US20100087508A1 - Compositions and Methods for Inhibiting Expression of Eg5 and VEGF Genes - Google Patents

Compositions and Methods for Inhibiting Expression of Eg5 and VEGF Genes Download PDF

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US20100087508A1
US20100087508A1 US12552207 US55220709A US2010087508A1 US 20100087508 A1 US20100087508 A1 US 20100087508A1 US 12552207 US12552207 US 12552207 US 55220709 A US55220709 A US 55220709A US 2010087508 A1 US2010087508 A1 US 2010087508A1
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dsrna
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vegf
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David Bumcrot
Dinah Wen-Yee Sah
Ivanka Toudjarska
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Alnylam Pharmaceuticals Inc
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Alnylam Pharmaceuticals Inc
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Abstract

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA) in a SNALP formulation, and methods of using the compositions to inhibit the expression of the Eg5 and Vascular Endothelial Growth Factor (VEGF), and methods of using the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.

Description

    RELATED APPLICATIONS
  • [0001]
    This application is a continuation of PCT Application No. PCT/US2009/036233, filed Mar. 5, 2009 which claims the benefit of U.S. Provisional Application No. 61/034,019, filed Mar. 5, 2008, and U.S. Provisional Application No. 61/083,367, filed Jul. 24, 2008, and U.S. Provisional Application No. 61/086,381, filed Aug. 5, 2008, and U.S. Provisional Application No. 61/112,079, filed Nov. 6, 2008, and U.S. Provisional Application No. 61/150,664, filed Feb. 6, 2009 which are hereby incorporated in their entirety by reference.
  • FIELD OF THE INVENTION
  • [0002]
    This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA), and their use in mediating RNA interference to inhibit the expression of a combination of genes, e.g., the Eg5 and Vascular Endothelial Growth Factor (VEGF) genes formulated in SNALP, and the use of the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.
  • BACKGROUND OF THE INVENTION
  • [0003]
    The maintenance of cell populations within an organism is governed by the cellular processes of cell division and programmed cell death. Within normal cells, the cellular events associated with the initiation and completion of each process is highly regulated. In proliferative disease such as cancer, one or both of these processes may be perturbed. For example, a cancer cell may have lost its regulation (checkpoint control) of the cell division cycle through either the overexpression of a positive regulator or the loss of a negative regulator, perhaps by mutation.
  • [0004]
    Alternatively, a cancer cell may have lost the ability to undergo programmed cell death through the overexpression of a negative regulator. Hence, there is a need to develop new chemotherapeutic drugs that will restore the processes of checkpoint control and programmed cell death to cancerous cells.
  • [0005]
    One approach to the treatment of human cancers is to target a protein that is essential for cell cycle progression. In order for the cell cycle to proceed from one phase to the next, certain prerequisite events must be completed. There are checkpoints within the cell cycle that enforce the proper order of events and phases. One such checkpoint is the spindle checkpoint that occurs during the metaphase stage of mitosis. Small molecules that target proteins with essential functions in mitosis may initiate the spindle checkpoint to arrest cells in mitosis. Of the small molecules that arrest cells in mitosis, those which display anti-tumor activity in the clinic also induce apoptosis, the morphological changes associated with programmed cell death. An effective chemotherapeutic for the treatment of cancer may thus be one which induces checkpoint control and programmed cell death. Unfortunately, there are few compounds available for controlling these processes within the cell. Most compounds known to cause mitotic arrest and apoptosis act as tubulin binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function/structure of the mitotic spindle thereby causing mitotic arrest. Because most of these compounds specifically target the tubulin protein which is a component of all microtubules, they may also affect one or more of the numerous normal cellular processes in which microtubules have a role. Hence, there is also a need for agents that more specifically target proteins associated with proliferating cells.
  • [0006]
    Eg5 is one of several kinesin-like motor proteins that are localized to the mitotic spindle and known to be required for formation and/or function of the bipolar mitotic spindle. Recently, there was a report of a small molecule that disturbs bipolarity of the mitotic spindle (Mayer, T. U. et. al. 1999. Science 286 (5441) 971-4, herein incorporated by reference). More specifically, the small molecule induced the formation of an aberrant mitotic spindle wherein a monoastral array of microtubules emanated from a central pair of centrosomes, with chromosomes attached to the distal ends of the microtubules. The small molecule was dubbed “monastrol” after the monoastral array. This monoastral array phenotype had been previously observed in mitotic cells that were immunodepleted of the Eg5 motor protein. This distinctive monoastral array phenotype facilitated identification of monastrol as a potential inhibitor of Eg5. Indeed, monastrol was further shown to inhibit the Eg5 motor-driven motility of microtubules in an in vitro assay. The Eg5 inhibitor monastrol had no apparent effect upon the related kinesin motor or upon the motor(s) responsible for golgi apparatus movement within the cell. Cells that display the monoastral array phenotype either through immunodepletion of Eg5 or monastrol inhibition of Eg5 arrest in M-phase of the cell cycle. However, the mitotic arrest induced by either immunodepletion or inhibition of Eg5 is transient (Kapoor, T. M., 2000. J Cell Biol 150 (5) 975-80). Both the monoastral array phenotype and the cell cycle arrest in mitosis induced by monastrol are reversible. Cells recover to form a normal bipolar mitotic spindle, to complete mitosis and to proceed through the cell cycle and normal cell proliferation. These data suggest that an inhibitor of Eg5 which induced a transient mitotic arrest may not be effective for the treatment of cancer cell proliferation. Nonetheless, the discovery that monastrol causes mitotic arrest is intriguing and hence there is a need to further study and identify compounds which can be used to modulate the Eg5 motor protein in a manner that would be effective in the treatment of human cancers. There is also a need to explore the use of these compounds in combination with other antineoplastic agents.
  • [0007]
    VEGF (also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF can be produced by a wide variety of tissues, and its overexpression or aberrant expression can result in a variety disorders, including cancers and retinal disorders such as age-related macular degeneration and other angiogenic disorders.
  • [0008]
    Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
  • SUMMARY OF THE INVENTION
  • [0009]
    Disclosed are compositions having two double-stranded ribonucleic acids (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) and a human VEGF gene in a cell. The dsRNAs are formulated in a stable nucleic acid lipid particle (SNALP). Also disclosed are method for using the composition to decrease expression of Eg5/KSP and/or VEGF in a cell, and method of treatment of a disease, e.g., liver cancer, using the compositions of the invention.
  • [0010]
    Accordingly, disclosed herein is a composition having a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in a cell, wherein both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALP); said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand has a first sequence and said first antisense strand has a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1311 (5′-UCGAGAAUCUAAACUAACU-3′), wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and said second dsRNA consists of a second sense strand and a second antisense strand, said second sense strand having a third sequence and said second antisense strand having a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′), wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in length.
  • [0011]
    In some embodiments, the first antisense strand has a second sequence complementary to SEQ ID NO:1311 (5′-UCGAGAAUCUAAACUAACU-3′) and the second antisense strand has a fourth sequence complementary to SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′). In other embodiments, the first dsRNA consists of a sense strand consisting of SEQ ID NO:1534 (5′-UCGAGAAUCUAAACUAACUTT-3′) and an antisense strand consisting of SEQ ID NO:1535 (5′-AGUUAGUUUAGAUUCUCGATT-3′) and the second dsRNA consists of a sense strand consisting of SEQ ID NO:1536 (5′-GCACAUAGGAGAGAUGAGCUU-3′), and an antisense strand consisting of SEQ ID NO:1537 (5′-AAGCUCAUCUCUCCUAUGUGCUG-3′). In further embodiments, each strand is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”: the first dsRNA consists of a sense strand consisting of SEQ ID NO:1240 (5′-ucGAGAAucuAAAcuAAcuTsT-3′), and an antisense strand consisting of SEQ ID NO:1241 (5′-AGUuAGUUuAGAUUCUCGATsT); the second dsRNA consists of a sense strand consisting of SEQ ID NO:1242 (5′-GcAcAuAGGAGAGAuGAGCUsU-3′) and an antisense strand consisting of SEQ ID NO:1243 (5′-AAGCUcAUCUCUCCuAuGuGCusG-3′).
  • [0012]
    In some embodiments, the first dsRNA contains two overhangs and the second dsRNA contains an overhang at the 3′ of the antisense and a blunt end at the 5′ end of the antisense strand.
  • [0013]
    The first and second dsRNA can have at least one modified nucleotide. For example, each dsRNA can have at least one modified nucleotide chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide having a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. The modified nucleotide can be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base having nucleotide. In some embodiments, the first and second dsRNA each comprise at least one 2′-O-methyl modified ribonucleotide and at least one nucleotide having a 5′-phosphorothioate group.
  • [0014]
    Each strand of each dsRNA can be, e.g., 19-23 bases in length, or, alternatively 21-23 bases in length. In one embodiment, each strand of the first dsRNA is 21 bases in length and the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length.
  • [0015]
    In some embodiments, the first and second dsRNA are present in an equimolar ratio.
  • [0016]
    As described herein, the dsRNAs are formulated as SNALPS. In some embodiments, the SNALP formulation includes DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA. For example, the SNALP can have the components in the proportions listed in Table 17.
  • [0017]
    The composition of the invention can be used to reduce expression of Eg5 and/or VAGF. In some embodiments, the composition of the invention, upon contact with a cell expressing Eg5, inhibits expression of Eg5 by at least 40, 50, 60, 70, 80, or by at least 90%. In other embodiments, the composition of the invention, upon contact with a cell expressing VEGF, inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or by at least 90%. Administration of the composition to a cell can expression of both Eg5 and VEGF in said cell. The composition of claims 1-17, wherein the composition is administered in a nM concentration.
  • [0018]
    Administration of the composition of the invention to a cell can result in, e.g., an increase in mono-aster formation in the cell. Administration of the composition to a mammal can result in at least one effect selected from the group consisting of prevention of tumor growth, reduction in tumor growth, or prolonged survival in said mammal. The effect can be measured using at least one assay selected from the group consisting of determination of body weight, determination of organ weight, visual inspection, mRNA analysis, serum AFP analysis and survival monitoring. Included are compositions with these effect when administered in a nM concentration.
  • [0019]
    In a further embodiment the composition of the invention includes Sorafenib.
  • [0020]
    Also included in the invention are methods of suing the compositions of the invention. In one embodiment is are methods for inhibiting the expression of Eg5/KSP and VEGF in a cell by administering any of the compositions of the invention to the cell. Other embodiments are methods for preventing tumor growth, reducing tumor growth, or prolonging survival in a mammal in need of treatment for cancer by administering the composition to said mammal. In some embodiments the mammal has liver cancer, e.g., the mammal is a human with liver cancer. The method can include a further step of administering Sorafenib.
  • BRIEF DESCRIPTION OF THE FIGURES
  • [0021]
    FIG. 1 is a graph showing liver weights as percentage of body weight following administration of SNALP-siRNAs in a Hep3B mouse model.
  • [0022]
    FIGS. 2A-2D are graphs showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.
  • [0023]
    FIG. 3 is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.
  • [0024]
    FIG. 4 is a graph showing the body weight in untreated control animals.
  • [0025]
    FIG. 5 is a graph showing the effects of control luciferase-SNALP siRNAs on body weight in a Hep3B mouse model.
  • [0026]
    FIG. 6 is a graph showing the effects of VSP-SNALP siRNAs on body weight in a Hep3B mouse model.
  • [0027]
    FIG. 7A is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.
  • [0028]
    FIG. 7B is a graph showing the effects of SNALP-siRNAs on serum AFP levels as measured by serum ELISA in a Hep3B mouse model.
  • [0029]
    FIG. 8 is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.
  • [0030]
    FIG. 9 is a graph showing the effects of SNALP-siRNAs on human KSP levels normalized to human GAPDH levels in a Hep3B mouse model.
  • [0031]
    FIG. 10 is a graph showing the effects of SNALP-siRNAs on human VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.
  • [0032]
    FIG. 11A is a graph showing the effects of SNALP-siRNAs on mouse VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.
  • [0033]
    FIG. 11B is a set of graphs showing the effects of SNALP-siRNAs on human GAPDH levels and serum AFP levels in a Hep3B mouse model.
  • [0034]
    FIGS. 12A-12C are graphs showing the effects of SNALP-siRNAs on tumor KSP, VEGF and GAPDH levels in a Hep3B mouse model.
  • [0035]
    FIG. 13A and FIG. 13B are graphs showing the effects of SNALP-siRNAs on survival in mice with hepatic tumors. Treatment was started at 18 days (FIG. 13A) and 26 days (FIG. 13B) after tumor cell seeding.
  • [0036]
    FIG. 14 is a graph showing the effects of SNALP-siRNAs on serum alpha fetoprotein (AFP) levels.
  • [0037]
    FIGS. 15A and 15B are images of H&E stained sections in tumor bearing animals (three weeks after Hep3B cell implantation) were administered 2 mg/kg SNALP-VSP (A) or 2 mg/kg SNALP-Luc (B). Twenty four hours later, tumor bearing liver lobes were processed for histological analysis. Arrows indicate mono asters.
  • [0038]
    FIG. 16 is a flow diagram illustrating the manufacturing process of ALN-VSPDS01.
  • [0039]
    FIG. 17 is a cryo-transmission electron microscope (cryo-TEM) image of ALN-VSP02.
  • [0040]
    FIG. 18 is a flow diagram illustrating the manufacturing process of ALN-VSP02.
  • [0041]
    FIG. 19 is a graph illustrating the effects on survival of administration SNALP formulated siRNA and Sorafenib.
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0042]
    The invention provides compositions and methods for inhibiting the expression of the Eg5 gene and VEGF gene in a cell or mammal using the dsRNAs. The dsRNAs are preferably packaged in a stable nucleic acid particle (SNALP). The invention also provides compositions and methods for treating pathological conditions and diseases, such as liver cancer, in a mammal caused by the expression of the Eg5 gene and VEGF genes. The dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
  • [0043]
    The following detailed description discloses how to make and use the compositions containing dsRNAs to inhibit the expression of the Eg5 gene and VEGF genes, respectively, as well as compositions and methods for treating diseases and disorders caused by the expression of these genes, such as cancer. The pharmaceutical compositions featured in the invention include a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the Eg5 gene, together with a pharmaceutically acceptable carrier. The compositions featured in the invention also include a dsRNA having an antisense strand having a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the VEGF gene.
