagga ggcagaggga gcctcatatg 21480 cacccacatt tccagggatc tagagacag aaggaggaaaccaccatca tgttaaagca 21540 gabagttagg taacacatcc tgtaatacaa gttattttt ccacatctaa aggctaaaaa 21600 tagttgttag aatttaaaga taattgtaa atgagtttct atccttctag tttcacatca 21660 aatggaatca tgctgccttc acatcactag tgcccgttat ttgtgtttta tttccacaat 21720 gttgtctaat tccactcttt gggcttcccc agggatccag cctccctcac tcgcccatcg 21780 cagggagatg ctttattcat ctttgtgtct tctgtgccgg gcatagcgca tggcacagaa 21840 taagcactca gtaattgatt cacgagtgaa taaatggatg agtgggtgag ttcaatattg 21900 actacaaaaa ccctaaggcc acactggtga gtggctgcgc ctgtagtccc agctgctggg 21960 gaatctgagg caggaggatc tcttgagccc aggagtttga aactagcctg ggcgatatag 22020 cgagaacctg tctcaaatga caaaaacagg gccaggtgca gtggctcacg cctggaatcc 22080 cagcacttta ggaggccaag atgggaggat cacttgaggc caggagtccg agaccagcct 22140 gggcaacata gggagaccct gtctctacaa aaaatttttt aaaaattagc tgggcatggc 22200 ggtgtgcgct tgtagtccca gctactcagg aggctgaggc aggaggatca cttgagccca 22260 ggaaattgag gctgcagcga gccatgatgg caccactgca ctgcagcctg ggcgtcagaa 22320 cgagacctgc tctcaaaaaa acaaacaaac aacaaaaaaa aaggctttct taaagagact 22380 tgagaacaga aaggggaaca gatacataac ttatatattt atttgttcat ctttccacct 22440 tcctggaggg tggaggggaa caggtctgta tttggagttt tgaatgctaa aagtgggaat 22500 acatgtactg tttgccatga tctgttcaaa agttaagcca aatgccttag attctcctga 22560 aaactggaat gccactgtaa actataagcc ccacttcaaa gataaaagat cttgatgaac 22620 agggctgggt ctgtggactg ggcctctccc caccacacaa ggaagggtgg tgccagttga 22680 aggaaaatca cttaaatcct tgctgtctcc tataaggtg tggtcccagg tagggctgtc 22740 agaattagca aattaaaaca cagggcatct gtgaaaatta gaatttcaga taacaaaaa 22800 taattggcat aggctgcata atgtccctca aagatatcag gtcctaatct ccagaacctg 22860 taaatgtgat cttatttgga aaaggggtct ttgtagatgt ggttaaatta aggattttga 22920 gatgggggga ttatcctgta tttctaggt aggtcctaaa tgcagtcaca ctcatccttg 22980 taagaggaag gaagagagag atggaaaaca cagaagagaa gacaatgtgg tgatggaggc 23040 agagattgga gtgaggtggc cacaagccaa ggactgctgg cagctaccag cagccagaaa 23100 agtccaggaa ccaattctct cttggagctc cagagggagt gtggccctgc tgacacctta 23160 gcttcaacct agtgatcctg attttggact ttggccttca gaagtgtgag ggaatgaata 23220 tctgttgttt taagccacca agtttatggt catttcctac agcagccaca ggaatcaaaa 23280 acagtaagta tgtcccatgc aatgtttgtg acacacacca aaaatattac ttgttgttca 23340 cctgaaattc aaatttaact gggtctcctg tattttattt ggccaaccta gttcccaggc 23400 ccaaagaaag aggcttttga aatttgcaag aaagctggtt ggagctgtca gaaagtggac 23460 tttgtaaaca cagtaccacc gaaccaattt gaactgtact acctctagac aaaagagagg 23520 gcagtcagac agttgttcgt gatttcttct ttcaacagtc atttgagcac ttactacaaa 23580 acagaagcta tgtgtaaggg tggaggcgtt agctgttaat caggacctcc aggctaagtt 23640 tctgtattag tccgttttca cgctgctgat aaagacatac ccgagactgg ggaatttaca 23700 aaagaaagag gtttaattgg acttacagtt ccaagtggct ggggaagcct cacaatcatg 23760 gcagaaggca aggaggagca agccacatct tacatggatg gcagcagaca gacagggaga gagagcttgt gcaggggac tcctcttttt aaaaccatca gatctcgtta gacttattca ctatcaagag aacagcacag aaaagacctg cccccatgat tcagttactt cccaccagat ccctcccaca acatgtggga attcaagatg agatttgtta ccatatcagt taccaaccct 24060. tccagataaa tcacgtgaaa tatcgccatt aacagagtga gctcaggtgg ttcttcagtg catttctgat acctgaacct tccctgggaa tttcacagac catcaggctc tccacccttt gatagcagga tagcagggcc caggttctgc aggaggagat gttaccacag gcctgaaagg gagggagggg cagatgctac aggaagatgc tggctctgga ttcgctggag gagctttcaa gggaagtaga tacacactgt ctccatcatt tcatgtccat cacactctaa aatgctttgg acaagaagca aatgttaaag acaaatgtgg cccattttcc tgtacaaaga gggctgctcc 24420. catgccaggc tattggcact ggttgggcatg aggcttctct gctgccctgg ccggggggtt ctctcactca ccattggctc tctgacacct ggagagacca ccacccttgg gctttcatga tgctcacaga atccacactg ttggagcttt aaggagcctg gatcaactgg aacaggcagg 24540 gagtactagg acagcccagc attgcccca atatccagg cctgataaaa gagaaaaaca 24600 ggtagctcac aggaaagga taaaaaaagg aggaggtt taacatgaa aggtgcttga 24660 tctccctcat aaaaaaga ctgctgattc catccaggca agtgacagaa aaaaaaaatt 247720 taatttaaaa agactgctga taaaaccaca gcgagacact gctgctcagg gatctgaggg 24780 tgtgggcagc caggctgcca cgcatcatgg gtcggagagg agaccacac ccctggagca 24840 gaggggcggct gatctgtcag atgccttg acagcaccctc agcttccaag aattaaccct 24900 ttctatgtga gcagaggcat ccatgggggg acacactggt gatcatctg ttatgtagaa 24960 gtctggaaaa catcaggatg gaactggtga ataagtgtg gcctctgacg gatggagcg 25020 gtccgtctgc actgctgcgg gtgcccctca gatcctgtgg gtcagtgaga aaagcagtga 25080 ggaacaaggc aggtactgtg tactgtcctc tgcgtgcaag gaaggccagc gcatgcaaca 25140 gagtccacac agacatagcc taactctgga aggaagaatg agaatgcagt ttcagtggtg 25200 gcctctggtg gggagaaact gggtgaaggg agatgtcatt tccattctc tactattaat 25260 tttgtattac catgcttaaa tgttactttt tacctttttt tttttttg agacagggtc 25320 tctctctgtt gcccaggcag gagtgcagtg gtacaatcat ggttcactgc agcctgaacc 25380 tcccaggctc aagcaatcct cccacctcag cctcctgagt agctgggact ataggcacgc 25440 ataccaccgt gcccagctat ttttttaat caagatggag tttttctatg ttgcccaggc 25500 tggtctcaag ctcctggact caagcaatcc tcctgcctca gcctcccaaa gggctgagat 25560 taaaacgtga gtcaccctgc ccagccaatt gctttttaaa aaagattaaa tgcatgtata 25620 cgctcaggca tcagcacact tggaaaggat gaaaatatcc ggaagaaggg ttcttttaaa 25680 aggctcctca agtgatgctg gcaggcatga cgaatgtccc tggtcacaaa agctctgatc 25740 tggcctaacc ctgtcatgtt agagactgga gtgcgtgtgt gtgcgcgcaa agtgtggggg 25800 gatgggggtg agtgtgtgtg gtgtgtaagc atgagtgtgt atgtgtgtgg tgtgggggtg 25860 tgtgctgtgt gagcgtgtgt gagtctgtgt gtgtagtgtg tgtgtgaagt atgtggtgtg 25920 tatgtgtgac gtgaggtgtg tgtggtgtgt gagttgtgta tggtgtgtgc atgagcatgt 25980 gtgtgggcat gtgatgtgtg tgtggtgtgt aagcatgtgt gagtgtgtat gtttgagcat 26040 gtgtggtgtg ttgtgatatg tgtgtggtgt gtgagcatgt gtgtgtgatg tgtctgtgtg 26100 tggtgtgtgt gagcatgtgt gttgtgtgtg tggtgcatgt gtgtggcgtg tgagcgtgtg 26160 tgtgcattgt gtctgtgagc atgtgtgagt gtgtgtgtgt tcagcatata taaggcatgt 26220 aactgaacac agcactttag agggctctcc tggagtcaga gggggtgggt aggaggagaa 26280 gggaggtggg ctagtgtgct gaagtatcta ctccttgtca tagtctgtga caacccagac 26340 tagcccatga gccaccctgt tccctgcatt tccaatgaga cctcggtgga catgttccct 26400 gaggtgaggc tgactgatgt catttgacga tcttgatgcc aaatcctttt atatcaaaaa 26460 caaccagaac actctctttt ctcttagtgc tttcacccag atgaccacat ttcatcctcc 26520 cagccactct gggccaggtg gcactgctgg tttgaaaggg aggtctcccc tggagtaact 26580 tccgtgggcg gattcacacc ctgcccacag tcctgtccca gtcagcccac catggtggtc 26640 tccggttcct ccagaattcc cgcttttcag ctcatcccca cattcccgga gggactgaga 26700 gcgcagcccc agggccctgc tctttggggg ccgtctctac acccagagaa gcagcaaggc 26760 attcctaggt ttctctttca gatgcagaac ttcagtgttc agagatgttc ccactggtcc 26820 tgagagggct cagttcagct ttaatgactg cgctgttgcg tgtgctctgc agagggcggg 26880 tggcccagcg tggctgactg cagttttcct gacgtggagc ccgagcctgc cccgctgttt 26940 attaattaag gatcactctg cttgcagaac cctgaactcc ccagaactgt gaggtgggag 27000 aaccccgaga ggccacctgg ccccacttcc cacctgctgc ccaaaccccc tctctgcctt 27060 cctgacagtc accccaactc ccagtgatcc ccatcaacca tctgacaagg ggactgagag 27120 ggaagagaaa ggaggggccc aaagaggaag gtaaaactgt cgggaacagc ccccaaatgt 27180 gtgacagcct tcagtggagt tgcccacttt cccttttctc ctccctgcag gacctccctt 27240 ctccccagtc ctccccaact tctgaggtta cattgagaaa agtctgcaga gaggtgccag 27300 catcacaagg tgttaaggac cacgagtttg gcattttaac agatgccaga gccacttgag 27360 aaatgtggta actaagccca gagaggtaca gttaacctcc ccagagtcac acagcaggtt 27420 catggcaaag ctggactagc acaggtgtcc ttcccctgca gatccccttc tgtgccccac 27480 atcacctccc tccagtgtct gggccacctg gagatgggcc ctcagactca cccggccaga 27540 ggtgccatct catgggagag gtctggccag gaagcatcga tatttgagat cccaagaaat 27600 gaagacttgg cctgtcagat gacagacttc ggtcatggga acacgtgatc tgttttacac 27660 atgcgtcccc tcagcagcag ctttccagaa cattcccact ttcttctgta gtgagaagaa 27720 ctctttccct gcagcctcct gcccaactcc tccttcagtg tctttgcttc agtgtctttg 27780 ataaaccatt ctgctttgca gagtgcgagc tctgccttgc agggttcgca tctgcctgtg 27840 ctgagtaacc aacgctaagg tcgagtggtc ggtcacctct cataagagct agggttgtct 27900 catgctgatg actaggactt gccctcaagg agaaaaataa atcaaaacaa aagcaaaaac 27960 agcaaacatg catctcttaa agaaggctct gagtccaggt aaatttcctt ccactgaagc 28020 agccaggctg aattcgaatt atctttgccc ctgcttaaaa actaatgcaa atttcctag 28080 agaatatcca ctaattcctg gagggggcat gggcattcct gatgcccatg agaggaccat 28140 ttgctcttcc ctcagtatgc taaataacag aagcgacatt tgttgctgga aagtatcagt 28200 gaagttaata aggttttct tgcccagggt gagggaacag ttcccaatga caaatgctgt 28260 atgggaaggg gctgtagaac tgccagcccc tttggtccat ccgtaaagtg aactctgtgg 28320 atcctggagg attccagcgt ctttttttt ttttcttttt ttttaagaca gagccttgct 28380 gtcacccagg ctggagtgca gtggcacgat ctcagttcac tgcaacctcc gcctcccggg 28440 ttcaagcgat tctcatgtct cggcctcccg agcagcaaga ctacaggtgc gcaccaccat 28500 gcccgactaa tttttgtatt attagtagag acgggggttt cactctgttg gccaggctgg 28560 tctcaaactc ctgacctcag gtgatccacc cgcctcagcc tcccaaagtg ctgggattac 28620 aggcatgagc caccatgccc agccagcatc tttcattttt ctgtctgctt tggccctttc 28680 ctctctcact gtcttccttt tccatttcca aagtcagtcc atctcactat tagcacaaaa 28740 actgctagag cgcttgtcat tggtcatctc tccctgcacc tggctggtct gttcttggcc 28800 actgaagcgt ttcccccagc tgttgcttta atcattttat tgttattatg ccttacttaa 28860 gaaatggata tgagatgcat ttacctgtct cttcctgcca ctctgcagag ccagtaagat 28920 gtggtggaaa gggcccaggc tttggaggag ggctggctgg ggttggatct tggctgcccc 28980 ctactagctg tgtgaccttg ggtaagtagc tggacctctc tgagcctggt tcggaatcat 29040 agcacctctc tttcagggct gctgtaagga atagcagtgg tgtgtataaa gcagagcgca 29100 cagccagcaa ctggccccta gccacactgc tgagcaccta ctgtgataag ctgccattgt 29160 ggtgtgtgaa gcaaagggga aacatgcctg ctgtagtgag cttcctgtag ggcaggttgt 29220 agaaccagag gtgggttcca aggttacaaa gggactctta gtgtattagt ctgttctcac 29280 attactataa agacctacct gagactggat catttataaa gaaaagaggt ttaattggct 29340 cacattggct gggtgcggtg gctcacgcct gtaatcccag cattttggga ggccaaggcc 29400 ggcggatcac ttgaggtcag gaatttgaga ccagcctggc caacatggtg aaaccctgtc 29460 tcttctaaaa taaaatacaa aaattagctg gccatggtgg tgtgcgcctg gaatcccagc 29520 tactcaggag gctgaggtgg aagaattgct tgagcccggg aggtggaggt tgcagtgagc 29580 caagatcgcc ccactgcact ctagcctggg cagcagactg agactctgtc tcaataaaaa 29640 aaaaaaaaaa gaaaagaaaa agaattgcaa gaaataaatt attgtttatg agctatatgg 29700 tctgtggtac cttgttgtgg gactgggagt cttggcgtct ccctgaccct gcctgttgct 29760 gcagcaccgc tcagccctgc ctgctcccta cctgcctccc ctcggcctct cctgcctcca 29820 ccgggcccct ggtgcctcct ctagagacag tcctcctggg accgattgtg ttctcactta 29880 cacgaggcat ccaggactac agataaccag aggaaggggc gccccccccg cctgccctcc 29940 tccctggcat cctcacgctg cagaggtcag agcctcatcc cagcccctta cctgccccta 30000 ctctgtggag aaccgtggtc agttcgccag gccggatcca cgaacggcct tgtggaagat 30060 ggtgagctca cacccagagc tggctccgat gaccctgtct cctttacatg tttctacctt 30120 cccctcccta ccttccccca ctgctgggcg cagagtggag gcagatgagg tttaaagctc 30180 agaagggctt aaacgggttg gggcgcagtg gctcatgcct gtaatcccgg cactttggga 30240 ggccaaggca gaggatcact tgagcccagg agttcgagac caacctgagc aacatagtga 30300 gaccgcgtct ctacaaaaaa taaaataaat aaaattagct ttgcagggtg gcatgcacct 30360 gcagtccctg ctactcagaa ggctgaggtg ggaggatcgc ttgtgcccag gagtttgagg 30420 ctgcagtgag ctatgctggc accacagcac tccagcctga gtaacagaat gagatcctgt 30480 ctcaaaacaa acaaacaaac aaacaaaaga aggcttaaag ggggctccag gtgggcttgg 30540 cagcacaaag ctatgaagtt ctatcttaga cacaagttct gttactgggc ctttgcaggc 30600 tggcctgggt acctggctgc catagacagg gaaccttcca gatgagctgc aggcgtggag 30660 cacaggagcc agggtgctct tcctgggctc tgtccacagg cagaacgtac acagtctttg 30720 tacacgtccg gcggctctgg tgcctatttt tgtttgtgtt tttcttttgt ttggggggat 30780 ggatttggtt tcccccgagc cctctgtcct cctgtcacct ggctggtgct cggcaatgtt 30840 gaccagctgc ctggctggag ttggcagtgg ctaaggctgt gacagctaac atgttcctga 30900 gtcctctcat ttcttcacca taatgccctg ttgagtttgc agatactgtc tctgttttta 30960 tctcccgggg aaactgaggc tcagagtggc taggccacct tcccatggtc cctcagctca 31020 tgagggccac acagggcatt gcggtggcct tctcctcagc cttgaccctc cggccccagc 31080 attgctgcct caaggggtct cctctgctga gccgtgcacc ttctgcctgg cagctccaac 31140 tctgtggctg tgttcagtgg ctcagcactg ccccttgacc ctccctggcc ttctgcggat 31200 gccagactgg agcactctga caaggtctgg ggtggttgta tgggtcctgt gacctctata 31260 cacctcccag tgcctgggaa tcctgcagat acaccctcct tagccgtccc taaccataga 31320 ggacatttct gaggtccccg agagagtggg gcacccctgc aggatccaac tgctgggccc 31380 aggaaggata gcagcagcat gaggggttcc attagccaca aactcacggc atggaacctt 31440 cacccacctc gcccctcatc tgctgtttag cacctggcac gccgtgtata cttactgatt 31500 attacatttt aatggcaaat tatagtggca aacgtatgca tctttgcaca attgttgtac 31560 agcatgatga acaagtcatt aatagtaaag aataaatgtg aaagtgagaa aaatctgact 31620 gccaaagttt ttactccttc cttccctccc cagactttta aatgaaagtt tagggataat 31680 cccttagttg tcctgctagt aggacttgca attaaaagaa ttgggccaag aacacttcta 31740 cgcttctcct tttaggtttg ggtgtaaatt cggggtattt ctcactgatg aaagcctggt 31800 gcagggcaga ccgtgggaag ctttcatttc cggaatggac catcaacatc ccttggagaa 31860 gaattctctt ctccagaccc agacctggtg tcctggcacc cattgggcaa gtgggtccta 31920 gaagacaaac ctggtcagag cctggaggct gcttagcatt ccccacgcac attagcagct 31980 cggagagctc aggaagccgc agcccctcct tgcctcacca gcctggatca ggacagcatc 32040 ccctggaaga cacacaggggc ctggctctg attaccagc ctggaggga agctcaatcg 32100 agcatcatgt cacccggtgc cccatgcag ggtggcactg gtgagacccc caagccaatg 32160 ataccaccctc acaggagtgc aggcccattg tggccagatc atctgactt ttcaagataa 32220 atcagaaatc gtatttccat gagatatccc tatttgcaag tgatggtgac taaattagaa 32280 gttttgaat attgtaacat gttcgtaggc tgtttgtctg gtttaactc tatctggagg 32340 aattcaagct agacttcagg aattcagct tgaggcagg atttgagac cttagggaa 32400 gaaggacgtc tggggtat tctgactgtt gtcctcctgg aagggaaa cagagaacta 32460 gaagactgcc cttagcgaag ttcaagcac ctaagcccgg gaccctcagc aagtgttctt 32520 gagtcacaga ttctccctga ggcgccctt tctggctcca tagaatggct gattctgtaa 32580 ctcggtgagt tgcttttt ttttcctcc atcaccagg ctggagtgca gtgaagctgg 32640 agtgccgtgg agcgatcact gcaactctg tctcccaggt tcagcatt ctccttcctc 