RNA agents for GST-Pi gene modulation
10047110 ยท 2018-08-14
Assignee
Inventors
- Kenjirou Minomi (Osaka, JP)
- Hirokazu Takahashi (Osaka, JP)
- Erika Terada (Osaka, JP)
- Jens Harborth (Carlsbad, CA, US)
- Jun Zhang (San Diego, CA)
- Mohammad Ahmadian (Carlsbad, CA)
- Wenbin Ying (San Diego, CA)
Cpc classification
C12N2310/344
CHEMISTRY; METALLURGY
C12N2320/32
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
C07D311/30
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
C12N15/1135
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
A61K49/0021
HUMAN NECESSITIES
C12N2320/35
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
This invention provides compounds, compositions and methods for modulating the expression of human GST-? using RNA interference. The RNA interference molecules can be used in methods for preventing or treating diseases such as malignant tumor. A nucleic acid molecule can have a) a polynucleotide sense strand and a polynucleotide antisense strand; b) each strand of the molecule being from 15 to 30 nucleotides in length; c) a contiguous region of from 15 to 30 nucleotides of the antisense strand being complementary to a sequence of an mRNA encoding GST-?; and d) at least a portion of the sense strand can be complementary to at least a portion of the antisense strand, and the molecule has a duplex region of from 15 to 30 nucleotides in length.
Claims
1. A nucleic acid molecule, wherein: the molecule has a polynucleotide sense strand and a polynucleotide antisense strand; wherein a contiguous region of the antisense strand is complementary to a sequence of an mRNA encoding GST-?; and wherein the antisense strand is UAGGGUCUCAAAAGGCUUCag (SEQ ID NO:1341), and the sense strand is GAAGCCUUUUGAGACCCUAtt (SEQ ID NO:1276); or wherein the antisense strand is ACAGCAGGGUCUCAAAAGGct (SEQ ID NO:1305), and the sense strand is CCUUUUGAGACCCUGCUGUtt (SEQ ID NO:1240) wherein upper case letters refer to ribonucleotides and lower case letters refer to deoxyribonucleotides.
2. The nucleic acid molecule of claim 1, wherein the polynucleotide sense strand and the polynucleotide antisense strand are connected as a single strand, and form a duplex region connected at one end by a loop.
3. The nucleic acid molecule of claim 1, wherein the molecule is an RNAi molecule active for gene silencing.
4. The nucleic acid molecule of claim 1, wherein the molecule is active for inhibiting expression of GST-?.
5. The nucleic acid molecule of claim 1, wherein the molecule has an IC50 for knockdown of GST-? in A549 cells of less than 100 pM.
6. A composition comprising one or more nucleic acid molecules of claim 1 and a pharmaceutically acceptable carrier.
7. The composition of claim 6, wherein the carrier is a lipid molecule or liposome.
8. A method for treating pancreatic cancer or lung cancer, the method comprising administering to a subject in need a composition of claim 6.
9. The method of claim 8, wherein the disease is pancreatic cancer.
10. The nucleic acid molecule of claim 1, wherein at least one or more nucleotides in any strand is chemically modified.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(5) This invention relates to compounds, compositions and methods for nucleic acid based therapeutics for modulating expression of GST-?.
(6) In some embodiments, this invention provides molecules active in RNA interference, as well as structures and compositions that can silence expression of GST-?.
(7) The structures and compositions of this disclosure can be used in preventing or treating various diseases such as malignant tumor.
(8) In further embodiments, this invention provides compositions for delivery and uptake of one or more therapeutic RNAi molecules of this invention, as well as methods of use thereof. The RNA-based compositions of this invention can be used in methods for preventing or treating malignant tumors, such as cancers.
(9) Therapeutic compositions of this invention include nucleic acid molecules that are active in RNA interference. The therapeutic nucleic acid molecules can be targeted to GSTP1 (GST-?) for gene silencing.
(10) In various embodiments, this invention provides a range of molecules that can be active as a small interfering RNA (siRNA), and can regulate or silence GST-? gene expression.
(11) The siRNAs of this invention can be used for preventing or treating malignant tumors.
(12) Embodiments of this invention further provide a vehicle, formulation, or lipid nanoparticle formulation for delivery of the inventive siRNAs to subjects in need of preventing or treating a malignant tumor. This invention further contemplates methods for administering siRNAs as therapeutics to mammals.
(13) The therapeutic molecules and compositions of this invention can be used for RNA interference directed to preventing or treating a GST-? associated disease, by administering a compound or composition to a subject in need.
(14) The methods of this invention can utilize the inventive compounds for preventing or treating malignant tumor. The malignant tumor can be presented in various diseases, for example, cancers that highly expressing GST-?, cancers caused by cells expressing mutated KRAS, sarcomas, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, carcinomas, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, colorectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, kidney cancer, urethral cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovary cancer, skin cancer, leukemia, malignant lymphoma, epithelial malignant tumors, and non-epithelial malignant tumors.
(15) In certain embodiments, a combination of therapeutic molecules of this invention can be used for silencing or inhibiting GST-? gene expression.
(16) This invention provides a range of RNAi molecules, where each molecule has a polynucleotide sense strand and a polynucleotide antisense strand; each strand of the molecule is from 15 to 30 nucleotides in length; a contiguous region of from 15 to 30 nucleotides of the antisense strand is complementary to a sequence of an mRNA encoding GST-?; and at least a portion of the sense strand is complementary to at least a portion of the antisense strand, and the molecule has a duplex region of from 15 to 30 nucleotides in length.
(17) A RNAi molecule of this invention can have a contiguous region of from 15 to 30 nucleotides of the antisense strand that is complementary to a sequence of an mRNA encoding GST-?, which is located in the duplex region of the molecule.
(18) In some embodiments, a RNAi molecule can have a contiguous region of from 15 to 30 nucleotides of the antisense strand that is complementary to a sequence of an mRNA encoding GST-?.
(19) Embodiments of this invention may further provide methods for preventing, treating or ameliorating one or more symptoms of malignant tumor, or reducing the risk of developing malignant tumor, or delaying the onset of malignant tumor in a mammal in need thereof.
(20) GST-? and RNAi Molecules
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(22) One of ordinary skill in the art would understand that a reported sequence may change over time and to incorporate any changes needed in the nucleic acid molecules herein accordingly.
(23) Embodiments of this invention can provide compositions and methods for gene silencing of GST-? expression using small nucleic acid molecules. Examples of nucleic acid molecules include molecules active in RNA interference (RNAi molecules), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, as well as DNA-directed RNAs (ddRNA), Piwi-interacting RNAs (piRNA), and repeat associated siRNAs (rasiRNA). Such molecules are capable of mediating RNA interference against GST-? gene expression.
(24) The composition and methods disclosed herein can also be used in treating various kinds of malignant tumors in a subject.
(25) The nucleic acid molecules and methods of this invention may be used to down regulate the expression of genes that encode GST-?.
(26) The compositions and methods of this invention can include one or more nucleic acid molecules, which, independently or in combination, can modulate or regulate the expression of GST-? protein and/or genes encoding GST-? proteins, proteins and/or genes encoding GST-? associated with the maintenance and/or development of diseases, conditions or disorders associated with GST-?, such as malignant tumor.