  • [0044]
    Accordingly, certain aspects of the invention provide pharmaceutical compositions containing the Eg5 and VEGF dsRNAs and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Eg5 gene and the VEGF gene respectively, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Eg5 and VEGF genes.
  • I. DEFINITIONS
  • [0045]
    For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
  • [0046]
    “G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences comprising such replacement moieties are embodiments of the invention.
  • [0047]
    As used herein, “Eg5” refers to the human kinesin family member 11, which is also known as KIF11, Eg5, HKSP, KSP, KNSL1 or TRIPS. Eg5 sequence can be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeq ID number:NM004523. The terms “Eg5” and “KSP” and “Eg5/KSP are used interchangeably
  • [0048]
    As used herein, VEGF, also known as vascular permeability factor, is an angiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The VEGF gene contains 8 exons that express a 189-amino acid protein isoform. A 165-amino acid isoform lacks the residues encoded by exon 6, whereas a 121-amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform predicted to contain 145 amino acids and to lack exon 7. VEGF can act on endothelial cells by binding to an endothelial tyrosine kinase receptor, such as Flt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is involved in endothelial cell differentiation and vasculogenesis. A third receptor, VEGFR-3, has been implicated in lymphogenesis.
  • [0049]
    The various isoforms have different biologic activities and clinical implications. For example, VEGF145 induces angiogenesis and like VEGF189 (but unlike VEGF165) VEGF145 binds efficiently to the extracellular matrix by a mechanism that is not dependent on extracellular matrix-associated heparin sulfates. VEGF displays activity as an endothelial cell mitogen and chemoattractant in vitro and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a wide variety of cancer cell types and promotes the growth of tumors by inducing the development of tumor-associated vasculature Inhibition of VEGF function has been shown to limit both the growth of primary experimental tumors as well as the incidence of metastases in immunocompromised mice. Various dsRNAs directed to VEGF are described in co-pending U.S. Ser. No. 11/078,073 and 11/340,080, which are hereby incorporated by reference in their entirety.
  • [0050]
    As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the Eg5/KSP and/or VEGF gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • [0051]
    As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
  • [0052]
    As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • [0053]
    This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.
  • [0054]
    “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.
  • [0055]
    The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
  • [0056]
    As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding Eg5/KSP and/or VEGF) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a Eg5 mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding Eg5.
  • [0057]
    The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.
  • [0058]
    As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. In some embodiments the dsRNA can have a nucleotide overhang at one end of the duplex and a blunt end at the other end.
  • [0059]
    The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally the most tolerated mismatches are in the terminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
  • [0060]
    The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
  • [0061]
    As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which an iRNA agent is transcribed.
  • [0062]
    “Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
  • [0063]
    The terms “silence” and “inhibit the expression of” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to the Eg5 and/or VEGF gene, herein refer to the at least partial suppression of the expression of the Eg5 gene, as manifested by a reduction of the amount of Eg5 mRNA and/or VEGF mRNA which may be isolated from a first cell or group of cells in which the Eg5 and/or VEGF gene is transcribed and which has or have been treated such that the expression of the Eg5 and/or VEGF gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of
  • [0000]
    ( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in control cells ) · 100 %
  • [0064]
    Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Eg5 and/or VEGF gene expression, e.g. the amount of protein encoded by the Eg5 and/or VEGF gene which is produced by a cell, or the number of cells displaying a certain phenotype, e.g. apoptosis. In principle, target gene silencing can be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of the Eg5 gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
  • [0065]
    For example, in certain instances, expression of the Eg5 gene (or VEGF gene) is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In other embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. The Tables and Example below provides values for inhibition of expression using various Eg5 and/or VEGF dsRNA molecules at various concentrations.
  • [0066]
    As used herein in the context of Eg5 expression (or VEGF expression), the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by Eg5 and/or VEGF expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by Eg5 and/or VEGF expression), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, such as the slowing and progression of hepatic carcinoma.
  • [0067]
    As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by Eg5 and/or VEGF expression or an overt symptom of pathological processes mediated by Eg5 and/or VEGF expression. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by Eg5 and/or VEGF expression, the patient's history and age, the stage of pathological processes mediated by Eg5 and/or VEGF expression, and the administration of other anti-pathological processes mediated by Eg5 and/or VEGF expression agents.
  • [0068]
    As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
  • [0069]
    The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. As described in more detail below, such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • [0070]
    As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
  • II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)
  • [0071]
    As described in more detail below, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 and/or VEGF gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the Eg5 and/or VEGF gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said Eg5 and/or VEGF gene, inhibits the expression of said Eg5 and/or VEGF gene.
  • [0072]
    The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
  • [0073]
    The dsRNA comprises two strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the Eg5 and/or VEGF gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. In other embodiments the duplex structure is 25-30 base pairs in length.
  • [0074]
    In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ. For example, a composition can include a first dsRNA targeted to Eg5 with a duplex length of 19 base pairs and a second dsRNA targeted to VEGF with a duplex length of 21 base pairs.
  • [0075]
    Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. In other embodiments the region of complementarity is 25-30 nucleotides in length.
  • [0076]
    In one embodiment the region of complementarity is 19 nucleotides in length. In another embodiment the region of complementarity is 21 nucleotides in length. When two different siRNAs are used in combination, the region of complementarity can be identical or can differ. For example, a composition can include a first dsRNA targeted to Eg5 with a region of complementarity of 19 nucleotides and a second dsRNA targeted to VEGF with a region of complementarity of 21 nucleotides.
  • [0077]
    Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In other embodiments, each is strand is 25-30 base pairs in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ. For example, a composition can include a dsRNA targeted to Eg5 with a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides, and a second dsRNA targeted to VEGF with a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.
  • [0078]
    The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand.
  • [0079]
    A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
  • [0080]
    As described in more detail herein, the composition of the invention includes a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF. The first and second dsRNA can have the same overhang architecture, e.g., number of nucleotide overhangs on each strand, or each dsRNA can have a different architecture. In one embodiment, the first dsRNA targeting Eg5 includes a 2 nucleotide overhang at the 3′ end of each strand and the second dsRNA targeting VEGF includes a 2 nucleotide overhang on the 3′ end of the antisense strand and a blunt end at the 5′ end of the antisense strand (e.g., the 3′ end of the sense strand).
  • [0081]
    In one embodiment, the Eg5 gene targeted by the dsRNA of the invention is the human Eg5 gene. In one embodiment, the antisense strand of the dsRNA targeting Eg5 comprises at least 15 contiguous nucleotides of one of the antisense sequences of Table 1-3. In specific embodiments, the first sequence of the dsRNA is selected from one of the sense strands of Tables 1-3 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1-3. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1-3 can readily be determined using the target sequence and the flanking Eg5 sequence. In some embodiments the dsRNA targeted to Eg5 will comprise at least two nucleotide sequence selected from the groups of sequences provided in Tables 1-3. One of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of the Eg5 gene. As such, the dsRNA will comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1-3 and the second oligonucleotide is described as the antisense strand in Tables 1-3
  • [0082]
    In embodiments using a second dsRNA targeting VEGF, such agents are exemplified in the Examples, Tables 4a and 4b, and in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference. In one embodiment the dsRNA targeting VEGF has an antisense strand complementary to at least 15 contiguous nucleotides of the VEGF target sequences described in Table 4a. In other embodiments, the dsRNA targeting VEGF comprises one of the antisense sequences of Table 4b, or one of the sense sequences of Table 4b, or comprises one of the duplexes (sense and antisense strands) of Table 4b.
  • [0083]
    The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1-3, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1-3 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1-3, and differing in their ability to inhibit the expression of the Eg5 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1-3 can readily be made using the Eg5 sequence and the target sequence provided. Additional dsRNA targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.
  • [0084]
    In addition, the RNAi agents provided in Tables 1-3 identify a site in the Eg5 mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents, e.g., dsRNA, that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1-3 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the Eg5 gene. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target Eg5 gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1-3. Additional RNAi agents, e.g., dsRNA, targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.
  • [0085]
    The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the Eg5 gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Eg5 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the Eg5 gene is important, especially if the particular region of complementarity in the Eg5 gene is known to have polymorphic sequence variation within the population.
  • [0086]
    Modifications
  • [0087]
    In yet another embodiment, the dsRNA is chemically modified to enhance stability.
  • [0088]
    The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • [0089]
    Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
  • [0090]
    Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference
  • [0091]
    Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
  • [0092]
    Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
  • [0093]
    In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
  • [0094]
    Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
  • [0095]
    Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
  • [0096]
    Other preferred modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
  • [0097]
    DsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine's, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
  • [0098]
    Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
  • [0099]
    Conjugates
  • [0100]
    Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • [0101]
    Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
  • [0102]
    It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
  • [0103]
    In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
  • [0104]
    In some cases, a ligand can be multifunctional and/or a dsRNA can be conjugated to more than one ligand. For example, the dsRNA can be conjugated to one ligand for improved uptake and to a second ligand for improved release.
  • [0105]
    Vector Encoded RNAi Agents
  • [0106]
    In another aspect of the invention, Eg5 and VEGF specific dsRNA molecules that are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
  • [0107]
    The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
  • [0108]
    The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
  • [0109]
    Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
  • [0110]
    For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
  • [0111]
    Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
  • [0112]
    Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
  • [0113]
    A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
  • [0114]
    Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
  • [0115]
    The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
  • [0116]
    In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
  • [0117]
    Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell. dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single EG5 gene (or VEGF gene) or multiple Eg5 genes (or VEGF genes) over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
  • [0118]
    The Eg5 specific dsRNA molecules and VEGF specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • [0119]
    Pharmaceutical Compositions Containing dsRNA
  • [0120]
    In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a Eg5/KSP and/or VEGF gene, such as pathological processes mediated by Eg5/KSP and/or VEGF expression, e.g., liver cancer. Such pharmaceutical compositions are formulated based on the mode of delivery.
  • [0121]
    Dosage
  • [0122]
    The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of EG5/KSP and/or VEGF genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.
  • [0123]
    The pharmaceutical composition can be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on EG5/KSP AND/OR VEGF levels is long lasting, such that subsequent doses are administered at not more than 7 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
  • [0124]
    In some embodiments the dsRNA is administered using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
  • [0125]
    The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
  • [0126]
    Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by EG5/KSP AND/OR VEGF expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human EG5/KSP AND/OR VEGF. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human EG5/KSP AND/OR VEGF.
  • [0127]
    Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
  • [0128]
    The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • [0129]
    In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
  • [0130]
    Administration
  • [0131]
    The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, and subdermal, oral or parenteral, e.g., subcutaneous.
  • [0132]
    Typically, when treating a mammal with hyperlipidemia, the dsRNA molecules are administered systemically via parental means. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. For example, dsRNAs, conjugated or unconjugate or formulated with or without liposomes, can be administered intravenously to a patient. For such, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers). Formulations are described in more detail herein.
  • [0133]
    The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
  • [0134]
    Formulations
  • [0135]
    The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • [0136]
    The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
  • [0137]
    Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. In one aspect are formulations that target the liver when treating hepatic disorders such as hyperlipidemia.
  • [0138]
    In addition, dsRNA that target the EG5/KSP AND/OR VEGF gene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the Eg5/KSP and/or VEGF gene can contain other therapeutic agents such as other cancer therapeutics or one or more dsRNA compounds that target non-EG5/KSP AND/OR VEGF genes.
  • [0139]
    Oral, Parenteral, Topical, and Biologic Formulations
  • [0140]
    Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. patent publication. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
  • [0141]
    Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which 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.
  • [0142]
    Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference. In addition, dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including Lipofectin™ (Invitrogen/Life Technologies, Carlsbad, Calif) and Effectene™ (Qiagen, Valencia, Calif). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am. Soc. Nephrol. 7: 1728 (1996)). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat. Biotechnol. 23(8):1002-7.
  • [0143]
    Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNA molecules to liver cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.
  • [0144]
    Characterization of Formulated dsRNAs
  • [0145]
    Formulations prepared by either the in-line mixing or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
  • [0146]
    Liposomal Formulations
  • [0147]
    There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
  • [0148]
    Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
  • [0149]
    In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
  • [0150]
    Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245) Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • [0151]
    Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
  • [0152]
    Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • [0153]
    Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
  • [0154]
    Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
  • [0155]
    Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
  • [0156]
    One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • [0157]
    Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
  • [0158]
    Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
  • [0159]
    Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
  • [0160]
    Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al).
  • [0161]
    Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
  • [0162]
    A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
  • [0163]
    Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
  • [0164]
    Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
  • [0165]
    If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • [0166]
    If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • [0167]
    If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • [0168]
    If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • [0169]
    The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
  • [0170]
    SNALPs
  • [0171]
    In one embodiment, a dsRNA featured in the invention is fully encapsulated in the lipid formulation to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
  • [0172]
    In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.
  • [0173]
    The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
  • [0174]
    In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
  • [0175]
    In one embodiment, the lipid-siRNA particle includes 40% 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
  • [0176]
    The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
  • [0177]
    The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
  • [0178]
    In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
  • [0179]
    LNP01
  • [0180]
    In one embodiment, the lipidoid ND98·4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
  • [0000]
    Figure US20100087508A1-20100408-C00001
  • [0181]
    LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
  • [0182]
    Emulsions
  • [0183]
    The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • [0184]
    Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • [0185]
    Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
  • [0186]
    Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • [0187]
    A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • [0188]
    Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • [0189]
    Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • [0190]
    The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
  • [0191]
    In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
  • [0192]
    The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • [0193]
    Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • [0194]
    Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.
  • [0195]
    Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
  • [0196]
    Penetration Enhancers
  • [0197]
    In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • [0198]
    Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
  • [0199]
    Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
  • [0200]
    Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
  • [0201]
    Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
  • [0202]
    Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
  • [0203]
    Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
  • [0204]
    Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
  • [0205]
    Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • [0206]
    Carriers
  • [0207]
    dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
  • [0208]
    Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extra-circulatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
  • [0209]
    Excipients
  • [0210]
    In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
  • [0211]
    Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • [0212]
    Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • [0213]
    Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • [0214]
    Other Components
  • [0215]
    The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • [0216]
    Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
  • [0217]
    Combination Therapy
  • [0218]
    In one aspect, a composition of the invention can be used in combination therapy. The term “combination therapy” includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention. The compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.