32700 agcctcccaa gtagctggga ttacaagcat gcagcaccac acctggctaa ttttgtgtt 32760 tttaatagag acggcccgaa gtgctaggat tacaggcatg agccaccgcg gccagccata 32820 actctgtgac tcttgttaca aaggccttat attttgctct tgaggtgg ttttggttg 32880 atgcctgttg gttgccatct tttaactagg gatgttttat caaatgccc agccaagtg 32940 tccaaacaaa ttatacctta aagtttgaaa atgtctggca cttctaattc aatgcctgtt 33000 gtgccaggca ctgggctgct gaggaactga gtcccgtccc tgcaggctag ctagagaaca 33060 cacacacacacacacacacacacaca gagtggtctt acaagtcagt ttatattct 33120 acctatatgc aaaaggta ttattatgtt gaggtgcctt gattaaaa ttttcttta 33180 agggaggat gcctaaaaca ggcattacct gaaacctcct ctctccagca tggttgtct 33240 tctgtcatga ctcagggttt tcactgagaa tgggatggaa atgtggtcta aagatagggc 33300 caatgttggg actggatccc ctctgggaag tcagaccagg ctaggcagg tccttgaagc 33360 catcaggaaa agcctctctgga gccagaaaaaaaaaaa aaaatggtgt taactaact 33420 cagtctcaaa tcctgaatag gactcaagtc aagcaaaata attaaaggag ttagcaaagg 33480 gcaagtcaga gagaccgagc aacaccaatg tcttccggga gccctgtggc gagtgacaga 33540 gcctggactc tggagtagaa ctcatcttgt gtcttcttct gccactcgtt agctgggtga 33600 ccttgagcca agccccttaa cctcttggac cctatgttct tatctctaag taggggctgg 33660 taatatcttc ccctttgagg aatgccctct aaggggtgtt gtgaagattc ggtaaggtgg 33720 caggggtagg actcctggcc agaaacaggc acataataaa tgctaagtct ctccttctct 33780 ccacctgctg gatgctgtag atactaagga tttcgatgtg aatgagacaa aacccctgcc 33840 ttccaggagc ctttgagaat cagagaacta gacccatttc cagaacaagg ggatgcaggg 33900 tctggataaa gttttgggga tcaatagagc agagggctcc cagaggatcc catagggttg 33960 actcctaact caagggcatg agacaacccc caggaagggc accctggaag gggtccggct 34020 gtccctgatt tacttgtggg cactggggga atgcccggag ccatccagcc ctcagggctc 34080 tgtgtgattc tgggttcctc ccataaaaga taatcagatt ctttcacgtt aatgtctttc 34140 tccacctcat tgcacatcat gcagctattc attgactcag caagtatcag ctttgcatgc gaccttggcc tacccacttt agcttttagt aatagctccc ttcttgaata attackaccag 34320. tggggaaaca gaacctaact cttacctctg ggaggcttat ttgctttgag aacatatgtc ctgcagtttt gttcatatgg cagtgaagtt tcgtgcacac actctagagc caggcagcct 34380 gggttcaaag cgcagctctg ccaggtccta actgcatgaa tttgggcaag tcgctcaacc tctccatgcc tgagtttcct catctgtaag attggagcaa tggtaatacc tgctttttag ggttgagaag agaattaaat gggtaaagtg cttagagtgg agctttgcaa gtagtaagtg ctatgtaagt gttcgattta aaatgaaaga cccttaaata cattctttgt 34680. tcatttcac agcccttcat ttcacaacct tacatttcac aaccaagctc tgtctcccct ggaatccagc cataactctg ctcacaagtg tgagacaggc cccagcagag ctgcacgaag aggagagag gcagcccccc agactccca ccccctgtcc aagatggca aaccagaca cagcctctgt accaccccag caggtattca gaatctgcaa tctccaaagc ccacttcaat 34860 tgtaaatgta gagccacgtg cgctttaagt cacctgtcac tctggaggct cttttgctca 34920 gttcctcacc attagcaggg atgacaggga gtgcaggagt gcggtcgact cccagatatt 34980 ggagagcgct gggctagctg cccattctcc cggcctccac tcctctttgc tgtccagcca 35040 tcacttgctc tttgaaggca aacaaaacag aaaacagtgc caaaagtatg ggaagaaagc 35100 cagcttctcc cctggggtgc ctgtgatgcc atgcccaccc tccctgacca cgcagcccct 35160 gtggaccctc agggccccaa gcccccattt ccatcacatg cgtacaccca tgtgtgtcca 35220 tagccgccca tctcagtcaa taaggctgct cctgcccact tggaatagtg gtgacaacca 35280 ggagtggctt atgggaacta tcccaatggc ctgacagcat gtccgctgca aaccgctgag 35340 gtaggacact gccctcatgt ctagctgatc agcaagaggc gcagttgctt tcttaggtaa 35400 cattgctgct gtgtcctggc cattgctggg gggtggcact taatctacac cagatttttc 35460 cctcctgtat cttccaagct gcttggatct tggtgctgaa ttaggttgga ctttgtcttg 35520 tggggaaggg aggactatag accctcaacg taagcaatgg tcagactatt ctaagaaaac 35580 tcgccgaatt aaagcatgag gtaaatttag ttctgacttc tgtccacccc actgccactg 35640 tcccctttta tcccatgatc ccttgctttt cttttcctcc tctctcccta tctcttgtgt 35700 ttgacgcatg ataggaattc agaaatatat gtttgtggat ttgtttattc acgtagcaaa 35760 ccatttcttg agtgcctacc atgggccagg tagaatgggc ggccccgggc tgcagtggtt 35820 tcttcagccc ctctccaggg tttacactgt gcaagacggt ttgtgatggg tcctcccatc 35880 gaggaccaca ctcttctttc tctgtgcccc ttggtcctca gtctctgacc ccacttcaaa 35940 ggcagcattc actcagggaa gctcccatac aatgctagtc agagtaaaag tttggacaaa 36000 ttgccaggaa gcagcttgtc agtatgcata aacagccttt aaaatattac tactctttga 36060 cccagaattt cacttctagg aatctgtcct aaggaagtag tcacatgcaa aagatttatg 36120 taccaagatg ttcatcaaag tgttgtttta taacaggaag tctcagaagc tggataaata 36180 tccaactct ggaaatggtt agatagaata gtatgtagcc attagaaat tatgtctatg 36240 gggtttaaaa tgtcatggga aaacacttct gataaaag agcatgagaa ctgtatattt 36300 agcataatct taactatgtt ttagaatgca caggaaaaaa atgtacaaac atattcatag 36360 tgatgtctct ggtggtagga ttatgatcag taagtacttc tgtctctca tattttcctg 36420 tatttgataa tacatgcata tgttgttttt aaaatagaa aaattttaag tttaaaattg 36480 gagctgaaaa gtgttttag gtcaggcgag gtggctcaca cctgtaatag caccacttg 36540 ggaggctgag gcagtcagat cactgagcc caggagttcg agaccagcct ggccacatg 36600 gtgaaacccc atctcta aaaaaaaa aattagccat gtgtgtgc acacatctgt 36660 aatcccagct acttggggagg ctgaggcatg agaattgctt gaacccagga ggtggaggtt 36720 gcagtgagcc aagatcgtgc cactgcactc tagtctgggc aacagagtaa gacttagt 36780 aaagaaaaaaaaaaaga aaagcctttt aaacagtagc agacatact atataatccct 36840 tactaagctg tcggtcaaat tttattat atatttattt tattcattta tattttttag 36900 acagggtctc actctgttgc ccaggctgga gtacagtggc gtgatcatgg ctctcttcaa 36960 acttgacctc ccgggctcaa gtgatcctcc catcttagcc tcccaagtag atgggaccac 37020 aggtgcatac caccacacct ggctaatttt tttattttt tatttttaga gatggtgttt 37080 actatgttgc ccaggctagt ctcaaactcc tgggctcaag ctatcctccc acctcggcct 37140 cccgaagtgc tggggttacc agcatgagcc actgtaccca gccctcaaat tttaaaaaat 37200 ctataagaga cattattgga caattagaga aattcacata tggacttata atagtatcag 37260 agtgtgtggt gtgatggttc tggagggaat ggactttttc tttggagaca ggcttttcta 37320 tgcccaccct tttatcttgc taacttatca tcatccaggt tccagcagaa acattacttc 37380 ccccaggaaa tttcttaagg gtgcagtatc atgatgtctg cagcaaattc tcaaatagct 37440 caggaaaaaa gtacgtgtgt ggtatgagtg tgtgtatgta tgtgtgtata tatatacaca 37500 tatatacaca tatatataca tatatgttta tatatataca tatatgtta tatatataca 37560 crack catatata crack crack tatttta tattatata 37620 tgcagagt gcaatgttg ccaagttaaa gattggtgag tctaggtgaa gggaatatgg 37680 tattattgt attatttgtg skyttttct taagttttga aattttcaa acaaaaaatt 37740 ggaggaagaa ggcatgccag tctaccccaa gccctccatt ggaatgctga aaatctaaac 37800 aatgtgattt ggcaatttca tttctttct gttgtggggcc agtagtcctt agatgttggg 37860 gaagggggta gtcgctgagg tgtggttgac ttaggatgga agaagcagaa gtcaagactc 37920 ccagggtcaa agtggtttgc tctgctgacc caagtgtggg aggcccagag tcagcgtttc 37980 aggtgtgcta attcagcatg gttctattca cggcaaagt ccaccctggg cacctctctg 38040 gcagcaatct tgggtgactc tactaggcc aggcctccat gacctagt ctggatccca 38100 38160 tcttctttgc cagcatgtag aaagtttaaa taattcccct ctttacaaca aaaaaaaca 38220 taccccttc agtcaccac cctagctctc ttctcctttt cccagccaga tttttaaa 38280 agcatcctag gccaggcgcg gtgactcacg cctgtaattc cagcactttg ggaggccaag 38340 gtgggtggat cacaaggtca ggagatcgag accatcctgg ctaacatggt gaaaccccat 38400 ctctactaaa aatacaaaaa agtagccggg agtggtggca ggtgcctgta gtcccagcta 38460 ctcgggaggc tgaggcagga gaatggcgtg aacctggtag gcggaggttg cagtgagccg 38520 agatggcgcc actgcactcc agcctgggtg acagagtgag actccgtctc aggaaaaaaa 38580 aaaaaaaaaa aaaaaaaagc atcctcagca ctttggcaac tccatctcct cccaacatgt 38640 ccctgttact ggaatccagc caggactcag ccccgatctt tctactctaa ccagttgtct 38700 cagttaacaa ggacaggttt atgctgcagt gacaaacaag atcccaaatt cttgtggctt 38760 cacacatctg gcaccacctc atcttccagc cttaggagtc atcttttagt tccttgaaaa 38820 ctctttacag ttttctgttg gggccttgtc atatactatt cccctggaat gttctttcct 38880 atcccctccc tttcaccttg ctaacttgtg cccatccttc aggtctcagc agaaacatca 38940 cttccttggg gaagttttct ccaacaccca cactacacag gtgtcccatc tacactccta 39000 tgactttgtg gtacttgtct cacttcattt tccactgcct tccccacaag gcacctgcac 39060 aagggcaagg accgtaccac tgtacctatg tcactcattg ctgtggtcac ctgcactctg 39120 gctgcctacc ttaactacac attagaatca cctgaggagc ttttaaagcc acaatgcaag 39180 actccaccct aggccaattg gatccaaatc cctggggtag ggccagacat cagtggagtt 39240 atatatacat atatatattt tgtttgtttg tttgtttgtt ttttgagaca gagttttgct 39300 ctgtcaccca ggctggagtg cagtggcgcg atcttggctc actgcaagct ccgcctctcg 39360 ggttcacacc attctcctgc ctcagcctcc tgagtggctg gaactacaag tgctcgccac 39420 cacgcccagc taattttttt gtgtttttag tagagatggg gtttcaccgt gttagccagg 39480 atggtctcga tctcctgacc tcatgatctg cctgcctcat cagcctccca gagtgctggg 39540 attacaggca tgagccactg cacccggcca tcagtggata tattttaaa gcactgcaga 39600 gaattctgtt gcatcagctt gagaaccact gatctgcctt gtgcttcaca tttaaaactt 39660 ttttttaatg aataaataaa ccccaaaaaa ttaatctccc taagcctccc tagaagatag 39720 gatggtaagg atattttcct aggtaaaaat atgttaattt catatttcat gaaatttcat 39780 gttcatttc aatcaagctc tgtcatacac cttacatggg gcaagcccag tgcctgggca 39840 gggtgtaatt atactcatta cacaggcaag gaaaagtcac attaggtgat ggagcacaaa 39900 taggcagtta atggtttcag ggctagttag gatatgtttg tctttcaatt gcaagtaata 39960 gaagcccaaa gaaattggtt atttatataa tataattgat tggttcccaa atttgaaaaa 40020 ttcaggaata gacccagctt aggtacagct ggatccagtc actcaaacaa tgtcacaaag 40080 aaccctttga caggaatgta tcctgtgttg actctacttt gctctgagta gtctttcccc 40140 aggtgatgat aaaaatggtc atcatcgcca ggcttgtgtc ctgtttagta ggaatataca 40200 agaagagctc agtaaatgct ggccccacca ctaagcaaaa acaaaacttt tgttgttgtt 40260 attgttgttt taaataacag cttagacctt tcttctttcc ttgttattct cttcatctg 40320 taatccagtt ttctacttct gaagtataga atgttctgat gatttattct tcattaccca 40380 caacttgcac atgtttattt aaaaatgcca ggattgcctg gccgttgtgt gctgttaacc 40440 tttgtttgct gttagtggat ccctgaagtt caggctccca ggggagcaga taatgggtat 40500 ccagttcctg caatatccac cctctggcaa gccaagttcc ttcctgggta aggttttgcc 40560 tacctgcatt cctagggaag tttctgggcc tgaccaccaa gccagctctg agaaggggtg 40620 cataagcccc accatgcttt ggctctgtcc ctatagaata tttatgttg ttactgaaaa 40680 ctaaaggaag atgggtgcgg tggctcatgc ctgtaatccc agcactttgg gaggccaaga 40740 cagattgatc actcgatgcc aggagttcaa gaccagcctg gccaacatgg tgaaaccttg 40800 tctctacaaa aacaaaaaa aaaaaatt agccgggtat ggtggcatgc acctgtggta 40860 ccagctactc aagaggctga ggcacaagaa tctcttgaac ctgggaggta gaggttgcag 40920 tgagccgaga tcgcactact gcattccagc ctgggtgaca gagcaagatt ctgtctccaa 40980 aaaaaaaaa aaaaagaaaa ggaaagctaa aggagagaga ctaaaatgat atcaggttcc 41040 tggagaacaa acagacatga ttttgcttca tggcaggaca gccggaagaa gtgggattat 41100 atcctcacat tacaaataag aaaactgaga ctcagaatgg ttaagtcact tgtcccaggc 41160 cacacagcca gtaaattaca gaaacagaat ttgaacccaa atcttccagc tccaaagctt 41220 gtgttctttt cactacctcc tgcttaattt tttaatttct aagattagac ccttcatcta 41280 tccatgacac ctgcctgtca tccctgaaa aaaggtgaac gccgttcaga aatttttcta 41340 gcctgagctc actcccagtt cacttatttt tgctttgtca tggctgccca gtccccactt 41400 gtagaccagg aataggtcat ggctgcgggg actacacgct gtcgctgctg caagggccgg 41460 cctctgtttc cggggctgag tgggggccag acctgccagg agcaccatct tctgtgggtc 41520 ctgcctggat gtcacatccc ggccccaaga agtcactgca aaccttcgta ttattgagct 41580 tcacatccta gaatttgctg tcactgtggc tgctgcatga agttgtcctg agagaaacgg 41640 gcattgtcat taacagggaa attgatggtc tgggggaaaa gtcatcctca ttctcttgca 41700 gatctatggg tgattgagac tggctgatgt tgaaggggtt tctcagccat cgtgtgccat 41760 gttatggaac agtggtgtag ccagccattt gacacccagc gctgaccttt gtttaacaac 41820 ctcacctata tatgacaaaa tgattgtcag aaataatcgt gtaatgaaat gactgtaata 41880 atggccagaa aagaaacgca gatagtaaaa tgtttctctt gttgaactct gtacatataa 41940 ttgcaccagg atttttttca aataaaaagt aaatattata ctacaaaaaa gggaaaaagc 42000 acaagcattt attaaatagc tttctatatc tttctgagtt ttgatccttt gattgcagac 42060 tgatgtaata ttttatgtaa atcattgctt ggttactaag tgaactttaa gaaaagtgag 42120 acgtctgcag aagttgccca taatttagca gctactgtat tgtaccattg atgtacggct 42180 ttattttctt gattaattat ttaaacaata taattcacaa ttttaaaata ataaatttcc 42240 acttaaaatg gtatttaaac tcagcaaaat atatcatcta tgagtaaaat ttgtatttac 42300 caagcaaaaa tattacagtt tgtggttcac atgctgtctc actgttttaa attttaaata 42360 caaaaactcc aagtaggctg ggtgtggtgg ctcacacctg taatcccagt actttgggag 42420 gctgaggcag gcatatcgct tgagttcagg agttcaagat ttgcctgggc aacatagtga 42480 gatcctgtct ctactgaaaa caattagctg ggtgtggtgg cacatgcctg cggtcccagc 42540 tactcaggag gctgagatag gaggatcact tgaaccctgg gggacagagg ttgcagtgag 42600 gcaagattgc accactgcac tccagcctgg gtgacagatt gagaccctgt ctcaaaaaaa 42660 gaaaaaaaaa aaagaaacac aaaaactcca ggtggtcgca cagaatgaca ggactgaagt 42720 aacttagctc caatttctgt cttcataatc actgtcctac cattgtctgt gcttagaatc 42780 tacttgctta atgcaggaac atgtgttctc acagagatgg aaaatgcaaa tggcgccaga 42840 agcaagctgg aaattctgaa ccattaagaa tttactctct gccaggcacg gtggctcacg 42900 cctgtaatcc caggactttg ggaggctgag gcaggcagat catctgaggt caggagttca 42960 agaccagcct ggccaacatg gtgaaacttc atctctacaa aaatacaaaa attagccagg 43020 catgatggtg ggtgcctgta atcccagcta ctcgggaggc tgaggcagga gaatcgcttg 43080 cacctgagag gtggaggttg cagtgagccg agatctatct gcaccattgc acttcagcct 43140 gggagacaga gtaagactcc atctcaaaaa aaaaaaaaaa aaaaaagaac ttactctcaa 43200 aataaatacg tgtggctgac tccacatatg gtagggccaa ctgtataact agaagttctc 43260 caaataactt ctgtggagaa aaaaaagttt attaaaggtt aactttttta aagtgctaac 43320 tagaacctta ctaacactga gatcgcacca attgtttata acttagacag ggccgggtgc 43380 agtggctcat gcctataatc ccaacacttt gggaggccga ggcaggtgga tcacttgatg 43440 tcaggagttc gagaccagcc taaccaacat gatgaaaccc catctctact aaaaatacaa 43500 aaattagcca ggcacggtgg tacacgcctg taatcccagc tactggggag ggtgaggcag 43560 gagaatctct tgaacccagg aggcggagat tgcagtgggc caagatcgca ccattgcact 43620 ctagccccag caacaagagt gaaactctgt ttcaaacaaa caaacaaaaa aaaaaacctc 43680 ttggaccagg aaaatatttt ttaagggagg agtattttat cactggcatt gtttaggatt 43740 gcaggcacat gatgctaatg aaaagcagac taactattag ttggttttat tactgttttt 43800 gaactctctc tctcccttt tttttttttt gagacagagt ctctctctct gtcacccagg 43860 ctggaatgca gtgactgcag tctcagctca ctacatcctc tgcctcctca gttcaagtga 43920 ttctcgtgcc tcagcctccc gagtagctgg gattacaggg caccacacca ggctaagtttt 43980 ttgtattttt agtagaggca gggtttcacc atgttgccca ggctggtctc