(27) The compositions and methods of this invention are described with reference to exemplary sequences of GST-?. A person of ordinary skill in the art would understand that various aspects and embodiments of the invention are directed to any related GST-? genes, sequences, or variants, such as homolog genes and transcript variants, and polymorphisms, including single nucleotide polymorphism (SNP) associated with any GST-? genes.
(28) In some embodiments, the compositions and methods of this invention can provide a double-stranded short interfering nucleic acid (siRNA) molecule that downregulates the expression of a GST-? gene, for example human GST-?.
(29) A RNAi molecule of this invention can be targeted to GST-? and any homologous sequences, for example, using complementary sequences or by incorporating non-canonical base pairs, for example, mismatches and/or wobble base pairs, that can provide additional target sequences.
(30) In instances where mismatches are identified, non-canonical base pairs, for example, mismatches and/or wobble bases can be used to generate nucleic acid molecules that target more than one gene sequence.
(31) For example, non-canonical base pairs such as UU and CC base pairs can be used to generate nucleic acid molecules that are capable of targeting sequences for differing GST-? targets that share sequence homology. Thus, a RNAi molecule can be targeted to a nucleotide sequence that is conserved between homologous genes, and a single RNAi molecule can be used to inhibit expression of more than one gene.
(32) In some aspects, the compositions and methods of this invention include RNAi molecules that are active against GST-? mRNA, where the RNAi molecule includes a sequence complementary to any mRNA encoding a GST-? sequence.
(33) In some embodiments, a RNAi molecule of this disclosure can have activity against GST-? RNA, where the RNAi molecule includes a sequence complementary to an RNA having a variant GST-? encoding sequence, for example, a mutant GST-? gene known in the art to be associated with malignant tumor.
(34) In further embodiments, a RNAi molecule of this invention can include a nucleotide sequence that can interact with a nucleotide sequence of a GST-? gene and mediate silencing of GST-? gene expression.
(35) Examples of RNAi molecules of this invention targeted to GST-? mRNA are shown in Tables 1 and 2.
(36) TABLE-US-00001 TABLE1 RNAimoleculesequencesforGST-7c SEQ SENSESTRAND SEQ ANTISENSESTRAND Ref ID (5-->3) ID (5-->3) Pos NO SEQIDNOS:2to608 NO SEQIDNOS:609to1215 232 2 GCCGCAGUCUUCGCCACCAtt 609 UGGUGGCGAAGACUGCGGCgg 233 3 CCGCAGUCUUCGCCACCAUtt 610 AUGGUGGCGAAGACUGCGGcg 234 4 CGCAGUCUUCGCCACCAUGtt 611 CAUGGUGGCGAAGACUGCGgc 235 5 GCAGUCUUCGCCACCAUGCtt 612 GCAUGGUGGCGAAGACUGCgg 236 6 CAGUCUUCGCCACCAUGCCtt 613 GGCAUGGUGGCGAAGACUGcg 237 7 AGUCUUCGCCACCAUGCCGtt 614 CGGCAUGGUGGCGAAGACUgc 238 8 GUCUUCGCCACCAUGCCGCtt 615 GCGGCAUGGUGGCGAAGACtg 239 9 UCUUCGCCACCAUGCCGCCtt 616 GGCGGCAUGGUGGCGAAGAct 240 10 CUUCGCCACCAUGCCGCCCtt 617 GGGCGGCAUGGUGGCGAAGac 241 11 UUCGCCACCAUGCCGCCCUtt 618 AGGGCGGCAUGGUGGCGAAga 242 12 UCGCCACCAUGCCGCCCUAtt 619 UAGGGCGGCAUGGUGGCGAag 243 13 CGCCACCAUGCCGCCCUACtt 620 GUAGGGCGGCAUGGUGGCGaa 244 14 GCCACCAUGCCGCCCUACAtt 621 UGUAGGGCGGCAUGGUGGCga 245 15 CCACCAUGCCGCCCUACACtt 622 GUGUAGGGCGGCAUGGUGGcg 246 16 CACCAUGCCGCCCUACACCtt 623 GGUGUAGGGCGGCAUGGUGgc 247 17 ACCAUGCCGCCCUACACCGtt 624 CGGUGUAGGGCGGCAUGGUgg 248 18 CCAUGCCGCCCUACACCGUtt 625 ACGGUGUAGGGCGGCAUGGtg 249 19 CAUGCCGCCCUACACCGUGtt 626 CACGGUGUAGGGCGGCAUGgt 250 20 AUGCCGCCCUACACCGUGGtt 627 CCACGGUGUAGGGCGGCAUgg 251 21 UGCCGCCCUACACCGUGGUtt 628 ACCACGGUGUAGGGCGGCAtg 252 22 GCCGCCCUACACCGUGGUCtt 629 GACCACGGUGUAGGGCGGCat 253 23 CCGCCCUACACCGUGGUCUtt 630 AGACCACGGUGUAGGGCGGca 254 24 CGCCCUACACCGUGGUCUAtt 631 UAGACCACGGUGUAGGGCGgc 255 25 GCCCUACACCGUGGUCUAUtt 632 AUAGACCACGGUGUAGGGCgg 256 26 CCCUACACCGUGGUCUAUUtt 633 AAUAGACCACGGUGUAGGGcg 257 27 CCUACACCGUGGUCUAUUUtt 634 AAAUAGACCACGGUGUAGGgc 258 28 CUACACCGUGGUCUAUUUCtt 635 GAAAUAGACCACGGUGUAGgg 259 29 UACACCGUGGUCUAUUUCCtt 636 GGAAAUAGACCACGGUGUAgg 260 30 ACACCGUGGUCUAUUUCCCtt 637 GGGAAAUAGACCACGGUGUag 261 31 CACCGUGGUCUAUUUCCCAtt 638 UGGGAAAUAGACCACGGUGta 262 32 ACCGUGGUCUAUUUCCCAGtt 639 CUGGGAAAUAGACCACGGUgt 263 33 CCGUGGUCUAUUUCCCAGUtt 640 ACUGGGAAAUAGACCACGGtg 264 34 CGUGGUCUAUUUCCCAGUUtt 641 AACUGGGAAAUAGACCACGgt 265 35 GUGGUCUAUUUCCCAGUUCtt 642 GAACUGGGAAAUAGACCACgg 266 36 UGGUCUAUUUCCCAGUUCGtt 643 CGAACUGGGAAAUAGACCAcg 267 37 GGUCUAUUUCCCAGUUCGAtt 644 UCGAACUGGGAAAUAGACCac 268 38 GUCUAUUUCCCAGUUCGAGtt 645 CUCGAACUGGGAAAUAGACca 269 39 UCUAUUUCCCAGUUCGAGGtt 646 CCUCGAACUGGGAAAUAGAcc 270 40 CUAUUUCCCAGUUCGAGGCtt 647 GCCUCGAACUGGGAAAUAGac 271 41 UAUUUCCCAGUUCGAGGCCtt 648 GGCCUCGAACUGGGAAAUAga 272 42 AUUUCCCAGUUCGAGGCCGtt 649 CGGCCUCGAACUGGGAAAUag 273 43 UUUCCCAGUUCGAGGCCGCtt 650 GCGGCCUCGAACUGGGAAAta 274 44 UUCCCAGUUCGAGGCCGCUtt 651 AGCGGCCUCGAACUGGGAAat 275 45 UCCCAGUUCGAGGCCGCUGtt 652 CAGCGGCCUCGAACUGGGAaa 276 46 CCCAGUUCGAGGCCGCUGCtt 653 GCAGCGGCCUCGAACUGGGaa 277 47 CCAGUUCGAGGCCGCUGCGtt 654 CGCAGCGGCCUCGAACUGGga 278 48 CAGUUCGAGGCCGCUGCGCtt 655 GCGCAGCGGCCUCGAACUGgg 279 49 AGUUCGAGGCCGCUGCGCGtt 656 CGCGCAGCGGCCUCGAACUgg 280 50 GUUCGAGGCCGCUGCGCGGtt 657 CCGCGCAGCGGCCUCGAACtg 281 51 UUCGAGGCCGCUGCGCGGCtt 658 GCCGCGCAGCGGCCUCGAAct 282 52 UCGAGGCCGCUGCGCGGCCtt 659 GGCCGCGCAGCGGCCUCGAac 283 53 CGAGGCCGCUGCGCGGCCCtt 660 GGGCCGCGCAGCGGCCUCGaa 284 54 GAGGCCGCUGCGCGGCCCUtt 661 AGGGCCGCGCAGCGGCCUCga 285 55 AGGCCGCUGCGCGGCCCUGtt 662 CAGGGCCGCGCAGCGGCCUcg 286 56 GGCCGCUGCGCGGCCCUGCtt 663 GCAGGGCCGCGCAGCGGCCtc 287 57 GCCGCUGCGCGGCCCUGCGtt 664 CGCAGGGCCGCGCAGCGGCct 288 58 CCGCUGCGCGGCCCUGCGCtt 665 GCGCAGGGCCGCGCAGCGGcc 289 59 CGCUGCGCGGCCCUGCGCAtt 666 UGCGCAGGGCCGCGCAGCGgc 290 60 GCUGCGCGGCCCUGCGCAUtt 667 AUGCGCAGGGCCGCGCAGCgg 291 61 CUGCGCGGCCCUGCGCAUGtt 668 CAUGCGCAGGGCCGCGCAGcg 292 62 UGCGCGGCCCUGCGCAUGCtt 669 GCAUGCGCAGGGCCGCGCAgc 293 63 GCGCGGCCCUGCGCAUGCUtt 670 AGCAUGCGCAGGGCCGCGCag 294 64 CGCGGCCCUGCGCAUGCUGtt 671 CAGCAUGCGCAGGGCCGCGca 295 65 GCGGCCCUGCGCAUGCUGCtt 672 GCAGCAUGCGCAGGGCCGCgc 296 66 CGGCCCUGCGCAUGCUGCUtt 673 AGCAGCAUGCGCAGGGCCGcg 297 67 GGCCCUGCGCAUGCUGCUGtt 674 CAGCAGCAUGCGCAGGGCCgc 298 68 GCCCUGCGCAUGCUGCUGGtt 675 CCAGCAGCAUGCGCAGGGCcg 299 69 CCCUGCGCAUGCUGCUGGCtt 676 GCCAGCAGCAUGCGCAGGGcc 300 70 CCUGCGCAUGCUGCUGGCAtt 677 UGCCAGCAGCAUGCGCAGGgc 301 71 CUGCGCAUGCUGCUGGCAGtt 678 CUGCCAGCAGCAUGCGCAGgg 302 72 UGCGCAUGCUGCUGGCAGAtt 679 UCUGCCAGCAGCAUGCGCAgg 303 73 GCGCAUGCUGCUGGCAGAUtt 680 AUCUGCCAGCAGCAUGCGCag 304 74 CGCAUGCUGCUGGCAGAUCtt 681 GAUCUGCCAGCAGCAUGCGca 305 75 GCAUGCUGCUGGCAGAUCAtt 682 UGAUCUGCCAGCAGCAUGCgc 306 76 CAUGCUGCUGGCAGAUCAGtt 683 CUGAUCUGCCAGCAGCAUGcg 307 77 AUGCUGCUGGCAGAUCAGGtt 684 CCUGAUCUGCCAGCAGCAUgc 308 78 UGCUGCUGGCAGAUCAGGGtt 685 CCCUGAUCUGCCAGCAGCAtg 309 79 GCUGCUGGCAGAUCAGGGCtt 686 GCCCUGAUCUGCCAGCAGCat 310 80 CUGCUGGCAGAUCAGGGCCtt 687 GGCCCUGAUCUGCCAGCAGca 311 81 UGCUGGCAGAUCAGGGCCAtt 688 UGGCCCUGAUCUGCCAGCAgc 312 82 GCUGGCAGAUCAGGGCCAGtt 689 CUGGCCCUGAUCUGCCAGCag 313 83 CUGGCAGAUCAGGGCCAGAtt 690 UCUGGCCCUGAUCUGCCAGca 314 84 UGGCAGAUCAGGGCCAGAGtt 691 CUCUGGCCCUGAUCUGCCAgc 315 85 GGCAGAUCAGGGCCAGAGCtt 692 GCUCUGGCCCUGAUCUGCCag 316 86 GCAGAUCAGGGCCAGAGCUtt 693 AGCUCUGGCCCUGAUCUGCca 317 87 CAGAUCAGGGCCAGAGCUGtt 694 CAGCUCUGGCCCUGAUCUGcc 318 88 AGAUCAGGGCCAGAGCUGGtt 695 CCAGCUCUGGCCCUGAUCUgc 319 89 GAUCAGGGCCAGAGCUGGAtt 696 UCCAGCUCUGGCCCUGAUCtg 320 90 AUCAGGGCCAGAGCUGGAAtt 697 UUCCAGCUCUGGCCCUGAUct 321 91 UCAGGGCCAGAGCUGGAAGtt 698 CUUCCAGCUCUGGCCCUGAtc 322 92 CAGGGCCAGAGCUGGAAGGtt 699 CCUUCCAGCUCUGGCCCUGat 323 93 AGGGCCAGAGCUGGAAGGAtt 700 UCCUUCCAGCUCUGGCCCUga 324 94 GGGCCAGAGCUGGAAGGAGtt 701 CUCCUUCCAGCUCUGGCCCtg 325 95 GGCCAGAGCUGGAAGGAGGtt 702 CCUCCUUCCAGCUCUGGCCct 326 96 GCCAGAGCUGGAAGGAGGAtt 703 UCCUCCUUCCAGCUCUGGCcc 327 97 CCAGAGCUGGAAGGAGGAGtt 704 CUCCUCCUUCCAGCUCUGGcc 328 98 CAGAGCUGGAAGGAGGAGGtt 705 CCUCCUCCUUCCAGCUCUGgc 329 99 AGAGCUGGAAGGAGGAGGUtt 706 ACCUCCUCCUUCCAGCUCUgg 330 100 GAGCUGGAAGGAGGAGGUGtt 707 CACCUCCUCCUUCCAGCUCtg 330 101 GAGCUGGAAGGAGGAGGUAtt 708 UACCUCCUCCUUCCAGCUCtg 331 102 AGCUGGAAGGAGGAGGUGGtt 709 CCACCUCCUCCUUCCAGCUct 332 103 GCUGGAAGGAGGAGGUGGUtt 710 ACCACCUCCUCCUUCCAGCtc 