  • [0219]
    In one aspect of the invention, the subject compounds may be administered in combination with one or more separate agents that modulate protein kinases involved in various disease states. Examples of such kinases may include, but are not limited to: serine/threonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen activated protein kinases (MAPK), meiosis specific kinase (MEK), RAF and aurora kinase. Examples of receptor kinase families include epidermal growth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor (e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA, SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK, EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g. Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR) (e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but are not limited to, BCR-ABL (e.g. p43abl, ARG); BTK (e.g. ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.
  • [0220]
    In another aspect of the invention, the subject compounds may be administered in combination with one or more agents that modulate non-kinase biological targets or processes. Such targets include histone deacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins (e.g., HSP90), and proteosomes.
  • [0221]
    In one embodiment, subject compounds may be combined with antineoplastic agents (e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibit one or more biological targets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar, Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 and AEE788. Such combinations may enhance therapeutic efficacy over efficacy achieved by any of the agents alone and may prevent or delay the appearance of resistant mutational variants.
  • [0222]
    In certain preferred embodiments, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease for the purposes of shrinking tumors, destroying remaining cancer cells left over after surgery, inducing remission, maintaining remission and/or alleviating symptoms relating to the cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents such as mustard gas derivatives (Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecan analogs (Irinotecan and Topotecan); anti-tumor antibiotics such as Chromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such as folic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, and Gemcitabine), purine antagonists (6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors (Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin); topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab, Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotide reductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor (Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubule agents (Estramustine); and retinoids (Bexarotene, Isotretinoin, Tretinoin (ATRA). In certain preferred embodiments, the compounds of the invention are administered in combination with a chemoprotective agent. Chemoprotective agents act to protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane.
  • [0223]
    In one aspect of the invention, the subject compounds are administered in combination with radiation therapy. Radiation is commonly delivered internally (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • [0224]
    It will be appreciated that compounds of the invention can be used in combination with an immunotherapeutic agent. One form of immunotherapy is the generation of an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a site distant from the tumor. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the subject to be treated, or a derivative of such cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr. et al. claim a method for treating a resectable carcinoma to prevent recurrence or metastases, comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of about 107 cells.
  • [0225]
    It will be appreciated that the compounds of the invention may advantageously be used in conjunction with one or more adjunctive therapeutic agents. Examples of suitable agents for adjunctive therapy include steroids, such as corticosteroids (amcinonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflucortolone valerate, dexamethasone, dexamethasone sodium phosphate, desonide, furoate, fluocinonide, fluocinolone acetonide, halcinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, methyl prednisolone, mometasone, prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, and halobetasol proprionate); a 5HTi agonist, such as a triptan (e.g. sumatriptan or naratriptan); an adenosine A1 agonist; an EP ligand; an NMDA modulator, such as a glycine antagonist; a sodium channel blocker (e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); a cannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; a leukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentin and related compounds; a tricyclic antidepressant (e.g. amitryptilline); a neurone stabilizing antiepileptic drug; a mono-aminergic uptake inhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; a nitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOS inhibitor; an inhibitor of the release, or action, of tumour necrosis factor α; an antibody therapy, such as a monoclonal antibody therapy; an antiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or an immune system modulator (e.g. interferon); an opioid analgesic; a local anaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g. ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g. aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); a decongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, or dextramethorphan); a diuretic; or a sedating or non-sedating antihistamine.
  • [0226]
    The compounds of the invention can be co-administered with siRNA that target other genes. For example, a compound of the invention can be co-administered with an siRNA targeted to a c-Myc gene. In one example, AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myc targeted siRNAs are disclosed in U.S. patent application Ser. No. 12/373,039 which is herein incorporated by reference.
  • [0227]
    Methods for Treating Diseases Caused by Expression of the Eg5 and VEGF Genes
  • [0228]
    The invention relates in particular to the use of a composition containing at least two dsRNAs, one targeting an Eg5 gene, and one targeting a VEGF gene, for the treatment of a cancer, such as liver cancer, e.g., for inhibiting tumor growth and tumor metastasis. For example, a composition, such as pharmaceutical composition, may be used for the treatment of solid tumors, like intrahepatic tumors such as may occur in cancers of the liver. A composition containing a dsRNA targeting Eg5 and a dsRNA targeting VEGF may also be used to treat other tumors and cancers, such as breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma and for the treatment of skin cancer, like melanoma, for the treatment of lymphomas and blood cancer. The invention further relates to the use of a composition containing an Eg5 dsRNA and a VEGF dsRNA for inhibiting accumulation of ascites fluid and pleural effusion in different types of cancer, e.g., liver cancer, breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer, melanoma, lymphomas and blood cancer. Owing to the inhibitory effects on Eg5 and VEGF expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
  • [0229]
    In one embodiment, a patient having a tumor associated with AFP expression, or a tumor secreting AFP, e.g., a hepatoma or teratoma, is treated. In certain embodiments, the patient has a malignant teratoma, an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, a hepatoblastoma, a heptocellular carcinoma, testicular cancer or ovarian cancer.
  • [0230]
    The invention furthermore relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating cancer or for preventing tumor metastasis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.
  • [0231]
    The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of a specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.
  • [0232]
    Methods for Inhibiting Expression of the Eg5 Gene and the VEGF Gene
  • [0233]
    In yet another aspect, the invention provides a method for inhibiting the expression of the Eg5 gene and the VEGF gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target Eg5 gene and the target VEGF gene is silenced.
  • [0234]
    In one embodiment, a method for inhibiting Eg5 gene expression and VEGF gene expression includes administering a composition containing two different dsRNA molecules, one having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the Eg5 gene and the other having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the VEGF gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.