aaactcctgg 44040 cctcaagcga tctgcccatc ttgacctccc aaagtgttgg gattacaggc gtgagccacc 44100 gtgcctagcc ctgttttga actctctaga gacagtccag ccccttatta cttgtcctga 44160 ggcagctgct cccttcacct ggccccccgc attgtgttcc ggacccttgt cctggtggtg 44220 ctaaagaata tctctgtcga tcctttgggg actggggaaa ctgaggccca gtgccacgcg 44280 atgccattg ttcagggaag attaggtcat ctgctaggtc cccagtcact tgaccttctt 44340 cccagacagg aagaagctgc tctgggtctc tcagtgctcc acgtgtcttt gcacattgaa 44400 atgttttctg attttttttt tttttttttt gctgttacat ttacttttaa aaaataacaa 44460 gcaataaaat gttacatttg agaaggttga aatgagaatt gatttgagtt aaattctagc 44520 agatttttct tagagaaatg atatcatcat ctccagctac ctgcaattga tctactctga 44580 attaagaaag agacttccat ttgttgttta tattttgcac tcttgatgtg tttctttaaa 44640 ttatggtcat gggccaggtg taggagctca cacctgtaat cccagcacct tgggactctg 44700 aggagggagg atcactggag gccaggagtt caagacctcg tctgtacagt aaattttaaa 44760 aattagccag gcatggtagc attcacctgt agtcttagct acttgggagg ctgagatggg 44820 aggattgctt gagccagaac tttgaggcta cagtgagtta ttttcacgcc actgccctct 44880 agcctggctg acagagcaag acctgcctca aaaaaataag taaaaaataa attaaatttc 44940 aatcattagc agtcattagg atatttaaat acagtatgtt gaatcaaagt tacgcatgtg 45000 tgtattttt tttccagaga gttgtttatc atgtgggttt taatttaact ttaaaaaaaat 45060 gttggctgga cagttgccca aatggtatca tcagccattt ggttgagaac gtatgtcctg 45120 cgggctcctc tgtcactgga gttttgctag ctgacagcca ctggctagtt agagactgca 45180 gtcagcacag atgcaggcgt ggacttgcgc acgtaaccat gtcaatgcaa agccatcact 45240 tcttaaaaat tctgaaccct gctgtctgag atggtggtgc agcggataga actctgctct 45300 aagaggcagt agctaattcc atgtcttctt tgcccttgac tagctgagtg actttgcaca 45360 tggggcttgc ctctctgttg ccttgtctgc aaagtggaat catcttttcc ttgctagaca 45420 gaaggtggac cctggaccta tggccttttt gagtttcccc cccgcttctt agaaggacct 45480 ctgatcctac tgagtttaat acccacgggt taataattgg gaaaagcaaa ggaagcgctt 45540 ctgtttaggt aattatatgc atgtttttgt ctttttctgg ctggaaagat atccaagcca 45600 ctgggaaggt ccgtggctac ccagggtagc cctctctggg gagggctgct atatccaaga 45660 gcccctcatg agaatttgaa aatcgaccat ggtagggcct gctgactttt gacagctaat 45720 ggtgtgctga gaattgtccc tccaaagatg cctttccatt ccctcgggag agtctgggca 45780 gcccctactg ggggctggga tgctggctct tccctcagcc tccaccccaa ctgctctctt 45840 ccctcctccc ctccccagcc ccctaatttc tctcacaagg ctttgttctg cagcaacctt tcctaatgca gtcctggcct cttcgcagct tcattacata accttccgtg gactcctggt 45960 ccaaggatca ccccagaaag ccagtcagag gtaggcacgc agctggggtc catttactta ccttccccac cccctcgga ctcagaggtg gtgcaggat ttggactcca agaattaca gctccaccac catcaccaga gccaaaactc aggatgcatg tgcttcatct gctgcttatt tccagctgag agccagtggt gccatggttc cttagggagc cggtcccctg atgccggctc 46200 ctggccccaa atctctctga tccgggctct tccagaatgt cttgtctcca ccatcgcctt 46320. tgaccaatgg tgtccctttg cctggtaatg tccccttttgc ctgatgatgg ccctgtcact cctctcttta gcacagagga ggctgtttca tcccttcaag cctgccctcc cttcaagtct 46440. tagctcaagt tcaccttctc cgcagagcct tctccaatct tcttgactc gtctcctctc agctccagca acctctgtct ctggcactga ttccttactt agctaagaga atcacagaca cttggggctc aggacaatct gctttctctc ttcttaccca tggccttgga ctgtgtgtac 46560 ctctttgtct ccactcccaa acccaacccc cagagggcag agagcatgtt gtctgtccct 46620 ttgctcagca tgaagccatg cgtgtggtag atcggcagag ttccataact tgtgttgacc 46680 gaggggtcac tttgctctga aattacccct gtgtccttca gtatttgcac agatagcttc 46740 ctggccagac cgaatatatc caagggcatg gcccacctct gctcctgttt ccaggtccct 46800 ggtgggggt agttcatgcc ttcctcataa tctgcccact ggcctggtcc tcaaggtctt 46860 cccaactgct cagccagagt tgagaaaatg ggtcgctcca tcctgtttgt gtcgttctct 46920 ccttcctggc ccactctcct gcccacaggt atccaggggc tgcctgtagc attagaggac 46980 atacatgcac atgcgtgggc atgggacact cacgtagcct ccaagcacag catcaataat 47040 gcattctgtg cttatagca tggaaagctg ctctaaactt tattacacag tggacatgtc 47100 tgaagcagct cccaaatcca cccctgagtg tgttggaatt ggcaagccta tcacttggga 47160 gtctagtttt tttgttcgtt aataatagat gcttcctgtg gccccagctt ggcaattttg 47220 atttaaagtg atcttaactg aagagactaa tggacgggtc tgaatttgtg ccttttaagc 47280 acaaagtatt gctcttaatt aactggattc tatcctttga gcaggcagag gccttccccc 47340 aagggcgtca ttaacgatcc acatctggac atcttccaaa gccttcttct gtttcaggcc 47400 aaccgcaggt gtgttcctga acacccagga ggctatgaga gccacatatg cctcccaaat 47460 acacacagtg tgcatgccca gggacataga gcagtgtgca aagtcccatt ccatctctct 47520 ccacctggga gaggatggct cttctgtctg attcatggct caaagtggta aaggagctcc 47580 ccactccccg tcccacgcct actcagagtc tgcaaatatg tatgcgatat gagagctcgt 47640 cagttagctg tcttcagtgt ggcgcacatt tgaggagtct gactcccctc cagcacaggc 47700 caatgtgcac tgctctccta tctttgtacc cccactgttg cactgtgcag aggttggagc 47760 catagaagta ccagagctgt gaaaggagag gccccctctc acctctgccc tggtctccat 47820 ccccactttc tctaggaagc tagtaggtgc tgacagggga gagaagggag gggaggggtc 47880 cagaaacagt ggctcatgcc tgcaatccta gcactttggg aggctgaggc aggaggatca 47940 tttgaggtca ggagtttgag accagcctgg gcaatgtagc aagaccctat ctctacaaaa 48000 agaaaaaatg taattagctg ggtgtggtgg tgggcacctg tagtcctagc tacttgggag 48060 gatgaggtgg gaggattgct tgagcccaag agtttgaggt tacagtaagc tgtgattgca 48120 ccactgcact ccagcctggg caacagagct gagaccctat ctcaaaaaaa gaaaaaaaaa 48180 aagaaaggag agagagaa agaaaaagaaa agaaaaaaaaa aaaagaaggg aagggaaagc 48240 ccagaagagt gtggggagag gaggcggccg tcattctggg gccctcagtg tgcacaacca 48300 gataacacat gctctgtggg cttttgtacc attttgcttg agcataaaga aaggaaggct 48360 gcccctaaat agaaagcact ctggaggcaa aaaatctga ctccaatcct ggccctgcca 48420 ctttcccagc tgaggactta gacaagcacc ctagcctctt ggacattctc agagccatct 48480 gctgcaagtg ggtgctgcca tacccacctt actgggcagg cttggggac caagggtggt 48540 aaatggctca gtctttcatg atgcggccac acagcaggtg cgccatccag gtccatttct 48600 ttccttcctt tcccccaaat caagttgtca ttaaagtact agtccacatt aatgaaatca actgtattaa ttttctattt gctgctataa taaatcatca gaatttagt ggcttaaacc aacacaaatg tattacctta cagttctgga ggccagaagc cctccatagg tgtcactggg ctgaaatcaa ggttttggca aggttgcggt cctttctgga gggtccaggg gagaatccat tttcttcctt tttccagctt ctaaaggtttt catgcattcc ttggctcatg atcttctata 48900. gctatagtca gaaaatttt ccatcaatca tcttcaaagc cagcaatggc aggatgagtc ctcacatcac cttgctctga caccagttct ctgcctccct cttccacatg tcaggaccct catgattact ttgggctcac tctgataatc tgggatgatc tctctatttt agagtcagct 49140. gactgggac cttaattcca tctacaaccc caattcctct ttgccatgta cagtgacata ttcacaggtt ctggggatta ggacgagcct gtctctgaaa ggctacttta catgaaaatt cattttttta attack ttttttcctc ttgagacaag gtctcactct atggttcagg ctggagtgca gtggtatgat cacagctcac tgcagcctcg acgtctctgg gctcaggtga tcctcccacc tcagcttccc tagtagctgg aactacaggg gtgagccccc atgcccagct 49380 aattttttt tttttttt tttgagacag agtctcactc agtcacccag gctggtgtgc 49440 agtggtgcaa tctcagctca cagcaacctc cgcctcctgg gttcaagtga ttcttgtgcc 49500 tcagcctccc aaggagctgg gactacaggt gtgcaccacc acgcccgact aatttttgta 49560 tttttagtaa agatggggtt tcaccatgtt ggccaggctg gtctcaaact cctgatctca 49620 agtgatccac caacctcagc ctctcaaagt gctgggatta caggtgtaag ccaacatgcc 49680 cggccccagc taatttttaa atattttttt tgtagagatg gggttttacc attttgtcta 49740 ggctggtctt gaactcctgg gctcaagcaa acctcccacc ttggtctccc aaagtgctgg 49800 gattacagca tgagccactg cactcggcct taagagaaga tttaataatt aatactttac 49860 aacaagatct ggaagaggtg ggatgagtaa ctaaatgagg atacaagtaa cccgggtcat 49920 atttgctaat acccttggtc acattgaact tgatatctta tcagattttc ctaatcagct 49980 cctttagcag cagtgttgca gcatcttatc tcattttgtt ttttgttttt ttgcctagca catgcctgta aatcactgga ttgaggtgtt tagtgtttg ttgtccttg gatgcttctt 50100 ataaatccat atttcatggc tccctggaaa gtgctatgca aatgataagc tgcaaggatg gaaaggaaat tgcagtgctc ctgaattgta aatgggcttt tcgaggagg tttctaatta ctcgctcttt ctcttgaact gaggagttga agtgtaggtg gcagatccat aacagataat catgtgtgtg atgtgacttc agcctgagcg tcgaggacca agtcacagag caggaacagc 50340 cactctccag tgtccttggg gctacgtctg aggagaacct gggatttcat atatgacctg 50400. cactggctgg ggggctctct tgacgtaacg tgttccctct gagcatgtta cagattctga cattcttatg ttccttctgt ggagagacat gtacttagtg acctaactca ctttagcata tttttgctca tcgtttgtgt agcttaagg aatcagataa ttaccccctc cccactactt tcggaagcac aaatgcaatg ccctagaatt gtactgggga ctcaaaaaga aaagagagta gtaaaatcta ttaaagggga caaagacagc ctatatacta caagctttct atttttatgg cagagaatgc catttcttaa gtaaacagag aactgcattt gacctgcaat atcaaatgca 50760 tggatttgat gctttggaaa gcaactgttt tctgcgttaa tctgggtgtc ttccgtgaaa 50820 tgtcctcctg cctttggctt aaacactagc tttgtctaca gccattccat cctgaacctg 50880 cccaatcttg tctgaatcct ggtttcacca ctgacaagct gtgtgtcctt gggcaagtta 50940 cttcacctgt ctgtgcttca gagtcctcat ctgtgagttg gggaatctgg acagaatcta 51000 ccccataggg cgtagtgagg atgtgttgaa ttatcccaag tggctacaca gagtaagcac 51060 tcaaatgatg tcatcgttgt catgattgct gttaccagag cctagagttc attctgatac 51120 tcgagtctgt ggcccatcca gcccaggtaa ggaatagttg gaggagttgg gcatgttcag 51180 cttgaagagg agacgacagg ggatatggga tagttgaatc tgtgaagggc cccctgggat 51240 gaagaactgg catgttctgt gtggctccag ggcactgagc aggaccatt tgccaaagtc 51300 tcagggacac agtttctagc tatagacaga aaaattttct gtcactcaga ggatgaaaat 51360 agaatgagcc cccttaagag gtaatgagct ccctgtcatt ggaaggattc cagaagagct 51420 aggtaaccac tttaggtgct atcaaggggc ttttttcttt aaagtccttt ccaaaagctt 51480 ctgagattgc ataaata ggaagccatc ttggtgcttt aacacaaact ctccccagtg 51540 atgagggttg agccaaagcc agattggcaa ccagagagga gacttgtgta caaggagttc 51600 ctcgagtcaa ttgctttttc cttgttctag ccagccagag ggctcctgtt ggaaaacagg 51660 agaccggaga ggctgaggcc tgaccaaacc agcttctgca ggccagctgg gaggccacaa 51720 ctcctaccta cgggaaaaact gaagggcatc tctattttta gattagcaaa agaaaataa 51780 tttaagtttg agtctccttt gcaactttta aaagacatct ttattgagat gatcattcac 51840 attctataaa attcccccac tttgagttac aattcagtgg ttttagtctt ccttgatgat 51900 tttgatggtc ttttcttaag gctcttggaa gacccagaag cctctcagac acaggtgggt 51960 gtggagggcg tagcacagag gcagacttct catttcctgg gtctcccctt taatgactct 52020 cagagacccc tccttcccc tgcccctggc ttctacccca ggggtgtaga gttttgccat 52080 tttccaagca gaacttcatt tcctcttctg tgtctacact ctttgtgctt ctttcttgcc 52140 agctttttct cctttgcccg cccttccttc cttccttccc tccctccctc cttccctcct 52200 tccctctttc cctccttccc cccttccacc cttcccccct tccccccttc cctccttcct 52260 tccttccctc cttccttcct tccttcctgc cttccttcct tcctgccttc cttccttcct 52320 gccttccttc cttccttcct tccttccttc cttccttcct ggtatgtgac taatttctgt 52380 ttcaggacat aaatgttgtc caggctgttc tttggtcttt ctgttggata atggacattt ggcattgaga gaggctgctt tttctgaaat catgttcttg gggcccagaa cctaggtgtg tgcttctgac tttgttttct tcctgatcca aattctgata tgtccattta aattgatcta gacccacagg gcactgtggg acagatcctc agtggaacat gactctgtaa cgagagcatt ttgttttgtc aaaatgagaa catattattg cctttcatct gattgtaaac fathercatg tttataaaac agtataatga gacaaaaatg tagacacta taggaaa tctccctaat tgtatttctc ttcacagaga aagcccctgt tgggcatata tactctt tgtttatttg 52800 tttgactaca catatatgta ttctttctt atgtataaaa attctgaaca tgcacatttc 52860 tgcaactact gtttcactt gatgatgcat ggacctct agagtgtacg tttctcttc 52920 cttacaaagc agttggcttc gccagggta caccaggaca cggttttggc tctgtcccca 52980 gggtgtcacg ggaccagggg atgatctcac agggtctgcc atctgccctg cctggccgga 53040 ggctgcatcg agagggccaa ggggcaccac gtgtcgtggg tactgtcaaa caagagcctt 53100 cagagccttc cacagtcttt ctttgctc ccagcattgc ttcccgctg gtggactctg 53160 aatctagaac tagctccagg cgcctctcca aattcagacg ggagctgggg cactatata 53220 atgcaaatct aggcaaagcc ctcccatac caggatccag aatggggtgg ggccttttgc 53280 cctgaaaagc tgtttagttt gaaaataca acaggaca gaaagtttg gctaaattta 53340 tggataagt tttaacgatg gtaaccatag taggttcat cgacagccag cgatggttct 53400 gaacacttga catgtattaa ctcacctaat ccccacattt tacagacaat gcaaggagg 53460 ctctgggagg ttgagtgact tgccccaaag tcgcacagct cctaagtgaa ggattcggag 53520 tggactccag gcagcctggt ctgactccct gcactgcgct gtgcttatct ctggccccaa 53580 tgccgccatg cagaagtgtc tgggggcact ttgtctctgt cagacagaat tcggagatgt 53640 gtatgcttgc cctggtatgg cacttctctt tttttgagac agaatctcac tctgtcaccc 53700 tggctggagt gcagtggcat gatctcagct cactgcaacc tccgcctccc aggttcaagc 53760 aattcttgtg cctcagcctc ccaagtagct gggattatag atgtgcacca tcgtgcctag 53820 ctaaattttt gtacttttag taaagatgtt gttttgctgt gttggccaag ctgatctcga 53880 acttttggcc tcaagtgatc tgcctacctc agcctcccaa agtgctggga ttacaggcat 53940 gagccaccat gcctggcagt gtggcacttc ttacgtgtgt tcagcggaca ctgtttatct 54000 tctgtccctc caagacggtg ctgagctcag gtcgttcatt actggcagac aactgctgat 54060 ttccaacaga attgccatcc tcttctcccc tgcgactttc agagtgtgac ctcagactca 54120 aaaattagaa gtgaaaacat cttaaaaact atcacctttt cttcctaatc ctcctctccc 54180 ctccctgtct tccttgttgt ccccatctaa tgaactatca tggcaaaaag agcccatttc 54240 tggtcatttt ctgtggcctt tcaaactccc acctacccca ctgctcctgg gtgcattacc 54300 cgaaagctga gacttcagtg cagaaagtgc caggccctct gtccccccag atcgccttcc 54360 ttgtcttccc tgtgcttgcc tgtcacattg tgtgggttcc agcgctggaa ggaatgagga 54420 acagattctc tggttctcct tttgaagttt accttcgctc caccacttct gagaccttcc 54480 cggaagttgc cccttgtttc tctcctctcc agggctgccc cagagctgcc tctcacctct 54540 tcctgctgtc accccaccac catcagggca gaagttggga caaagcctct cctactggct 54600 cctgcttttc tcccttaggt ccagcctcct cttctccatc ttcaggagtc tccttctcca 54660 ctcacacgtc atgacttcag cacctcgcat cagtccagaa tatgactgct tgttcaagtg 54720 ccacctttct catgcatttt tttctagtga caatcacagc caccctgtgg ggcaggagtg 54780 tcatcatccc catgtttcaa atgaagaatt gcagttcaga gagggcaagt gactggccca 54840 gcctcaacag ctagccagtg gaccccacca gggcttctga ctccagtccg ggttcccttt 54900 ccacccaaat ccatggaggg agctgagccg agaacaggtg tccttcagga agacgtgaag 54960 ccaaagcctc cacctccaaa ctcaggggcc cagggagtcc aggcacccat ccactcacaa 55020 ggctggatat ggtgcattcc aggagagggg ttgggggcga gtggcctctc tgtgtacccg 55080 tggggataga tgcgcaagtg gcatcgccac atcgtgagtc ctggcttcat gggtgagctc 55140 caggtccaac gagaagccaa gcagggggcc cttcaagctc agctttgggc ccgggtcggg 55200 gtacagggta gagcgggcct ccccagcccc tgccatgagg ccaaggcagt gcatcgttcg 55260 cagcgtacat tcagaaacca aagcctagga gctggttatc attccggttt acagctgatg 55320 gaagagcagg tgcttccgag aacccacagt gctctttggc cagtgaccca agggtgcctc 55380 