333 104 CUGGAAGGAGGAGGUGGUGtt 711 CACCACCUCCUCCUUCCAGct 334 105 UGGAAGGAGGAGGUGGUGAtt 712 UCACCACCUCCUCCUUCCAgc 335 106 GGAAGGAGGAGGUGGUGACtt 713 GUCACCACCUCCUCCUUCCag 336 107 GAAGGAGGAGGUGGUGACCtt 714 GGUCACCACCUCCUCCUUCca 337 108 AAGGAGGAGGUGGUGACCGtt 715 CGGUCACCACCUCCUCCUUcc 338 109 AGGAGGAGGUGGUGACCGUtt 716 ACGGUCACCACCUCCUCCUtc 339 110 GGAGGAGGUGGUGACCGUGtt 717 CACGGUCACCACCUCCUCCtt 340 111 GAGGAGGUGGUGACCGUGGtt 718 CCACGGUCACCACCUCCUCct 341 112 AGGAGGUGGUGACCGUGGAtt 719 UCCACGGUCACCACCUCCUcc 342 113 GGAGGUGGUGACCGUGGAGtt 720 CUCCACGGUCACCACCUCCtc 343 114 GAGGUGGUGACCGUGGAGAtt 721 UCUCCACGGUCACCACCUCct 344 115 AGGUGGUGACCGUGGAGACtt 722 GUCUCCACGGUCACCACCUcc 345 116 GGUGGUGACCGUGGAGACGtt 723 CGUCUCCACGGUCACCACCtc 346 117 GUGGUGACCGUGGAGACGUtt 724 ACGUCUCCACGGUCACCACct 347 118 UGGUGACCGUGGAGACGUGtt 725 CACGUCUCCACGGUCACCAcc 348 119 GGUGACCGUGGAGACGUGGtt 726 CCACGUCUCCACGGUCACCac 349 120 GUGACCGUGGAGACGUGGCtt 727 GCCACGUCUCCACGGUCACca 350 121 UGACCGUGGAGACGUGGCAtt 728 UGCCACGUCUCCACGGUCAcc 351 122 GACCGUGGAGACGUGGCAGtt 729 CUGCCACGUCUCCACGGUCac 352 123 ACCGUGGAGACGUGGCAGGtt 730 CCUGCCACGUCUCCACGGUca 353 124 CCGUGGAGACGUGGCAGGAtt 731 UCCUGCCACGUCUCCACGGtc 354 125 CGUGGAGACGUGGCAGGAGtt 732 CUCCUGCCACGUCUCCACGgt 355 126 GUGGAGACGUGGCAGGAGGtt 733 CCUCCUGCCACGUCUCCACgg 356 127 UGGAGACGUGGCAGGAGGGtt 734 CCCUCCUGCCACGUCUCCAcg 357 128 GGAGACGUGGCAGGAGGGCtt 735 GCCCUCCUGCCACGUCUCCac 358 129 GAGACGUGGCAGGAGGGCUtt 736 AGCCCUCCUGCCACGUCUCca 359 130 AGACGUGGCAGGAGGGCUCtt 737 GAGCCCUCCUGCCACGUCUcc 360 131 GACGUGGCAGGAGGGCUCAtt 738 UGAGCCCUCCUGCCACGUCtc 361 132 ACGUGGCAGGAGGGCUCACtt 739 GUGAGCCCUCCUGCCACGUct 362 133 CGUGGCAGGAGGGCUCACUtt 740 AGUGAGCCCUCCUGCCACGtc 363 134 GUGGCAGGAGGGCUCACUCtt 741 GAGUGAGCCCUCCUGCCACgt 364 135 UGGCAGGAGGGCUCACUCAtt 742 UGAGUGAGCCCUCCUGCCAcg 365 136 GGCAGGAGGGCUCACUCAAtt 743 UUGAGUGAGCCCUCCUGCCac 366 137 GCAGGAGGGCUCACUCAAAtt 744 UUUGAGUGAGCCCUCCUGCca 367 138 CAGGAGGGCUCACUCAAAGtt 745 CUUUGAGUGAGCCCUCCUGcc 368 139 AGGAGGGCUCACUCAAAGCtt 746 GCUUUGAGUGAGCCCUCCUgc 369 140 GGAGGGCUCACUCAAAGCCtt 747 GGCUUUGAGUGAGCCCUCCtg 370 141 GAGGGCUCACUCAAAGCCUtt 748 AGGCUUUGAGUGAGCCCUCct 371 142 AGGGCUCACUCAAAGCCUCtt 749 GAGGCUUUGAGUGAGCCCUcc 372 143 GGGCUCACUCAAAGCCUCCtt 750 GGAGGCUUUGAGUGAGCCCtc 373 144 GGCUCACUCAAAGCCUCCUtt 751 AGGAGGCUUUGAGUGAGCCct 374 145 GCUCACUCAAAGCCUCCUGtt 752 CAGGAGGCUUUGAGUGAGCcc 375 146 CUCACUCAAAGCCUCCUGCtt 753 GCAGGAGGCUUUGAGUGAGcc 376 147 UCACUCAAAGCCUCCUGCCtt 754 GGCAGGAGGCUUUGAGUGAgc 377 148 CACUCAAAGCCUCCUGCCUtt 755 AGGCAGGAGGCUUUGAGUGag 378 149 ACUCAAAGCCUCCUGCCUAtt 756 UAGGCAGGAGGCUUUGAGUga 379 150 CUCAAAGCCUCCUGCCUAUtt 757 AUAGGCAGGAGGCUUUGAGtg 380 151 UCAAAGCCUCCUGCCUAUAtt 758 UAUAGGCAGGAGGCUUUGAgt 381 152 CAAAGCCUCCUGCCUAUACtt 759 GUAUAGGCAGGAGGCUUUGag 382 153 AAAGCCUCCUGCCUAUACGtt 760 CGUAUAGGCAGGAGGCUUUga 383 154 AAGCCUCCUGCCUAUACGGtt 761 CCGUAUAGGCAGGAGGCUUtg 384 155 AGCCUCCUGCCUAUACGGGtt 762 CCCGUAUAGGCAGGAGGCUtt 385 156 GCCUCCUGCCUAUACGGGCtt 763 GCCCGUAUAGGCAGGAGGCtt 386 157 CCUCCUGCCUAUACGGGCAtt 764 UGCCCGUAUAGGCAGGAGGct 387 158 CUCCUGCCUAUACGGGCAGtt 765 CUGCCCGUAUAGGCAGGAGgc 388 159 UCCUGCCUAUACGGGCAGCtt 766 GCUGCCCGUAUAGGCAGGAgg 389 160 CCUGCCUAUACGGGCAGCUtt 767 AGCUGCCCGUAUAGGCAGGag 390 161 CUGCCUAUACGGGCAGCUCtt 768 GAGCUGCCCGUAUAGGCAGga 391 162 UGCCUAUACGGGCAGCUCCtt 769 GGAGCUGCCCGUAUAGGCAgg 392 163 GCCUAUACGGGCAGCUCCCtt 770 GGGAGCUGCCCGUAUAGGCag 409 164 CCCAAGUUCCAGGACGGAGtt 771 CUCCGUCCUGGAACUUGGGga 410 165 CCAAGUUCCAGGACGGAGAtt 772 UCUCCGUCCUGGAACUUGGgg 411 166 CAAGUUCCAGGACGGAGACtt 773 GUCUCCGUCCUGGAACUUGgg 412 167 AAGUUCCAGGACGGAGACCtt 774 GGUCUCCGUCCUGGAACUUgg 413 168 AGUUCCAGGACGGAGACCUtt 775 AGGUCUCCGUCCUGGAACUtg 414 169 GUUCCAGGACGGAGACCUCtt 776 GAGGUCUCCGUCCUGGAACtt 415 170 UUCCAGGACGGAGACCUCAtt 777 UGAGGUCUCCGUCCUGGAAct 416 171 UCCAGGACGGAGACCUCACtt 778 GUGAGGUCUCCGUCCUGGAac 417 172 CCAGGACGGAGACCUCACCtt 779 GGUGAGGUCUCCGUCCUGGaa 418 173 CAGGACGGAGACCUCACCCtt 780 GGGUGAGGUCUCCGUCCUGga 419 174 AGGACGGAGACCUCACCCUtt 781 AGGGUGAGGUCUCCGUCCUgg 420 175 GGACGGAGACCUCACCCUGtt 782 CAGGGUGAGGUCUCCGUCCtg 421 176 GACGGAGACCUCACCCUGUtt 783 ACAGGGUGAGGUCUCCGUCct 422 177 ACGGAGACCUCACCCUGUAtt 784 UACAGGGUGAGGUCUCCGUcc 423 178 CGGAGACCUCACCCUGUACtt 785 GUACAGGGUGAGGUCUCCGtc 424 179 GGAGACCUCACCCUGUACCtt 786 GGUACAGGGUGAGGUCUCCgt 425 180 GAGACCUCACCCUGUACCAtt 787 UGGUACAGGGUGAGGUCUCcg 426 181 AGACCUCACCCUGUACCAGtt 788 CUGGUACAGGGUGAGGUCUcc 427 182 GACCUCACCCUGUACCAGUtt 789 ACUGGUACAGGGUGAGGUCtc 428 183 ACCUCACCCUGUACCAGUCtt 790 GACUGGUACAGGGUGAGGUct 429 184 CCUCACCCUGUACCAGUCCtt 791 GGACUGGUACAGGGUGAGGtc 430 185 CUCACCCUGUACCAGUCCAtt 792 UGGACUGGUACAGGGUGAGgt 431 186 UCACCCUGUACCAGUCCAAtt 793 UUGGACUGGUACAGGGUGAgg 432 187 CACCCUGUACCAGUCCAAUtt 794 AUUGGACUGGUACAGGGUGag 433 188 ACCCUGUACCAGUCCAAUAtt 795 UAUUGGACUGGUACAGGGUga 434 189 CCCUGUACCAGUCCAAUACtt 796 GUAUUGGACUGGUACAGGGtg 435 190 CCUGUACCAGUCCAAUACCtt 797 GGUAUUGGACUGGUACAGGgt 436 191 CUGUACCAGUCCAAUACCAtt 798 UGGUAUUGGACUGGUACAGgg 437 192 UGUACCAGUCCAAUACCAUtt 799 AUGGUAUUGGACUGGUACAgg 438 193 GUACCAGUCCAAUACCAUCtt 800 GAUGGUAUUGGACUGGUACag 439 194 UACCAGUCCAAUACCAUCCtt 801 GGAUGGUAUUGGACUGGUAca 440 195 ACCAGUCCAAUACCAUCCUtt 802 AGGAUGGUAUUGGACUGGUac 441 196 CCAGUCCAAUACCAUCCUGtt 803 CAGGAUGGUAUUGGACUGGta 442 197 CAGUCCAAUACCAUCCUGCtt 804 GCAGGAUGGUAUUGGACUGgt 443 198 AGUCCAAUACCAUCCUGCGtt 805 CGCAGGAUGGUAUUGGACUgg 444 199 GUCCAAUACCAUCCUGCGUtt 806 ACGCAGGAUGGUAUUGGACtg 445 200 UCCAAUACCAUCCUGCGUCtt 807 GACGCAGGAUGGUAUUGGAct 446 201 CCAAUACCAUCCUGCGUCAtt 808 UGACGCAGGAUGGUAUUGGac 447 202 CAAUACCAUCCUGCGUCACtt 809 GUGACGCAGGAUGGUAUUGga 448 203 AAUACCAUCCUGCGUCACCtt 810 GGUGACGCAGGAUGGUAUUgg 449 204 AUACCAUCCUGCGUCACCUtt 811 AGGUGACGCAGGAUGGUAUtg 450 205 UACCAUCCUGCGUCACCUGtt 812 CAGGUGACGCAGGAUGGUAtt 451 206 ACCAUCCUGCGUCACCUGGtt 813 CCAGGUGACGCAGGAUGGUat 452 207 CCAUCCUGCGUCACCUGGGtt 814 CCCAGGUGACGCAGGAUGGta 453 208 CAUCCUGCGUCACCUGGGCtt 815 GCCCAGGUGACGCAGGAUGgt 454 209 AUCCUGCGUCACCUGGGCCtt 816 GGCCCAGGUGACGCAGGAUgg 455 210 UCCUGCGUCACCUGGGCCGtt 817 CGGCCCAGGUGACGCAGGAtg 456 211 CCUGCGUCACCUGGGCCGCtt 818 GCGGCCCAGGUGACGCAGGat 457 212 CUGCGUCACCUGGGCCGCAtt 819 UGCGGCCCAGGUGACGCAGga 458 213 UGCGUCACCUGGGCCGCACtt 820 GUGCGGCCCAGGUGACGCAgg 459 214 GCGUCACCUGGGCCGCACCtt 821 GGUGCGGCCCAGGUGACGCag 460 215 CGUCACCUGGGCCGCACCCtt 822 GGGUGCGGCCCAGGUGACGca 461 216 GUCACCUGGGCCGCACCCUtt 823 AGGGUGCGGCCCAGGUGACgc 462 217 UCACCUGGGCCGCACCCUUtt 824 AAGGGUGCGGCCCAGGUGAcg 463 218 CACCUGGGCCGCACCCUUGtt 825 CAAGGGUGCGGCCCAGGUGac 464 219 ACCUGGGCCGCACCCUUGGtt 826 CCAAGGGUGCGGCCCAGGUga 465 220 CCUGGGCCGCACCCUUGGGtt 827 CCCAAGGGUGCGGCCCAGGtg 466 221 CUGGGCCGCACCCUUGGGCtt 828 GCCCAAGGGUGCGGCCCAGgt 467 222 UGGGCCGCACCCUUGGGCUtt 829 AGCCCAAGGGUGCGGCCCAgg 468 223 GGGCCGCACCCUUGGGCUCtt 830 GAGCCCAAGGGUGCGGCCCag 469 224 GGCCGCACCCUUGGGCUCUtt 831 AGAGCCCAAGGGUGCGGCCca 470 225 GCCGCACCCUUGGGCUCUAtt 832 UAGAGCCCAAGGGUGCGGCcc 471 226 CCGCACCCUUGGGCUCUAUtt 833 AUAGAGCCCAAGGGUGCGGcc 472 227 CGCACCCUUGGGCUCUAUGtt 834 CAUAGAGCCCAAGGGUGCGgc 473 228 GCACCCUUGGGCUCUAUGGtt 835 CCAUAGAGCCCAAGGGUGCgg 474 229 CACCCUUGGGCUCUAUGGGtt 836 CCCAUAGAGCCCAAGGGUGcg 475 230 ACCCUUGGGCUCUAUGGGAtt 837 UCCCAUAGAGCCCAAGGGUgc 476 231 CCCUUGGGCUCUAUGGGAAtt 838 UUCCCAUAGAGCCCAAGGGtg 477 232 CCUUGGGCUCUAUGGGAAGtt 839 CUUCCCAUAGAGCCCAAGGgt 478 233 CUUGGGCUCUAUGGGAAGGtt 840 CCUUCCCAUAGAGCCCAAGgg 479 234 UUGGGCUCUAUGGGAAGGAtt 841 UCCUUCCCAUAGAGCCCAAgg 480 235 UGGGCUCUAUGGGAAGGACtt 842 GUCCUUCCCAUAGAGCCCAag 481 236 GGGCUCUAUGGGAAGGACCtt 843 GGUCCUUCCCAUAGAGCCCaa 482 237 GGCUCUAUGGGAAGGACCAtt 844 UGGUCCUUCCCAUAGAGCCca 483 238 GCUCUAUGGGAAGGACCAGtt 845 CUGGUCCUUCCCAUAGAGCcc 484 239 CUCUAUGGGAAGGACCAGCtt 846 GCUGGUCCUUCCCAUAGAGcc 485 240 UCUAUGGGAAGGACCAGCAtt 847 UGCUGGUCCUUCCCAUAGAgc 486 241 CUAUGGGAAGGACCAGCAGtt 848 CUGCUGGUCCUUCCCAUAGag 487 242 UAUGGGAAGGACCAGCAGGtt 849 CCUGCUGGUCCUUCCCAUAga 488 243 AUGGGAAGGACCAGCAGGAtt 850 UCCUGCUGGUCCUUCCCAUag 489 244 UGGGAAGGACCAGCAGGAGtt 851 CUCCUGCUGGUCCUUCCCAta 490 245 GGGAAGGACCAGCAGGAGGtt 852 CCUCCUGCUGGUCCUUCCCat 491 246 GGAAGGACCAGCAGGAGGCtt 853 GCCUCCUGCUGGUCCUUCCca 492 247 GAAGGACCAGCAGGAGGCAtt 854 UGCCUCCUGCUGGUCCUUCcc 493 248 AAGGACCAGCAGGAGGCAGtt 855 CUGCCUCCUGCUGGUCCUUcc 494 249 AGGACCAGCAGGAGGCAGCtt 856 GCUGCCUCCUGCUGGUCCUtc 495 250 GGACCAGCAGGAGGCAGCCtt 857 GGCUGCCUCCUGCUGGUCCtt 496 251 GACCAGCAGGAGGCAGCCCtt 858 GGGCUGCCUCCUGCUGGUCct 