  • [0235]
    Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • EXAMPLES Example 1 dsRNA Synthesis
  • [0236]
    Source of Reagents
  • [0237]
    Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
  • [0238]
    siRNA Synthesis
  • [0239]
    For screening of dsRNA, single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
  • [0240]
    Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.
  • [0241]
    Conjugates
  • [0242]
    The following is a prophetic description of the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
  • Diethyl-2-azabutane-1,4-dicarboxylate AA
  • [0243]
    Figure US20100087508A1-20100408-C00002
  • [0244]
    A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
  • 3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB
  • [0245]
    Figure US20100087508A1-20100408-C00003
  • [0246]
    Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
  • 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC
  • [0247]
    Figure US20100087508A1-20100408-C00004
  • [0248]
    3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.
  • 3-({6-[1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD
  • [0249]
    Figure US20100087508A1-20100408-C00005
  • [0250]
    The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
  • 1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE
  • [0251]
    Figure US20100087508A1-20100408-C00006
  • [0252]
    Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
  • [6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF
  • [0253]
    Figure US20100087508A1-20100408-C00007
  • [0254]
    Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
  • (6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG
  • [0255]
    Figure US20100087508A1-20100408-C00008
  • [0256]
    Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
  • Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH
  • [0257]
    Figure US20100087508A1-20100408-C00009
  • [0258]
    Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stiffed at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
  • [0259]
    Cholesterol Derivatised CPG AI
  • [0000]
    Figure US20100087508A1-20100408-C00010
  • [0260]
    Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
  • [0261]
    The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
  • [0262]
    dsRNA Targeting the Eg5 Gene
  • [0263]
    Initial Screening Set
  • [0264]
    siRNA design was carried out to identify siRNAs targeting Eg5 (also known as KIF11, HSKP, KNSL1 and TRIPS). Human mRNA sequences to Eg5, RefSeq ID number:NM004523, was used.
  • [0265]
    siRNA duplexes cross-reactive to human and mouse Eg5 were designed. Twenty-four duplexes were synthesized for screening. (Table 1a). A second screening set was defined with 266 siRNAs targeting human Eg5, as well as its rhesus monkey ortholog (Table 2a). An expanded screening set was selected with 328 siRNA targeting human Eg5, with no necessity to hit any Eg5 mRNA of other species (Table 3a).
  • [0266]
    The sequences for human and a partial rhesus Eg5 mRNAs were downloaded from NCBI Nucleotide database and the human sequence was further on used as reference sequence (Human EG5:NM004523.2, 4908 bp, and Rhesus EG5: XM001087644.1, 878 bp (only 5′ part of human EG5)
  • [0267]
    For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine: u,c-2′-O-methyl nucleotides: s-phosphorothioate linkage.
  • [0000]
    TABLE 1a
    Sequences of Eg5/KSP dsRNA duplexes
    position in
    human SEQ SEQ SEQ
    Eg5/KSP ID sequence of 23mer target ID ID duplex
    sequence NO: site NO: sense sequence (5′-3′) No: antisense sequence (5′-3′) name
    385-407 1244 ACCGAAGUGUUGUUUGUC 1 cGAAGuGuuGuuuGuccA 2 UUGGAcAAAcAAcACUUCG AL-DP-
    CAAUU ATsT TsT 6226
    347-369 1245 UAUGGUGUUUGGAGCAUC 3 uGGuGuuuGGAGcAucuA 4 GuAGAUGCUCcAAAcACcA AL-DP-
    UACUA cTsT TsT 6227
    1078-1100 1246 AAUCUAAACUAACUAGAA 5 ucuAAAcuAAcuAGAAuc 6 GGAUUCuAGUuAGUUuAGA AL-DP-
    UCCUC cTsT TsT 6228
    1067-1089 1247 UCCUUAUCGAGAAUCUAA 7 cuuAucGAGAAucuAAAc 8 AGUUuAGAUUCUCGAuAAG AL-DP-
    ACUAA uTsT TsT 6229
    374-396 1248 GAUUGAUGUUUACCGAAG 9 uuGAuGuuuAccGAAGuG 10 AcACUUCGGuAAAcAUcAA AL-DP-
    UGUUG uTsT TsT 6230
    205-227 1249 UGGUGAGAUGCAGACCAU 11 GuGAGAuGcAGAccAuuu 12 uAAAUGGUCUGcAUCUcAC AL-DP-
    UUAAU ATsT TsT 6231
    1176-1198 1250 ACUCUGAGUACAUUGGAA 13 ucuGAGuAcAuuGGAAuA 14 AuAUUCcAAUGuACUcAGA AL-DP-
    UAUGC uTsT TsT 6232
    386-408 1251 CCGAAGUGUUGUUUGUCC 15 GAAGuGuuGuuuGuccAA 16 AUUGGAcAAAcAAcACUUC AL-DP-
    AAUUC uTsT TsT 6233
    416-438 1252 AGUUAUUAUGGGCUAUAA 17 uuAuuAuGGGcuAuAAuu 18 cAAUuAuAGCCcAuAAuAA AL-DP-
    UUGCA GTsT TsT 6234
    485-507 1253 GGAAGGUGAAAGGUCACC 19 AAGGuGAAAGGucAccuA 20 UuAGGUGACCUUUcACCUU AL-DP-
    UAAUG ATsT TsT 6235
    476-498 1254 UUUUACAAUGGAAGGUGA 21 uuAcAAuGGAAGGuGAAA 22 CUUUcACCUUCcAUUGuAA AL-DP-
    AAGGU GTsT TsT 6236
    486-508 1255 GAAGGUGAAAGGUCACCU 23 AGGuGAAAGGucAccuAA 24 AUuAGGUGACCUUUcACCU AL-DP-
    AAUGA uTsT TsT 6237
    487-509 1256 AAGGUGAAAGGUCACCUA 25 GGuGAAAGGucAccuAAu 26 cAUuAGGUGACCUUUcACC AL-DP-
    AUGAA GTsT TsT 6238
    1066-1088 1257 UUCCUUAUCGAGAAUCUA 27 ccuuAucGAGAAucuAAA 28 GUUuAGAUUCUCGAuAAGG AL-DP-
    AACUA cTsT TsT 6239
    1256-1278 1258 AGCUCUUAUUAAGGAGUA 29 cucuuAuuAAGGAGuAuA 30 GuAuACUCCUuAAuAAGAG AL-DP-
    UACGG cTsT TsT 6240
    2329-2351 1259 CAGAGAGAUUCUGUGCUU 31 GAGAGAuucuGuGcuuuG 32 CcAAAGcAcAGAAUCUCUC AL-DP-
    UGGAG GTsT TsT 6241
    1077-1099 1260 GAAUCUAAACUAACUAGA 33 AucuAAAcuAAcuAGAAu 34 GAUUCuAGUuAGUUuAGAU AL-DP-
    AUCCU cTsT TsT 6242
    1244-1266 1261 ACUCACCAAAAAAGCUCU 35 ucAccAAAAAAGcucuuA 36 AuAAGAGCUUUUUUGGUGA AL-DP-
    UAUUA uTsT TsT 6243
    637-659 1262 AAGAGCUUUUUGAUCUUC 37 GAGcuuuuuGAucuucuu 38 uAAGAAGAUcAAAAAGCUC AL-DP-
    UUAAU ATsT TsT 6244
    1117-1139 1263 GGCGUACAAGAACAUCUA 39 cGuAcAAGAAcAucuAuA 40 UuAuAGAUGUUCUUGuACG AL-DP-
    UAAUU ATsT TsT 6245
    373-395 1264 AGAUUGAUGUUUACCGAA 41 AuuGAuGuuuAccGAAGu 42 cACUUCGGuAAAcAUcAAU AL-DP-
    GUGUU GTsT TsT 6246
    1079-1101 1265 AUCUAAACUAACUAGAAU 43 cuAAAcuAAcuAGAAucc 44 AGGAUUCuAGUuAGUUuAG AL-DP-
    CCUCC uTsT TsT 6247
    383-405 1266 UUACCGAAGUGUUGUUUG 45 AccGAAGuGuuGuuuGuc 46 GGAcAAAcAAcACUUCGGU AL-DP-
    UCCAA cTsT TsT 6248
    200-222 1267 GGUGGUGGUGAGAUGCAG 47 uGGuGGuGAGAuGcAGAc 48 GGUCUGcAUCUcACcACcA AL-DP-
    ACCAU cTsT TsT 6249
  • [0000]
    TABLE 1b
    Analysis of Eg5/KSP ds duplexes
    single
    dose
    screen @
    25 nM [% SDs 2nd screen
    duplex residual (among
    name mRNA] quadruplicates)
    AL-DP-6226 23% 3%
    AL-DP-6227 69% 10%
    AL-DP-6228 33% 2%
    AL-DP-6229 2% 2%
    AL-DP-6230 66% 11%
    AL-DP-6231 17% 1%
    AL-DP-6232 9% 3%
    AL-DP-6233 24% 6%
    AL-DP-6234 91% 2%
    AL-DP-6235 112% 4%
    AL-DP-6236 69% 4%
    AL-DP-6237 42% 2%
    AL-DP-6238 45% 2%
    AL-DP-6239 2% 1%
    AL-DP-6240 48% 2%
    AL-DP-6241 41% 2%
    AL-DP-6242 8% 2%
    AL-DP-6243 7% 1%
    AL-DP-6244 6% 2%
    AL-DP-6245 12% 2%
    AL-DP-6246 28% 3%
    AL-DP-6247 71% 4%
    AL-DP-6248 5% 2%
    AL-DP-6249 28% 3%
  • [0000]
    TABLE 2a
    Sequences of Eg5/KSP dsRNA duplexes
    SEQ SEQ SEQ
    ID sequence of 19-mer ID ID antisense sequence (5′- duplex
    NO: target site NO. sense sequence (5′-3′) NO. 3′) name
    1268 CAUACUCUAGUCGUUCCCA 49 cAuAcucuAGucGuucccATsT 50 UGGGAACGACuAGAGuAUGTsT AD-12072
    1269 AGCGCCCAUUCAAUAGUAG 51 AGcGcccAuucAAuAGuAGTsT 52 CuACuAUUGAAUGGGCGCUTsT AD-12073
    1270 GGAAAGCUAGCGCCCAUUC 53 GGAAAGcuAGcGcccAuucTsT 54 GAAUGGGCGCuAGCUUUCCTsT AD-12074
    1271 GAAAGCUAGCGCCCAUUCA 55 GAAAGcuAGcGcccAuucATsT 56 UGAAUGGGCGCuAGCUUUCTsT AD-12075
    1272 AGAAACUACGAUUGAUGGA 57 AGAAAcuAcGAuuGAuGGATsT 58 UCcAUcAAUCGuAGUUUCUTsT AD-12076
    1273 UGUUCCUUAUCGAGAAUCU 59 uGuuccuuAucGAGAAucuTsT 60 AGAUUCUCGAuAAGGAAcATsT AD-12077
    1274 CAGAUUACCUCUGCGAGCC 61 cAGAuuAccucuGcGAGccTsT 62 GGCUCGcAGAGGuAAUCUGTsT AD-12078
    1275 GCGCCCAUUCAAUAGUAGA 63 GcGcccAuucAAuAGuAGATsT 64 UCuACuAUUGAAUGGGCGCTsT AD-12079
    1276 UUGCACUAUCUUUGCGUAU 65 uuGcAcuAucuuuGcGuAuTsT 66 AuACGcAAAGAuAGUGcAATsT AD-12080
    1277 CAGAGCGGAAAGCUAGCGC 67 cAGAGcGGAAAGcuAGcGcTsT 68 GCGCuAGCUUUCCGCUCUGTsT AD-12081
    1278 AGACCUUAUUUGGUAAUCU 69 AGAccuuAuuuGGuAAucuTsT 70 AGAUuACcAAAuAAGGUCUTsT AD-12082
    1279 AUUCUCUUGGAGGGCGUAC 71 AuucucuuGGAGGGcGuAcTsT 72 GuACGCCCUCcAAGAGAAUTsT AD-12083
    1280 GGCUGGUAUAAUUCCACGU 73 GGcuGGuAuAAuuccAcGuTsT 74 ACGUGGAAUuAuACcAGCCTsT AD-12084
    1281 GCGGAAAGCUAGCGCCCAU 75 GcGGAAAGcuAGcGcccAuTsT 76 AUGGGCGCuAGCUUUCCGCTsT AD-12085
    1282 UGCACUAUCUUUGCGUAUG 77 uGcAcuAucuuuGcGuAuGTsT 78 cAuACGcAAAGAuAGUGcATsT AD-12086