tgagaggcct cgcagcaccc ggaggtgctg ctgaggcaac gccctgactg taagaaggac 55440 cattcatcct cagagagtgg ccgtgatgct gctgcgacag tcccaccatc cctcccgact 55500 ctcactccca acagacttcc cactgtaaag ctgaactctc cagcaaatca cctctcgcca 55560 gactctctcc tcactctctc tgggtccact agaggttcct cagcctctct ttgccttggt 55620 tttcccagct gtaaaatgga gcaaagaggg cctatgtacc cacaaaggtg tggttggagc 55680 gactcctcct acattagggc ctcgagtggg gcttcatgat tggttggtgg aggtctccaa 55740 acccacccag tgccaccgaa ggctgagact gcagatgcaa tgccacaggt gtccttctc 55800 agcctgggca gctgaacatc atgtgtaaaa cggggataat aagataataa cagccccttg 55860 cacctatgtg gctgtgagga ttaaacaaga taaatgtgta acagtgcctg gctatagaaa 55920 tatttactct tgttattaag ggaagaatat gtgtggctaa aaagggatcg aagatgtaaa 55980 agccaatccc tccccctcta gcatatttaa gggtaatgtt gagttggttt gtggaccatt 56040 tgctgcctgt tagagctgga aggtagggac cccctctcaa cagcgatgct acaaattata 56100 cccattggag gtcaaccaaa agacaaagct tattggctgg acatggtggc tcacacctgt 56160 aatcctagca ctttgggagg ccaaggcagg cggatcactt gagatcagga gttcgagacc 56220 agcctggcca acatggtgaa accccatccc tactaaaaat acaaaaatta gctgggcgtg 56280 gtggtgcaca cctgtaatcc cagctactca ggaggctgag gcaggagaat cactagaacc 56340 caggaggtga aggttgcagt gagccgagat cgcaccactg tactcaaacc gaggcaacag 56400 agggagacgc aatctcaaaa aaaagaaaaa aagacaaagc ttgttaatac cagcatattg 56460 ttaaggaat aaagtaggct gcagaacaac tggtgtaata tggtgccatg tagggaaaat 56520 tacatgtgtg cataggagag gggtctgcaa ggttgtgccc taagatgtta gagtggttcc 56580 tttgcttttc tcttttataa tttgtattt gacttttaaa taaggaccat aaatcacttt 56640 tataaaatac attctctcca gcccctacta ctcctttaaa gaataagagt ggtttgccca 56700 agaaagacag ttttttttgc tctggttttc ttgattctga catcagagga aactccttct 56760 catccacttg gggctctggg ttcaggggat tcatttcagg cagattaaag tggtgaccag 56820 gggcattcgt ggacacaggg aggcacagga gcaccatcag tttgtctcac acaaccactg 56880 tcatcctcac tgaggctgt tgctgatca aaaacagtat tgggccaggc acggtggctc 56940 acacctgtaa taccaccact tall tall gatcacttga tall 57000 tcgagatcaa cctggccaac atggtgaac cttgtctcta ctaaaagttc aaaaattagc 57060 caggcgtggt gggtgcctgt agtcccagct acttgggagg ctgaggcagg agaattgctt 57120 gaacccgaga ggtagaggtt gcagtgagcc gagatggcac caccacactc cagcctggggc 57180 gaccgagggg gactctgtct taaaaaaaa aaaaaaaaa aaaatatat atatatat 57240 atgtcaaaaa tggggtagtt tttagatcta tagtagttct aaaaacaaag gccatccaag 57300 catgacagat ttacaagcac tattggctat tccagtagtt acatggagg agagaagctt 57360 ttagttaaaaaaaaaaaaaaaaacc ttaggtcaaaaaaaattg 57420 tcctctcaga cacaatctgg gatttctc atgacagtgg gcattagcca actgacatca 57480 gcagcaacca tccgtgtgca cacagtggca ccacctccctc ccaaaaagca gccttcatct 57540 atgccctcat acatcgttg attattctt ttggattg gcccggaattt atttaagttt 57600 cttcttgcca gcatgagtct ttccttctg tatgctcctt atcttctctc tttaattgg cagttctgct tgaaatctgg gtctttcatt agtagtagtt caatttggtt ccagaacatt ctgtggtgtg atgcaatgtg accagagctc acacttcaga gctcttcaag ggccagtctt actgagcacc tcccagtggc tgcctgtgtg ctgggcgcca cttgtggtgg gcaggagaga 57840 ggaggggaca caaaggaga cacagctcct tcttagaagc tcaaagttgg ggaccagctg 57960. ccacagaaga gtatgtttag catctgagac accaagatcc agcgtcacaa gggtgtttat tagcctcct catctctttc tttttcttttt tttttttttt tttcctcagg cagtcttact58020 ctgtcaccca ggctggagtg cagtggcatg atctcggctc actgcatgca accaccacct cccgggttta agcaattctc ctgcctcagc ctccccagta gctgggatta caggtgccca 58140. ccaccacacc cagctaattt ttgtgttttt stable gggtttcacc atgttggtca ggctggtctc gaactcctga cctcagatga ttcacccacc tcggcctccc agtgtgctgg 58260 gattacaggt gtgagccacc gcgcctggcc tgctgttga ttcatctata gtatgtttga 58320 cttgatgacc tccagttacc ttagacagag gttctcatct aagctccaac tttccattc 58380 ctttgtcctc gtctttcccc ttaacccctc cacatttctc tcaaatcac cccactcta 58440 aaaaatactg tttatttttc ttttaaattt caattatct attackcattg aaataaatca 58500 aaatagcatg gaataagcga aaaaaatgga tcccacctt ccccactccc attccctagg 58560 gctaaccata gttaaccatt taatgactag gttttttttgt tgttgttatt ttttattat 58620 ttatttgag acagagtctt actctgtcac ccaggctgga gtgcagtgt gtgatctcgg 58680 ctcactgcaa cctctgcctc ccaggttca gcattctcct gcctctgcct cctgagtagc 58740 tgggattaca gtgcctgcc accacacctg gctaatttt gtactttgg tagagacagg 58800 gtttctcaat gttagccagg ctgtctcga actcctggcc tcaagtgatc tgcccacctt 58860 ggccttccaa atactggga ttaggtatg agccaccgca cccagccctc ctgggctctt 58920 ttcctttagt tgcactcgct ccccgctcct ggagtagagg gatttccgag agactgtggg 58980 ctccagcctt cacctaggcc caggactagg atgcctgccc taacatttat ctttatacct 59040 taaagcaaaa cagctggacc ataagcattc aagaaaac tgtgaataag gagaaagttc 59100 tcccaggaaa caagcttt agttatgttg ggccagccct tatattcctt agctgttacc 59160 agtcactgct tgatttaatc tcggctatca cttggcctga caggtctgct gctggtgcca 59220 ggatgtctgg gtttgaagc ctggctccat tacatacttc ctgtgtgacc ttgggcaact 59280 tactcaacct gtctgttcct cagttcccc agctgtatta tgtcagcata atagtttgtt 59340 gtgtgaatta atgaggtaa taactggaaa tgctcaac atggttccta tcatgagaaa 59400 tcctgctttc cgcctaaatg tgctggaaaa ttcctggtgg tgcagacag gagaccagag 59460 haaggaaag acagggtgca gaagccaaaaattaccttgg agacaaagc gcatgttaag 59520 gttattttg gattctaggt ttctctgc ttggtctca gttacctaca agagatccat 59580 ttaggggatt ttgttgtt tttaacgata gctttattga gatataattc atatgccata 59640 aaagtcactc ttttaaaatg tttccggtat attcacaagg ctgtgcagcc ttccctgtcc 59700 ttgattccag tctgagtttt taactgaagg gataaggagg accacgcttt ccccagacca 59760 gaaccgcggg ccagggggcg attccgctga gtcaccgcgg gcgcctggtg cgcggcggcg 59820 gagcccggga ccttccttgg ctgcccccta gcgagggccg cagcgcagcc tgagacaccc 59880 gccggggccg ctccacggcc gtcggattta gactggaagc tcggtccagg tccccagctt 59940 gatgcgcccg cggtgtagga gaccagcccg actcgagctt cccctgagcc cctggactct 60000 tgactccagc agggcctggg taatgaacgt cagctcccct ttcccaaagg ggttgctctg 60060 ttgggaaggc acccgtttga tacagtagca tagagatggg ttttagcatc aaaatatcag 60120 aattcaagcc ttgctctctg cttactagct gtgtgaccct aaaaaggttt ctgaacgtct 60180 ctgagcttca gtttcctcat cattccttct cacggggtgg ttgtgagcat tacagagatc 60240 ctctctgtga agcccctgtg agtggctcat cctgagggct gaaataaaca tgttattaat 60300 aatccaaaac tggcaaggga tgttgactgg tccccctccc ttgcccaagg agctttctag 60360 aacctgagtt atcattacca aactgtactg ccttgagtaa gaaagttaga aggaatggga 60420 aggatggtgg caggtggagg aaggcggatt ggtcatcacc tccttgcagc aagaaacagc 60480 cccagatcgt gggaaaccta cagacctgct agacagacta ggagcaaaag ctggggcttt 60540 aagaatcccc agggaggttc tcctgagaga gtagccagtt ggattttgta agcagagatt 60600 tgtttgggga ggaggtgaca acgtagggag cagaggggca aagctgtcgg gaatcctgcc 60660 ttgagggcag ggatgtgtgt tggggggagt tgggtcactg gggctcggtg gccttgggca 60720 agtttctacc tctcaggtcc tttacccacc tagggtcgcc atcctgccca cctcacaggt 60780 tacagtgagc ctggatgcac tgtcatgggc aggtgcccag gaaaatggca gacatgttcc 60840 aaacagcacg cagcattccc cagtgatgcc cagggtcacc ttggaggtgg gcgagatgcc 60900 tggggtttct cgtccacccc acaacacctc aggggacagc caaagctgtc ccttcaggta 60960 agctgcacag aagatgtgaa ctctgctgca aagactctat tctttgggag caaaagggac 61020 ccagggtctc acctgcacat ccctgtccct gagggcctag gggttcttgg aggccccagc 61080 cttggcaaaa tgaggaagaa ggtgaaggtt gtctgggccc ctgccaggct ccttcctcgg 61140 ccacgcactc cccttcctgc acacacaccc ttctccctcc accccatctc cattgttgtc 61200 agaaaagtca caataaaaag gtccatattg tctagttccc atacttttaa tttttaaaat 61260 tttatttatt tatttattta tgtatttttt gagacagagt cttaacccag gctggagttc 61320 agtggcatga tctaggctca ctgcaacctc tccctcctgg gttcaagtga ttctcatgcc 61380 tcagcctccc gagtagctga gattacagat atgtgccact atgcccagct aatttttgta 61440 tttttagtag agacggggtt tcaccatgtt ggccaggctg gtctcgaact cctggcctca 61500 agtgatctgc ctgcctgagc ctccggaagt gctgggattt caggtgtgag ccaccgcact 61560 cggctccaca cttttcactt attaaaagac tgtggtgtcc atcaatggat gaatgaataa 61620 accaatgtgg actatccctc ccattaccca aggaatgaag cacggagccg tgccaagatc 61680 tggattcaca gtgaaagaag ccagtcacca aaagccacgt gctgtgtgac ttcccttata 61740 cgaaatatcc agaagagata catccatggt gacagaaagt agatgagcag ctggggactg 61800 gcgaagggga gaagggggag cagctgtcta tgaggtccag cctttcttct gggttttggtg 61860 agaatgtttt ggaactagat agaggtgata gttgtacaac attgtgaatg tactaaatgc 61920 cactgaatca ttcattttaa atcgttcttt acgttgcatg aattttaagt caatcaaaaa 61980 cagttgtttg aaaagagaaa agcctatggg tagcggcagc agtgattgga tttatgattc 62040 gattccatgg ctcatccctc ccctgcctca cccctcgcc ctccgacgtc ttcttcttt 62100 actctgaact gttatctttg ttctcatctc tctctctct tctcaaccct gcagacactt 62160 ttccctttct ttgtctgccc ccaccctcca gatttccgtg tctccagtgt ctccctacga 62220 ggcatgaatt gagactggga gggtgtgatt ctgaagaagg caccaacagt gactcagcta 62280 gccccttccc ccaccccgcc ccccgggcct caatttagct aaaaaaccac agggacggac 62340 tcaggaggca atacctttcc aagggtccct aaaaaatgtc ccattttagt gtccaggttt 62400 cactcaactt tagtgcctcc cctaaaatgt gttccttacc tcccaccca ctgcatctaa 62460 gtcactgcct gagaaaacag gattgagaa gagaaagg gagagaga gagagaga 62520 gagagaga gagggaa ggctgatgga tttagaaag aaaaaaaa gtggtctgag 62580 gaaaacagcc tggtgtgtt tattttcctg tctgtgtac gctctcggc cttttggcta 62640 agatcaagtg tattttcctg tctgtgtgtc tcgcttagat tacaggtc tgtggttgat 62700 gandacgtctg gtccaggctg cgtagtcacc tcaagggcat gcttattgat gtgttttca 62760 attcactatc tttgcatggg agtcccaggc caagaggcac agctgcgcca ttgtctgtt 62820 gttttagata tcctttatcc agttctcca gagaaatcat cctgccctc tggaggt 62880 gggcagcagg ggtcagagat gggagggaa ggaggagcc aggtccttgg ctaggatgcc 62940 agggtcccct gcctctcacc tggctgggc tggaggctc ctgctgtcct gtcactgatc 63000 actaccccgc cccagcctcc tgagttagaa gacaggct aaagtagagt atttcttcat 63060 tgaaaaaccc atacaaata aaggttcata aaaaaaaaa atttagactg ggtgctgtgg 63120 ctcacacctg tgatcccagc actttgggag gccaaggcag gtggatcgct tgagccctgg 63180 ggttcatgac cagcctgggc aacatagtga aaccccatct ctacaaaaaa tacaaaaaat 63240 tagccaggca tggtggtgca tacctgtggt cccagcttct cagcctatgg acccacatag 63300 aatacaatgt cagcataaga agggagccct ggggtcacca aatggtttgg gcggcaaaga 63360 acctgaaggt tgagagaagt ggcttggtta cccagctgtt ggatgtgaga cctggccact 63420 gcttcttcca taccctagac ctgcaccctg acatctcaag taaaaagttg ggggatgttt 63480 tatggtccag gatgaaggaa gggcagtgag gggcagcgga gcatcacttt gcatttctgt 63540 ctgcctctta ctggctgtgt gacctggggc aggtaacttc ccagactcct gggaatcata 63600 acacctatga tgatgatgat gatgatgatg atgatgatga tgacacctac ctcaaggatt 63660 gccctgaagg gtcacagaga tgcctgcaag gcacctgcat ggagcaagcg ccccttctct 63720 ggcaggtgct gggtgagcac tacctgctgc caggccctgg ggctatggca ctgcgtgacc 63780 ctgcaagtcc tacctggcga agctgtcgtt cttgtgctca gtcagtgttg gttgtaagac 63840 tgagaagagt cacttcattt tgctctccag ggacatcttt ctgggtccta ttttctgcct 63900 atgtcaagta gcgcctcaag gatgctcctg aaaatgggct tgtctttctt aacatggcag 63960 gtaggtccca aagcattagc atggggcagc tgacctagcc cagccaatgc agtgcagtga 64020 ctcttgcaac cgagtctaat cagaaggtcc atgaacctac gagcatttcc tgtcccagga 64080 tcagggtgga ggctgagcct ccctgcttag agattcttcc catgcattcc acttttttcc 64140 ccaaaagaaa atattgaccc ttgagaggca cacagtttat ttattttgca tagtaaatag 64200 tagcctgtat tttaaggatg agttgatttc tgcatcagcc cctgtaggtc atcagccttc 64260 tattggtgca tctgactctc tctagccctg cagggatggt ggaggggag gggaaggagg 64320 gatctttat ggaaaccagg acagtgagac tcattgccct gtcatctgct ctgtggtgct 64380 gaatgaggca gcccaagaga gaataccct gagcgagcat ccccagcctc caaaacagtg 64440 gcgcattgcc ctgagtcctg ggaatgacct ttgattctcc tgctcctgac ttggaaccca 64500 tggaacctc tagaagcagc tgaggaaaac ccaacatgaa aagcagaact ccacactgag 64560 aatataggag gtgatcggaa catacaatga ttcttgctaa gaccgattca cagtttttct 64620 ttttttcga tcgaagaaat actggagaag cctaaagaag gagtctaaaa actctggcac 64680 gtgggccaaa actgtccttg agctaagaat gattttcaca tttttaagtg gttgaaaaat 64740 gaaataaaat aagatgatgt tttgtgacac atgaaagcta tgggaaattc aaattctaat 64800 atctataaat agtgtttat cagaacacag tcatgctcat ttatttatgc tcgatggctg 64860 ctttcccgct acaattacgt tgagcagtta caacagagac cacgtggccc acaaagcctt 64920 acaatattta ctatctggcc ctttccagaa aaaaatgtgc cgactcttga ccttaacctc 64980 agcaatttgg gaggccgagg caggcggatc gcttgagctc tggagttcat gaccagcctg 65040 ggcaacatag taagactcca tctctacaaa aaatacaaaa cattagccag gcatggtggt 65100 gcacacctgt ggtcctagcc actcgggaga ctgaggtggg aggatcgcct gagcccagga 65160 agtcgaggct gcagtgagct gtgatggcac cactgcacct cagcctgggc gacagagcaa 65220 gaccttgtct ccaataaat aaataatgca aagtaaaata aataaacca tataaaaagg 65280 aatcaattta aaattataat gaaagctggc cgggcatggt ggctcacgcc tgtaatccca 65340 gcactttggg aggctgaggt gggtggatca cgaggccagg agatcgagac catcttggct 65400 aacacggtga aaccccgtct ctactaaaaaa tacaaaaaaa aaattagccg ggcacagtgg 65460 cgggcgcctg tagtcccagc tactcgggag gctgaggcag gagaatgtct tgaacccggg 65520 aggtggagct tgcagtgagc cgagatcgtg ccacttgcag tccagcctgg gcgaaagagc 65580 gagactccgt ctcaaaaca aaaacaaaaa caaaaaacaaa aaaaaattat aatgaaagcc 65640 aaggggcata gtagaacaaa ttttctagag ctcattaagt caaatgagtc accagttagt 65700 aaaacgcagt cacggggaag agagggcagg attctttgaa gcagcggctc tcctaaaaac 65760 aacccaccct tgtccagctg ccttccctcc tgagggtgtt ccctttgact gtgtgacccc 65820 catcccctat ttcccaaccg tccaagccca cctctagcat aatacgagct tttaatccct 65880 ctccctgacc ccaacccgat tttgaagccc agtctagtat tttctcaaat acacttcttg 65940 gctccattcc ttcctttcca tcacctctgc cttttcactg catgcttgga ccactgcagt 66000 cagctcccta tgaacagttg ctctctaccc atccaatcgg ccccgcctgc tgctgccaaa 66060 ttcaccgagg gcacctctgt ggtgctgcct gtggacaaag tccaagccag ccacctcacc 66120 cacctacagg tgagtggga gcagccagcg tgtccagtgg tttaccccat cgccacagac 66180 ttggtgatgt gtcgatgtgc agaagggg tgttggcagc cacaacacaa gcaaccccgc 66240 cccatgtgag atctaagatg ggcgtgctgg gagccacctc tgagaatcca aagaaggca 66300 gaggggagaa cggctcacac ggcacaaaca ctccttcctt ttttttttt ctttttcctt 66360 tttgaaagga gtctcactct attgcccagg caggagtgca gtggtgcaat ctcagctcac 66420 tgcaacctcc gcctcctagg ttcaagcgat tctccagcct cagcttccca agtagctggg 66480 attacaggta cactccacca tgcccggcta attttgtgt ttttagtaga gacggggttt 66540 ccctatgttg gccaggctgg tcttgagctc ctgacctcag gtgatctgcc tgccttggcc 66600 tcccaaagtg ctgggattac aggtgtgagc catggggcct agcctccttc catttaaatg 66660 tatgcctaat ttgcccattg