497 252 ACCAGCAGGAGGCAGCCCUtt 859 AGGGCUGCCUCCUGCUGGUcc 498 253 CCAGCAGGAGGCAGCCCUGtt 860 CAGGGCUGCCUCCUGCUGGtc 499 254 CAGCAGGAGGCAGCCCUGGtt 861 CCAGGGCUGCCUCCUGCUGgt 500 255 AGCAGGAGGCAGCCCUGGUtt 862 ACCAGGGCUGCCUCCUGCUgg 501 256 GCAGGAGGCAGCCCUGGUGtt 863 CACCAGGGCUGCCUCCUGCtg 502 257 CAGGAGGCAGCCCUGGUGGtt 864 CCACCAGGGCUGCCUCCUGct 503 258 AGGAGGCAGCCCUGGUGGAtt 865 UCCACCAGGGCUGCCUCCUgc 504 259 GGAGGCAGCCCUGGUGGACtt 866 GUCCACCAGGGCUGCCUCCtg 505 260 GAGGCAGCCCUGGUGGACAtt 867 UGUCCACCAGGGCUGCCUCct 506 261 AGGCAGCCCUGGUGGACAUtt 868 AUGUCCACCAGGGCUGCCUcc 507 262 GGCAGCCCUGGUGGACAUGtt 869 CAUGUCCACCAGGGCUGCCtc 508 263 GCAGCCCUGGUGGACAUGGtt 870 CCAUGUCCACCAGGGCUGCct 509 264 CAGCCCUGGUGGACAUGGUtt 871 ACCAUGUCCACCAGGGCUGcc 510 265 AGCCCUGGUGGACAUGGUGtt 872 CACCAUGUCCACCAGGGCUgc 511 266 GCCCUGGUGGACAUGGUGAtt 873 UCACCAUGUCCACCAGGGCtg 512 267 CCCUGGUGGACAUGGUGAAtt 874 UUCACCAUGUCCACCAGGGct 513 268 CCUGGUGGACAUGGUGAAUtt 875 AUUCACCAUGUCCACCAGGgc 514 269 CUGGUGGACAUGGUGAAUGtt 876 CAUUCACCAUGUCCACCAGgg 515 270 UGGUGGACAUGGUGAAUGAtt 877 UCAUUCACCAUGUCCACCAgg 516 271 GGUGGACAUGGUGAAUGACtt 878 GUCAUUCACCAUGUCCACCag 517 272 GUGGACAUGGUGAAUGACGtt 879 CGUCAUUCACCAUGUCCACca 518 273 UGGACAUGGUGAAUGACGGtt 880 CCGUCAUUCACCAUGUCCAcc 519 274 GGACAUGGUGAAUGACGGCtt 881 GCCGUCAUUCACCAUGUCCac 520 275 GACAUGGUGAAUGACGGCGtt 882 CGCCGUCAUUCACCAUGUCca 521 276 ACAUGGUGAAUGACGGCGUtt 883 ACGCCGUCAUUCACCAUGUcc 522 277 CAUGGUGAAUGACGGCGUGtt 884 CACGCCGUCAUUCACCAUGtc 523 278 AUGGUGAAUGACGGCGUGGtt 885 CCACGCCGUCAUUCACCAUgt 524 279 UGGUGAAUGACGGCGUGGAtt 886 UCCACGCCGUCAUUCACCAtg 525 280 GGUGAAUGACGGCGUGGAGtt 887 CUCCACGCCGUCAUUCACCat 526 281 GUGAAUGACGGCGUGGAGGtt 888 CCUCCACGCCGUCAUUCACca 527 282 UGAAUGACGGCGUGGAGGAtt 889 UCCUCCACGCCGUCAUUCAcc 528 283 GAAUGACGGCGUGGAGGACtt 890 GUCCUCCACGCCGUCAUUCac 529 284 AAUGACGGCGUGGAGGACCtt 891 GGUCCUCCACGCCGUCAUUca 530 285 AUGACGGCGUGGAGGACCUtt 892 AGGUCCUCCACGCCGUCAUtc 531 286 UGACGGCGUGGAGGACCUCtt 893 GAGGUCCUCCACGCCGUCAtt 532 287 GACGGCGUGGAGGACCUCCtt 894 GGAGGUCCUCCACGCCGUCat 533 288 ACGGCGUGGAGGACCUCCGtt 895 CGGAGGUCCUCCACGCCGUca 534 289 CGGCGUGGAGGACCUCCGCtt 896 GCGGAGGUCCUCCACGCCGtc 535 290 GGCGUGGAGGACCUCCGCUtt 897 AGCGGAGGUCCUCCACGCCgt 536 291 GCGUGGAGGACCUCCGCUGtt 898 CAGCGGAGGUCCUCCACGCcg 537 292 CGUGGAGGACCUCCGCUGCtt 899 GCAGCGGAGGUCCUCCACGcc 538 293 GUGGAGGACCUCCGCUGCAtt 900 UGCAGCGGAGGUCCUCCACgc 539 294 UGGAGGACCUCCGCUGCAAtt 901 UUGCAGCGGAGGUCCUCCAcg 540 295 GGAGGACCUCCGCUGCAAAtt 902 UUUGCAGCGGAGGUCCUCCac 541 296 GAGGACCUCCGCUGCAAAUtt 903 AUUUGCAGCGGAGGUCCUCca 542 297 AGGACCUCCGCUGCAAAUAtt 904 UAUUUGCAGCGGAGGUCCUcc 543 298 GGACCUCCGCUGCAAAUACtt 905 GUAUUUGCAGCGGAGGUCCtc 544 299 GACCUCCGCUGCAAAUACAtt 906 UGUAUUUGCAGCGGAGGUCct 545 300 ACCUCCGCUGCAAAUACAUtt 907 AUGUAUUUGCAGCGGAGGUcc 546 301 CCUCCGCUGCAAAUACAUCtt 908 GAUGUAUUUGCAGCGGAGGtc 547 302 CUCCGCUGCAAAUACAUCUtt 909 AGAUGUAUUUGCAGCGGAGgt 548 303 UCCGCUGCAAAUACAUCUCtt 910 GAGAUGUAUUUGCAGCGGAgg 549 304 CCGCUGCAAAUACAUCUCCtt 911 GGAGAUGUAUUUGCAGCGGag 550 305 CGCUGCAAAUACAUCUCCCtt 912 GGGAGAUGUAUUUGCAGCGga 551 306 GCUGCAAAUACAUCUCCCUtt 913 AGGGAGAUGUAUUUGCAGCgg 552 307 CUGCAAAUACAUCUCCCUCtt 914 GAGGGAGAUGUAUUUGCAGcg 553 308 UGCAAAUACAUCUCCCUCAtt 915 UGAGGGAGAUGUAUUUGCAgc 554 309 GCAAAUACAUCUCCCUCAUtt 916 AUGAGGGAGAUGUAUUUGCag 555 310 CAAAUACAUCUCCCUCAUCtt 917 GAUGAGGGAGAUGUAUUUGca 556 311 AAAUACAUCUCCCUCAUCUtt 918 AGAUGAGGGAGAUGUAUUUgc 557 312 AAUACAUCUCCCUCAUCUAtt 919 UAGAUGAGGGAGAUGUAUUtg 558 313 AUACAUCUCCCUCAUCUACtt 920 GUAGAUGAGGGAGAUGUAUtt 559 314 UACAUCUCCCUCAUCUACAtt 921 UGUAGAUGAGGGAGAUGUAtt 560 315 ACAUCUCCCUCAUCUACACtt 922 GUGUAGAUGAGGGAGAUGUat 561 316 CAUCUCCCUCAUCUACACCtt 923 GGUGUAGAUGAGGGAGAUGta 562 317 AUCUCCCUCAUCUACACCAtt 924 UGGUGUAGAUGAGGGAGAUgt 563 318 UCUCCCUCAUCUACACCAAtt 925 UUGGUGUAGAUGAGGGAGAtg 563 319 GCUCCCUCAUCUACACCAAtt 926 UUGGUGUAGAUGAGGGAGCtg 564 320 CUCCCUCAUCUACACCAACtt 927 GUUGGUGUAGAUGAGGGAGat 565 321 UCCCUCAUCUACACCAACUtt 928 AGUUGGUGUAGAUGAGGGAga 565 322 CUCCCUCAUCUACACCAAAtt 929 UUUGGUGUAGAUGAGGGAGat 566 323 CCCUCAUCUACACCAACUAtt 930 UAGUUGGUGUAGAUGAGGGag 567 324 CCUCAUCUACACCAACUAUtt 931 AUAGUUGGUGUAGAUGAGGga 567 325 CCUCAUCUACACCAACUAAtt 932 UUAGUUGGUGUAGAUGAGGga 568 326 CUCAUCUACACCAACUAUGtt 933 CAUAGUUGGUGUAGAUGAGgg 569 327 UCAUCUACACCAACUAUGAtt 934 UCAUAGUUGGUGUAGAUGAgg 570 328 CAUCUACACCAACUAUGAGtt 935 CUCAUAGUUGGUGUAGAUGag 571 329 AUCUACACCAACUAUGAGGtt 936 CCUCAUAGUUGGUGUAGAUga 572 330 UCUACACCAACUAUGAGGCtt 937 GCCUCAUAGUUGGUGUAGAtg 573 331 CUACACCAACUAUGAGGCGtt 938 CGCCUCAUAGUUGGUGUAGat 574 332 UACACCAACUAUGAGGCGGtt 939 CCGCCUCAUAGUUGGUGUAga 575 333 ACACCAACUAUGAGGCGGGtt 940 CCCGCCUCAUAGUUGGUGUag 576 334 CACCAACUAUGAGGCGGGCtt 941 GCCCGCCUCAUAGUUGGUGta 577 335 ACCAACUAUGAGGCGGGCAtt 942 UGCCCGCCUCAUAGUUGGUgt 578 336 CCAACUAUGAGGCGGGCAAtt 943 UUGCCCGCCUCAUAGUUGGtg 579 337 CAACUAUGAGGCGGGCAAGtt 944 CUUGCCCGCCUCAUAGUUGgt 580 338 AACUAUGAGGCGGGCAAGGtt 945 CCUUGCCCGCCUCAUAGUUgg 581 339 ACUAUGAGGCGGGCAAGGAtt 946 UCCUUGCCCGCCUCAUAGUtg 582 340 CUAUGAGGCGGGCAAGGAUtt 947 AUCCUUGCCCGCCUCAUAGtt 583 341 UAUGAGGCGGGCAAGGAUGtt 948 CAUCCUUGCCCGCCUCAUAgt 584 342 AUGAGGCGGGCAAGGAUGAtt 949 UCAUCCUUGCCCGCCUCAUag 585 343 UGAGGCGGGCAAGGAUGACtt 950 GUCAUCCUUGCCCGCCUCAta 586 344 GAGGCGGGCAAGGAUGACUtt 951 AGUCAUCCUUGCCCGCCUCat 587 345 AGGCGGGCAAGGAUGACUAtt 952 UAGUCAUCCUUGCCCGCCUca 588 346 GGCGGGCAAGGAUGACUAUtt 953 AUAGUCAUCCUUGCCCGCCtc 589 347 GCGGGCAAGGAUGACUAUGtt 954 CAUAGUCAUCCUUGCCCGCct 590 348 CGGGCAAGGAUGACUAUGUtt 955 ACAUAGUCAUCCUUGCCCGcc 591 349 GGGCAAGGAUGACUAUGUGtt 956 CACAUAGUCAUCCUUGCCCgc 592 350 GGCAAGGAUGACUAUGUGAtt 957 UCACAUAGUCAUCCUUGCCcg 593 351 GCAAGGAUGACUAUGUGAAtt 958 UUCACAUAGUCAUCCUUGCcc 594 352 CAAGGAUGACUAUGUGAAGtt 959 CUUCACAUAGUCAUCCUUGcc 595 353 AAGGAUGACUAUGUGAAGGtt 960 CCUUCACAUAGUCAUCCUUgc 596 354 AGGAUGACUAUGUGAAGGCtt 961 GCCUUCACAUAGUCAUCCUtg 597 355 GGAUGACUAUGUGAAGGCAtt 962 UGCCUUCACAUAGUCAUCCtt 598 356 GAUGACUAUGUGAAGGCACtt 963 GUGCCUUCACAUAGUCAUCct 599 357 AUGACUAUGUGAAGGCACUtt 964 AGUGCCUUCACAUAGUCAUcc 600 358 UGACUAUGUGAAGGCACUGtt 965 CAGUGCCUUCACAUAGUCAtc 601 359 GACUAUGUGAAGGCACUGCtt 966 GCAGUGCCUUCACAUAGUCat 602 360 ACUAUGUGAAGGCACUGCCtt 967 GGCAGUGCCUUCACAUAGUca 603 361 CUAUGUGAAGGCACUGCCCtt 968 GGGCAGUGCCUUCACAUAGtc 604 362 UAUGUGAAGGCACUGCCCGtt 969 CGGGCAGUGCCUUCACAUAgt 605 363 AUGUGAAGGCACUGCCCGGtt 970 CCGGGCAGUGCCUUCACAUag 606 364 UGUGAAGGCACUGCCCGGGtt 971 CCCGGGCAGUGCCUUCACAta 607 365 GUGAAGGCACUGCCCGGGCtt 972 GCCCGGGCAGUGCCUUCACat 608 366 UGAAGGCACUGCCCGGGCAtt 973 UGCCCGGGCAGUGCCUUCAca 609 367 GAAGGCACUGCCCGGGCAAtt 974 UUGCCCGGGCAGUGCCUUCac 610 368 AAGGCACUGCCCGGGCAACtt 975 GUUGCCCGGGCAGUGCCUUca 611 369 AGGCACUGCCCGGGCAACUtt 976 AGUUGCCCGGGCAGUGCCUtc 612 370 GGCACUGCCCGGGCAACUGtt 977 CAGUUGCCCGGGCAGUGCCtt 613 371 GCACUGCCCGGGCAACUGAtt 978 UCAGUUGCCCGGGCAGUGCct 614 372 CACUGCCCGGGCAACUGAAtt 979 UUCAGUUGCCCGGGCAGUGcc 615 373 ACUGCCCGGGCAACUGAAGtt 980 CUUCAGUUGCCCGGGCAGUgc 616 374 CUGCCCGGGCAACUGAAGCtt 981 GCUUCAGUUGCCCGGGCAGtg 617 375 UGCCCGGGCAACUGAAGCCtt 982 GGCUUCAGUUGCCCGGGCAgt 618 376 GCCCGGGCAACUGAAGCCUtt 983 AGGCUUCAGUUGCCCGGGCag 619 377 CCCGGGCAACUGAAGCCUUtt 984 AAGGCUUCAGUUGCCCGGGca 620 378 CCGGGCAACUGAAGCCUUUtt 985 AAAGGCUUCAGUUGCCCGGgc 621 379 CGGGCAACUGAAGCCUUUUtt 986 AAAAGGCUUCAGUUGCCCGgg 622 380 GGGCAACUGAAGCCUUUUGtt 987 CAAAAGGCUUCAGUUGCCCgg 623 381 GGCAACUGAAGCCUUUUGAtt 988 UCAAAAGGCUUCAGUUGCCcg 624 382 GCAACUGAAGCCUUUUGAGtt 989 CUCAAAAGGCUUCAGUUGCcc 625 383 CAACUGAAGCCUUUUGAGAtt 990 UCUCAAAAGGCUUCAGUUGcc 626 384 AACUGAAGCCUUUUGAGACtt 991 GUCUCAAAAGGCUUCAGUUgc 627 385 ACUGAAGCCUUUUGAGACCtt 992 GGUCUCAAAAGGCUUCAGUtg 627 386 ACUGAAGCCUUUUGAGACAtt 993 UGUCUCAAAAGGCUUCAGUtg 628 387 CUGAAGCCUUUUGAGACCCtt 994 GGGUCUCAAAAGGCUUCAGtt 629 388 UGAAGCCUUUUGAGACCCUtt 995 AGGGUCUCAAAAGGCUUCAgt 630 389 GAAGCCUUUUGAGACCCUGtt 996 CAGGGUCUCAAAAGGCUUCag 631 390 AAGCCUUUUGAGACCCUGCtt 997 GCAGGGUCUCAAAAGGCUUca 631 391 GAAGCCUUUUGAGACCCUAtt 998 UAGGGUCUCAAAAGGCUUCag 632 392 AGCCUUUUGAGACCCUGCUtt 999 AGCAGGGUCUCAAAAGGCUtc 632 393 CGCCUUUUGAGACCCUGCAtt 1000 UGCAGGGUCUCAAAAGGCGtc 632 394 AGCCUUUUGAGACCCUGCAtt 1001 UGCAGGGUCUCAAAAGGCUtc 633 395 GCCUUUUGAGACCCUGCUGtt 1002 CAGCAGGGUCUCAAAAGGCtt 634 396 CCUUUUGAGACCCUGCUGUtt 1003 ACAGCAGGGUCUCAAAAGGct 634 397 CCUUUUGAGACCCUGCUGAtt 1004 UCAGCAGGGUCUCAAAAGGct 635 398 CUUUUGAGACCCUGCUGUCtt 1005 GACAGCAGGGUCUCAAAAGgc 635 399 CUUUUGAGACCCUGCUGUAtt 1006 