    1283 GUAUAAUUCCACGUACCCU 79 GuAuAAuuccAcGuAcccuTsT 80 AGGGuACGUGGAAUuAuACTsT AD-12087
    1284 AGAAUCUAAACUAACUAGA 81 AGAAucuAAAcuAAcuAGATsT 82 UCuAGUuAGUUuAGAUUCUTsT AD-12088
    1285 AGGAGCUGAAUAGGGUUAC 83 AGGAGcuGAAuAGGGuuAcTsT 84 GuAACCCuAUUcAGCUCCUTsT AD-12089
    1286 GAAGUACAUAAGACCUUAU 85 GAAGuAcAuAAGAccuuAuTsT 86 AuAAGGUCUuAUGuACUUCTsT AD-12090
    1287 GACAGUGGCCGAUAAGAUA 87 GAcAGuGGccGAuAAGAuATsT 88 uAUCUuAUCGGCcACUGUCTsT AD-12091
    1288 AAACCACUUAGUAGUGUCC 89 AAAccAcuuAGuAGuGuccTsT 90 GGAcACuACuAAGUGGUUUTsT AD-12092
    1289 UCCCUAGACUUCCCUAUUU 91 ucccuAGAcuucccuAuuuTsT 92 AAAuAGGGAAGUCuAGGGATsT AD-12093
    1290 UAGACUUCCCUAUUUCGCU 93 uAGAcuucccuAuuucGcuTsT 94 AGCGAAAuAGGGAAGUCuATsT AD-12094
    1291 GCGUCGCAGCCAAAUUCGU 95 GcGucGcAGccAAAuucGuTsT 96 ACGAAUUUGGCUGCGACGCTsT AD-12095
    1292 AGCUAGCGCCCAUUCAAUA 97 AGcuAGcGcccAuucAAuATsT 98 uAUUGAAUGGGCGCuAGCUTsT AD-12096
    1293 GAAACUACGAUUGAUGGAG 99 GAAAcuAcGAuuGAuGGAGTsT 100 CUCcAUcAAUCGuAGUUUCTsT AD-12097
    1294 CCGAUAAGAUAGAAGAUCA 101 ccGAuAAGAuAGAAGAucATsT 102 UGAUCUUCuAUCUuAUCGGTsT AD-12098
    1295 UAGCGCCCAUUCAAUAGUA 103 uAGcGcccAuucAAuAGuATsT 104 uACuAUUGAAUGGGCGCuATsT AD-12099
    1296 UUUGCGUAUGGCCAAACUG 105 uuuGcGuAuGGccAAAcuGTsT 106 cAGUUUGGCcAuACGcAAATsT AD-12100
    1297 CACGUACCCUUCAUCAAAU 107 cAcGuAcccuucAucAAAuTsT 108 AUUUGAUGAAGGGuACGUGTsT AD-12101
    1298 UCUUUGCGUAUGGCCAAAC 109 ucuuuGcGuAuGGccAAAcTsT 110 GUUUGGCcAuACGcAAAGATsT AD-12102
    1299 CCGAAGUGUUGUUUGUCCA 111 ccGAAGuGuuGuuuGuccATsT 112 UGGAcAAAcAAcACUUCGGTsT AD-12103
    1300 AGAGCGGAAAGCUAGCGCC 113 AGAGcGGAAAGcuAGcGccTsT 114 GGCGCuAGCUUUCCGCUCUTsT AD-12104
    1301 GCUAGCGCCCAUUCAAUAG 115 GcuAGcGcccAuucAAuAGTsT 116 CuAUUGAAUGGGCGCuAGCTsT AD-12105
    1302 AAGUUAGUGUACGAACUGG 117 AAGuuAGuGuAcGAAcuGGTsT 118 CcAGUUCGuAcACuAACUUTsT AD-12106
    1303 GUACGAACUGGAGGAUUGG 119 GuAcGAAcuGGAGGAuuGGTsT 120 CcAAUCCUCcAGUUCGuACTsT AD-12107
    1304 ACGAACUGGAGGAUUGGCU 121 AcGAAcuGGAGGAuuGGcuTsT 122 AGCcAAUCCUCcAGUUCGUTsT AD-12108
    1305 AGAUUGAUGUUUACCGAAG 123 AGAuuGAuGuuuAccGAAGTsT 124 CUUCGGuAAAcAUcAAUCUTsT AD-12109
    1306 UAUGGGCUAUAAUUGCACU 125 uAuGGGcuAuAAuuGcAcuTsT 126 AGUGcAAUuAuAGCCcAuATsT AD-12110
    1307 AUCUUUGCGUAUGGCCAAA 127 AucuuuGcGuAuGGccAAATsT 128 UUUGGCcAuACGcAAAGAUTsT AD-12111
    1308 ACUCUAGUCGUUCCCACUC 129 AcucuAGucGuucccAcucTsT 130 GAGUGGGAACGACuAGAGUTsT AD-12112
    1309 AACUACGAUUGAUGGAGAA 131 AAcuAcGAuuGAuGGAGAATsT 132 UUCUCcAUcAAUCGuAGUUTsT AD-12113
    1310 GAUAAGAGAGCUCGGGAAG 133 GAuAAGAGAGcucGGGAAGTsT 134 CUUCCCGAGCUCUCUuAUCTsT AD-12114
    1311 UCGAGAAUCUAAACUAACU 135 ucGAGAAucuAAAcuAAcuTsT 136 AGUuAGUUuAGAUUCUCGATsT AD-12115
    1312 AACUAACUAGAAUCCUCCA 137 AAcuAAcuAGAAuccuccATsT 138 UGGAGGAUUCuAGUuAGUUTsT AD-12116
    1313 GGAUCGUAAGAAGGCAGUU 139 GGAucGuAAGAAGGcAGuuTsT 140 AACUGCCUUCUuACGAUCCTsT AD-12117
    1314 AUCGUAAGAAGGCAGUUGA 141 AucGuAAGAAGGcAGuuGATsT 142 UcAACUGCCUUCUuACGAUTsT AD-12118
    1315 AGGCAGUUGACCAACACAA 143 AGGcAGuuGAccAAcAcAATsT 144 UUGUGUUGGUcAACUGCCUTsT AD-12119
    1316 UGGCCGAUAAGAUAGAAGA 145 uGGccGAuAAGAuAGAAGATsT 146 UCUUCuAUCUuAUCGGCcATsT AD-12120
    1317 UCUAAGGAUAUAGUCAACA 147 ucuAAGGAuAuAGucAAcATsT 148 UGUUGACuAuAUCCUuAGATsT AD-12121
    1318 ACUAAGCUUAAUUGCUUUC 149 AcuAAGcuuAAuuGcuuucTsT 150 GAAAGcAAUuAAGCUuAGUTsT AD-12122
    1319 GCCCAGAUCAACCUUUAAU 151 GcccAGAucAAccuuuAAuTsT 152 AUuAAAGGUUGAUCUGGGCTsT AD-12123
    1320 UUAAUUUGGCAGAGCGGAA 153 uuAAuuuGGcAGAGcGGAATsT 154 UUCCGCUCUGCcAAAUuAATsT AD-12124
    1321 UUAUCGAGAAUCUAAACUA 155 uuAucGAGAAucuAAAcuATsT 156 uAGUUuAGAUUCUCGAuAATsT AD-12125
    1322 CUAGCGCCCAUUCAAUAGU 157 cuAGcGcccAuucAAuAGuTsT 158 ACuAUUGAAUGGGCGCuAGTsT AD-12126
    1323 AAUAGUAGAAUGUGAUCCU 159 AAuAGuAGAAuGuGAuccuTsT 160 AGGAUcAcAUUCuACuAUUTsT AD-12127
    1324 UACGAAAAGAAGUUAGUGU 161 uAcGAAAAGAAGuuAGuGuTsT 162 AcACuAACUUCUUUUCGuATsT AD-12128
    1325 AGAAGUUAGUGUACGAACU 163 AGAAGuuAGuGuAcGAAcuTsT 164 AGUUCGuAcACuAACUUCUTsT AD-12129
    1326 ACUAAACAGAUUGAUGUUU 165 AcuAAAcAGAuuGAuGuuuTsT 166 AAAcAUcAAUCUGUUuAGUTsT AD-12130
    1327 CUUUGCGUAUGGCCAAACU 167 cuuuGcGuAuGGccAAAcuTsT 168 AGUUUGGCcAuACGcAAAGTsT AD-12131
    1328 AAUGAAGAGUAUACCUGGG 169 AAuGAAGAGuAuAccuGGGTsT 170 CCcAGGuAuACUCUUcAUUTsT AD-12132
    1329 AUAAUUCCACGUACCCUUC 171 AuAAuuccAcGuAcccuucTsT 172 GAAGGGuACGUGGAAUuAUTsT AD-12133
    1330 ACGUACCCUUCAUCAAAUU 173 AcGuAcccuucAucAAAuuTsT 174 AAUUUGAUGAAGGGuACGUTsT AD-12134
    1331 CGUACCCUUCAUCAAAUUU 175 cGuAcccuucAucAAAuuuTsT 176 AAAUUUGAUGAAGGGuACGTsT AD-12135
    1332 GUACCCUUCAUCAAAUUUU 177 GuAcccuucAucAAAuuuuTsT 178 AAAAUUUGAUGAAGGGuACTsT AD-12136
    1333 AACUUACUGAUAAUGGUAC 179 AAcuuAcuGAuAAuGGuAcTsT 180 GuACcAUuAUcAGuAAGUUTsT AD-12137
    1334 UUCAGUCAAAGUGUCUCUG 181 uucAGucAAAGuGucucuGTsT 182 cAGAGAcACUUUGACUGAATsT AD-12138
    1335 UUCUUAAUCCAUCAUCUGA 183 uucuuAAuccAucAucuGATsT 184 UcAGAUGAUGGAUuAAGAATsT AD-12139
    1336 ACAGUACACAACAAGGAUG 185 AcAGuAcAcAAcAAGGAuGTsT 186 cAUCCUUGUUGUGuACUGUTsT AD-12140
    1337 AAGAAACUACGAUUGAUGG 187 AAGAAAcuAcGAuuGAuGGTsT 188 CcAUcAAUCGuAGUUUCUUTsT AD-12141
    1338 AAACUACGAUUGAUGGAGA 189 AAAcuAcGAuuGAuGGAGATsT 190 UCUCcAUcAAUCGuAGUUUTsT AD-12142
    1339 UGGAGCUGUUGAUAAGAGA 191 uGGAGcuGuuGAuAAGAGATsT 192 UCUCUuAUcAAcAGCUCcATsT AD-12143
    1340 CUAACUAGAAUCCUCCAGG 193 cuAAcuAGAAuccuccAGGTsT 194 CCUGGAGGAUUCuAGUuAGTsT AD-12144
    1341 GAAUAUGCUCAUAGAGCAA 195 GAAuAuGcucAuAGAGcAATsT 196 UUGCUCuAUGAGcAuAUUCTsT AD-12145
    1342 AUGCUCAUAGAGCAAAGAA 197 AuGcucAuAGAGcAAAGAATsT 198 UUCUUUGCUCuAUGAGcAUTsT AD-12146
    1343 AAAAAUUGGUGCUGUUGAG 199 AAAAAuuGGuGcuGuuGAGTsT 200 CUcAAcAGcACcAAUUUUUTsT AD-12147
    1344 GAGGAGCUGAAUAGGGUUA 201 GAGGAGcuGAAuAGGGuuATsT 202 uAACCCuAUUcAGCUCCUCTsT AD-12148
    1345 GGAGCUGAAUAGGGUUACA 203 GGAGcuGAAuAGGGuuAcATsT 204 UGuAACCCuAUUcAGCUCCTsT AD-12149
    1346 GAGCUGAAUAGGGUUACAG 205 GAGcuGAAuAGGGuuAcAGTsT 206 CUGuAACCCuAUUcAGCUCTsT AD-12150
    1347 AGCUGAAUAGGGUUACAGA 207 AGcuGAAuAGGGuuAcAGATsT 208 UCUGuAACCCuAUUcAGCUTsT AD-12151
    1348 GCUGAAUAGGGUUACAGAG 209 GcuGAAuAGGGuuAcAGAGTsT 210 CUCUGuAACCCuAUUcAGCTsT AD-12152
    1349 CCAAACUGGAUCGUAAGAA 211 ccAAAcuGGAucGuAAGAATsT 212 UUCUuACGAUCcAGUUUGGTsT AD-12153
    1350 GAUCGUAAGAAGGCAGUUG 213 GAucGuAAGAAGGcAGuuGTsT 214 cAACUGCCUUCUuACGAUCTsT AD-12154
    1351 ACCUUAUUUGGUAAUCUGC 215 AccuuAuuuGGuAAucuGcTsT 216 GcAGAUuACcAAAuAAGGUTsT AD-12155
    1352 UUAGAUACCAUUACUACAG 217 uuAGAuAccAuuAcuAcAGTsT 218 CUGuAGuAAUGGuAUCuAATsT AD-12156
    1353 AUACCAUUACUACAGUAGC 219 AuAccAuuAcuAcAGuAGcTsT 220 GCuACUGuAGuAAUGGuAUTsT AD-12157
    1354 UACUACAGUAGCACUUGGA 221 uAcuAcAGuAGcAcuuGGATsT 222 UCcAAGUGCuACUGuAGuATsT AD-12158
    1355 AAAGUAAAACUGUACUACA 223 AAAGuAAAAcuGuAcuAcATsT 224 UGuAGuAcAGUUUuACUUUTsT AD-12159
    1356 CUCAAGACUGAUCUUCUAA 225 cucAAGAcuGAucuucuAATsT 226 UuAGAAGAUcAGUCUUGAGTsT AD-12160
    1357 UUGACAGUGGCCGAUAAGA 227 uuGAcAGuGGccGAuAAGATsT 228 UCUuAUCGGCcACUGUcAATsT AD-12161
    1358 UGACAGUGGCCGAUAAGAU 229 uGAcAGuGGccGAuAAGAuTsT 230 AUCUuAUCGGCcACUGUcATsT AD-12162
    1359 GCAAUGUGGAAACCUAACU 231 GcAAuGuGGAAAccuAAcuTsT 232 AGUuAGGUUUCcAcAUUGCTsT AD-12163
    1360 CCACUUAGUAGUGUCCAGG 233 ccAcuuAGuAGuGuccAGGTsT 234 CCUGGAcACuACuAAGUGGTsT AD-12164
    1361 AGAAGGUACAAAAUUGGUU 235 AGAAGGuAcAAAAuuGGuuTsT 236 AACcAAUUUUGuACCUUCUTsT AD-12165
    1362 UGGUUUGACUAAGCUUAAU 237 uGGuuuGAcuAAGcuuAAuTsT 238 AUuAAGCUuAGUcAAACcATsT AD-12166
    1363 GGUUUGACUAAGCUUAAUU 239 GGuuuGAcuAAGcuuAAuuTsT 240 AAUuAAGCUuAGUcAAACCTsT AD-12167
    1364 UCUAAGUCAAGAGCCAUCU 241 ucuAAGucAAGAGccAucuTsT 242 AGAUGGCUCUUGACUuAGATsT AD-12168
    1365 UCAUCCCUAUAGUUCACUU 243 ucAucccuAuAGuucAcuuTsT 244 AAGUGAACuAuAGGGAUGATsT AD-12169
    1366 CAUCCCUAUAGUUCACUUU 245 cAucccuAuAGuucAcuuuTsT 246 AAAGUGAACuAuAGGGAUGTsT AD-12170
    1367 CCCUAGACUUCCCUAUUUC 247 cccuAGAcuucccuAuuucTsT 248 GAAAuAGGGAAGUCuAGGGTsT AD-12171
    1368 AGACUUCCCUAUUUCGCUU 249 AGAcuucccuAuuucGcuuTsT 250 AAGCGAAAuAGGGAAGUCUTsT AD-12172
    1369 UCACCAAACCAUUUGUAGA 251 ucAccAAAccAuuuGuAGATsT 252 UCuAcAAAUGGUUUGGUGATsT AD-12173
    1370 UCCUUUAAGAGGCCUAACU 253 uccuuuAAGAGGccuAAcuTsT 254 AGUuAGGCCUCUuAAAGGATsT AD-12174
    1371 UUUAAGAGGCCUAACUCAU 255 uuuAAGAGGccuAAcucAuTsT 256 AUGAGUuAGGCCUCUuAAATsT AD-12175
    1372 UUAAGAGGCCUAACUCAUU 257 uuAAGAGGccuAAcucAuuTsT 258 AAUGAGUuAGGCCUCUuAATsT AD-12176
    1373 GGCCUAACUCAUUCACCCU 259 GGccuAAcucAuucAcccuTsT 260 AGGGUGAAUGAGUuAGGCCTsT AD-12177
    1374 UGGUAUUUUUGAUCUGGCA 261 uGGuAuuuuuGAucuGGcATsT 262 UGCcAGAUcAAAAAuACcATsT AD-12178
    1375 AGUUUAGUGUGUAAAGUUU 263 AGuuuAGuGuGuAAAGuuuTsT 264 AAACUUuAcAcACuAAACUTsT AD-12179
    1376 GCCAAAUUCGUCUGCGAAG 265 GccAAAuucGucuGcGAAGTsT 266 CUUCGcAGACGAAUUUGGCTsT AD-12180
    1377 AAUUCGUCUGCGAAGAAGA 267 AAuucGucuGcGAAGAAGATsT 268 UCUUCUUCGcAGACGAAUUTsT AD-12181