agaacggctg agacgcattt taagtggcca gggtctactt 66720 agagttagtg ctcatgacca ggcccaggtc aagcctggct ggccagatgg tgcctttgac 66780 ctgctctgtc tctgtgcaaa ggaatgagct gaaggatggg ggtgcagtgt gtgggcagtg 66840 ggctggggct ggcaggactc agtgactaag ggaagagaac tttcctcact accagcctgt 66900 cttttcaggg caccgcgggg ggctttggga cttggtgatg aacacagcac agagagctgt 66960 ccagcatgcg ggtccctggc ttctcacact tcccaggctc cttcagaggc tctctccaaa 67020 gggagctgct ctctctagaa cccatgaatt tggaatatag gcaaccactg cattggggac 67080 cactgacctc aaacatagag accagagcaa atggggctca tcacgtgaaa ctcatctgga 67140 actctagcag gttcttttat atatatatat atatatatat attttttatt attatacttt 67200 aagttctagg gtacatgtgc acaacatgca ggtttgttac atatgtatac atgtgccatg 67260 ttggtgtgct gcacccatta attcatcatt tacattaggt atatctccta atgctatccc 67320 tccccactcc ccccacccca caacaggccc cagtgtgtga tgttcccctt cctgtgtcca 67380 agtgttctca ttgttcaatt cccacctacg agtgagaaca tgctgtgttt ggtttttttg 67440 tccttgcgat agtttgctga gaatgatggt ttccagcttc atccatgtcc ctacaaagga 67500 catgaactca tcatttttta tggctgcata gtattccatg gtgtatatgt gccacatttt 67560 cttaatccag tctatcattg ttggacattt gggttggttc caagtctttg ctattgtgaa 67620 tagtgccgca ataaacatac gtgtgcatgt gtctttataa cagcatgatt tatattcctt 67680 tggttatata cccagtaatg agatggctgg gtcaaatggt atttctagtt ctagatccct 67740 gaggaatcgc cacactgtct tccacaatgg ttgaactagt ttacagtcct accaacagtg 67800 taaaagtgtt cctatttctc cacatcctct ccagcagctg ttgtttcctg actttttaat 67860 gatcgccatt ctaactggtg tgagatgtta tctcatggtg gttttgattt gcatttctct 67920 gatggccagt gatgatgagc atttttcac atgtctgttg gcgaactcta gcagctctt 67980 ttcacaagtt catggagaga ggttcccac tgaggaatc acatctgtct gatcaaaga 68040 ggcttgggaa atggctctcc tgttcattcc ctgaaaacct ctgatggaac cactgccact 68100 gtggcagccc cagcactggc accccagcca tgattgtgc cccagccaca tctctgctgt 68160 gagccccaga gccctggtta attackcatc cacgtgttga tggggagg cccattcaca 68220 aaagcgacat aaagcccagg gagacgtggc cgtggcaaga agggtgtggg actacattcc 68280 gcccccaact gagagattca gaaaccagaa aaaaatggaa aacatactg tgctcttggg 68340 tgggaaaact aaatatcatg aagggagcaa tttttagt tttggcctat atacaattc 68400 cagccgaaat cccagtggaa ctttgagaat ttgcaggaaaaaaaaatg tctaaagtac 68460 atctggaga storm agaggtca atattttga aaagaaat gatatctag 68520 cccacctaga gaatagact tgagatccaa agctaatca ggaggctcta gcaaattga 68580 cagataagca ggacagagtg catggtgcat tcacctgggg aagagggcag attggtctac 68640 aaataggcct gggtccactg actttagctg ttatatttgg ggagaaactt ttcaacctca 68700 ctccatctta aacctaaaaa tattccagat gaattaataa atataaaaaa ttagaccact 68760 aaaaatgtag aagaaaatgg atgatctttc tataccatag agcaatggaa taaatcacaa 68820 aggaaaacag atttgactat ataaaactta aaccctgccc atcaaaaacc atcagaaacc 68880 aaaataaaag gcaaccaact ggagaagata gttgccacaa atatgatcaa gggttaatgt 68940 tattcataaa ttaagagccc acacaagtca ttagaataag cactgagacc tgaacagaca 69000 agcaaaaaga atgagagtgg gtcggcgcgg cggctcatgc ctgtaatccc agcactttgg 69060 aaggctgaag caggcggatc acttgatccc aggagttcca acaccagcct gagcaacatg 69120 gtgaaaccct gcctctacaa aagtcataaa tattagccgg gtgtgatggc acacgcctgt 69180 agtcccagct actcaggagg ctgaggtggg tggatcactt gagcccgggga ggtagagtct 69240 gcagtgagcc aagatcacac cgctgcactc cagctggagc aacagagtga gaccctgact 69300 taaaaaaaa aaaaaaaaa agaggaaa atgctgatc tcactagtaa ttaaaacatc 69360 aggccaggcg cagtggctca cacctttaat cccagcactc tgggaggctg aggcaggcag 69420 atcacttgag atcaggagtt ctagaccagc ttggccaca tggtgaatc ccgtctctac 69480 aaaaaataca aaaattcgcc aagcgtggtg gcacatgcct gtgatcccag ctactcggga 69540 ggctgagaca ggagaattgc ttgaacacgg gaggcagagg ttgcagtaag ctgagatcgt 69600 accattccag tccagcctgg gctacagagc gagactctgt cccagaaaaa attaaaacat 69660 cacatattta aaaactcta ggatatcatt taaaaaaaa ttatagact gttttttaga 69720 gcactttag gttcacagtg aactgagtg gaagtacag agactcccg tatgttccct 69780 gccctccacg tacagcctcc cccactgcca acgtcctgca ccagtgt acacttgtta 69840 scacaatga atcctcatta accattacatt atcaccag ttcatagttt acattagtaa 69900 aacatcatct tcatctata agcacaaaa she does it she does she does she tgtatgatta actcagtgtt gaagactc acacttcata cccacttgca ctgcatctga gaagcaattg 70020 gtgtctacag ccgctacacc ctcaacaagc ccgatcttgt ttgaaaagca attggtgatg 70080 cttctcaaaa ttctatggac aaagtcagcc gggcatggtg gctcatgcct gtaatcccta 70140 aactttggga ggccgaggca ggcagatcac ctgaggtctg gtgaaaccct gtctctacta 70200 aaaatgcaaa aattacccag gcatggtggc tggggcctgt aatcccagct actcgggagg 70260 ctgaggcagg agaatcgctt gaagcaagga ggcggaggtt tcagtgagcc aagattgcac 70320 cactgcactc cagcctgggt gacaagagtg aaactccatc taaaaaaaaa aaattatgga 70380 caaagttttt caaaaagata tttaatgcaa ctttatttgt aatattggaa catctgaggc 70440 catttcagtg ctaactatta ggggatggtt aggaaaatat ggtacatatg tggaaaggaa 70500 catttggtag ttagtgcccc tgatgtttac aaaggctttt agtgaccaac aaatgctcat 70560 gctataatct tatgtgaaaa aagcaagtag cataattgca actatatttt taatgcatag 70620 aataaaggc tagaaggaaa tatcacagat ccttgacata cattcccaaa cctttgtaaa 70680 tccgcggatt catgaaaaca gacacatttg cacaagtgcc tgatcttttc tgttatacat 70740 tcattagaag tcaagccctg gtgccacaaa gtatctgcct tttcaaatgt gatcagaatg 70800 ttctcttttg cttcaaggcc atttttcacg aagcagtggc atttttgcct cttcatcaga 70860 gtcaccgtgt gccctggagg actgagaaca gcagagccgt tttaggatgg gacagggcag 70920 ccaggaggat tgggctcact ccctactgag tgcctcactc ccgtacagcc cccatagagg 70980 aagaggggtt caaatttatt cctcagccag atggcatgtg ccgcctgtcc tggaatttca 71040 catcacttat gatggaccaa aattccaaaa gctgaatcca tgattgtcaa agtctggtat 71100 ggcaggatgt caacagtaat cgtttctggg cagagggatg attttctctt cccatcttgc 71160 tttgtataaa tacattttct ataataaggt tgtattactt ttctcatcaa gaaatagcaa 71220 agtactgttt tactcaaaat atgaatagag ccaggcatgg tggcagctta tgcctgtaat 71280 cccaacactt tgagaggcgg atatgggagg atcactttag cccaggagtt tgagaccagc 71340 ctgggcaaca tagtgagacc cccgtcccca ctcccccaaa gaaaacccac aaagcattta 71400 tcctggatta ttcacagggg ccaaaaaaaa aaaaaattc aggcctccta tagccatgag 71460 ctacgaatat gaaaatatgc aaatgtgtaa gaaaagccag cacatccgat ttttacttttt 71520 actttcacac ctctgtccac catgttccaa gagaagaaac ttggtcattg aaaggaatag 71580 atcaaatcca aagaacaaaa cactgtgct cattaaactt cttagtgttc acaaagcttt 71640 agctgcaggt tgaatggggc aacccgaatt ggctggctca cctgggctgc agggagcaga 71700 gatcgcgaca ctgcactcca gcctgggcaa caaagcgaga ctctatctca aaaaaaaaaa 71760 agttcataaa ttcaaagtta tgaattattt ttaaaataat aataatttac aataaagatg 71820 aggacaaagt gtgagtaaat ggtggttct atccagctct gttgagctga agtggcatct 71880 ccctgctggg gcttttgggg aagaagggtg tgtgttgctc ttcagatccc aagcctcatg 71940 cccctactgg gccctgtggg gtgcttctca gcccaccagg agagccaccg ttggaacaca 72000 cacgtggggg acctggtggg tgccggtgtg gtgaatgggg gccacagcct gactccagga 72060 agccagcaaa ctcggagctg gaggagtcag gacacccccg atgagtcaag agttggtttt 72120 gctgccagtt gacatctgat tgaaccatct cttcacttct ccgtgcctca ctttccttac 72180 cagacaggct ctgctgatgc tgtccctctc ctgttcagtc gtgccctcac cgttaaagag 72240 aaagagcaaa ctgctgggca gcagcattga tttttttaat gaagtggaaa gagagctggg 72300 aataacaagt cgggcccacc tcacctgcct cacctggtgg gtttatttgt tttgtttttt 72360 tttttttgtt ttgagacaga gtttcaccct gtcacccagg ctggagtgca gtggtgtaat 72420 ctcagctcac tgcaacctcc acctgccagg ttcaattgat tctcctgcct cagcctcccc 72480 agtagctggg attacaggca cctgccacat gcctggctaa ttattgtatt tttagtagag 72540 atggggtttt accatgttgg ccaggctggt ctcgatctcc tgacctcagg tgatccaccc 72600 acctcggcct cccaaagtgc tgagatcaca ggcgtgagcc accatgcctg gccgtcacct 72660 ggtggtgttg aatatgaact gctgcggtgt tggtaaatta agcaagcaga tagatgtaaa 72720 taacgcttgg gcaggaatat ggagcacggg atgaggatgg gcggccaact gttagagagg 72780 gtagcaggga ggctgagatc tgcctgccat gaactgggag gagaggctcc tctctctctt 72840 cacccccact ctgcccccca acactcctca gaacttatcc tctcctcttc tttccccagg 72900 tgaactttga accaggatgg ctgagccccg ccaggagttc gaagtgatgg aagatcacgc 72960 tgggacgtac gggttggggg acaggaaaga tcaggggggc tacaccatgc accaagacca 73020 agagggtgac acggacgctg gcctgaaagg ttagtggaca gccatgcaca gcaggcccag 73080 atcactgcaa gccaaggggt ggcgggaaca gtttgcatcc agaattgcaa agaaatttta 73140 aatacattat tgtcttagac tgtcagtaaa gtaaagcctc attaatttga gtgggccaag 73200 ataactcaag cagtgagata atggccagac acggtggctc acgcctgtaa tcccagcact 73260 ttggaaggcc caggcaggag gatcccttga ggccaggaat ttgagaccgg cctgggcaac 73320 atagcaagac cccgtctcta aaataattta aaaattagcc aggtgttgtg gtgcatgtct 73380 atagtcctag ctactcagga tgctgaggca gaaggatcac ttgagcccag gagttcaagg 73440 ttgcagtaag ctgtgattat aaaactgcac tccagcctga gcaacagagc aagaccctgt 73500 caaaaaaaaa agaaaagaaa aaaaaaaaa agaaatttac cttgagttac ccacatgagt 73560 gaatgtaggg acagagattt tagggcctta acaatctctc aaatacaggg tactttttga 73620 ggcattagcc acacctgtta gcttataaat cagtggtatt gattagcatg taaaatatgt 73680 gactttaaac attgcttttt atctcttact tagatcaggc ctgagtggcc tctctttagc 73740 aagagttggt tagccctggg attcttactg tagccacatt ataaaaac atcgacttct 73800 aaacattcta taataccatc ttttggccaa attgacttcg cctcttctc tctctttcca 73860 aatgaaatgt gtttcatttc actgtcagac cacatggttg gggaccccac agagcacaca 73920 gccctccctc tgccttccca tgctggccct tcaccactg ctggagtgcc aggttggtcc 73980 aagggttgga ccaagttgtc tgaggttgtc tcaaggttgg tcgaggctgt ctccgcgctg 74040 ggttgtgcta caaggagccc ttctttccat gggtgtggct ggcagtgagt gctcacagca 74100 acagcccaca gtgcagcccg agggcaggat ggactcagtc cctgcctcca tacccatttc 74160 taggaggca aaatggcaaa cactctactt ttctcttttta atgctaaaaa tagaaaaca 74220 ccttgcagcc cagggtatgg gtagtgcatg gaagccgtgg agttgtgagg tgggaagtga 74280 cctctgctgg atatgtctat tcaggaagat tgctggagtg ggtggggtct ctgggaggtc 74340 ccctgagtgt gggaagctgg gaccaccagc tttctcgcac agggagtggc catcccagct 74400 tggagaggtt ccaggactgg ttgggaggca cgtttcagat ttctatctgt tgaatcagcg 74460 aagatattgg attatgagga atttgggaat taggaaagtg ggtgcaggtg ggttgggggt 74520 aggtgaagga agacatgggc gtattgggg agcaggggct gctcagaggt gttccagaag 74580 ctctgggtga ggaggtgaga gggaccgggg aatgcagctc ggcccagcct ccctgcctga 74640 ggtcagccat cacgtggtga tggcaagatg gaaatgtgct ttctgactgc tccagccagt 74700 gctgccagat tcagctcccc agggaggca cctgagaggc tccaagccag gagatctgtt 74760 ttctcctttg ttttgttttt ttttgttttg ttttgtttta ttatacttta agttctaggg 74820 tacatgtgca caacgtgcag gtttgttaca tatgtataca tgtgccatgt tggtgtgctg 74880 cacccatcaa cttgtcattt acattaggta tatctcctaa tgctatccct cccccctccc 74940 cccaccccct gttttctcct ttgaatcctt cttagaggcc gggtgcggtg gctcacgcct 75000 gtaatcccag cactttggga ggctgcggca ggaggattgc ttgagcccag gagttccaga 75060 ccagcctggg caacatagtg agacctcgtc tctacagata ataattttaa aaattatccg 75120 ggcatagtgg catgcaccta tagtcccagc tactcaagag gcagaggcag gaggatcact 75180 tgagcccagg aggcggaggt tgccgtgagc caagatccca ccactgcact ccagcctggg 75240 cgacagagac ccccatgtca aataataata ataataaata aatccttctc agtcccttcc 75300 tcactgtgtc cccctccact gaatttttcc acctcctctc cacttcccc cactcccgct 75360 ttccctctcc ttctctcccc actccatcttt ttctttctc tgctgtttct cgtccctccc 75420 tcctctccat cccacaacac tgcctaccct gtccctgccc caccctggtg ctcaggatgt 75480 gtgaagtgag gggtggtagc ccccaagacc tcaaccccga aggttagcct gttgaaacca 75540 ctttctccca gctgcccccc tggcagttgg tgctgctggg ggaaactggg attgggggcc 75600 agattttgcc tcttttcctg acaaagagag atgaagagtt ctctcaccag gtgcctggga 75660 ctggggtgtg ggtgtcccag cctatcccag cgcatctgtt ctgcatcatg attaatagtg 75720 ctgctttcag ccgggcgcgg tggctcacac ctgtaatccc agcactttgg gaggctaagg 75780 tgggcagatc acaaggtcag gagttcgaga ccagcctggc caacatggtg aaacctcgtc 75840 tctactaaaa atacaaaaat taaccaggtg tggtggtggg tgcctgtagt cccagctact 75900 tgggaggctg aggcaggaga atcacttgaa tctgggaagc agaggttgca gtgagccaag 75960 atcgtgccac tgcactccag cctgggtgac agagcgagac tccgtcctaa aaaaaaagga 76020 gttttgctct gtcgcccagg ctggagtgta gtggcgccat ctcggctcac cgcaacctgc 76080 gcctcccggg tgcaagcgat tctcctgcct cagcctccca agtagctagg attacaggcg 76140 cctaccacca cgcccggcca gttcttgtat ttttagaaga gacggggttt caccctgttg 76200 gccaggctcg tctgggactc ctgacctcag gtaatccgcc cacctcagcc tcccaaagtg 76260 ctgggattgc aggcatgagc caccgtgccc agtcaactcc ttctcaaaaa aaaaaaata 76320 gtgctgcttt ctctttcaag tgtcctgatt tgggtgatag taaatgccac tctacttata 76380 agggatctac ctcagaatgc taattgggac atttttgtag cactctactg ttggcagcag 76440 gtgatgctca caacagcccg tgagggtgga tgacgtccgc ttcacagatg acaaaggagc 76500 ctcatgctca gaccgtgggc tgccagagca ggtccatggc tgcagcccca catggaccat 76560 atttccccct tgtcactctt tccaccaagc tcccttggaa cttcagttat taagctctct 76620 tgggtggaat ccaagttaga atcacaacat gtgcctcata tggattgtgc cagtgaaaaa 76680 tgacattcta tttagaggca gggcagcctg gcttagagtc agtttaaaat atgtattatg 76740 ctgcaacaaa tgtaccatga tcctgtaaga tgttcacaac aagggaactg gatgtggggt 76800 atactgtctg tactaacttc acaagttttc tgtaaatcta aaactgttc aaaataacaa 76860 gttcgtttaa aattaactcc aggagaccag gtacggtagc taatgcctat aatcccagca 76920 cttcggaagg ctgaggcagg tggattgctt gagcccagga gtttgagaca agcctggggca 76980 acatggtgaa atcctgtctc taaaaaaaat cacaaaaatt agccaggtgt ggtggcgcat 77040 tcctgtagtc ccagctactt gcggggctga ggtgggagaa tcatctgagc ccaggattt 77100 gaggctgcag tgagctgtga ttgtaccact gcactccac ctgggcaca gagcagacc 77160 ctgtctcaa aaaaaaaat gaaataagt ccaggaaga agtaggtttt accactctta 77220 ttttctgaag agaaactaa atttaatgtg taaagtgagg acagttcac caagttagtg 77280 tttgagttgc ctaaaatatg ttgctaaaa ctattcaaag ctttcacata aaacatgatc 77340 agaagttcta tgccaaaaca tatgtgtgtg tatatatata tgcactatat atactgtata 77400 taaaaatgca aaatctaaat tgccacctt ttagaattg ctctgaagg aaagcatttc 77460 aagataattt gcttacccaa agaatact ttccaagaa gcaagtaata cttaaggtgt 77520 tcataatcct catcaaatta attcttgcta ctgaagctt acaaggagct gttttgatgt 77580 cgggtgtgac aggtttgact tggcagaagg tgtcacttta ctaacaacat tttaaataag 77640 tgacagaaga caagaaacta cacgttaaat gccagaacaa agagtgtcta agtggatgct 77700 aagagttgaa atatggctgg atacctgccc aagagagctg aaaagtagat gaaagttggt 77760 tacctataaa ctagtgcacc ctaatgaatt aaaaggtgtt gatgagttaa cttgttatgc 77820 cttccagata agacatgcaa atggggcttc ttcctccttc actacttcca agggatttaa 77880 caaggagacc aatgcaaatg