UACAGCAGGGUCUCAAAAGgc 636 400 UUUUGAGACCCUGCUGUCCtt 1007 GGACAGCAGGGUCUCAAAAgg 637 401 UUUGAGACCCUGCUGUCCCtt 1008 GGGACAGCAGGGUCUCAAAag 638 402 UUGAGACCCUGCUGUCCCAtt 1009 UGGGACAGCAGGGUCUCAAaa 639 403 UGAGACCCUGCUGUCCCAGtt 1010 CUGGGACAGCAGGGUCUCAaa 640 404 GAGACCCUGCUGUCCCAGAtt 1011 UCUGGGACAGCAGGGUCUCaa 641 405 AGACCCUGCUGUCCCAGAAtt 1012 UUCUGGGACAGCAGGGUCUca 642 406 GACCCUGCUGUCCCAGAACtt 1013 GUUCUGGGACAGCAGGGUCtc 643 407 ACCCUGCUGUCCCAGAACCtt 1014 GGUUCUGGGACAGCAGGGUct 643 408 ACCCUGCUGUCCCAGAACAtt 1015 UGUUCUGGGACAGCAGGGUct 644 409 CCCUGCUGUCCCAGAACCAtt 1016 UGGUUCUGGGACAGCAGGGtc 645 410 CCUGCUGUCCCAGAACCAGtt 1017 CUGGUUCUGGGACAGCAGGgt 646 411 CUGCUGUCCCAGAACCAGGtt 1018 CCUGGUUCUGGGACAGCAGgg 647 412 UGCUGUCCCAGAACCAGGGtt 1019 CCCUGGUUCUGGGACAGCAgg 648 413 UGCUGUCCCAGAACCAGGAtt 1020 UCCUGGUUCUGGGACAGCAgg 648 414 GCUGUCCCAGAACCAGGGAtt 1021 UCCCUGGUUCUGGGACAGCag 649 415 CUGUCCCAGAACCAGGGAGtt 1022 CUCCCUGGUUCUGGGACAGca 650 416 UGUCCCAGAACCAGGGAGGtt 1023 CCUCCCUGGUUCUGGGACAgc 651 417 GUCCCAGAACCAGGGAGGCtt 1024 GCCUCCCUGGUUCUGGGACag 652 418 UCCCAGAACCAGGGAGGCAtt 1025 UGCCUCCCUGGUUCUGGGAca 653 419 CCCAGAACCAGGGAGGCAAtt 1026 UUGCCUCCCUGGUUCUGGGac 654 420 CCAGAACCAGGGAGGCAAGtt 1027 CUUGCCUCCCUGGUUCUGGga 655 421 CAGAACCAGGGAGGCAAGAtt 1028 UCUUGCCUCCCUGGUUCUGgg 656 422 AGAACCAGGGAGGCAAGACtt 1029 GUCUUGCCUCCCUGGUUCUgg 657 423 GAACCAGGGAGGCAAGACCtt 1030 GGUCUUGCCUCCCUGGUUCtg 658 424 AACCAGGGAGGCAAGACCUtt 1031 AGGUCUUGCCUCCCUGGUUct 659 425 ACCAGGGAGGCAAGACCUUtt 1032 AAGGUCUUGCCUCCCUGGUtc 660 426 CCAGGGAGGCAAGACCUUCtt 1033 GAAGGUCUUGCCUCCCUGGtt 661 427 CAGGGAGGCAAGACCUUCAtt 1034 UGAAGGUCUUGCCUCCCUGgt 662 428 AGGGAGGCAAGACCUUCAUtt 1035 AUGAAGGUCUUGCCUCCCUgg 663 429 GGGAGGCAAGACCUUCAUUtt 1036 AAUGAAGGUCUUGCCUCCCtg 664 430 GGAGGCAAGACCUUCAUUGtt 1037 CAAUGAAGGUCUUGCCUCCct 665 431 GAGGCAAGACCUUCAUUGUtt 1038 ACAAUGAAGGUCUUGCCUCcc 666 432 AGGCAAGACCUUCAUUGUGtt 1039 CACAAUGAAGGUCUUGCCUcc 667 433 GGCAAGACCUUCAUUGUGGtt 1040 CCACAAUGAAGGUCUUGCCtc 668 434 GCAAGACCUUCAUUGUGGGtt 1041 CCCACAAUGAAGGUCUUGCct 669 435 CAAGACCUUCAUUGUGGGAtt 1042 UCCCACAAUGAAGGUCUUGcc 670 436 AAGACCUUCAUUGUGGGAGtt 1043 CUCCCACAAUGAAGGUCUUgc 671 437 AGACCUUCAUUGUGGGAGAtt 1044 UCUCCCACAAUGAAGGUCUtg 672 438 GACCUUCAUUGUGGGAGACtt 1045 GUCUCCCACAAUGAAGGUCtt 673 439 ACCUUCAUUGUGGGAGACCtt 1046 GGUCUCCCACAAUGAAGGUct 674 440 CCUUCAUUGUGGGAGACCAtt 1047 UGGUCUCCCACAAUGAAGGtc 675 441 CUUCAUUGUGGGAGACCAGtt 1048 CUGGUCUCCCACAAUGAAGgt 676 442 UUCAUUGUGGGAGACCAGAtt 1049 UCUGGUCUCCCACAAUGAAgg 677 443 UCAUUGUGGGAGACCAGAUtt 1050 AUCUGGUCUCCCACAAUGAag 678 444 CAUUGUGGGAGACCAGAUCtt 1051 GAUCUGGUCUCCCACAAUGaa 679 445 AUUGUGGGAGACCAGAUCUtt 1052 AGAUCUGGUCUCCCACAAUga 680 446 UUGUGGGAGACCAGAUCUCtt 1053 GAGAUCUGGUCUCCCACAAtg 681 447 UGUGGGAGACCAGAUCUCCtt 1054 GGAGAUCUGGUCUCCCACAat 682 448 GUGGGAGACCAGAUCUCCUtt 1055 AGGAGAUCUGGUCUCCCACaa 683 449 UGGGAGACCAGAUCUCCUUtt 1056 AAGGAGAUCUGGUCUCCCAca 684 450 GGGAGACCAGAUCUCCUUCtt 1057 GAAGGAGAUCUGGUCUCCCac 685 451 GGAGACCAGAUCUCCUUCGtt 1058 CGAAGGAGAUCUGGUCUCCca 686 452 GAGACCAGAUCUCCUUCGCtt 1059 GCGAAGGAGAUCUGGUCUCcc 687 453 AGACCAGAUCUCCUUCGCUtt 1060 AGCGAAGGAGAUCUGGUCUcc 688 454 GACCAGAUCUCCUUCGCUGtt 1061 CAGCGAAGGAGAUCUGGUCtc 689 455 ACCAGAUCUCCUUCGCUGAtt 1062 UCAGCGAAGGAGAUCUGGUct 690 456 CCAGAUCUCCUUCGCUGACtt 1063 GUCAGCGAAGGAGAUCUGGtc 691 457 CAGAUCUCCUUCGCUGACUtt 1064 AGUCAGCGAAGGAGAUCUGgt 692 458 AGAUCUCCUUCGCUGACUAtt 1065 UAGUCAGCGAAGGAGAUCUgg 693 459 GAUCUCCUUCGCUGACUACtt 1066 GUAGUCAGCGAAGGAGAUCtg 694 460 AUCUCCUUCGCUGACUACAtt 1067 UGUAGUCAGCGAAGGAGAUct 695 461 UCUCCUUCGCUGACUACAAtt 1068 UUGUAGUCAGCGAAGGAGAtc 696 462 CUCCUUCGCUGACUACAACtt 1069 GUUGUAGUCAGCGAAGGAGat 697 463 UCCUUCGCUGACUACAACCtt 1070 GGUUGUAGUCAGCGAAGGAga 698 464 CCUUCGCUGACUACAACCUtt 1071 AGGUUGUAGUCAGCGAAGGag 699 465 CUUCGCUGACUACAACCUGtt 1072 CAGGUUGUAGUCAGCGAAGga 700 466 UUCGCUGACUACAACCUGCtt 1073 GCAGGUUGUAGUCAGCGAAgg 701 467 UCGCUGACUACAACCUGCUtt 1074 AGCAGGUUGUAGUCAGCGAag 702 468 CGCUGACUACAACCUGCUGtt 1075 CAGCAGGUUGUAGUCAGCGaa 703 469 GCUGACUACAACCUGCUGGtt 1076 CCAGCAGGUUGUAGUCAGCga 704 470 CUGACUACAACCUGCUGGAtt 1077 UCCAGCAGGUUGUAGUCAGcg 705 471 UGACUACAACCUGCUGGACtt 1078 GUCCAGCAGGUUGUAGUCAgc 706 472 GACUACAACCUGCUGGACUtt 1079 AGUCCAGCAGGUUGUAGUCag 707 473 ACUACAACCUGCUGGACUUtt 1080 AAGUCCAGCAGGUUGUAGUca 708 474 CUACAACCUGCUGGACUUGtt 1081 CAAGUCCAGCAGGUUGUAGtc 709 475 UACAACCUGCUGGACUUGCtt 1082 GCAAGUCCAGCAGGUUGUAgt 710 476 ACAACCUGCUGGACUUGCUtt 1083 AGCAAGUCCAGCAGGUUGUag 711 477 CAACCUGCUGGACUUGCUGtt 1084 CAGCAAGUCCAGCAGGUUGta 712 478 AACCUGCUGGACUUGCUGCtt 1085 GCAGCAAGUCCAGCAGGUUgt 713 479 ACCUGCUGGACUUGCUGCUtt 1086 AGCAGCAAGUCCAGCAGGUtg 714 480 CCUGCUGGACUUGCUGCUGtt 1087 CAGCAGCAAGUCCAGCAGGtt 715 481 CUGCUGGACUUGCUGCUGAtt 1088 UCAGCAGCAAGUCCAGCAGgt 716 482 UGCUGGACUUGCUGCUGAUtt 1089 AUCAGCAGCAAGUCCAGCAgg 717 483 GCUGGACUUGCUGCUGAUCtt 1090 GAUCAGCAGCAAGUCCAGCag 718 484 CUGGACUUGCUGCUGAUCCtt 1091 GGAUCAGCAGCAAGUCCAGca 719 485 UGGACUUGCUGCUGAUCCAtt 1092 UGGAUCAGCAGCAAGUCCAgc 720 486 GGACUUGCUGCUGAUCCAUtt 1093 AUGGAUCAGCAGCAAGUCCag 721 487 GACUUGCUGCUGAUCCAUGtt 1094 CAUGGAUCAGCAGCAAGUCca 722 488 ACUUGCUGCUGAUCCAUGAtt 1095 UCAUGGAUCAGCAGCAAGUcc 723 489 CUUGCUGCUGAUCCAUGAGtt 1096 CUCAUGGAUCAGCAGCAAGtc 724 490 UUGCUGCUGAUCCAUGAGGtt 1097 CCUCAUGGAUCAGCAGCAAgt 725 491 UGCUGCUGAUCCAUGAGGUtt 1098 ACCUCAUGGAUCAGCAGCAag 726 492 GCUGCUGAUCCAUGAGGUCtt 1099 GACCUCAUGGAUCAGCAGCaa 727 493 CUGCUGAUCCAUGAGGUCCtt 1100 GGACCUCAUGGAUCAGCAGca 728 494 UGCUGAUCCAUGAGGUCCUtt 1101 AGGACCUCAUGGAUCAGCAgc 729 495 GCUGAUCCAUGAGGUCCUAtt 1102 UAGGACCUCAUGGAUCAGCag 730 496 CUGAUCCAUGAGGUCCUAGtt 1103 CUAGGACCUCAUGGAUCAGca 731 497 UGAUCCAUGAGGUCCUAGCtt 1104 GCUAGGACCUCAUGGAUCAgc 732 498 GAUCCAUGAGGUCCUAGCCtt 1105 GGCUAGGACCUCAUGGAUCag 733 499 AUCCAUGAGGUCCUAGCCCtt 1106 GGGCUAGGACCUCAUGGAUca 750 500 CCCUGGCUGCCUGGAUGCGtt 1107 CGCAUCCAGGCAGCCAGGGgc 751 501 CCUGGCUGCCUGGAUGCGUtt 1108 ACGCAUCCAGGCAGCCAGGgg 752 502 CUGGCUGCCUGGAUGCGUUtt 1109 AACGCAUCCAGGCAGCCAGgg 753 503 UGGCUGCCUGGAUGCGUUCtt 1110 GAACGCAUCCAGGCAGCCAgg 754 504 GGCUGCCUGGAUGCGUUCCtt 1111 GGAACGCAUCCAGGCAGCCag 755 505 GCUGCCUGGAUGCGUUCCCtt 1112 GGGAACGCAUCCAGGCAGCca 773 506 CCCUGCUCUCAGCAUAUGUtt 1113 ACAUAUGCUGAGAGCAGGGgg 774 507 CCUGCUCUCAGCAUAUGUGtt 1114 CACAUAUGCUGAGAGCAGGgg 775 508 CUGCUCUCAGCAUAUGUGGtt 1115 CCACAUAUGCUGAGAGCAGgg 776 509 UGCUCUCAGCAUAUGUGGGtt 1116 CCCACAUAUGCUGAGAGCAgg 793 510 GGGCGCCUCAGUGCCCGGCtt 1117 GCCGGGCACUGAGGCGCCCca 794 511 GGCGCCUCAGUGCCCGGCCtt 1118 GGCCGGGCACUGAGGCGCCcc 795 512 GCGCCUCAGUGCCCGGCCCtt 1119 GGGCCGGGCACUGAGGCGCcc 796 513 CGCCUCAGUGCCCGGCCCAtt 1120 UGGGCCGGGCACUGAGGCGcc 797 514 GCCUCAGUGCCCGGCCCAAtt 1121 UUGGGCCGGGCACUGAGGCgc 798 515 CCUCAGUGCCCGGCCCAAGtt 1122 CUUGGGCCGGGCACUGAGGcg 799 516 CUCAGUGCCCGGCCCAAGCtt 1123 GCUUGGGCCGGGCACUGAGgc 800 517 UCAGUGCCCGGCCCAAGCUtt 1124 AGCUUGGGCCGGGCACUGAgg 801 518 CAGUGCCCGGCCCAAGCUCtt 1125 GAGCUUGGGCCGGGCACUGag 802 519 AGUGCCCGGCCCAAGCUCAtt 1126 UGAGCUUGGGCCGGGCACUga 803 520 GUGCCCGGCCCAAGCUCAAtt 1127 UUGAGCUUGGGCCGGGCACtg 804 521 UGCCCGGCCCAAGCUCAAGtt 1128 CUUGAGCUUGGGCCGGGCAct 805 522 GCCCGGCCCAAGCUCAAGGtt 1129 CCUUGAGCUUGGGCCGGGCac 806 523 CCCGGCCCAAGCUCAAGGCtt 1130 GCCUUGAGCUUGGGCCGGGca 807 524 CCGGCCCAAGCUCAAGGCCtt 1131 GGCCUUGAGCUUGGGCCGGgc 808 525 CGGCCCAAGCUCAAGGCCUtt 1132 AGGCCUUGAGCUUGGGCCGgg 809 526 GGCCCAAGCUCAAGGCCUUtt 1133 AAGGCCUUGAGCUUGGGCCgg 810 527 GCCCAAGCUCAAGGCCUUCtt 1134 GAAGGCCUUGAGCUUGGGCcg 811 528 CCCAAGCUCAAGGCCUUCCtt 1135 GGAAGGCCUUGAGCUUGGGcc 812 529 CCAAGCUCAAGGCCUUCCUtt 1136 AGGAAGGCCUUGAGCUUGGgc 813 530 CAAGCUCAAGGCCUUCCUGtt 1137 CAGGAAGGCCUUGAGCUUGgg 814 531 AAGCUCAAGGCCUUCCUGGtt 1138 CCAGGAAGGCCUUGAGCUUgg 815 532 AGCUCAAGGCCUUCCUGGCtt 1139 GCCAGGAAGGCCUUGAGCUtg 816 533 GCUCAAGGCCUUCCUGGCCtt 1140 GGCCAGGAAGGCCUUGAGCtt 817 534 CUCAAGGCCUUCCUGGCCUtt 1141 AGGCCAGGAAGGCCUUGAGct 818 535 UCAAGGCCUUCCUGGCCUCtt 1142 GAGGCCAGGAAGGCCUUGAgc 819 536 CAAGGCCUUCCUGGCCUCCtt 1143 GGAGGCCAGGAAGGCCUUGag 820 537 AAGGCCUUCCUGGCCUCCCtt 1144 GGGAGGCCAGGAAGGCCUUga 837 538 CCCUGAGUACGUGAACCUCtt 1145 GAGGUUCACGUACUCAGGGga 838 539 CCUGAGUACGUGAACCUCCtt 1146 GGAGGUUCACGUACUCAGGgg 839 540 CUGAGUACGUGAACCUCCCtt 1147 GGGAGGUUCACGUACUCAGgg 856 541 CCCAUCAAUGGCAACGGGAtt 1148 UCCCGUUGCCAUUGAUGGGga 857 542 CCAUCAAUGGCAACGGGAAtt 1149 UUCCCGUUGCCAUUGAUGGgg 858 543 CAUCAAUGGCAACGGGAAAtt 1150 UUUCCCGUUGCCAUUGAUGgg 859 544 AUCAAUGGCAACGGGAAACtt 1151 GUUUCCCGUUGCCAUUGAUgg 