    1378 UGAAAGGUCACCUAAUGAA 269 uGAAAGGucAccuAAuGAATsT 270 UUcAUuAGGUGACCUUUcATsT AD-12182
    1379 CAGACCAUUUAAUUUGGCA 271 cAGAccAuuuAAuuuGGcATsT 272 UGCcAAAUuAAAUGGUCUGTsT AD-12183
    1380 AGACCAUUUAAUUUGGCAG 273 AGAccAuuuAAuuuGGcAGTsT 274 CUGCcAAAUuAAAUGGUCUTsT AD-12184
    1381 AGUUAUUAUGGGCUAUAAU 275 AGuuAuuAuGGGcuAuAAuTsT 276 AUuAuAGCCcAuAAuAACUTsT AD-12185
    1382 GCUGGUAUAAUUCCACGUA 277 GcuGGuAuAAuuccAcGuATsT 278 uACGUGGAAUuAuACcAGCTsT AD-12186
    1383 AUUUAAUUUGGCAGAGCGG 279 AuuuAAuuuGGcAGAGcGGTsT 280 CCGCUCUGCcAAAUuAAAUTsT AD-12187
    1384 UUUAAUUUGGCAGAGCGGA 281 uuuAAuuuGGcAGAGcGGATsT 282 UCCGCUCUGCcAAAUuAAATsT AD-12188
    1385 UUUGGCAGAGCGGAAAGCU 283 uuuGGcAGAGcGGAAAGcuTsT 284 AGCUUUCCGCUCUGCcAAATsT AD-12189
    1386 UUUUACAAUGGAAGGUGAA 285 uuuuAcAAuGGAAGGuGAATsT 286 UUcACCUUCcAUUGuAAAATsT AD-12190
    1387 AAUGGAAGGUGAAAGGUCA 287 AAuGGAAGGuGAAAGGucATsT 288 UGACCUUUcACCUUCcAUUTsT AD-12191
    1388 UGAGAUGCAGACCAUUUAA 289 uGAGAuGcAGAccAuuuAATsT 290 UuAAAUGGUCUGcAUCUcATsT AD-12192
    1389 UCGCAGCCAAAUUCGUCUG 291 ucGcAGccAAAuucGucuGTsT 292 cAGACGAAUUUGGCUGCGATsT AD-12193
    1390 GGCUAUAAUUGCACUAUCU 293 GGcuAuAAuuGcAcuAucuTsT 294 AGAuAGUGcAAUuAuAGCCTsT AD-12194
    1391 AUUGACAGUGGCCGAUAAG 295 AuuGAcAGuGGccGAuAAGTsT 296 CUuAUCGGCcACUGUcAAUTsT AD-12195
    1392 CUAGACUUCCCUAUUUCGC 297 cuAGAcuucccuAuuucGcTsT 298 GCGAAAuAGGGAAGUCuAGTsT AD-12196
    1393 ACUAUCUUUGCGUAUGGCC 299 AcuAucuuuGcGuAuGGccTsT 300 GGCcAuACGcAAAGAuAGUTsT AD-12197
    1394 AUACUCUAGUCGUUCCCAC 301 AuAcucuAGucGuucccAcTsT 302 GUGGGAACGACuAGAGuAUTsT AD-12198
    1395 AAAGAAACUACGAUUGAUG 303 AAAGAAAcuAcGAuuGAuGTsT 304 cAUcAAUCGuAGUUUCUUUTsT AD-12199
    1396 GCCUUGAUUUUUUGGCGGG 305 GccuuGAuuuuuuGGcGGGTsT 306 CCCGCcAAAAAAUcAAGGCTsT AD-12200
    1397 CGCCCAUUCAAUAGUAGAA 307 cGcccAuucAAuAGuAGAATsT 308 UUCuACuAUUGAAUGGGCGTsT AD-12201
    1398 CCUUAUUUGGUAAUCUGCU 309 ccuuAuuuGGuAAucuGcuTsT 310 AGcAGAUuACcAAAuAAGGTsT AD-12202
    1399 AGAGACAAUUCCGGAUGUG 311 AGAGAcAAuuccGGAuGuGTsT 312 cAcAUCCGGAAUUGUCUCUTsT AD-12203
    1400 UGACUUUGAUAGCUAAAUU 313 uGAcuuuGAuAGcuAAAuuTsT 314 AAUUuAGCuAUcAAAGUcATsT AD-12204
    1401 UGGCAGAGCGGAAAGCUAG 315 uGGcAGAGcGGAAAGcuAGTsT 316 CuAGCUUUCCGCUCUGCcATsT AD-12205
    1402 GAGCGGAAAGCUAGCGCCC 317 GAGcGGAAAGcuAGcGcccTsT 318 GGGCGCuAGCUUUCCGCUCTsT AD-12206
    1403 AAAGAAGUUAGUGUACGAA 319 AAAGAAGuuAGuGuAcGAATsT 320 UUCGuAcACuAACUUCUUUTsT AD-12207
    1404 AUUGCACUAUCUUUGCGUA 321 AuuGcAcuAucuuuGcGuATsT 322 uACGCAAAGAuAGUGcAAUTsT AD-12208
    1405 GGUAUAAUUCCACGUACCC 323 GGuAuAAuuccAcGuAcccTsT 324 GGGuACGUGGAAUuAuACCTsT AD-12209
    1406 UACUCUAGUCGUUCCCACU 325 uAcucuAGucGuucccAcuTsT 326 AGUGGGAACGACuAGAGuATsT AD-12210
    1407 UAUGAAAGAAACUACGAUU 327 uAuGAAAGAAAcuAcGAuuTsT 328 AAUCGuAGUUUCUUUcAuATsT AD-12211
    1408 AUGCUAGAAGUACAUAAGA 329 AuGcuAGAAGuAcAuAAGATsT 330 UCUuAUGuACUUCuAGcAUTsT AD-12212
    1409 AAGUACAUAAGACCUUAUU 331 AAGuAcAuAAGAccuuAuuTsT 332 AAuAAGGUCUuAUGuACUUTsT AD-12213
    1410 ACAGCCUGAGCUGUUAAUG 333 AcAGccuGAGcuGuuAAuGTsT 334 cAUuAAcAGCUcAGGCUGUTsT AD-12214
    1411 AAAGAAGAGACAAUUCCGG 335 AAAGAAGAGAcAAuuccGGTsT 336 CCGGAAUUGUCUCUUCUUUTsT AD-12215
    1412 CACACUGGAGAGGUCUAAA 337 cAcAcuGGAGAGGucuAAATsT 338 UUuAGACCUCUCcAGUGUGTsT AD-12216
    1413 CACUGGAGAGGUCUAAAGU 339 cAcuGGAGAGGucuAAAGuTsT 340 ACUUuAGACCUCUCcAGUGTsT AD-12217
    1414 ACUGGAGAGGUCUAAAGUG 341 AcuGGAGAGGucuAAAGuGTsT 342 cACUUuAGACCUCUCcAGUTsT AD-12218
    1415 CGUCGCAGCCAAAUUCGUC 343 cGucGcAGccAAAuucGucTsT 344 GACGAAUUUGGCUGCGACGTsT AD-12219
    1416 GAAGGCAGUUGACCAACAC 345 GAAGGcAGuuGAccAAcAcTsT 346 GUGUUGGUcAACUGCCUUCTsT AD-12220
    1417 CAUUCACCCUGACAGAGUU 347 cAuucAcccuGAcAGAGuuTsT 348 AACUCUGUcAGGGUGAAUGTsT AD-12221
    1418 AAGAGGCCUAACUCAUUCA 349 AAGAGGccuAAcucAuucATsT 350 UGAAUGAGUuAGGCCUCUUTsT AD-12222
    1419 GAGACAAUUCCGGAUGUGG 351 GAGAcAAuuccGGAuGuGGTsT 352 CcAcAUCCGGAAUUGUCUCTsT AD-12223
    1420 UUCCGGAUGUGGAUGUAGA 353 uuccGGAuGuGGAuGuAGATsT 354 UCuAcAUCcAcAUccGGAATsT AD-12224
    1421 AAGCUAGCGCCCAUUCAAU 355 AAGcuAGcGcccAuucAAuTsT 356 AUUGAAUGGGCGCuAGCUUTsT AD-12225
    1422 GAAGUUAGUGUACGAACUG 357 GAAGuuAGuGuAcGAAcuGTsT 358 cAGUUCGuAcACuAACUUCTsT AD-12226
    1423 UAUAAUUCCACGUACCCUU 359 uAuAAuuccAcGuAcccuuTsT 360 AAGGGuACGUGGAAUuAuATsT AD-12227
    1424 ACAGUGGCCGAUAAGAUAG 361 AcAGuGGccGAuAAGAuAGTsT 362 CuAUCUuAUCGGCcACUGUTsT AD-12228
    1425 UCUGUCAUCCCUAUAGUUC 363 ucuGucAucccuAuAGuucTsT 364 GAACuAuAGGGAUGAcAGATsT AD-12229
    1426 UUCUUGCUAUGACUUGUGU 365 uucuuGcuAuGAcuuGuGuTsT 366 AcAcAAGUcAuAGcAAGAATsT AD-12230
    1427 GUAAGAAGGCAGUUGACCA 367 GuAAGAAGGcAGuuGAccATsT 368 UGGUcAACUGCCUUCUuACTsT AD-12231
    1428 CAUUGACAGUGGCCGAUAA 369 cAuuGAcAGuGGccGAuAATsT 370 UuAUCGGCcACUGUcAAUGTsT AD-12232
    1429 AGAAACCACUUAGUAGUGU 371 AGAAAccAcuuAGuAGuGuTsT 372 AcAcuAcuAAGUGGUUUCUTsT AD-12233
    1430 GGAUUGUUCAUCAAUUGGC 373 GGAuuGuucAucAAuuGGcTsT 374 GCcAAUUGAUGAAcAAUCCTsT AD-12234
    1431 UAAGAGGCCUAACUCAUUC 375 uAAGAGGccuAAcucAuucTsT 376 GAAUGAGUuAGGCCUCUuATsT AD-12235
    1432 AGUUAGUGUACGAACUGGA 377 AGuuAGuGuAcGAAcuGGATsT 378 UCcAGUUcGuAcACuAACUTsT AD-12236
    1433 AGUACAUAAGACCUUAUUU 379 AGuAcAuAAGAccuuAuuuTsT 380 AAAuAAGGUCUuAUGuACUTsT AD-12237
    1434 UGAGCCUUGUGUAUAGAUU 381 uGAGccuuGuGuAuAGAuuTsT 382 AAUCuAuAcAcAAGGCUcATsT AD-12238
    1435 CCUUUAAGAGGCCUAACUC 383 ccuuuAAGAGGccuAAcucTsT 384 GAGUuAGGCCUCUuAAAGGTsT AD-12239
    1436 ACCACUUAGUAGUGUCCAG 385 AccAcuuAGuAGuGuccAGTsT 386 CUGGAcAcuAcuAAGUGGUTsT AD-12240
    1437 GAAACUUCCAAUUAUGUCU 387 GAAAcuuccAAuuAuGucuTsT 388 AGAcAuAAUUGGAAGUUUCTsT AD-12241
    1438 UGCAUACUCUAGUCGUUCC 389 uGcAuAcucuAGucGuuccTsT 390 GGAACGACuAGAGuAUGcATsT AD-12242
    1439 AGAAGGCAGUUGACCAACA 391 AGAAGGcAGuuGAccAAcATsT 392 UGUUGGUcAACUGCCUUCUTsT AD-12243
    1440 GUACAUAAGACCUUAUUUG 393 GuAcAuAAGAccuuAuuuGTST 394 cAAAuAAGGUCUuAUGuAcTsT AD-12244
    1441 UAUAAUUGCACUAUCUUUG 395 uAuAAuuGcAcuAucuuuGTsT 396 cAAAGAuAGUGcAAUuAuATsT AD-12245
    1442 UCUCUGUUACAAUACAUAU 397 ucucuGuuAcAAuAcAuAuTsT 398 AuAUGuAUUGuAAcAGAGATsT AD-12246
    1443 UAUGCUCAUAGAGCAAAGA 399 uAuGcucAuAGAGcAAAGATsT 400 UCUUUGCUCuAUGAGcAuATsT AD-12247
    1444 UGUUGUUUGUCCAAUUCUG 401 uGuuGuuuGuccAAuucuGTsT 402 cAGAAUUGGAcAAAcAAcATST AD-12248
    1445 ACUAACUAGAAUCCUCCAG 403 AcuAAcuAGAAuccuccAGTsT 404 CUGGAGGAUUCuAGUuAGUTsT AD-12249
    1446 UGUGGUGUCUAUACUGAAA 405 uGuGGuGucuAuAcuGAAATsT 406 UUUcAGuAuAGAcACcAcATsT AD-12250
    1447 UAUUAUGGGAGACCACCCA 407 uAuuAuGGGAGAccAcccATsT 408 UGGGUGGUCUCCcAuAAuATsT AD-12251
    1448 AAGGAUGAAGUCUAUCAAA 409 AAGGAuGAAGucuAucAAATsT 410 UUUGAuAGAcUUcAUCCUUTsT AD-12252
    1449 UUGAUAAGAGAGCUCGGGA 411 uuGAuAAGAGAGcucGGGATsT 412 UCCCGAGCUCUCUuAUcAATsT AD-12253
    1450 AUGUUCCUUAUCGAGAAUC 413 AuGuuccuuAucGAGAAucTsT 414 GAUUCUCGAuAAGGAAcAUTsT AD-12254
    1451 GGAAUAUGCUCAUAGAGCA 415 GGAAuAuGcucAuAGAGcATsT 416 UGCUCuAUGAGcAuAUUCCTsT AD-12255
    1452 CCAUUCCAAACUGGAUCGU 417 ccAuuccAAAcuGGAucGuTsT 418 ACGAUCcAGUUUGGAAUGGTsT AD-12256
    1453 GGCAGUUGACCAACACAAU 419 GGcAGuuGAccAAcAcAAuTsT 420 AUUGUGUUGGUcAACUGCCTsT AD-12257
    1454 CAUGCUAGAAGUACAUAAG 421 cAuGcuAGAAGuAcAuAAGTsT 422 CUuAUGuACUUCuAGcAUGTsT AD-12258
    1455 CUAGAAGUACAUAAGACCU 423 cuAGAAGuAcAuAAGAccuTsT 424 AGGUCUuAUGuACUUCuAGTsT AD-12259
    1456 UUGGAUCUCUCACAUCUAU 425 uuGGAucucucAcAucuAuTsT 426 AuAGAUGUGAGAGAUCcAATsT AD-12260
    1457 AACUGUGGUGUCUAUACUG 427 AAcuGuGGuGucuAuAcuGTsT 428 cAGuAuAGAcACcAcAGUUTsT AD-12261
    1458 UCAUUGACAGUGGCCGAUA 429 ucAuuGAcAGuGGccGAuATsT 430 uAUCGGCcACUGUcAAUGATsT AD-12262
    1459 AUAAAGCAGACCCAUUCCC 431 AuAAAGcAGAcccAuucccTsT 432 GGGAAUGGGUCUGCUUuAUTsT AD-12263
    1460 ACAGAAACCACUUAGUAGU 433 AcAGAAAccAcuuAGuAGuTsT 434 AcuAcuAAGUGGUUUCUGUTsT AD-12264
    1461 GAAACCACUUAGUAGUGUC 435 GAAAccAcuuAGuAGuGucTsT 436 GAcACuACuAAGUGGUUUCTsT AD-12265
    1462 AAAUCUAAGGAUAUAGUCA 437 AAAucuAAGGAuAuAGucATsT 438 UGAcuAuAUCCUuAGAUUUTsT AD-12266
    1463 UUAUUUAUACCCAUCAACA 439 uuAuuuAuAcccAucAAcATsT 440 UGUUGAUGGGuAuAAAuAATsT AD-12267
    1464 ACAGAGGCAUUAACACACU 441 AcAGAGGcAuuAAcAcAcuTsT 442 AGUGUGUuAAUGCCUCUGUTsT AD-12268
    1465 ACACACUGGAGAGGUCUAA 443 AcAcAcuGGAGAGGucuAATsT 444 UuAGACCUCUCcAGUGUGUTsT AD-12269
    1466 ACACUGGAGAGGUCUAAAG 445 AcAcuGGAGAGGucuAAAGTsT 446 CUUuAGACCUCUCcAGUGUTsT AD-12270
    1467 CGAGCCCAGAUCAACCUUU 447 cGAGcccAGAucAAccuuuTsT 448 AAAGGUUGAUCUGGGCUCGTsT AD-12271
    1468 UCCCUAUUUCGCUUUCUCC 449 ucccuAuuucGcuuucuccTsT 450 GGAGAAAGCGAAAuAGGGATsT AD-12272
    1469 UCUAAAAUCACUGUCAACA 451 ucuAAAAucAcuGucAAcATsT 452 UGUUGAcAGUGAUUUuAGATsT AD-12273
    1470 AGCCAAAUUCGUCUGCGAA 453 AGccAAAuucGucuGcGAATsT 454 UUCGcAGACGAAUUUGGCUTsT AD-12274
    1471 CCCAUUCAAUAGUAGAAUG 455 cccAuucAAuAGuAGAAuGTsT 456 cAUUCuACuAUUGAAUGGGTsT AD-12275
    1472 GAUGAAUGCAUACUCUAGU 457 GAuGAAuGcAuAcucuAGuTsT 458 ACuAGAGuAUGcAUUcAUCTsT AD-12276