ataaggactg tagggctcaa gctggggaca gattggggaa 77940 agggggacca tcatgcccat atagatgtcc ctgtgccctg gcagtcaagg ctgctgaaaa 78000 ataacaaaac ccagaagtct gcgtgatgct gcctctccat ttgtccaaag ccttcttgcg 78060 gcagtttgca ggctttgca aaagctccag gaccaaggag ctatgttcat gctggaagct 78120 tgttcaggat tagctgttct ttgtgggatg ggtgcagcca gggccaggtg tccagggaca 78180 gtgttttaac aaagggcatg aggtgtctga tctcacagtg gaactccact tgcctttttt 78240 tcatcttctc attctgcttc atgcacagaa ccagccccat cctgaaactg actctaaatt 78300 actcccgccc caggtggagt gcctttctcg gagttcaaca gagccttcct gtcgcccaag 78360 ggacaactcc actgaatgcc caagccacac ccaaaaccta acaagtaaaa accaaattct 78420 gtgctccccc atcctgggcc attcctggtt tctctactgc tgttggtgat accaccatca 78480 gcttgtccat catgaccctg gccagttcct cccacaaccc tccacagcac ccagggacct 78540 cacctccatt ccatccgaca cagatctcct caccacaaac cttggttttg caacagcagc 78600 catgagacct ttacaccctc cgcccttcat cctgtccccc actgaggccc cagagccatt 78660 ccttaaagca gcgcgccaca aactataacc cacaagccaa ttctggtacc cagcctgttt 78720 tgcacagcca gtgaactgac aatgatcttt tcatacagcc agaaaaacaa aacaaaacaa 78780 aaaacaacaa aaaaaaaccc caccattctg agcatgtgac ttccatgttc aagatgtctc 78840 atgttcagaa aggcccctgg aaaaggagga aggggagctg ggcacaaagg gagaccctct 78900 cagctgagct cctcccatcc agacattttc ctggacttcc tatccaatga cttcccttag 78960 cttcttatca gccacccctg tctgcccagg aggctggaag atgtggcctt ttaactgggc 79020 acagctctgt cctctatcat atcagggctc tgttcccaag gagggtagag agaatggaca 79080 ccaggtggac cctcagcagt ctgtgccaca gagggagtgt ttgcaatttc cagactaaaa 79140 gtccccatgt gcttgacggg gtatgtgact acaacgtgat gcttgacttt tcctcatatg 79200 accagagcca ctttgtccat ctggtacaat gtcagctatc tgctaggggc cctccaggat 79260 tcccagtcaa ttccatatct gcatcaccac cattggcact aaataaaata aaatactcaa 79320 gttcctgctg gtgagcatga gcagtgctac actgggccct tcaaccaagg tgacatgata 79380 atgactgaaa ataatcactg ccacttattg gggacgtctc atctgccagg catggtacaa 79440 agtgctttaa ataagcattc aacaatttca tgctgacaga agccctgtga gccagtggag 79500 ctactactat gcccattata caggggagaa aactgaggca gagagaggtt aggtaattcg 79560 ctcagcctca cacaaccaat aggtggtgga gccaggattt gggccccatc tgcctgactc 79620 tctagaggct ctatcttcca gtcttccaga gttgagtcta agccatgaat aggacaatta 79680 gacagcagag gaaacccatt cagccaccat gtgcatgaag agtaaggaat ttctgtcata 79740 cagaggggag tgaattcact gagctgagag ctgaggaacc attgatctga tggctgagac 79800 accactggga agactggaga ggcttttctg ggcatgcagt gccaggcaca gggaggagctg 79860 agggaagatg actaagaggt actggcaaag aattcagaaa ttctgatgga agctttacat 79920 gttaccatca catccatcca tctatccacc catccatcca cccatatctt cctccctcca 79980 cccaatcatg catacatcca gtcatctata caccacccac ccacccatcc atccatccat 80040 ccatcccttc atccatccca tcatccatcc aattatacat acatccaatc atatatctgt 80100 acataatcca ttcttccctc ggttcatcca tccatccatt catccatcca tccacccatc 80160 ccttccttca tccttcctat catccatcca atcatatatc tgtacataat ccattcttcc 80220 ctcggttcat ccatccatcc attcatccat ccatccaccc atcccttcct tcatccttcc 80280 tatcatccat ccaatcatac atatatccaa tcatacatct gcacatcacc agctcatcca 80340 tctatccatt tatccatcca tccttccttc catccatcat tcatccatca tacatacatc 80400 taaccataca tctctacatc attcattctt ccatcgattc atccaattat ccatcattcc 80460 ttcctccatc catcccatta tccatttgat catacatata tcatctatac atcatccatt 80520 catccatcca tccatccatc cacccatatc ttcatccaat caatcataca tacatcgaat 80580 catctacaca tcacccatcc atccatccat ccattcatct atccacccat ccatccatcc 80640 atccatccat tcatctatcc acccatccat ccatccatcc atccatccat ccatgtaacc 80700 atccagtcat atatccaatt acacatccat ccagttatac attcatacat gcatctaatc 80760 attcaattat acatacacac atccatataa ttctacatcc aattatacct ccatccaatt 80820 acacattcat acacccacct aataaattat taattcatat atccatccat aatatatac 80880 atcaattata catccatcta atcattcagt aattcaccca ccatccagtc atctatccaa 80940 tatacattc atccaatcat ccatccatcc atccacccat tcatccatcc atccgtccgt 81000 ccacccatca tggtatgagc catgatttac cacgatggtc ccctgtggac agcccaggtg 81060 gggcagaact gaagggaagc ccaggggctgc cccataac atttgcctcc tttacatgga 81120 tgagaactag atccacatgt ataaatcctc atgatttgaa ggtgcttta ccaacattca 81180 ctcatgggat tctcccagga gctctaggag gaggcaggta gagttgaggt catctcacgc 81240 atttacaga tgaggaacg gaggccctga gaggcaggtc gaggccacc tgaccagaa 81300 gaagtggaac tgggacttga acccagccat cttgcccctt ggtcccatgc tcttagcct 81360 gtaactcctg cttcctgtg gggcatctcc aggaggaccc tatcggctgg ccatgggcct 81420 gccctggagt cttttgctt gtgtggccat ccttcctcc tcaggagt gtgtgctccc 81480 agagcacagg ctgtactctc tgagcatttt gtccctccc agtacctagc actcagctct 81540 gtatacattg gggctctcaag aattctcac cttccagagt gtaggcctt gacctgctca 81600 gccctggata ctgcatgatg cattgataag cccataaaat aaccagggca gattgactcc 81660 cagtggccaa agtgccacag ggaagggaca attcagccct tctaggagga ggaggaggta 81720 gttttctcat ttctattaag gcaacaaaag ctgccttact aaggacattc ttggtggagg 81780 gcgtgactgt caaccactgt gatcatttgg gcctctcttg cccaggcttc ccattctgaa 81840 aggacagttt tattgtaggt acacatggct gccatttcaa atgtaactca cagcttgtcc 81900 atcagtcctt ggaggtcttt ctatgaaagg agcttggtgg cgtccaaaca ccacccaatg 81960 tccacttaga agtaagcacc gtgtctgccc tgagctgact ccttttccaa ggaaggggtt 82020 ggatcgctga gtgtttttcc aggtgtctac ttgttgttaa ttaatagcaa tgacaaagca 82080 gaaggttcat gcgtagctcg gctttctggt atttgctgcc cgttgaccaa tggaagataa 82140 acctttgcct caggtggcac cactagctgg ttaagaggca ctttgtcctt tcacccagga 82200 gcaaacgcac atcacctgtg tcctcatctg atggccctgg tgtggggcac agtcgtgttg 82260 gcagggaggg aggtggggtt ggtccccttt gtgggtttgt tgcgaggccg tgttccagct 82320 gtttccacag ggagcgattt tcagctccac aggacactgc tccccagttc ctcctgagaa 82380 caaaaggggg cgctggggag aggccaccgt tctgagggct cactgtatgt gttccagaat 82440 ctcccctgca gacccccact gaggacggat ctgaggaacc gggctctgaa acctctgatg 82500 ctaagagcac tccaacagcg gaaggtgggc cccccttcag acgccccctc catgcctcca 82560 gcctgtgctt agccgtgctt tgagcctccc tcctggctgc atctgctgct ccccctggct 82620 gagagatgtg ctcactcctt cggtgctttg caggacagcg tggtgggagc tgagccttgc 82680 gtcgatgcct tgcttgctgg tgctgagtgt gggcaccttc atcccgtgtg tgctctggag 82740 gcagccaccc ttggacagtc ccgcgcacag ctccacaaag ccccgctcca tacgattgtc 82800 ctcccacacc cccttcaaaa gccccctcct ctctctttct tcaggggcca gtaggtccca 82860 gagcagccat ttggctgagg gaaggggcag gtcagtggac atctgatctt ggtttagtat 82920 ccttcatttt gggggctctg ggtgtggcct gggcctctgg actttggcca cggtgtttgt 82980 tccagccctt ctcctaacct gtcctttcca gacactcggc atctaggtta ttagcacctc 83040 gcatactttc tgacatgctc ctcagtcctg attttgacca tcttctcttg cttcccatct 83100 gtgtcagtca agactgcatt tggctgtaag aaacagaaac cccaactaac tgtggcattt 83160 acatgaagag gtttactttt ctcacataat cagatgtcta gacttggcca gcacctcaag 83220 ggtcattgat gctctcctgt ctttattttc tgtcatcttt agtggttgga ttgttgcctc 83280 atggttacaa agtggctgct gcacttccag gcatcacatc tgcctttgaa gcaggaacaa 83340 gttgcaaagt aaagtggcca aaagggccct gaaactaaat gtgtcccctt aggaaagcag 83400 gagttttctt gcaagtggca atcttctgct tatgtctcat tggccagagc tgggtcttac 83460 ggccacccct tgctgcgagc aaggctggga cattgagcat tttgccgtcc aacctcttta 83520 gcagaataaa ccaaggggga agaacgttaa tagtggcttt tgagtcacta gttggcagta 83580 tctgcccctc tatctttcca tcctccccat ggagtttcaa ggttcctttc tcagtacttc 83640 ttcaggctct gcacgttcat ttggatcttg tgtcttgggg tgaaaaactg gcccaagtgt 83700 ctccccaagc atccaccttt ggattaattt ggaaaatggc tgtcaagtgc ccgcctcttg 83760 cttggtataa tgctacagct ttagaggacg cagcaggcat gggccttgcc gctgaggttc 83820 ttagcctcat gagaatatcc agatcagatt ctcttggctc cttcttagag ccagtgatgc 83880 aagacacttc ctgctcatct tgtcgggacg gttttacaag ttgcctgcca tcctgagaaa 83940 gtctacaaaa cgatgccaga cctcatgcca gcttcccaag ccttgactct cagtgctccc 84000 tcaacaggat tctggaagaa tctcccaaac aagtcgcaat cccctctgga ccctgtgcag 84060 gcatgagact caagagcatt ggctcccacc cctggtggag ggaacactgc tggggctggg 84120 atcttgcctg gttgctccgc ctgcacccaa gacaaccata attaaaatgt ccttcattga 84180 acttggaaag ccttcaaagc tgacaactcc tttgtgtac ccggaaaggc ctgggagtgt 84240 gccagggcat tgctcgggag ggacgctgat ttggaagcat ttacctgatg agagactgac 84300 agcagctcct ggtagccgag ctttccctcc tgcctctgct gtgaaggtgg acccatccaa 84360 cagtcaaatg cctgactctg gacaggagcg gacctattta ttgccatgca agggactctg 84420 cacttttgaa ttgtgggtca tgggcttgga tttaggggtt agagctggga gaagtcttgg 84480 aagtcaccta gagatgacac tgccattttg cagatgagga aaccgtccaa tcaaaatgga 84540 ccaaggactt gcccaaagcc tcacagcaaa accataggcc cccgcactaa ccccagagtc 84600 cctgtgctgt cttaagaatc aaatagttgt aagcaatcat ctggttttca gtatttcttc 84660 ttttaaaatg cctggggcca tgcccagcag tctgtttcac tgcagcgttt acacagggct 84720 gccgggcttt cctggtggat gagctgggcg gttcatgagc cagaaccact cagcagcatg 84780 tcagtgtgct tcctggggag ctggtagcag gggctccggg ccctacttca gggctgcttt 84840 ctggcatatg gctgatcccc tcctcactcc tcctccctgc attgctcctg cgcaagaagc 84900 aaaggtgagg ggctgggtat ggctcgtcct ggcccctcta aggtggatct cggtggtttc 84960 tagatgtgac agcaccctta gtggatgagg gagctcccgg caagcaggct gccgcgcagc 85020 cccacacgga gatcccagaa ggaaccacag gtgagggtaa gccccagaga cccccaggca 85080 gtcaaggccc tgctgggtgc cccagctgac ctgtgacaga agtgagggag ctttgcgtgt 85140 ttatcctcct gtggggcagg aacatgggtg gattctggct cctgggaatc ttgggttgtg 85200 agtagctcga tgccttggtg ctcagttacc tccctggctg cctgccagcc tctcagagca 85260 tttagggcct tctggacttc tagatgctcc tcatcttgcc tcagtcagcg cgtcagttcc 85320 agagacttct ctgcagggtt ttctggggca ggtggtggca gacccgtgcc ttcttgacac 85380 ctgaggtcag tccaccctcc tgctcagact gcccagcaca gggtcacctc ccaaggggtg 85440 gaccccaaga tcacctgagc gcacagaggg tgcagatgac tggaccacac cttttggtga 85500 tcttaatgag gtggtcccag aggagctcag acatgcaatc tagcatccag ttctgggact 85560 ctgtctcctt ttcaaacgta ttcatgtaga acaggcatga cgagaatgcc ttgtcaacat 85620 gggtgatggg gaatcaatca gacagggcgc cgggctcaag gctgcagtca cccaagagtg 85680 gctcagccca ccaggcccta ggaaacgcct gcacagcctg gagctcctgg agtcatttcc 85740 ttcatgtctt cttcactgca cttacgtaaa gatgccagcc attggtttgg tgatttggag 85800 ggtgcccagt tgcccaacaa gaaatgcaga agaggcctag ccaggatttc accagcagtg 85860 gagagtagag aagatgtggc cagaaaagag tttccttttcc ctcctaaaga tggtactccc 85920 tgcagctact ggggaagcct gcagcattct ctagggctct gtgtgttgag agcagcccca 85980 ccctggcccc ttctgagtgc atttctgctt tgtgacttga tccgtgaagt cccctgagat 86040 gggcagaggg gatgtcctcg aagctggggc agagcctcat ccttgaacgt gaaggacgtt 86100 tgaagactgt ggcatgatca caggatgaga tcacaggggaa cttgagtttc tctcctcctc 86160 tcccttcaca gttatttcac tgagggaaat ccctcccctg cccagaatga aaactctagc 86220 caactcttga cttttccatc actccaaagt agttgaaagt acattagtct ccacagtggc 86280 aaaacagtgt gcaaaagcta aataattaga acagccagtc ccatgtgaca gtcaaagctt 86340 ctaactccat tcaaagttgc agccattccc ctcgagggct ggcagggagg ggaggggtaa 86400 gagaaacagg aaggttctta ctgagttggt cctggtgtga gctgcgtcac actccctgca 86460 gaggtttcaa ggagactctc tctctcttg tctccatggg gaccttattt gaattcttct 86520 actcttaccc cagcctgcca tctccagcta tcctcccctg aagagccctt ctgctgcgct 86580 ggattctggt ggccatgtca tctcctcggc cccgtgggag tctgaagatc tggctgcagc 86640 ctcacctctg aggtcctgct agttgccacc tcttaaacat gatctgaggc tcccatgcac 86700 tctgacctgt gcccacatgg ggcccacggg aaacacgctg gcaagcaaac tgtgggtgtg 86760 cagacggttc tcagggctgc agcacctgtc ctttgctctg cccccaaagc aaggccagcc 86820 catcttccat cctctagtgt tccttggtgg ggccctgacc acagtccacc aggtccctaa 86880 ccagagggga cacacaccag gtgtcctcaa tgtattgcct tgaaacagtt gtgctgggac 86940 tgtgatgggg ggtggccatg tagccacccc caccaccccc aagccactct ctccaaggaa 87000 atcctcctaa agatcccttt acatcctcca tgtggtgggg aggttctaga gttgggtgca 87060 tgtgtcttca gctactgaca atgcagacct tagttggcac ctcgctctgg cctatcctgt 87120 ttgctgttct tggcgctcca gtgaaactcc ccatgggcca tccagttggg gtgcagtgtg 87180 gccaccccct tgcaggttcc tgccttgctg gagagcacag ggccctcctg gctcttgtaa 87240 aacactcccc atggtacaga gaggccagca gtgatgtgag gcccaacctc cctccatggt 87300 gttcccaagc agctcccttt ctggggtcaa ggggtggcaa agacagtgca gcgtccaatt 87360 tctgactcaa gccgggcctg gctatcgcag ctctgcactg tgtgtgacag caaggcaact 87420 cacccagtgc cgtggcagtg accgtgtccg aggaagcctc ctcacaccct ctgtctcaag 87480 gactctggca tttagctgga cttgctgtag ctctgagcct ttctgccatt gccatcacct 87540 tgtcagaaac tcaggccgaa tctgcactca gagttgtgcc caggcagttg agccaacact 87600 tgctcagcga tattgtcaca tgacaaggca ctgtcaccac tgggcgtcgt gggtagcgca 87660 gtgtcggctg gatggacccg gagggtgtct gtgtcatgct agtgctagtg atgggagccc 87720 cgtgagccca ttgcccgccc tcccatgccc tcagcagctg cctggggaca gccaatggcc 87780 tgggtgtttc tgaggctacc acatggcttc caggaaactc gagaaccttt ctctcccttg 87840 cctacactct tcacacaggc ctgtgctggc cagcggtggg gatccggcat tcctatctta 87900 ggtgcagaaa gtgactgact cattgcaggc ctgggagata agactgatgg cccagccagc 87960 aagatgtatg gatttctcag aggcagtggc ctctgtcatt gtcctcagga aatgctggtg 88020 attctggtgg cctgaggtca atgcatgtca acgtggccaa cttgccttat aaactttttt 88080 tctggacaat tgcgtgcact gtcctgtaac agtgtcctgt tgtttatgat gcagaaatag 88140 gtgtttttaa agcctattga ttttggtact attaatgtgg tcaggaactt tctcagtctt 88200 tcttgtttgg ggtgagctgt ggcttcctaa acaggaaccc aagacacccc caaaagctgc 88260 tcaccagcac tgccagcctc cctcttacca agtagcaccc gttcaggaca ttctgcgaaa 88320 ggcatttgcc cagaagttgg gaggaaggaa atgtaacatt ttggggcacc taccatatgc 88380 caggcaccag gctaaacgtg ttcacacaaa ttctcttact aaccctcacc atccttctac 88440 aagacaaact agtatcttca tcttggggtt caagatgagg aaatggaggc tcagagaggt 88500 tgaatgaatg ccggtgcctg gatatgaacc ccatctgcct gactccgcaa cccaggcaaa 88560 gtctttcctt gaacttccca gcagccactg cttagacaca gcctccacaa ccatggctca 88620 gcagcaaatt gcttctctga cctcactcag cctgtgtgtc cttgttgagt gaggcattca 88680 ggaccctggt cccaaagtgg agaaagtctt tcctactagg tcatagctac acctgcatgt 88740 gggtgctgtg ccttttgttt agtgaacttt tatcaccagc atcctcagca atgacatttg 88800 cagagaagcc agagctgagg caccttggta ttcttgggat gtgactttcc tgaatgttta 88860 agggaaaatg cccgaaggta cagagagctt ggtttctagt aaacaataac tgtcttgctt 88920 ttacccccct tcatttgctg acacatacac cagctgaaga agcaggcatt ggagacaccc 88980 ccagcctgga agacgaagct gctggtcacg tgacccaagg tcagtgaact ggaattgcct 89040 gccatgactt gggggttggg