860 545 UCAAUGGCAACGGGAAACAtt 1152 UGUUUCCCGUUGCCAUUGAtg 861 546 CAAUGGCAACGGGAAACAGtt 1153 CUGUUUCCCGUUGCCAUUGat 862 547 AAUGGCAACGGGAAACAGUtt 1154 ACUGUUUCCCGUUGCCAUUga 863 548 AUGGCAACGGGAAACAGUGtt 1155 CACUGUUUCCCGUUGCCAUtg 864 549 UGGCAACGGGAAACAGUGAtt 1156 UCACUGUUUCCCGUUGCCAtt 865 550 GGCAACGGGAAACAGUGAGtt 1157 CUCACUGUUUCCCGUUGCCat 866 551 GCAACGGGAAACAGUGAGGtt 1158 CCUCACUGUUUCCCGUUGCca 867 552 CAACGGGAAACAGUGAGGGtt 1159 CCCUCACUGUUUCCCGUUGcc 868 553 AACGGGAAACAGUGAGGGUtt 1160 ACCCUCACUGUUUCCCGUUgc 869 554 ACGGGAAACAGUGAGGGUUtt 1161 AACCCUCACUGUUUCCCGUtg 870 555 CGGGAAACAGUGAGGGUUGtt 1162 CAACCCUCACUGUUUCCCGtt 871 556 GGGAAACAGUGAGGGUUGGtt 1163 CCAACCCUCACUGUUUCCCgt 872 557 GGAAACAGUGAGGGUUGGGtt 1164 CCCAACCCUCACUGUUUCCcg 891 558 GGGACUCUGAGCGGGAGGCtt 1165 GCCUCCCGCUCAGAGUCCCcc 892 559 GGACUCUGAGCGGGAGGCAtt 1166 UGCCUCCCGCUCAGAGUCCcc 894 560 ACUCUGAGCGGGAGGCAGAtt 1167 UCUGCCUCCCGCUCAGAGUcc 896 561 UCUGAGCGGGAGGCAGAGUtt 1168 ACUCUGCCUCCCGCUCAGAgt 897 562 CUGAGCGGGAGGCAGAGUUtt 1169 AACUCUGCCUCCCGCUCAGag 898 563 UGAGCGGGAGGCAGAGUUUtt 1170 AAACUCUGCCUCCCGCUCAga 899 564 GAGCGGGAGGCAGAGUUUGtt 1171 CAAACUCUGCCUCCCGCUCag 900 565 AGCGGGAGGCAGAGUUUGCtt 1172 GCAAACUCUGCCUCCCGCUca 901 566 GCGGGAGGCAGAGUUUGCCtt 1173 GGCAAACUCUGCCUCCCGCtc 902 567 CGGGAGGCAGAGUUUGCCUtt 1174 AGGCAAACUCUGCCUCCCGct 903 568 GGGAGGCAGAGUUUGCCUUtt 1175 AAGGCAAACUCUGCCUCCCgc 904 569 GGAGGCAGAGUUUGCCUUCtt 1176 GAAGGCAAACUCUGCCUCCcg 905 570 GAGGCAGAGUUUGCCUUCCtt 1177 GGAAGGCAAACUCUGCCUCcc 906 571 AGGCAGAGUUUGCCUUCCUtt 1178 AGGAAGGCAAACUCUGCCUcc 907 572 GGCAGAGUUUGCCUUCCUUtt 1179 AAGGAAGGCAAACUCUGCCtc 908 573 GCAGAGUUUGCCUUCCUUUtt 1180 AAAGGAAGGCAAACUCUGCct 909 574 CAGAGUUUGCCUUCCUUUCtt 1181 GAAAGGAAGGCAAACUCUGcc 910 575 AGAGUUUGCCUUCCUUUCUtt 1182 AGAAAGGAAGGCAAACUCUgc 911 576 GAGUUUGCCUUCCUUUCUCtt 1183 GAGAAAGGAAGGCAAACUCtg 912 577 AGUUUGCCUUCCUUUCUCCtt 1184 GGAGAAAGGAAGGCAAACUct 913 578 GUUUGCCUUCCUUUCUCCAtt 1185 UGGAGAAAGGAAGGCAAACtc 914 579 UUUGCCUUCCUUUCUCCAGtt 1186 CUGGAGAAAGGAAGGCAAAct 915 580 UUGCCUUCCUUUCUCCAGGtt 1187 CCUGGAGAAAGGAAGGCAAac 916 581 UGCCUUCCUUUCUCCAGGAtt 1188 UCCUGGAGAAAGGAAGGCAaa 917 582 GCCUUCCUUUCUCCAGGACtt 1189 GUCCUGGAGAAAGGAAGGCaa 918 583 CCUUCCUUUCUCCAGGACCtt 1190 GGUCCUGGAGAAAGGAAGGca 919 584 CUUCCUUUCUCCAGGACCAtt 1191 UGGUCCUGGAGAAAGGAAGgc 920 585 UUCCUUUCUCCAGGACCAAtt 1192 UUGGUCCUGGAGAAAGGAAgg 921 586 UCCUUUCUCCAGGACCAAUtt 1193 AUUGGUCCUGGAGAAAGGAag 922 587 CCUUUCUCCAGGACCAAUAtt 1194 UAUUGGUCCUGGAGAAAGGaa 923 588 CUUUCUCCAGGACCAAUAAtt 1195 UUAUUGGUCCUGGAGAAAGga 924 589 UUUCUCCAGGACCAAUAAAtt 1196 UUUAUUGGUCCUGGAGAAAgg 925 590 UUCUCCAGGACCAAUAAAAtt 1197 UUUUAUUGGUCCUGGAGAAag 926 591 UCUCCAGGACCAAUAAAAUtt 1198 AUUUUAUUGGUCCUGGAGAaa 927 592 CUCCAGGACCAAUAAAAUUtt 1199 AAUUUUAUUGGUCCUGGAGaa 928 593 UCCAGGACCAAUAAAAUUUtt 1200 AAAUUUUAUUGGUCCUGGAga 929 594 CCAGGACCAAUAAAAUUUCtt 1201 GAAAUUUUAUUGGUCCUGGag 930 595 CAGGACCAAUAAAAUUUCUtt 1202 AGAAAUUUUAUUGGUCCUGga 931 596 AGGACCAAUAAAAUUUCUAtt 1203 UAGAAAUUUUAUUGGUCCUgg 932 597 GGACCAAUAAAAUUUCUAAtt 1204 UUAGAAAUUUUAUUGGUCCtg 933 598 GACCAAUAAAAUUUCUAAGtt 1205 CUUAGAAAUUUUAUUGGUCct 934 599 ACCAAUAAAAUUUCUAAGAtt 1206 UCUUAGAAAUUUUAUUGGUcc 935 600 CCAAUAAAAUUUCUAAGAGtt 1207 CUCUUAGAAAUUUUAUUGGtc 936 601 CAAUAAAAUUUCUAAGAGAtt 1208 UCUCUUAGAAAUUUUAUUGgt 937 602 AAUAAAAUUUCUAAGAGAGtt 1209 CUCUCUUAGAAAUUUUAUUgg 938 603 AUAAAAUUUCUAAGAGAGCtt 1210 GCUCUCUUAGAAAUUUUAUtg 939 604 UAAAAUUUCUAAGAGAGCUtt 1211 AGCUCUCUUAGAAAUUUUAtt 940 605 AAAAUUUCUAAGAGAGCUAtt 1212 UAGCUCUCUUAGAAAUUUUat 941 606 AAAUUUCUAAGAGAGCUAAtt 1213 UUAGCUCUCUUAGAAAUUUta 942 607 AAUUUCUAAGAGAGCUAAAtt 1214 UUUAGCUCUCUUAGAAAUUtt 943 608 AUUUCUAAGAGAGCUAAAAtt 1215 UUUUAGCUCUCUUAGAAAUtt
(37) Key for Table 1: Upper case A, G, C and U referred to for ribo-A, ribo-G, ribo-C and ribo-U respectively. The lower case letters a, g, c, t represent 2-deoxy-A, 2-deoxy-G, 2-deoxy-C and thymidine respectively.
(38) TABLE-US-00002 TABLE2 RNAimoleculesequencesforGST-7c SEQ SENSESTRAND SEQ ANTISENSESTRAND Ref ID (5-->3) ID (5-->3) ID Pos NO SEQIDNOS:1216to1280 NO SEQIDNOS:1281to1345 A1 652 1216 UCCCAGAACCAGGGAGGCAtt 1281 UGCCUCCCUGGUUCUGGGAca A10 635 1217 CUUUUGAGACCCUGCUGUCtt 1282 GACAGCAGGGUCUCAAAAGgc A11 649 1218 CUGUCCCAGAACCAGGGAGtt 1283 CUCCCUGGUUCUGGGACAGca A12 650 1219 UGUCCCAGAACCAGGGAGGtt 1284 CCUCCCUGGUUCUGGGACAgc A13 631 1220 AAGCCUUUUGAGACCCUGCtt 1285 GCAGGGUCUCAAAAGGCUUca A14 638 1221 UUGAGACCCUGCUGUCCCAtt 1286 UGGGACAGCAGGGUCUCAAaa A15 636 1222 UUUUGAGACCCUGCUGUCCtt 1287 GGACAGCAGGGUCUCAAAAgg A16 640 1223 GAGACCCUGCUGUCCCAGAtt 1288 UCUGGGACAGCAGGGUCUCaa A17 332 1224 GCUGGAAGGAGGAGGUGGUtt 1289 ACCACCUCCUCCUUCCAGCtc A18 333 1225 CUGGAAGGAGGAGGUGGUGtt 1290 CACCACCUCCUCCUUCCAGct A19 321 1226 UCAGGGCCAGAGCUGGAAGtt 1291 CUUCCAGCUCUGGCCCUGAtc A2 639 1227 UGAGACCCUGCUGUCCCAGtt 1292 CUGGGACAGCAGGGUCUCAaa A20 323 1228 AGGGCCAGAGCUGGAAGGAtt 1293 UCCUUCCAGCUCUGGCCCUga A21 331 1229 AGCUGGAAGGAGGAGGUGGtt 1294 CCACCUCCUCCUUCCAGCUct A22 641 1230 AGACCCUGCUGUCCCAGAAtt 1295 UUCUGGGACAGCAGGGUCUca A23 330 1231 GAGCUGGAAGGAGGAGGUGtt 1296 CACCUCCUCCUUCCAGCUCtg A25 647 1232 UGCUGUCCCAGAACCAGGGtt 1297 CCCUGGUUCUGGGACAGCAgg A26 653 1233 CCCAGAACCAGGGAGGCAAtt 1298 UUGCCUCCCUGGUUCUGGGac A3 654 1234 CCAGAACCAGGGAGGCAAGtt 1299 CUUGCCUCCCUGGUUCUGGga A4 637 1235 UUUGAGACCCUGCUGUCCCtt 1300 GGGACAGCAGGGUCUCAAAag AS 642 1236 GACCCUGCUGUCCCAGAACtt 1301 GUUCUGGGACAGCAGGGUCtc A6 319 1237 GAUCAGGGCCAGAGCUGGAtt 1302 UCCAGCUCUGGCCCUGAUCtg A7 632 1238 AGCCUUUUGAGACCCUGCUtt 1303 AGCAGGGUCUCAAAAGGCUtc A8 633 1239 GCCUUUUGAGACCCUGCUGtt 1304 CAGCAGGGUCUCAAAAGGCtt A9 634 1240 CCUUUUGAGACCCUGCUGUtt 1305 ACAGCAGGGUCUCAAAAGGct AG7 632 1241 CGCCUUUUGAGACCCUGCAtt 1306 UGCAGGGUCUCAAAAGGCGtc AK1 257 1242 CCUACACCGUGGUCUAUUUtt 1307 AAAUAGACCACGGUGUAGGgc AK10 681 1243 UGUGGGAGACCAGAUCUCCtt 1308 GGAGAUCUGGUCUCCCACAat AK11 901 1244 GCGGGAGGCAGAGUUUGCCtt 1309 GGCAAACUCUGCCUCCCGCtc AK12 922 1245 CCUUUCUCCAGGACCAAUAtt 1310 UAUUGGUCCUGGAGAAAGGaa AK13/ 643 1246 ACCCUGCUGUCCCAGAACCtt 1311 GGUUCUGGGACAGCAGGGUct A24 AK2 267 1247 GGUCUAUUUCCCAGUUCGAtt 1312 UCGAACUGGGAAAUAGACCac AK3 512 1248 CCCUGGUGGACAUGGUGAAtt 1313 UUCACCAUGUCCACCAGGGct AK4 560 1249 ACAUCUCCCUCAUCUACACtt 1314 GUGUAGAUGAGGGAGAUGUat AK5 593 1250 GCAAGGAUGACUAUGUGAAtt 1315 UUCACAUAGUCAUCCUUGCcc AK6 698 1251 CCUUCGCUGACUACAACCUtt 1316 AGGUUGUAGUCAGCGAAGGag AK7 313 1252 CUGGCAGAUCAGGGCCAGAtt 1317 UCUGGCCCUGAUCUGCCAGca AK8 421 1253 GACGGAGACCUCACCCUGUtt 1318 ACAGGGUGAGGUCUCCGUCct AK9 590 1254 CGGGCAAGGAUGACUAUGUtt 1319 ACAUAGUCAUCCUUGCCCGcc AU10 635 1255 CUUUUGAGACCCUGCUGUAtt 1320 UACAGCAGGGUCUCAAAAGgc AU23 330 1256 GAGCUGGAAGGAGGAGGUAtt 1321 UACCUCCUCCUUCCAGCUCtg AU24 643 1257 ACCCUGCUGUCCCAGAACAtt 1322 UGUUCUGGGACAGCAGGGUct AU25 648 1258 UGCUGUCCCAGAACCAGGAtt 1323 UCCUGGUUCUGGGACAGCAgg AU7 632 1259 AGCCUUUUGAGACCCUGCAtt 1324 UGCAGGGUCUCAAAAGGCUtc AU9 634 1260 CCUUUUGAGACCCUGCUGAtt 1325 UCAGCAGGGUCUCAAAAGGct B1 629 1261 UGAAGCCUUUUGAGACCCUtt 1326 AGGGUCUCAAAAGGCUUCAgt B10 627 1262 ACUGAAGCCUUUUGAGACCtt 1327 GGUCUCAAAAGGCUUCAGUtg B11 596 1263 AGGAUGACUAUGUGAAGGCtt 1328 GCCUUCACAUAGUCAUCCUtg B12 597 1264 GGAUGACUAUGUGAAGGCAtt 1329 UGCCUUCACAUAGUCAUCCtt B13 598 1265 GAUGACUAUGUGAAGGCACtt 1330 GUGCCUUCACAUAGUCAUCct B14 564 1266 CUCCCUCAUCUACACCAACtt 1331 GUUGGUGUAGAUGAGGGAGat B2 630 1267 GAAGCCUUUUGAGACCCUGtt 1332 CAGGGUCUCAAAAGGCUUCag B3 563 1268 UCUCCCUCAUCUACACCAAtt 1333 UUGGUGUAGAUGAGGGAGAtg B4 567 1269 CCUCAUCUACACCAACUAUtt 1334 AUAGUUGGUGUAGAUGAGGga B5 566 1270 CCCUCAUCUACACCAACUAtt 1335 UAGUUGGUGUAGAUGAGGGag B6 625 1271 CAACUGAAGCCUUUUGAGAtt 1336 UCUCAAAAGGCUUCAGUUGcc B7 626 1272 AACUGAAGCCUUUUGAGACtt 1337 GUCUCAAAAGGCUUCAGUUgc B8 628 1273 CUGAAGCCUUUUGAGACCCtt 1338 GGGUCUCAAAAGGCUUCAGtt B9 565 1274 UCCCUCAUCUACACCAACUtt 1339 AGUUGGUGUAGAUGAGGGAga BG3 563 1275 GCUCCCUCAUCUACACCAAtt 1340 UUGGUGUAGAUGAGGGAGCtg BU02 631 1276 GAAGCCUUUUGAGACCCUAtt 1341 UAGGGUCUCAAAAGGCUUCag BU10 627 1277 ACUGAAGCCUUUUGAGACAtt 1342 UGUCUCAAAAGGCUUCAGUtg BU14 565 1278 CUCCCUCAUCUACACCAAAtt 1343 UUUGGUGUAGAUGAGGGAGat BU4 567 1279 CCUCAUCUACACCAACUAAtt 1344 UUAGUUGGUGUAGAUGAGGga Cl- 934 1280 ACCAAUAAAAUUUCUAAGAtt 1345 UCUUAGAAAUUUUAUUGGUcc 934
(39) Key for Table 2: Upper case A, G, C and U referred to for ribo-A, ribo-G, ribo-C and ribo-U respectively. The lower case letters a, g, c, t represent 2-deoxy-A, 2-deoxy-G, 2-deoxy-C and thymidine respectively.