    1473 CUCAUGUUCCUUAUCGAGA 459 cucAuGuuccuuAucGAGATsT 460 UCUCGAuAAGGAAcAUGAGTsT AD-12277
    1474 GAGAAUCUAAACUAACUAG 461 GAGAAucuAAAcuAAcuAGTsT 462 CuAGUuAGUUuAGAUUCUCTsT AD-12278
    1475 UAGAAGUACAUAAGACCUU 463 uAGAAGuAcAuAAGAccuuTsT 464 AAGGUCUuAUGuACUUCuATsT AD-12279
    1476 CAGCCUGAGCUGUUAAUGA 465 cAGccuGAGcuGuuAAucATsT 466 UcAUuAAcAGCUcAGGCUGTsT AD-12280
    1477 AAGAAGAGACAAUUCCGGA 467 AAGAAGAGAcAAuuccGGATsT 468 UCCGGAAUUGUCUCUUCUUTsT AD-12281
    1478 UGCUGGUGUGGAUUGUUCA 469 uGcuGGuGuGGAuuGuucATsT 470 UGAAcAAUCcAcACcAGcATsT AD-12282
    1479 AAAUUCGUCUGCGAAGAAG 471 AAAuucGucuGcGAAGAAGTsT 472 CUUCUUCGcAGACGAAUUUTsT AD-12283
    1480 UUUCUGGAAGUUGAGAUGU 473 uuucuGGAAGuuGAGAuGuTsT 474 AcAUCUcAACUUCcAGAAATsT AD-12284
    1481 UACUAAACAGAUUGAUGUU 475 uAcuAAAcAGAuuGAuGuuTsT 476 AAcAUcAAUCUGUUuAGuATsT AD-12285
    1482 GAUUGAUGUUUACCGAAGU 477 GAuuGAuGuuuAccGAAGuTsT 478 ACUUCGGuAAAcAUcAAUCTsT AD-12286
    1483 GCACUAUCUUUGCGUAUGG 479 GcAcuAucuuuGcGuAuGGTsT 480 CcAuACGcAAAGAuAGUGCTsT AD-12287
    1484 UGGUAUAAUUCCACGUACC 481 uGGuAuAAuuccAcGuAccTsT 482 GGuACGUGGAAUuAuACcATsT AD-12288
    1485 AGCAAGCUGCUUAACACAG 483 AGcAAGcuGcuuAAcAcAGTsT 484 CUGUGUuAAGcAGCUUGCUTsT AD-12289
    1486 CAGAAACCACUUAGUAGUG 485 cAGAAAccAcuuAGuAGuGTsT 486 cACuACuAAGUGGUUUCUGTsT AD-12290
    1487 AACUUAUUGGAGGUUGUAA 487 AAcuuAuuGGAGGuuGuAATsT 488 UuAcAACCUCcAAuAAGUUTsT AD-12291
    1488 CUGGAGAGGUCUAAAGUGG 489 cuGGAGAGGucuAAAGuGGTsT 490 CcACUUuAGACCUCUCcAGTsT AD-12292
    1489 AAAAAAGAUAUAAGGCAGU 491 AAAAAAGAuAuAAGGcAGuTsT 492 ACUGCCUuAuAUCUUUUUUTsT AD-12293
    1490 GAAUUUUGAUAUCUACCCA 493 GAAuuuuGAuAucuAcccATsT 494 UGGGuAGAuAUcAAAAUUCTsT AD-12294
    1491 GUAUUUUUGAUCUGGCAAC 495 GuAuuuuuGAucuGGcAAcTsT 496 GUUGCcAGAUcAAAAAuACTsT AD-12295
    1492 AGGAUCCCUUGGCUGGUAU 497 AGGAucccuuGGcuGGuAuTsT 498 AuACcAGCcAAGGGAUCCUTsT AD-12296
    1493 GGAUCCCUUGGCUGGUAUA 499 GGAucccuuGGcuGGuAuATsT 500 uAuACcAGCcAAGGGAUCCTsT AD-12297
    1494 CAAUAGUAGAAUGUGAUCC 501 cAAuAGuAGAAuGuGAuccTsT 502 GGAUcAcAUUCuACuAUUGTsT AD-12298
    1495 GCUAUAAUUGCACUAUCUU 503 GcuAuAAuuGcAcuAucuuTsT 504 AAGAuAGUGcAAUuAuAGcTsT AD-12299
    1496 UACCCUUCAUCAAAUUUUU 505 uAcccuucAucAAAuuuuuTsT 506 AAAAAUUUGAUGAAGGGuATsT AD-12300
    1497 AGAACAUAUUGAAUAAGCC 507 AGAAcAuAuuGAAuAAGccTsT 508 GGCUuAUUcAAuAUGUUCUTsT AD-12301
    1498 AAAUUGGUGCUGUUGAGGA 509 AAAuuGGuGcuGuuGAGGATsT 510 UCCUcAAcAGcACcAAUUUTsT AD-12302
    1499 UGAAUAGGGUUACAGAGUU 511 uGAAuAGGGuuAcAGAGuuTsT 512 AACUCUGuAACCCuAUUcATsT AD-12303
    1500 AAGAACUUGAAACCACUCA 513 AAGAAcuuGAAAccAcucATsT 514 UGAGUGGUUUcAAGUUCUUTsT AD-12304
    1501 AAUAAAGCAGACCCAUUCC 515 AAuAAAGcAGAcccAuuccTsT 516 GGAAUGGGUCUGCUUuAUUTsT AD-12305
    1502 AUACCCAUCAACACUGGUA 517 AuAcccAucAAcAcuGGuATsT 518 uACcAGUGUUGAUGGGuAUTsT AD-12306
    1503 UGGAUUGUUCAUCAAUUGG 519 uGGAuuGuucAucAAuuGGTsT 520 CcAAUUGAUGAAcAAUCcATsT AD-12307
    1504 UGGAGAGGUCUAAAGUGGA 521 uGGAGAGGucuAAAGuGGATsT 522 UCcACUUuAGACCUCUCcATsT AD-12308
    1505 GUCAUCCCUAUAGUUCACU 523 GucAucccuAuAGuucAcuTsT 524 AGUGAACuAuAGGGAUGACTsT AD-12309
    1506 AUAAUGGCUAUAAUUUCUC 525 AuAAuGGcuAuAAuuucucTsT 526 GAGAAAUuAuAGCcAUuAUTsT AD-12310
    1507 AUCCCUUGGCUGGUAUAAU 527 AucccuuGGcuGGuAuAAuTsT 528 AUuAuACcAGCcAAGGGAUTsT AD-12311
    1508 GGGCUAUAAUUGCACUAUC 529 GGGcuAuAAuuGcAcuAucTsT 530 GAuAGUGcAAUuAuAGCCCTsT AD-12312
    1509 GAUUCUCUUGGAGGGCGUA 531 GAuucucuuGGAGGGcGuATsT 532 uACGCCCUCcAAGAGAAUCTsT AD-12313
    1510 GCAUCUCUCAAUCUUGAGG 533 GcAucucucAAucuuGAGGTsT 534 CCUcAAGAUUGAGAGAUGCTsT AD-12314
    1511 CAGCAGAAAUCUAAGGAUA 535 cAGcAGAAAucuAAGGAuATsT 536 uAUCCUuAGAUUUCUGCUGTsT AD-12315
    1512 GUCAAGAGCCAUCUGUAGA 537 GucAAGAGccAucuGuAGATsT 538 UCuAcAGAUGGCUCUUGACTsT AD-12316
    1513 AAACAGAGGCAUUAACACA 539 AAAcAGAGGcAuuAAcAcATsT 540 UGUGUuAAUGCCUCUGUUUTsT AD-12317
    1514 AGCCCAGAUCAACCUUUAA 541 AGcccAGAucAAccuuuAATsT 542 UuAAAGGUUGAUCUGGGCUTsT AD-12318
    1515 UAUUUUUGAUCUGGCAACC 543 uAuuuuuGAucuGGcAAccTsT 544 GGUUGCcAGAUcAAAAAuATsT AD-12319
    1516 UGUUUGGAGCAUCUACUAA 545 uGuuuGGAGcAucuAcuAATsT 546 UuAGuAGAUGCUCcAAAcATsT AD-12320
    1517 GAAAUUACAGUACACAACA 547 GAAAuuAcAGuAcAcAAcATsT 548 UGUUGUGuACUGuAAUUUCTsT AD-12321
    1518 ACUUGACCAGUGUAAAUCU 549 AcuuGAccAGuGuAAAucuTsT 550 AGAUUuAcACUGGUcAAGUTsT AD-12322
    1519 ACCAGUGUAAAUCUGACCU 551 AccAGuGuAAAucuGAccuTsT 552 AGGUcAGAUUuAcACUGGUTsT AD-12323
    1520 AGAACAAUCAUUAGCAGCA 553 AGAAcAAucAuuAGcAGcATsT 554 UGCUGCuAAUGAUUGUUCUTsT AD-12324
    1521 CAAUGUGGAAACCUAACUG 555 cAAuGuGGAAAccuAAcuGTsT 556 cAGUuAGGUUUCcAcAUUGTsT AD-12325
    1522 ACCAAGAAGGUACAAAAUU 557 AccAAGAAGGuAcAAAAuuTsT 558 AAUUUUGuACCUUCUUGGUTsT AD-12326
    1523 GGUACAAAAUUGGUUGAAG 559 GGuAcAAAAuuGGuuGAAGTsT 560 CUUcAACcAAUUUUGuACCTsT AD-12327
    1524 GGUGUGGAUUGUUCAUCAA 561 GGuGuGGAuuGuucAucAATsT 562 UUGAUGAAcAAUCcAcACCTsT AD-12328
    1525 AGAGUUCACAAAAAGCCCA 563 AGAGuucAcAAAAAGcccATsT 564 UGGGCUUUUUGUGAACUCUTsT AD-12329
    1526 UGAUAGCUAAAUUAAACCA 565 uGAuAGcuAAAuuAAAccATsT 566 UGGUUuAAUUuAGCuAUcATsT AD-12330
    1527 AAUAAGCCUGAACUGAAUC 567 AAuAAGccuGAAGuGAAucTsT 568 GAUUcACUUcAGGCUuAUUTsT AD-12331
    1528 CAGUUGACCAACACAAUGC 569 cAGuuGAccAAcAcAAuGcTsT 570 GcAUUGUGUUGGUcAACUGTsT AD-12332
    1529 UGGUGUGGAUUGUUCAUCA 571 uGGuGuGGAuuGuucAucATsT 572 UGAUGAAcAAUCcAcACcATsT AD-12333
    1530 AUUCACCCUGACACAGUUC 573 AuucAcccuGAcAGAGuucTsT 574 GAACUCUGUcAcGGUGAAUTsT AD-12334
    1531 UAAGACCUUAUUUGGUAAU 575 uAAGAccuuAuuuGGuAAuTsT 576 AUuACcAAAuAAGGUCUuATsT AD-12335
    1532 AAGCAAUGUGGAAACCUAA 577 AAGcAAuGuGGAAAccuAATsT 578 UuAGGUUUccAcAUUGCUUTsT AD-12336
    1533 UCUGAAACUGGAUAUCCCA 579 ucuGAAAcuGGAuAucccATsT 580 UGGGAuAUCcAGUUUcAGATsT AD-12337
  • [0000]
    TABLE 2b
    Analysis of Eg5/KSP dsRNA duplexes
    1st single 2nd single
    dose dose 3rd
    screen @ screen @ single
    Eg5/KSP 50 nm [% SDs 1st screen 25 nM [% SDs 2nd screen dose SDs 3rd screen
    duplex resudual (among resudual (among screen (among
    name mRNA] quadruplicates) mRNA] quadruplicates) @ 25 nM quadruplicates)
    AD-12072 65% 2% 82% 5%
    AD-12073 84% 1% 61% 6%
    AD-12074 51% 3% 36% 9%
    AD-12075 56% 4% 36% 4%
    AD-12076 21% 4% 13% 3%
    AD-12077 11% 2% 6% 1%
    AD-12078 22% 3% 9% 2%
    AD-12079 22% 10% 15% 7%
    AD-12080 68% 4% 52% 13%
    AD-12081 34% 8% 35% 24%
    AD-12082 20% 2% 92% 5%
    AD-12083 85% 6% 63% 10%
    AD-12084 18% 6% 17% 4%
    AD-12085 13% 4% 12% 4%
    AD-12086 26% 5% 17% 3%
    AD-12087 95% 4% 80% 4%
    AD-12088 29% 6% 29% 2%
    AD-12089 69% 5% 64% 7%
    AD-12090 46% 15% 34% 5%
    AD-12091 16% 6% 17% 3%
    AD-12092 82% 26% 63% 5%
    AD-12093 84% 4% 70% 4%
    AD-12094 46% 3% 34% 1%
    AD-12095 14% 2% 13% 1%
    AD-12096 26% 11% 17% 1%
    AD-12097 23% 2% 21% 1%
    AD-12098 41% 14% 17% 3%
    AD-12099 57% 2% 48% 6%
    AD-12100 101% 11% 98% 8%
    AD-12101 46% 7% 32% 2%
    AD-12102 96% 17% 88% 18%
    AD-12103 19% 5% 20% 2%
    AD-12104 40% 8% 24% 2%
    AD-12105 39% 2% 36% 10%
    AD-12106 87% 6% 79% 19%
    AD-12107 29% 2% 32% 16%
    AD-12108 38% 4% 39% 8%
    AD-12109 49% 3% 44% 10%
    AD-12110 85% 5% 80% 14%
    AD-12111 64% 6% 71% 18%
    AD-12112 48% 4% 41% 5%
    AD-12113 13% 0% 14% 3%
    AD-12114 32% 6% 16% 4%
    AD-12115 8% 4% 7% 5%
    AD-12116 74% 5% 61% 7%
    AD-12117 21% 4% 20% 2%
    AD-12118 44% 4% 42% 6%
    AD-12119 37% 4% 24% 3%
    AD-12120 22% 2% 15% 4%
    AD-12121 32% 1% 22% 2%
    AD-12122 36% 16% 19% 5%
    AD-12123 28% 1% 16%
    AD-12124 28% 2% 16%
    AD-12125 15% 1% 14%
    AD-12126 51% 22% 27%
    AD-12127 54% 4% 42% 9%
    AD-12128 29% 1% 20% 2%
    AD-12129 22% 3% 19% 3%
    AD-12130 53% 6% 42% 7%
    AD-12131 28% 5% 22% 3%
    AD-12132 88% 2% 90% 18%
    AD-12133 34% 2% 26% 6%
    AD-12134 18% 3% 14% 2%
    AD-12135 50% 6% 37% 4%
    AD-12136 42% 19% 22% 2%
    AD-12137 85% 12% 92% 4%
    AD-12138 47% 6% 49% 1%
    AD-12139 80% 5% 72% 4%
    AD-12140 97% 22% 67% 9%
    AD-12141 120% 4% 107% 10%
    AD-12142 55% 8% 33% 4%
    AD-12143 64% 34% 19% 2%
    AD-12144 58% 29% 17% 2%
    AD-12145 27% 8% 18% 2%
    AD-12146 19% 20% 15% 1%
    AD-12147 29% 9% 35% 3%
    AD-12148 30% 3% 56% 5%
    AD-12149 8% 2% 12% 3%
    AD-12150 31% 2% 31% 7%
    AD-12151 9% 5% 14% 2%
    AD-12152 3% 3% 23% 3%
    AD-12153 20% 6% 34% 4%
    AD-12154 24% 7% 44% 3%
    AD-12155 33% 6% 53% 11%
    AD-12156 35% 5% 40% 5%
    AD-12157 8% 3% 23% 4%
    AD-12158 13% 2% 22% 5%
    AD-12159 34% 6% 46% 5%
    AD-12160 19% 3% 31% 4%
    AD-12161 88% 4% 83% 7%
    AD-12162 26% 7% 32% 7%
    AD-12163 55% 9% 40% 3%
    AD-12164 21% 3%
    AD-12165 30% 3% 41% 4%
    AD-12166 9% 10% 22% 9%
    AD-12167 26% 3% 30% 2%
    AD-12168 54% 4% 59% 20%
    AD-12169 41% 4% 51% 16%
    AD-12170 43% 4% 52% 20%
    AD-12171 67% 3% 73% 25%
    AD-12172 53% 15% 37% 2%
    AD-12173 39% 0% 39% 0%
    AD-12174 41% 5% 27% 0%