gggagggaca tggggtgggc tctgccctga aaagatcatt 89100 tggacctgag ctctaattca caagtccagg agattttagg gagttggttc ttatcaaagg 89160 ttggctactc agatatagaa agagccctag tggttttttt ctaataccat ttctgggtaa 89220 ttcctaaggc atttagtgtt ctgaaagatg ctagccttgt ccagcctggg agttgagaat 89280 gaatgtctaa cagaaactct aggccgggcg tggtggctca cgcctctaat cccagcacta 89340 tgggagaccc aggtgggcag atcacctgag gtcaggagtt tgagaccagc ctggccaaca 89400 tgtgaaatcc tgtctcacta caaataaaaa aattagccgg gtgtggtggt aggtgcctat 89460 aatcccagct actcaggagg ctgaggcagg aaatcgctc gaacccagga ggtggacgtt 89520 gcagtgagcc gagatcgcat cattgcactc cagcctgggc aaacaaaagca aaactccgtc 89580 tcaaaaaaaa aaaagaaact caaatatgtg tgacaggcga ttctcactgc aggctgccct 89640 gtggctgatc caggagcaag gccttaacca tgtcatcccc aagcgattgc ttgtaaactt 89700 tcttctgtgc agccttcaac ccttattatg attttcttct caggaaccaa actgctgtat 89760 tcaagaaagg cagctttgtg taatcattta tcataaatat cttaagaaaa atcctagaga 89820 ttcctaattt taggaaatgg gagacctatg gtactgatat aatgtgggct gggcttgttt 89880 tctgtcattt gctagataaa tgaacttgag agcctactgt aaaatgtgga agcttctaga 89940 ttgcagaagg gctgggaaaga cactgttctt ttctcccgag tgatgggatc tgtccagtat 90000 ttagagctgc ctctgaggcc atctgattct aggagactct gcctcgttga ggatattttg aggcctaact acacattcct gcccccagag aggtcacagc ctatagcagg ctgatgtttc tcatgtcaca tggcacaga aggcacattt tcgttctcag gctaacaaag agcttcaaaa actattagaa gggacagtgg ctaattagaa agaacctcag tcaatgtgtg aaattaacta ggacctggc tcctgtttct tttaggtcat gttttttcagc ttaggtaaaa ctagaggctt 90300. tgataaagca tgacctctag aaatcattgc ttttcataaa tggaagtggg tttgagtttt ttctactgat tgttagtgca ggtgatgtct acatgccccc agaacatatt ccatgcaaca aaaaaagccc aggtcaccgt ctttgctggg aacttgactt ttgtgctcac tgaattttaa gctttctgac agcagcctgg aatcatggag ggataaagta cctattagta agatggaaaa aggtgtttca ggttggagct gcagtctgtt gagagtaagc tatggagg cctgtatacg aggggtggac ttttcttctg tagtgtcca gagaccaggc ctcctgaga gggcatgggg gcttaactta cctggactac tgtgtttaca atactcattt atcttgaact cctcctaacc cctgagaatt gctacattta gtatttgctg agtacttcct agcatcctag ggaatcaata 90780 gaacattctc ccaaccaggc tggtgcggt ggctcatgtc tgtaatccca gcactttggg 90840 aggccaaggt aggcagatcc cttgaggcca ggagtgcaag actagcctgg ctgacatggt 90900 gaaaccccgt cttactaaa aatacaaaag ttagccaggc atggtggtac acacctgtaa 90960 tcccagctac atgggaggag taggaggcag gagaattgct tgaacctggg aggtggaggt 91020 tgctgtgagc cgagatcatg ccactgcact ccagcctggg cgacagagtg agtgagactc 91080 tgttaaaaa aaaaaaaaa aaagaacatt ctcctaacct ggcttcttcc tccaggggtg 91140 taattaatca tgtcagtttc ctcattgata cacacacaca cacactacaa tcctgtatcc 91200 attactttc aaggtacatt tactatttac gtttggggtc cttgtctctt ttttaatagt 91260 gtttcttaaa gtcttgtatt atatcagagt acagtaacat cccagtcaag agcactctag 91320 tagctctag gaggaaagcg acttccggaa ggcagtggag acctgtcctg ttggggcagc 91380 ataggggcag cccctgcctc tggtcagttc tggcgctcag gctcagggtt gcctctgggc 91440 tgttcttccc agagactgac aaagggctcc cataaggcac ctgcagagcc tgtgagaagc 91500 tgaagtcaat gttttcctga caccagttga tctgtgcagg atccattgat ttaaccacct 91560 gctgtgtggc atgcactgtg gtcgatgcca ggaacaggaa ttggaggggc ccatgagcat 91620 ggccagtatc acaggctgga ggtgctgctg cgctctgacc gggcctcttg gggatgagcc 91680 catgtcaacc accttgcctc cgatggggtc gggcccacag gttacctttg tgtgtccatg 91740 accacacctt cctccccgac ctcatccaaa tctctttctt ttccaagccc ctgaatcctt 91800 cagggctgca ggttttgttt aaagcagagc tggtgagttg cataggttgt tgcgttggga 91860 ctagatgggg tgttcaaaga gttgggagtt aaaaaacata aagggtattt attaggagaa 91920 ccaaggagtg taattctcct gttcttaata tgcggccagg ttaatgaatg tcacgtgaat 91980 gaaccagaaa aaaatgaagt gtgcccttga tcagctgggt tggtgtgcag caagctgtgt 92040 gaccagggga cagcagtggt cctgagggcc gtcactgtct gccgtgcaga gcccttcctc 92100 ccacgggggc ctacctcacc tgtgccaagg gcttgtctgt ggtcagtgac ctggatagat 92160 ctgaatgggg cttctttttc gaggagtctt atggcaggtc tctcagtaaa gactccattc 92220 ttgatgatca cacattttgg attttccaaa tctgtcagag aatgggcttg aggcggggtt 92280 tgtgggcact agtttcactg gtttcattta ccaaaaaggg gagcagaagt caagtatggt 92340 ggctcatccc tgtaatccca gaggcaagag aattgcttga gcccaggagt tcgagaccag 92400 cctgagcaac ataaggagac cccgtctcca caaaaatgaa aaataacatt ttagtcagac 92460 gtggtggcat gcatctgtgg tcccagctgc ttgggagggt gagatgggag ggttgtttga 92520 gccctggagt taaagttgca atgagctgtg attgcaccac tgcactctag cctgggtgac 92580 agaacgagac cctgtctcaa aaaaaaaaaa aaagaaagaa aaaaaggaaa aaaaaaactc 92640 atgcctgtaa tcccagcact ttggggaccg gggtgggcag atcacgaggt caggagatca 92700 agactatcct agccaacatg gtgaaacccc gtttctacta aaaatacaaa aattagccag 92760 gtgtggtggc acgtgcctgt aatcccagtt actcgggagg ctgaggcagg agaatcgctt 92820 gaaccaggga gtcagaggtt gcagtgagct gagatcgtgc cactgtactc cagcctgggc 92880 gacagagtga gactctgtct caaaccaaaa aaaaggggtg gggggcgggg gcaggagaac 92940 agtgagaggt agggagagga aaggggattc tcgctacacc caaaccagat accatctaga 93000 ggctagaatc tttgggaggc tcaaattccc tagaaagcag gagaagcttc tgtagccctc 93060 ccgctttccc agtagattaa gcccagggcg gctccagatg tgtgacatgc tctgtgccca 93120 accagagccc atcataggca gaggaataac acccacacca gaagggccct cggaggtcac 93180 cacgtccaag aaccctcttt acagatgagg aaactgaggc ccagagaggg gagagccacc 93240 tagcgagctg gtggcggcta gaccaggaga gctgtcattc caagcaagca aaggcaacga 93300 gacgagccca gagctgtgct cccatctctt tgttaggggg cctgggatgc cctctcagtg 93360 tcattttgtc caggatgatg ctccctctct taagcgatta atgcgccctt gctaaccttt 93420 tgctatcgct gcctcttcaa accagaggag ttgagagttc cgggccggca gaggaaggcg 93480 cctgaaaggc ccctggccaa tgagattagc gcccacgtcc agcctggacc ctgcggagag 93540 gcctctgggg tctctgggcc gtgcctcggg gagaaagagc cagaagctcc cgtcccgctg accgcgagcc ttcctcagca ccgtcccgtt tgcccagcgc ctcctccaac aggaggccct 93660 93720. caggagccct ccctggagtg gggacaaaaa ggcggggact gggccgaga gggtccggcc tttccgaagc ccgccaccac tgcgtatctc cacacagagc ctgaaagtgg taaggtggtc caggaaggct tcctccgaga gccaggcccc ccaggtctga gccaccagct catgtccggc atgcctgggg ctcccctcct gcctgagggc cccagagagg ccacacgcca accttcgggg93900 acaggacctg aggacacaga gggcggccgc cacgcccctg agctgctcaa gcaccagctt 93960 ctaggagacc tgcaccagga ggggccgccg ctgaaggggg cagggggcaa agagaggccg gggagcaagg aggaggtgga tgaagaccgc gacgtcgatg agtcctcccc ccaagactcc cctccctcca aggcctcccc agcccaagat gggcggcctc cccagacagc cgccagagaa gccaccagca tcccaggctt cccagcggag ggtgccatcc ccctccctgt ggatttcctc 94200 tccaaagttt ccacagagat cccagcctca gagcccgacg ggcccagtgt agggcgggcc 94260 aaagggcagg atgcccccct ggagttcacg tttcacgtgg aaatcacacc caacgtgcag 94320 aaggagcagg cgcactcgga ggagcatttg ggaagggctg catttccagg ggcccctgga 94380 gaggggccag aggcccgggg cccctctttg ggagaggaca caaaagaggc tgaccttcca 94440 gagccctctg aaaagcagcc tgctgctgct ccgcggggga agcccgtcag ccgggtccct 94500 caactcaaag gtctgtgtct tgagcttctt cgctccttcc ctggggacct cccaggcctc 94560 ccaggctgcg ggcactgcca ctgagcttcc aggcctcccg actcctgctg cttctgacgt 94620 tcctaggacg ccactaaatc gacacctggg tgcagctgct ccactccctc ggcctcctcc 94680 cgtgctcagg ctgtggccgc acgcgcccct cacgcttgcc cgccactctg catgtcacca 94740 gcacccccgc tccgtgctcc ccaccttgtt tgactctctg gccacttgat ttgtccacaa 94800 cggcccatca gcccacagga ggtttggtgg gtgccttcca ccgacaggat gacgggtgcc 94860 ctcatgtgt ctagaactct ccaccctcc catgtaggca taagcagccc cacttgcag 94920 atgaggaaac ggaggctcag agaagtacag taacttgccg aaggccaatg agtagtagt 94980 gabagagcca ggtttgggat ccaggtaggt tgtctctgaa agacacgcct gtcctgcatc 95040 ccacaacgcc tcccaggagg gtgtgtg tgcgccta accacagagat gtgtgggc 95100 cacacagcag gtgacacaca cagcatccag aggtggccca gagctcatgc tgtgcctttg 95160 gcccagtgcc ctgcccccac ccactgcc tgtggcagg agacaagga gcagacacaa 95220 gatctccctg gtccacatgc caccacctcc ctctgcagag vakaagggga tcctcatgct 95280 ggcattggag ggggttgagc agggcccacc ttgagccctc aggagcacga ccacagcagc 95340 cctgcaggga gggattggtg ggaggagt cccaagtatc agggagagga gagttggtgt 95400 cccacaggag acctcagagc cacaaggcga gcttgttcat aaatttggga cccttagcat 95460 ttcacagtta tttgcagc ccagaaatgg atgttactga agctcacagt tgcaagcatc 95520 tgttaaattt ttattagatt ttacttttag ggaaaacttt gaaatgctat aaagaagcct 95640. gtgtttaaaa gttaagacag aggctggggg cgatggctca cgcctgtaat ctcagcactt tgggaggcca aggcaggtgg atcatttgag gttaggagtt cgagaccagc ctggccaaca tggtgagacc ctgtctctac taaaattaca aaaaattagc tgggcgtggt ggcgggcacc tgtagtccca gctactgggg aggctgaagc aggataagtg cttgaaccca ggaggcggag gttacagtga gccaagatca caccactgta ccctaagcct gggcgacaga gtgagactct gtctcaaaaa ataaataa ataaagttaa gagagaaaaa aatatatcct atatcctttg ttaattcca aaacagtagg ggacaata ctgacttgac aggttactac aatattcct gaatgatgt tttcttgat actggcctac tagaggttca taggtgtgtt tggattaaaa aagagttcca tggcccagtg actggggga aaaaataaa gactaaagta agttaacag 96180. gcttttctgc tgcaggactt gtcagagcct ttaatgtact aatggccatt gtgaccctct gagaaggtca cagagtgggt ttcccaaact tacttgattc tacttgctaa catttcctgg aggaagtttg ggaaatgccg atttagcaga ttctttttgtt gtgccgtgga tggtgctggt 96300 tgatgtgggc aaaacaaaga acacgtgagt cagatccgcc tggggctctt actaaagtgc 96360 aggttcccag gtgccacttt aggcttacag acccagttgt ggggtaagcc tgggagtctt 96420 ttagcaggtg attctgccac atagtatagt tggaaaacct ctgggcatac tcattgctgg 96480 tccctctaga aatccaggtg acaatagcca atgagaagct ccaagagacc cagttgtcca 96540 tggggtagag ggaatgtgat attgaaacca aagaagaaaa tctatgatca gttttcagca 96600 gtgactgtca agagaaggag aagggtgagt tagcgctgat gctggctgac aggtcagcgg 96660 gttggttca ccaaggagtg tgatgaaggc tgatgttgtc tgtgggaatg tatgatggta 96720 actggtttgt agctaatttg gggaagcagt gagaattcgt gccctttgaa gaccagtaag 96780 tggcaagaaa cccaccaggc ctggctcagg gctgggctgg gcttggctcg tctcagagca 96840 gctggggctg gtggccaaag ccaccattag tgaggggcag gccctggggg tacaaccagc 96900 aactagggga caagacaac cctgccagcc tctcctattc tggaggcgtg tgaccagaaa 96960 tggagatgggg ttggtcagca tagatggcc aggaggtgg aaatcaggac tgctggcaat 97020 ctagccacat gggcagggga gccggggtggt tccaggcagt ttccaggcc aagaggttga 97080 gcaggcacct cacagggaat cagggccaag cctggctgca gtgtggagac aatgcaccca 97140 ccccatcct tggatctgc aggaggctgg gtcctcactg agctaccac atccatggcc 97200 ctgaggcttt taaaacaccc atccatggag tgggctggt cccagtgggg tgaggctgac 97260 cctggcagaa acggggcagg agcctgtggg ttagggac tgcaccttcc ttagatagcc 97320 tccatgccat catgtccccg tgacagttc tgctgcgtcc cctctgcatg gtcccaccct 97380 cggccagcct gctgccccct cttgccaggt tgcgctaatc agtgacccca gtgtgctgtg 97440 ttgatactaa caatgcgagg cctagcagat tcaagggaaa aggaaccaa ctgggttcc 97500 accagaccca actaaaaaa catggaccta tcccagagaa atccagctc accacagctg 97560 gctttctgtg aacagtgaaa atggagtgtg acagcattc ttttatta ttttatcagc 97620 tcgcatggtc agtaaaagca aagacgggac tggaagcgat gacaaaaaag ccaaggtaag 97680 ctgacgatgc cacggagctc tgcagctggt caagtttaca gagaagctgt gctttatgtc 97740 tgattcattc tcatatataa tgtggggagt atttgtcact aaagtacagc tgtcatttaa 97800 agtgctttgt atttggggc aggcttttaa aaagtccagc atttattagt tttgatactt 97860 accccaggga agagcagttg gcaggttcat gaagtcatgc tcctaattcc agctttctta 97920 gtgtactttc agtgagaccc tgacagtaaa tgaaggtgtg tttgaaaacc aaacccagga 97980 cagtaaatga aggtgtgttt gaaaaccagc cctaggacag taaatgaagc catcttctca 98040 ctgcataaac tgcacccaga tctttgccca tccttctcag tatttcactt cacccattgt 98100 ttactgtctc aatgactggg gaaatgtctg gggaaatgct cccgtaattg cacagtggcg 98160 ttttcctgg aaaatcccac catggctcta gataagacct atttttctta aaggtatcta 98220 aaatttccag cataaattct gtctgaaaca cctgaattttt aatcagtact ggagcccgga 98280 gggcatctcc agttgccaca tagctctgag cattcagtgg tgtgttgagg gctgctccccg 98340 gaagtgcctg cagagtcagg gctccccagc ctcatctagt gaggcagtgg aagggcctgt 98400 ggggatttgg agagctggcc tgggtctctg aagtgatagt gacagctgct tgtcaatcac 98460 ggtgcacatt tagtgccggg ggcaggggc agggaatacc agcctcatgc atgcatgcat 98520 tcatttgttc cttcttcat tcattcattc agtacacatg ggtacaacat ccctgccctg 98580 gagttgccca gagtctaggg aggggaaaga tctattaccc tgggcctcgg ccagctgggg 98640 agtgctgctg gtggagaggg gccgtgtgca gcgagggaag gaggagtcgt caataccccc 98700 accccagctt tgctttcttg tcatcagccc cagggcccca gcctgtgtcc ctcctctccc 98760 attgctactt catctcctgg gtcctcctta ccaagcctga ccacacagag ggccttggcc 98820 gcttccatgg ggaattggaa agcaataaga tagcatcccc tagaagccca gtgaagtctg 98880 ggacaggacc cttctctgag ctctgacttg ctcttggaaa cacttcgagg cttagcctcc 98940 ccactttgtt tcccaagagt gtgacctgtt cccctccaaa cacccccttc tcctccaggg 99000 ccatgcccac ccgtcaaaat cccccacggg caggacgaac tgtgggtgtc agtcaccatc 99060 tatcctgcat cctggttcca gggccccccc cagccccgcc tccataggga caggcgtgca 99120 gacacccgtc cctggctgct tcctcttgtg gaatgggttc aaaagtaagc agtgttgttt 99180 acactgacaa actgaaaaaa aaagaaaaag agataacatt ggaggcttgg cacagtggct 99240 catgcctgta atcccagcac tttgggaggc taaggtggga ggatgtcccc agcccaagag 99300 ttctagacca gcctgggcaa catagcaaga ccccatctca aaaaaaaaat ttaattggcc 99360 aggcagaggt gggaggatca cttgaaccca aagggtggag gctgcagtga gccgtgatgg 99420 caccactgca ctccagccag ggcaacagag ggagaccctg tctctaaaac aaacaaacaa 99480 acaaacaaac aaaagagtta acattggcca gattaggatt caccagatag tgttaatatt 99540 agtttgattt gagactttaa tcagaaagca catgtgtggt gggggtgggt gtaacctaag 99600 tcaggtagaa tctttccaac ttgggggggg cacactcctg attgtagcca tatgagtctg 99660 tcagtgtggt ggaagagacc atgggttaat gggcaggtaa aaaagcacct tgcctggaat 99720 tgagtagaaa gtaaggccct tcagaccccg tgacacactt ggggacattt tcttgagtaa 99780 catcctaaga ttcatgtacc ttgatgatct ccatcaactt actcatgtga agcaccttta 99840 aaccagtcgt ctccaaattc aggggcacag taacatccaa caggctggag aaagaacgta 99900 ctagaacttc cattcctttt tcatgtcctc ttctaaaagc tttgtcaggg ccaggcgcgg 99960 tggctcacgc ctgtaatccc agcactttgg gaggccgaga cgggtggatc acgaggtcag 100020 gagatcgaga ccatcctggc taacacagtg aaaccccatc tctactaaaa atacaaaaaa 100080 acgagccggg cgtggtggtg ggcgcctgta gtcccagcta ctcgggaggc tgaggcagga 100140 gaatggcgtg aacccaggag gcagagcttg cagtgagccg agattgcacc actgcagtcc 100200 agcctgggcg acagagcgag actccgtctc aaaaaagaaa aagaaaaaga aaaagaactg 100260 tgattgggga ggacggtcac tttcctgttc ttactgatca gaagggatat taagggtacc 100320 tgattcaaac agcctggaga tcactgcttt caaccattac ctgccttatt tatttttagt 100380 tac...