(40) For example, a siRNA of this invention may have an antisense strand which is SEQ ID NO:1341, and a sense strand which is SEQ ID NO:1276, or chemically modified strands thereof.
(41) For example, a siRNA of this invention may have an antisense strand which is SEQ ID NO:1305, and a sense strand which is SEQ ID NO:1240, or chemically modified strands thereof.
(42) Chemical modifications may comprise a 2-OMe substituent group on any nucleotide in any position in a strand, as well as other modifications known in the art.
(43) Methods for Modulating GST-? and Treating Malignant Tumor
(44) Embodiments of this invention can provide RNAi molecules that can be used to down regulate or inhibit the expression of GST-? and/or GST-? proteins.
(45) In some embodiments, a RNAi molecule of this invention can be used to down regulate or inhibit the expression of GST-? and/or GST-? proteins arising from GST-? haplotype polymorphisms that may be associated with a disease or condition such as malignant tumor.
(46) Monitoring of GST-? protein or mRNA levels can be used to characterize gene silencing, and to determine the efficacy of compounds and compositions of this invention.
(47) The RNAi molecules of this disclosure can be used individually, or in combination with other siRNAs for modulating the expression of one or more genes.
(48) The RNAi molecules of this disclosure can be used individually, or in combination, or in conjunction with other known drugs for preventing or treating diseases, or ameliorating symptoms of conditions or disorders associated with GST-?, including malignant tumor.
(49) The RNAi molecules of this invention can be used to modulate or inhibit the expression of GST-? in a sequence-specific manner.
(50) The RNAi molecules of this disclosure can include a guide strand for which a series of contiguous nucleotides are at least partially complementary to a GST-? mRNA.
(51) In certain aspects, malignant tumor may be treated by RNA interference using a RNAi molecule of this invention.
(52) Treatment of malignant tumor may be characterized in suitable cell-based models, as well as ex vivo or in vivo animal models.
(53) Treatment of malignant tumor may be characterized by determining the level of GST-? mRNA or the level of GST-? protein in cells of affected tissue.
(54) Treatment of malignant tumor may be characterized by non-invasive medical scanning of an affected organ or tissue.
(55) Embodiments of this invention may include methods for preventing, treating, or ameliorating the symptoms of a GST-? associated disease or condition in a subject in need thereof.
(56) In some embodiments, methods for preventing, treating, or ameliorating the symptoms of malignant tumor in a subject can include administering to the subject a RNAi molecule of this invention to modulate the expression of a GST-? gene in the subject or organism.
(57) In some embodiments, this invention contemplates methods for down regulating the expression of a GST-? gene in a cell or organism, by contacting the cell or organism with a RNAi molecule of this invention.
(58) RNA Interference
(59) RNA interference (RNAi) refers to sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Fire et al., Nature, 1998, Vol. 391, pp. 806811; Sharp, Genes & Development, 1999, Vol. 13, pp. 139-141.
(60) An RNAi response in cells can be triggered by a double stranded RNA (dsRNA), although the mechanism is not yet fully understood. Certain dsRNAs in cells can undergo the action of Dicer enzyme, a ribonuclease III enzyme. See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Hammond et al., Nature, 2000, Vol. 404, pp. 293-296. Dicer can process the dsRNA into shorter pieces of dsRNA, which are siRNAs.
(61) In general, siRNAs can be from about 21 to about 23 nucleotides in length and include a base pair duplex region about 19 nucleotides in length.
(62) RNAi involves an endonuclease complex known as the RNA induced silencing complex (RISC). An siRNA has an antisense or guide strand which enters the RISC complex and mediates cleavage of a single stranded RNA target having a sequence complementary to the antisense strand of the siRNA duplex. The other strand of the siRNA is the passenger strand. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex See, e.g., Elbashir et al., Genes & Development, 2001, Vol. 15, pp. 188-200.
(63) As used herein, the term sense strand refers to a nucleotide sequence of a siRNA molecule that is partially or fully complementary to at least a portion of a corresponding antisense strand of the siRNA molecule. The sense strand of a siRNA molecule can include a nucleic acid sequence having homology with a target nucleic acid sequence.
(64) As used herein, the term antisense strand refers to a nucleotide sequence of a siRNA molecule that is partially or fully complementary to at least a portion of a target nucleic acid sequence. The antisense strand of a siRNA molecule can include a nucleic acid sequence that is complementary to at least a portion of a corresponding sense strand of the siRNA molecule.
(65) RNAi molecules can down regulate or knock down gene expression by mediating RNA interference in a sequence-specific manner. See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al., Nature, 2001, Vol. 411, pp. 494-498; Kreutzer et al., WO2000/044895; Zernicka-Goetz et al., WO2001/36646; Fire et al., WO1999/032619; Plaetinck et al., WO2000/01846; Mello et al., WO2001/029058.
(66) As used herein, the terms inhibit, down-regulate, or reduce with respect to gene expression means that the expression of the gene, or the level of mRNA molecules encoding one or more proteins, or the activity of one or more of the encoded proteins is reduced below that observed in the absence of a RNAi molecule or siRNA of this invention. For example, the level of expression, level of mRNA, or level of encoded protein activity may be reduced by at least 1%, or at least 10%, or at least 20%, or at least 50%, or at least 90%, or more from that observed in the absence of a RNAi molecule or siRNA of this invention.
(67) RNAi molecules can also be used to knock down viral gene expression, and therefore affect viral replication.
(68) RNAi molecules can be made from separate polynucleotide strands: a sense strand or passenger strand, and an antisense strand or guide strand. The guide and passenger strands are at least partially complementary. The guide strand and passenger strand can form a duplex region having from about 15 to about 49 base pairs.
(69) In some embodiments, the duplex region of a siRNA can have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs.
(70) In certain embodiments, a RNAi molecule can be active in a RISC complex, with a length of duplex region active for RISC.
(71) In additional embodiments, a RNAi molecule can be active as a Dicer substrate, to be converted to a RNAi molecule that can be active in a RISC complex.
(72) In some aspects, a RNAi molecule can have complementary guide and passenger sequence portions at opposing ends of a long molecule, so that the molecule can form a duplex region with the complementary sequence portions, and the strands are linked at one end of the duplex region by either nucleotide or non-nucleotide linkers. For example, a hairpin arrangement, or a stem and loop arrangement. The linker interactions with the strands can be covalent bonds or non-covalent interactions.
(73) A RNAi molecule of this disclosure may include a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the nucleic acid to the antisense region of the nucleic acid. A nucleotide linker can be a linker of ?2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. The nucleotide linker can be a nucleic acid aptamer. By aptamer or nucleic acid aptamer as used herein refers to a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that includes a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule, where the target molecule does not naturally bind to a nucleic acid. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. See, e.g., Gold et al., Annu Rev Biochem, 1995, Vol. 64, pp. 763-797; Brody et al., J. Biotechnol., 2000, Vol. 74, pp. 5-13; Hermann et al., Science, 2000, Vol. 287, pp. 820-825.
(74) Examples of a non-nucleotide linker include an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds, for example polyethylene glycols such as those having from 2 to 100 ethylene glycol units. Some examples are described in Seela et al., Nucleic Acids Research, 1987, Vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, pp. 301; Arnold et al., WO1989/002439; Usman et al., WO1995/006731; Dudycz et al., WO1995/011910, and Ferentz et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 4000-4002.
(75) A RNAi molecule can have one or more overhangs from the duplex region. The overhangs, which are non-base-paired, single strand regions, can be from one to eight nucleotides in length, or longer. An overhang can be a 3-end overhang, wherein the 3-end of a strand has a single strand region of from one to eight nucleotides. An overhang can be a 5-end overhang, wherein the 5-end of a strand has a single strand region of from one to eight nucleotides.
(76) The overhangs of a RNAi molecule can have the same length, or can be different lengths.
(77) A RNAi molecule can have one or more blunt ends, in which the duplex region ends with no overhang, and the strands are base paired to the end of the duplex region.
(78) A RNAi molecule of this disclosure can have one or more blunt ends, or can have one or more overhangs, or can have a combination of a blunt end and an overhang end.
(79) A 5-end of a strand of a RNAi molecule may be in a blunt end, or can be in an overhang. A 3-end of a strand of a RNAi molecule may be in a blunt end, or can be in an overhang.
(80) A 5-end of a strand of a RNAi molecule may be in a blunt end, while the 3-end is in an overhang. A 3-end of a strand of a RNAi molecule may be in a blunt end, while the 5-end is in an overhang.
(81) In some embodiments, both ends of a RNAi molecule are blunt ends.
(82) In additional embodiments, both ends of a RNAi molecule have an overhang.
(83) The overhangs at the 5- and 3-ends may be of different lengths.
(84) In certain embodiments, a RNAi molecule may have a blunt end where the 5-end of the antisense strand and the 3-end of the sense strand do not have any overhanging nucleotides.
(85) In further embodiments, a RNAi molecule may have a blunt end where the 3-end of the antisense strand and the 5-end of the sense strand do not have any overhanging nucleotides.
(86) A RNAi molecule may have mismatches in base pairing in the duplex region.
(87) Any nucleotide in an overhang of a RNAi molecule can be a deoxyribonucleotide, or a ribonucleotide.
(88) One or more deoxyribonucleotides may be at the 5-end, where the 3-end of the other strand of the RNAi molecule may not have an overhang, or may not have a deoxyribonucleotide overhang.
(89) One or more deoxyribonucleotides may be at the 3-end, where the 5-end of the other strand of the RNAi molecule may not have an overhang, or may not have a deoxyribonucleotide overhang.
(90) In some embodiments, one or more, or all of the overhang nucleotides of a RNAi molecule may be 2-deoxyribonucleotides.
(91) Dicer Substrate RNAi Molecules
(92) In some aspects, a RNAi molecule can be of a length suitable as a Dicer substrate, which can be processed to produce a RISC active RNAi molecule. See, e.g., Rossi et al., US2005/0244858.
(93) A double stranded RNA (dsRNA) that is a Dicer substrate can be of a length sufficient such that it is processed by Dicer to produce an active RNAi molecule, and may further include one or more of the following properties: (i) the Dicer substrate dsRNA can be asymmetric, for example, having a 3 overhang on the antisense strand, and (ii) the Dicer substrate dsRNA can have a modified 3 end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA to an active RNAi molecule.
(94) In certain embodiments, the longest strand in a Dicer substrate dsRNA may be 24-30 nucleotides in length.
(95) A Dicer substrate dsRNA can be symmetric or asymmetric.
(96) In some embodiments, a Dicer substrate dsRNA can have a sense strand of 22-28 nucleotides and an antisense strand of 24-30 nucleotides.
(97) In certain embodiments, a Dicer substrate dsRNA may have an overhang on the 3 end of the antisense strand.
(98) In further embodiments, a Dicer substrate dsRNA may have a sense strand 25 nucleotides in length, and an antisense strand 27 nucleotides in length, with a 2 base 3-overhang. The overhang may be 1, 2 or 3 nucleotides in length. The sense strand may also have a 5 phosphate.
(99) An asymmetric Dicer substrate dsRNA may have two deoxyribonucleotides at the 3-end of the sense strand in place of two of the ribonucleotides.
(100) The sense strand of a Dicer substrate dsRNA may be from about 22 to about 30, or from about 22 to about 28; or from about 24 to about 30; or from about 25 to about 30; or from about 26 to about 30; or from about 26 and 29; or from about 27 to about 28 nucleotides in length.
(101) The sense strand of a Dicer substrate dsRNA may be 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
(102) In certain embodiments, a Dicer substrate dsRNA may have sense and antisense strands that are at least about 25 nucleotides in length, and no longer than about 30 nucleotides in length.
(103) In certain embodiments, a Dicer substrate dsRNA may have sense and antisense strands that are 26 to 29 nucleotides in length.
(104) In certain embodiments, a Dicer substrate dsRNA may have sense and antisense strands that are 27 nucleotides in length.
(105) The sense and antisense strands of a Dicer substrate dsRNA may be the same length as in being blunt ended, or different lengths as in having overhangs, or may have a blunt end and an overhang.
(106) A Dicer substrate dsRNA may have a duplex region of 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length.
(107) The antisense strand of a Dicer substrate dsRNA may have any sequence that anneals to at least a portion of the sequence of the sense strand under biological conditions, such as within the cytoplasm of a eukaryotic cell.
(108) A Dicer substrate with a sense and an antisense strand can be linked by a third structure, such as a linker group or a linker oligonucleotide. The linker connects the two strands of the dsRNA, for example, so that a hairpin is formed upon annealing.
(109) The sense and antisense strands of a Dicer substrate are in general complementary, but may have mismatches in base pairing.
(110) In some embodiments, a Dicer substrate dsRNA can be asymmetric such that the sense strand has 22-28 nucleotides and the antisense strand has 24-30 nucleotides.
(111) A region of one of the strands, particularly the antisense strand, of the Dicer substrate dsRNA may have a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21-nucleotide region adjacent to the 3 end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.
(112) An antisense strand of a Dicer substrate dsRNA can have from 1 to 9 ribonucleotides on the 5-end, to give a length of 22-28 nucleotides. When the antisense strand has a length of 21 nucleotides, then 1-7 ribonucleotides, or 2-5 ribonucleotides, or 4 ribonucleotides may be added on the 3-end. The added ribonucleotides may have any sequence.
(113) A sense strand of a Dicer substrate dsRNA may have 24-30 nucleotides. The sense strand may be substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions.
(114) Methods of Use of RNAi Molecules
(115) The nucleic acid molecules and RNAi molecules of this invention may be delivered to a cell or tissue by direct application of the molecules, or with the molecules combined with a carrier or a diluent.
(116) The nucleic acid molecules and RNAi molecules of this invention can be delivered or administered to a cell, tissue, organ, or subject by direct application of the molecules with a carrier or diluent, or any other delivery vehicle that acts to assist, promote or facilitate entry into a cell, for example, viral sequences, viral material, or lipid or liposome formulations.
(117) The nucleic acid molecules and RNAi molecules of this invention can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection.
(118) Delivery systems may include, for example, aqueous and nonaqueous gels, creams, emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers and permeation enhancers.
(119) Compositions and methods of this disclosure can include an expression vector that includes a nucleic acid sequence encoding at least one RNAi molecule of this invention in a manner that allows expression of the nucleic acid molecule.
(120) The nucleic acid molecules and RNAi molecules of this invention can be expressed from transcription units inserted into DNA or RNA vectors. Recombinant vectors can be DNA plasmids or viral vectors. Viral vectors can be used that provide for transient expression of nucleic acid molecules.
(121) For example, the vector may contain sequences encoding both strands of a RNAi molecule of a duplex, or a single nucleic acid molecule that is self-complementary and thus forms a RNAi molecule. An expression vector may include a nucleic acid sequence encoding two or more nucleic acid molecules.
(122) A nucleic acid molecule may be expressed within cells from eukaryotic promoters. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.
(123) In some aspects, a viral construct can be used to introduce an expression construct into a cell, for transcription of a dsRNA construct encoded by the expression construct.
(124) Lipid formulations can be administered to animals by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art.
(125) Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used.
EXAMPLES
Example 1
(126) In vitro transfection was performed in an A549 cell line to determine siRNA knockdown efficacy. Dose dependent knockdown for GST-? mRNA was observed with siRNAs as shown in Table 3.
(127) TABLE-US-00003 TABLE 3 Dose dependent knockdown for GST-? mRNA in an A549 cell line siRNA IC50 (pM) A9 27, 29 B2 121 B3 235 B4 229 B13 23, 34 BU02 21, 25, 34
Example 2
(128) Protocol for in vitro knockdown.
(129) One day before the transfection, plate the cells in a 96-well plate at 2?103 cells per well with 100 ?l of DMEM (HyClone Cat. #SH30243.01) containing 10% FBS and culture in a 37? C. incubator containing a humidified atmosphere of 5% CO2 in air. Before transfection, change medium to 90 ?l of Opti-MEM I Reduced Serum Medium (Life Technologies Cat. #31985-070) containing 2% FBS. Mix 0.2 ?l of Lipofectamine RNAiMax (Life Technologies Cat. #13778-100) with 4.8 ?l of Opti-MEM I for 5 minutes at room temperature. Mix 1 ?l of siRNA with 4 ?l of Opti-MEM I and combine with the LF2000 solution and then mix gently, without vortex. Wait for 5 minutes at room temperature. Incubate the mixture for 10 minutes at room temperature to allow the RNA-RNAiMax complexes to form. Add the 10 ?l of RNA-RNAiMax complexes to a well and shake the plate gently by hand. Incubate the cells in a 37? C. incubator containing a humidified atmosphere of 5% CO2 in air for 2 hours. Change medium to fresh-MEM I Reduced Serum Medium (Life Technologies Cat. #31985-070) containing 2% FBS. 24 hours after transfection, wash the cells with ice-cold PBS once. Lyse the cells with 50 ?l of Cell-to-Ct Lysis Buffer (Life Technologies Cat. #4391851 C) for 5-30 minutes at room temperature. Add 5 ?l of Stop Solution and incubate for 2 minutes at room temperature. Measure mRNA level by RT-qPCR with TAQMAN immediately. Alternatively, the samples can be frozen at ?80? C. and assayed at a later time.
Example 3
(130)
Example 4
(131)
Example 5
(132)
(133) In this experiment, A549 and PANC-1 cell lines were obtained from ATCC. The cell suspension was mixed well with ice thawed BD matrigel at 1:1 ratio for injection. Each mouse, athymic nude female mice, 6 to 8 weeks, Charles River, was inoculated subcutaneously in the right flank with 0.1 ml of an inoculum of 2?10.sup.6 (A549) or 2.5?10.sup.6 (PANC-1) cells using a 25 G needle and syringe (1 inoculum per mouse). Mice were anesthetized for inoculation. On the day when the established tumors reached approximately 250-350 mm.sup.3 (A549) or 150-250 mm.sup.3 (PANC-1) animals were subjected to bolus injection through tail vein. Animals were sacrificed by overdosed CO.sub.2 and tumors dissected at different time points following the dosing. Tumors were first wet weighted, and then separated into three parts for measurement of GST-? knockdown, biodistribution of siRNA, and biomarker analysis. The samples were snap frozen in liquid nitrogen and stored at ?80? C. until ready to be processed for bioanalysis.
Example 6: Orthotopic A549 Lung Cancer Mouse Model
(134) The GST-? siRNAs of this invention can exhibit profound reduction of orthotopic lung cancer tumors in vivo. In this example, a GST-? siRNA provided gene knockdown potency in vivo when administered in a liposomal formulation to the orthotopic lung cancer tumors in athymic nude mice.
(135) In general, an orthotopic tumor model can exhibit direct clinical relevance for drug efficacy and potency, as well as improved predictive ability. In the orthotopic tumor model, tumor cells are implanted directly into the same kind of organ from which the cells originated.
(136) The anti-tumor efficacy of the siRNA formulation against human lung cancer A549 was evaluated by comparing the final primary tumor weights measured at necropsy for the treatment group and the vehicle control group.
(137) Orthotopic lung cancer tumor inhibition was observed in vivo for a GST-? siRNA based on structure BU2 (SEQ ID NOs:1276 and 1341). An orthotopic A549 lung cancer mouse model was utilized with a relatively low dose at 2 mg/kg of the siRNA targeted to GST-?.
(138) The GST-? siRNA showed significant and unexpectedly advantageous lung tumor inhibition efficacy in this six-week study. After 43 days, the GST-? siRNA showed markedly advantageous tumor inhibition efficacy, with final tumor average weights significantly reduced by 2.8-fold as compared to control.
(139) For this study, male NCr nu/nu mice, 5-6 weeks old, were used. The experimental animals were maintained in a HEPA filtered environment during the experimental period. The siRNA formulations were stored at 4? C. before use, and warmed to room temperature 10 minutes prior to injection in mouse.
(140) For this A549 human lung cancer orthotopic model, on the day of surgical orthotopic implantation (SOI), the stock tumors were harvested from the subcutaneous site of animals bearing A549 tumor xenograft and placed in RPMI-1640 medium. Necrotic tissues were removed and viable tissues were cut into 1.5-2 mm.sup.3 pieces. The animals were anesthetized with isoflurane inhalation and the surgical area was sterilized with iodine and alcohol. A transverse incision approximately 1.5 cm long was made in the left chest wall of the mouse using a pair of surgical scissors. An intercostal incision was made between the third and the fourth rib and the left lung was exposed. One A549 tumor fragment was transplanted to the surface of the lung with an 8-0 surgical suture (nylon). The chest wall was closed with a 6-0 surgical suture (silk). The lung was re-inflated by intrathoracic puncture using a 3 cc syringe with a 25 G?1? needle to draw out the remaining air in the chest cavity. The chest wall was closed with a 6-0 surgical silk suture. All procedures of the operation described above were performed with a 7? magnification microscope under HEPA filtered laminar flow hoods.
(141) Three days after tumor implantation, the model tumor-bearing mice were randomly divided into groups of ten mice per group. For the group of interest, treatment of the ten mice was initiated three days after tumor implantation.
(142) For the group of interest, the formulation was (Ionizable lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K:DSPE-PEG-2K), a liposomal composition. The liposomes encapsulated the GST-? siRNA.
(143) For the study endpoint, the experimental mice were sacrificed forty-two days after treatment initiation. Primary tumors were excised and weighed on an electronic balance for subsequent analysis.
(144) For an estimation of compound toxicity, the mean body weight of the mice in the treated and control groups was maintained within the normal range during the entire experimental period. Other symptoms of toxicity were not observed in the mice.
Example 7
(145) Effect of small interfering RNA (siRNA) targeting GST-? on A549 cell growth in nude mice and angiogenesis on chorioallantoic membrane (CAM) assay. Three pairs of GST-? siRNA-plasmid and non-silencing-plasmid are constructed, and transfected into A549 cells through LIPOFECTAMINE 2000, respectively. The most effective pair of GST-? siRNA-plasmid is selected by ELISA and real-time RT-PCR. A549 cells are transfected with selected GST-? siRNA-plasmid, A549 cells are transfected with non-silencing-plasmid, and A549 cells without transfection are inoculated into nude mice, respectively. Chick embryos are randomly divided into four groups and CAM is treated by different solutions for 48 h: culture media DMEM as negative control group, un-transfected A549 cell culture supernatants as positive control group, GST-? siRNA A549 cell culture supernatants as GST-? siRNA group and non-silencing siRNA A549 cell culture supernatants as non-silencing siRNA group. The CAMs were harvested on day 12 for microscopic assays.
(146) Compared with control group, GST-? siRNA-plasmid induces reduction in GST-? secretion by A549 cells accompanied by reduction in GST-? mRNA. Compared with non-silencing siRNA group, the mean tumor volume of murine xenograft is reduced in GST-? siRNA group; time for xenografts growing to 50 mm.sup.3 is delayed. GST-? contents in xenograft are reduced. In CAM assays, GST-? content is zero in negative group, and in GST-? siRNA group is reduced by 20-70% compared to non-silencing siRNA group or positive group; vessels branch points of CAM in GST-? siRNA group or non-silencing siRNA group or positive group are increased compared with negative group; total vessel length of CAM in GST-? siRNA group is increased compared with negative group, while in non-silencing siRNA group or positive group it is increased. Compared with negative control group, the proliferation of microvessels is increased when cell culture supernatant with GST-? is added in GST-? siRNA group, significant proliferated vessels are observed in non-silencing siRNA group or positive group.
Example 8
(147) Cell culture. The human non-small cell lung carcinoma cell line, A549 is cultured in F-12K medium (ATCC) supplemented with 10% FBS (FBS, Invitrogen) at 37? C. in a humidified atmosphere with 5% CO.sub.2. The cells stably expressing control, or GST? siRNAs are generated by transducing A549TR cells with the respective lentiviral transduction particles as per manufacturer's instructions (Sigma-Aldrich). Resistant clones are selected in 2.5 ?g/mL puromycin (Invivogen) for 12 d, isolated using cloning cylinders, and subsequently expanded and maintained in puromycin-containing medium.
Example 9
(148) GST-? targeted siRNA results in profound regression of tumor volume in vivo.
(149) A lipid formulation is used to encapsulate and deliver siRNA in nanoparticles to xenografts of human A549 lung cancer cells in scid mice. The xenografts are tested to identify the presence of KRAS mutations or aberrant levels of expression compared to normal cells. When tumors became established (>100 mm.sup.3), mice are treated with either GST-? targeted siRNA or Control (non-specific) siRNA every 2 days for 2 weeks. The trial is halted when the control group has to be euthanized.
(150) Results: Treatment with GST-? targeted siRNA prevents tumor expansion and results in dramatic tumor volume reduction.
(151) The tumors that are recovered are sectioned and visualized by TUNEL staining. GST-? targeted siRNA-treated tumors display significantly higher levels of apoptosis. RNA is extracted from the tumors, and real-time PCR is performed to examine specific knockdown of GST-?.
(152) Results: Treatment with GST-? targeted siRNA dramatically reduces expression of GST-? in vivo.
Example 10
(153) The GST-? siRNAs of this invention exhibited increased serum stability.
(154) A GST-? siRNA was incubated in human serum and detection of remaining siRNA at various time points was done by HPLS/LCMS. The half-life (t.sub.1/2) in serum for both the sense strand and antisense strand of the GST-? siRNA (SEQ ID Nos:1276 and 1341) was about 100 minutes.
Example 11
(155) The GST-? siRNAs of this invention exhibited enhanced stability in formulation in plasma.
(156) A GST-? siRNA was incubated in a formulation in plasma and detection of remaining siRNA was done at various time points. The half-life (t.sub.1/4) in plasma of a formulation of GST-? siRNA (SEQ ID Nos:1276 and 1341) was significantly longer than 100 hours.
(157) The GST-? siRNA was prepared in a liposomal formulation having the composition (Ionizing lipid:cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5). The z-average size for the liposomal nanoparticles was 40.0 nm, and the siRNA was 91% encapsulated.
(158) The formulation was incubated in 50% human serum in PBS for 40 min, 1.5 h, 3 h, 24 h, and 96 h. The amount of the GST-? siRNA was determined by an ELISA-based assay.
Example 12
(159) The GST-? siRNAs of this invention can exhibit profound reduction of cancer xenograft tumors in vivo. The GST-? siRNAs can provide gene knockdown potency in vivo when administered in a liposomal formulation to the cancer xenograft tumors.
(160) Tumor inhibition efficacy was observed for a GST-? siRNA (SEQ ID NOs:1276 and 1341). Dose dependent knockdown of GST-? mRNA was observed in vivo with the siRNA targeted to GST-?. A cancer xenograft model was utilized with a siRNA targeted to GST-?.
(161) The GST-? siRNA showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days after administration. Treatment with a GST-? siRNA resulted in significant reduction of GST-? mRNA expression 4 days after injection in a lipid formulation. At the higher dose of 4 mg/kg, significant reduction of about 40% was detected 24 hours after injection.
(162) The GST-? siRNA was administered in a single injection of 10 mL/kg of a liposomal formulation having the composition (Ionizable lipid:Cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5).
(163) For the cancer xenograft model, an A549 cell line was obtained from ATCC. The cells were maintained in RPMI-1640 supplemented with 10% Fetal Bovine Serum and 100 U/ml penicillin and 100 ?g/ml streptomycin. Cells were split 48 hrs before inoculation so that cells were in log phase growth when harvested. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and determined in a hemocytometer in the presence of trypan blue (only viable cells are counted). The cells were resuspended to a concentration of 4?10.sup.7/ml in RPMI media without serum. Then the cell suspension was mixed well with ice thawed BD matrigel at 1:1 ratio for injection.
(164) Mice were Charles River Laboratory Athymic Nude (nu/nu) Female Mice, immuno-compromised, 6-8 weeks old, 3 mice per group.
(165) For tumor model preparation, each mouse was inoculated subcutaneously in the right flank with 0.1 ml an inoculum of 2?10.sup.6 of A549 cells using a 25 G needle and syringe, one inoculum per mouse. Mice were not anesthetized for inoculation.
(166) For tumor volume measurements and randomization, tumor size was measured to the nearest 0.1 mm. Tumor volumes were calculated using the formula: Tumor volume=length?width.sup.2/2. Tumor volumes were monitored twice a week. Once the established tumors reached approximately 350-600 mm.sup.3, the mice were assigned into groups with varied time points. On the same day, test articles were administered according to the dosing regimen.
(167) For dosage administration, on the day when the established tumors reached approximately 350-600 mm.sup.3, the test articles were taken out from 4? C. fridge. Before being applied to syringes, the bottle containing formulation was reverted by hand for a few times to make a homogeneous solution.
(168) For body weight, mice were weighed to the nearest 0.1 g. Body weights were monitored and recorded twice for weeks, for the rest of weeks, including the day of study termination.
(169) For tumors collection, animals were sacrificed by overdosed CO.sub.2 and tumors were dissected at 0, 24, 48, 72, 96 (optional), and 168 hours following the dosing. Tumors were first wet weighted, and then separated into three parts for KD, distribution and biomarker analysis. The samples were snap frozen in liquid nitrogen and stored at ?80? C. until ready to be processed.
(170) The embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying nucleic acid molecules with improved RNAi activity.
(171) All publications, patents and literature specifically mentioned herein are incorporated by reference in their entirety for all purposes.
(172) It is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the description disclosed herein without departing from the scope and spirit of the description, and that those embodiments are within the scope of this description and the appended claims.
(173) It must be noted that as used herein and in the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise. As well, the terms a (or an), one or more and at least one can be used interchangeably herein. It is also to be noted that the terms comprises, comprising, containing, including, and having can be used interchangeably, and shall be read expansively and without limitation.
(174) Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For Markush groups, those skilled in the art will recognize that this description includes the individual members, as well as subgroups of the members of the Markush group.
(175) Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
(176) All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.