Claims

1. A method for determining the acute neurotoxicity in vivo of a molecule containing a polynucleotide, the method comprising calculating a sequence score of the molecule, wherein the sequence score is calculated by the following formula: (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) / Total nucleotide length of polynucleotides ， When the sequence score is greater than or equal to 0.2, the molecule is predicted to have tolerable acute neurotoxicity in vivo.

2. A method for selecting a molecule comprising a polynucleotide having tolerable acute neurotoxicity in vivo, the method comprising calculating a sequence score of the molecule, wherein the sequence score is calculated by the following formula: (Number of C nucleotides or their analogues in a polynucleotide - Number of G nucleotides or their analogues in a polynucleotide) / Total nucleotide length of polynucleotides ， When the sequence score is greater than or equal to 0.2, the molecule is predicted to have tolerable acute neurotoxicity in vivo.

3. The method of claim 1 or 2, wherein the polynucleotide comprises DNA.

4. The method of claim 1 or 2, wherein the polynucleotide comprises RNA.

5. The method of claim 1 or 2, wherein the polynucleotide is single-stranded.

6. The method of claim 1 or 2, wherein the polynucleotide comprises an antisense oligonucleotide.

7. The method of claim 6, wherein the antisense oligonucleotide comprises at least one nucleotide analog.

8. The method of claim 7, wherein the at least one nucleotide analog is locked nucleic acid (LNA), 2'-O-alkyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleotide (ANA), 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalated nucleic acid (INA), restricted ethyl nucleoside (cEt), 2'-O-methyl nucleic acid (2'-OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), or any combination thereof.

9. The method of claim 6, wherein the antisense oligonucleotide comprises an internucleotide bond, wherein the internucleotide bond is a phosphodiester bond, a phosphotriester bond, a methylphosphonate bond, an aminophosphate bond, a thiophosphate bond, or a combination thereof.

10. The method of claim 6, wherein the antisense oligonucleotide is a gapmer, blockmer, mixer, or wingmer.

11. The method of claim 6, wherein the antisense oligonucleotide is a gapmer, blockmer, mixmer, headmer, tailmer, or totalmer.

12. The method of claim 1 or 2, the method further comprising measuring the microtubule strength of the molecule in a neuronal cell culture.

13. The method of claim 12, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than 25%.

14. The method of claim 12, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than 20%.

15. The method of claim 12, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than 15%.

16. The method of claim 12, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than 10%.

17. The method of claim 12, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than 5%.

18. The method of claim 12, wherein the molecule reduces the microtubule strength in neuronal cell cultures by less than 1%.

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