    AD-12175 29% 0% 38% 14%
    AD-12176 43% 2% 56% 25%
    AD-12177 68% 6% 74% 30%
    AD-12178 41% 4% 41% 6%
    AD-12179 53% 5% 44% 5%
    AD-12180 16% 2% 13% 4%
    AD-12181 19% 3% 14% 2%
    AD-12182 16% 4% 18% 8%
    AD-12183 26% 3% 19% 4%
    AD-12184 54% 2% 77% 8%
    AD-12185 8% 1% 9% 1%
    AD-12186 36% 3% 41% 6%
    AD-12187 34% 17% 27% 1%
    AD-12188 30% 3% 27% 4%
    AD-12189 51% 4% 48% 5%
    AD-12190 33% 2% 26% 4%
    AD-12191 20% 2% 13% 0%
    AD-12192 21% 1% 23% 10%
    AD-12193 64% 8% 98% 6%
    AD-12194 8% 2% 15% 4%
    AD-12195 34% 2% 48% 3%
    AD-12196 34% 2% 51% 3%
    AD-12197 75% 4% 93% 6%
    AD-12198 55% 5% 48% 2%
    AD-12199 102% 6% 118% 9%
    AD-12200 75% 6% 60% 12%
    AD-12201 42% 3% 16% 4%
    AD-12202 29% 4% 9% 3%
    AD-12203 114% 14% 89% 20%
    AD-12204 64% 7% 26% 5%
    AD-12205 66% 12% 35% 4%
    AD-12206 46% 3% 32% 12%
    AD-12207 57% 5% 40% 6%
    AD-12208 30% 8% 10% 5%
    AD-12209 101% 6% 102% 23%
    AD-12210 38% 11% 27% 14%
    AD-12211 16% 6% 10% 5%
    AD-12212 59% 8% 65% 5%
    AD-12213 24% 9% 12% 2%
    AD-12214 67% 14% 70% 12%
    AD-12215 29% 13% 13% 4%
    AD-12216 36% 4% 13% 1%
    AD-12217 36% 9% 11% 2%
    AD-12218 35% 5% 17% 3%
    AD-12219 41% 9% 14% 1%
    AD-12220 37% 5% 23% 3%
    AD-12221 58% 7% 39% 6%
    AD-12222 74% 9% 53% 3%
    AD-12223 74% 10% 67% 7%
    AD-12224 24% 2% 11% 2%
    AD-12225 75% 5% 76% 14%
    AD-12226 45% 8% 40% 3%
    AD-12227 61% 6% 47% 5%
    AD-12228 28% 3% 25% 5%
    AD-12229 54% 13% 37% 6%
    AD-12230 70% 17% 65% 4%
    AD-12231 32% 12% 22% 6%
    AD-12232 30% 3% 17% 2%
    AD-12233 38% 2% 32% 3%
    AD-12234 90% 5% 95% 7%
    AD-12235 57% 7% 46% 3%
    AD-12236 34% 8% 16% 2%
    AD-12237 42% 9% 32% 8%
    AD-12238 42% 6% 34% 6%
    AD-12239 42% 3% 40% 4%
    AD-12240 47% 6% 36% 5%
    AD-12241 69% 5% 70% 8%
    AD-12242 61% 2% 47% 3%
    AD-12243 26% 7% 15% 1%
    AD-12244 25% 6% 15% 1%
    AD-12245 65% 6% 83% 13%
    AD-12246 29% 7% 31% 6%
    AD-12247 57% 13% 50% 3%
    AD-12248 36% 8% 20% 3% 15% 7%
    AD-12249 44% 3% 70% 11% 103% 34%
    AD-12250 47% 5% 18% 5% 17% 4%
    AD-12251 121% 28% 35% 8% 60% 42%
    AD-12252 94% 19% 8% 3% 5% 3%
    AD-12253 94% 33% 42% 8% 49% 27%
    AD-12254 101% 58% 70% 5% 80% 32%
    AD-12255 163% 27% 28% 6% 36% 10%
    AD-12256 112% 62% 18% 3% 9% 4%
    AD-12257 10% 4% 9% 2% 6% 2%
    AD-12258 27% 9% 18% 3% 20% 6%
    AD-12259 20% 5% 12% 2% 13% 5%
    AD-12260 22% 7% 81% 7% 65% 13%
    AD-12261 122% 11% 66% 7% 80% 22%
    AD-12262 97% 30% 33% 6% 44% 18%
    AD-12263 177% 57% 85% 11% 84% 15%
    AD-12264 37% 6% 10% 1% 10% 4%
    AD-12265 40% 8% 17% 1% 20% 10%
    AD-12266 33% 9% 9% 1% 8% 4%
    AD-12267 34% 13% 11% 1% 6% 2%
    AD-12268 34% 6% 11% 1% 9% 2%
    AD-12269 54% 6% 33% 4% 29% 7%
    AD-12270 52% 5% 29% 4% 27% 6%
    AD-12271 53% 7% 27% 3% 19% 6%
    AD-12272 85% 15% 57% 7% 51% 16%
    AD-12273 36% 6% 26% 2% 30% 5%
    AD-12274 75% 21% 40% 2% 50% 19%
    AD-12275 29% 9% 8% 1% 8% 4%
    AD-12276 45% 19% 15% 2% 16% 12%
    AD-12277 58% 17% 32% 2% 55% 14%
    AD-12278 120% 35% 96% 10% 124% 38%
    AD-12279 47% 29% 17% 1% 12% 4%
    AD-12280 2% 0% 3% 1%
    AD-12281 2% 0% 5% 2%
    AD-12282 3% 0% 25% 5%
    AD-12283 3% 1% 35% 4%
    AD-12284 5% 2% 49% 8%
    AD-12285 7% 7% 21% 26%
    AD-12286 28% 34% 12% 7%
    AD-12287 40% 21% 51% 23%
    AD-12288 26% 7% 155% 146%
    AD-12289 43% 21% 220% 131%
    AD-12290 2% 1% 81% 23%
    AD-12291 4% 1% 70% 3%
    AD-12292 2% 1% 6% 2%
    AD-12293 4% 2% 36% 3%
    AD-12294 10% 6% 38% 3%
    AD-12295 29% 31% 37% 3%
    AD-12296 82% 4% 89% 2%
    AD-12297 75% 3% 65% 2%
    AD-12298 73% 4% 60% 3%
    AD-12299 76% 4% 66% 4%
    AD-12300 36% 4% 15% 1%
    AD-12301 33% 4% 18% 2%
    AD-12302 66% 5% 65% 3%
    AD-12303 35% 6% 17% 2%
    AD-12304 70% 8% 70% 6%
    AD-12305 63% 8% 80% 7%
    AD-12306 23% 6% 20% 3%
    AD-12307 78% 10% 58% 5%
    AD-12308 27% 8% 15% 2%
    AD-12309 58% 11% 42% 3%
    AD-12310 106% 23% 80% 2%
    AD-12311 73% 12% 60% 2%
    AD-12312 39% 3% 36% 3%
    AD-12313 64% 9% 49% 6%
    AD-12314 28% 7% 14% 6%
    AD-12315 31% 7% 13% 2%
    AD-12316 42% 5% 14% 2%
    AD-12317 34% 9% 15% 5%
    AD-12318 46% 4% 28% 4%
    AD-12319 77% 3% 56% 4%
    AD-12320 55% 7% 41% 3%
    AD-12321 21% 3% 10% 2%
    AD-12322 27% 8% 30% 12%
    AD-12323 26% 7% 35% 18%
    AD-12324 27% 8% 27% 14%
    AD-12325 32% 12% 32% 22%
    AD-12326 42% 22% 45% 41%
    AD-12327 36% 14% 37% 32%
    AD-12328 45% 2% 31% 3%
    AD-12329 61% 4% 34% 3%
    AD-12330 63% 5% 38% 4%
    AD-12331 50% 2% 26% 5%
    AD-12332 80% 4% 51% 7%
    AD-12333 34% 6% 12% 2%
    AD-12334 27% 2% 18% 3%
    AD-12335 84% 6% 60% 7%
    AD-12336 45% 4% 36% 4%
    AD-12337 30% 7% 19% 2%
  • [0000]
    TABLE 3
    Sequences and analysis of Eg5/KSP dsRNA duplexes
    single
    dose SDs
    screen @ 2nd
    SEQ SEQ 25 nM [% screen
    ID Antisense sequence (5′- ID duplex residual (among
    Sense sequence (5′-3′) NO. 3′) NO. name mRNA] quadruplicates)
    ccAuuAcuAcAGuAGcAcuTsT 582 AGUGCuACUGuAGuAAUGGTsT 583 AD-14085 19% 1%
    AucuGGcAAccAuAuuucuTsT 584 AGAAAuAUGGUUGCcAGAUTsT 585 AD-14086 38% 1%
    GAuAGcuAAAuuAAAccAATsT 586 UUGGUUuAAUUuAGCuAUCTsT 587 AD-14087 75% 10%
    AGAuAccAuuAcuAcAGuATsT 588 uACUGuAGuAAUGGuAUCUTsT 589 AD-14088 22% 8%
    GAuuGuucAucAAuuGGcGTsT 590 CGCcAAUUGAUGAAcAAUCTsT 591 AD-14089 70% 12%
    GcuuucuccucGGcucAcuTsT 592 AGuGAGCCGAGGAGAAAGCTsT 593 AD-14090 79% 11%
    GGAGGAuuGGcuGAcAAGATsT 594 UCUUGUcAGCcAAUCCUCCTsT 595 AD-14091 29% 3%
    uAAuGAAGAGuAuAccuGGTsT 596 CcAGGuAuACUCUUcAUuATsT 597 AD-14092 23% 2%
    uuucAccAAAccAuuuGuATsT 598 uAcAAAUGGUUUGGUGAAATsT 599 AD-14093 60% 2%
    cuuAuuAAGGAGuAuAcGGTsT 600 CCGuAuACUCCUuAAuAAGTsT 601 AD-14094 11% 3%
    GAAAucAGAuGGAcGuAAGTsT 602 CUuACGUCcAUCUGAUUUCTsT 603 AD-14095 10% 2%
    cAGAuGucAGcAuAAGcGATsT 604 UCGCUuAUGCUGAcAUCUGTsT 605 AD-14096 27% 2%
    AucuAAcccuAGuuGuAucTsT 606 GAuAcAACuAGGGUuAGAUTsT 607 AD-14097 45% 6%
    AAGAGcuuGuuAAAAucGGTsT 608 CCGAUUUuAAcAAGCUCUUTsT 609 AD-14098 50% 10%
    uuAAGGAGuAuAcGGAGGATsT 610 UCCUCCGuAuACUCCUuAATsT 611 AD-14099 12% 4%
    uuGcAAuGuAAAuAcGuAuTsT 612 AuACGuAUUuAcAUUGcAATsT 613 AD-14100 49% 7%
    ucuAAcccuAGuuGuAuccTsT 614 GGAuAcAACuAGGGUuAGATsT 615 AD-14101 36% 1%
    cAuGuAucuuuuucucGAuTsT 616 AUCGAGAAAAAGAuAcAUGTsT 617 AD-14102 49% 3%
    GAuGucAGcAuAAGcGAuGTsT 618 cAUCGCUuAUGCUGAcAUCTsT 619 AD-14103 74% 5%
    ucccAAcAGGuAcGAcAccTsT 620 GGUGUCGuACCUGUUGGGATsT 621 AD-14104 27% 3%
    uGcucAcGAuGAGuuuAGuTsT 622 ACuAAACUcAUCGUGAGcATsT 623 AD-14105 34% 4%
    AGAGcuuGuuAAAAucGGATsT 624 UCCGAUUUuAAcAAGCUCUTsT 625 AD-14106 9% 2%
    GcGuAcAAGAAcAucuAuATsT 626 uAuAGAUGUUCUUGuACGCTsT 627 AD-14107 5% 1%
    GAGGuuGuAAGccAAuGuuTsT 628 AAcAUUGGCUuAcAACCUCTsT 629 AD-14108 15% 1%
    AAcAGGuAcGAcAccAcAGTsT 630 CUGUGGUGUCGuACCUGUUTsT 631 AD-14109 91% 2%
    AAcccuAGuuGuAucccucTsT 632 GAGGGAuAcAACuAGGGUUTsT 633 AD-14110 66% 5%
    GcAuAAGcGAuGGAuAAuATsT 634 uAUuAUCcAUCGCUuAUGCTsT 635 AD-14111 33% 3%
    AAGcGAuGGAuAAuAccuATsT 636 uAGGuAUuAUCcAUCGCUUTsT 637 AD-14112 51% 3%
    uGAuccuGuAcGAAAAGAATsT 638 UUCUUUUCGuAcAGGAUcATsT 639 AD-14113 22% 3%
    AAAAcAuuGGccGuucuGGTsT 640 CcAGAACGGCcAAUGUUUUTsT 641 AD-14114 117% 8%
    cuuGGAGGGcGuAcAAGAATsT 642 UUCUUGuACGCCCUCcAAGTsT 643 AD-14115 50% 8%
    GGcGuAcAAGAAcAucuAuTsT 644 AuAGAUGUUCUUGuACGCCTsT 645 AD-14116 14% 3%
    AcucuGAGuAcAuuGGAAuTsT 646 AUUCcAAUGuACUcAGAGUTsT 647 AD-14117 12% 4%
    uuAuuAAGGAGuAuAcGGATsT 648 UCCGuAuACUCCUuAAuAATsT 649 AD-14118 26% 4%
    uAAGGAGuAuAcGGAGGAGTsT 650 CUCCUCCGuAuACUCCUuATsT 651 AD-14119 24% 5%
    AAAucAAuAGucAAcuAAATsT 652 UUuAGUUGACuAUUGAUUUTsT 653 AD-14120 8% 1%
    AAucAAuAGucAAcuAAAGTsT 654 CUUuAGUUGACuAUUGAUUTsT 655 AD-14121 24% 2%
    uucucAGuAuAcuGuGuAATsT 656 UuAcAcAGuAuACUGAGAATsT 657 AD-14122 10% 1%
    uGuGAAAcAcucuGAuAAATsT 658 UUuAUcAGAGUGUUUCAcATsT 659 AD-14123 8% 1%
    AGAuGuGAAucucuGAAcATsT 660 UGUUcAGAGAUUcAcAUCUTsT 661 AD-14124 9% 2%
    AGGuuGuAAGccAAuGuuGTsT 662 cAAcAUUGGCUuAcAACCUTsT 663 AD-14125 114% 6%
    uGAGAAAucAGAuGGAcGuTsT 664 ACGUCcAUCUGAUUUCUcATsT 665 AD-14126 9% 1%
    AGAAAucAGAuGGAcGuAATsT 666 UuACGUCcAUCUGAUUUCUTsT 667 AD-14127 57% 6%
    AuAucccAAcAGGuAcGAcTsT 668 GUCGuACCUGUUGGGAuAUTsT 669 AD-14128 104% 6%
    cccAAcAGGuAcGAcAccATsT 670 UGGUGUCGuACCUGUUGGGTsT 671 AD-14129 21% 2%
    AGuAuAcuGAAGAAccucuTsT 672 AGAGGUUCUUcAGuAuACUTsT 673 AD-14130 57% 6%
    AuAuAuAucAGccGGGcGcTsT 674 GCGCCCGGCUGAuAuAuAUTsT 675 AD-14131 93% 6%
    AAucuAAcccuAGuuGuAuTsT 676 AuAcAACuAGGGUuAGAUUTsT 677 AD-14132 75% 8%
    cuAAcccuAGuuGuAucccTsT 678 GGGAuAcAACuAGGGUuAGTsT 679 AD-14133 66% 4%
    cuAGuuGuAucccuccuuuTsT 680 AAAGGAGGGAuAcAACuAGTsT 681 AD-14134 44% 6%
    AGAcAucuGAcuAAuGGcuTsT 682 AGCcAUuAGUcAGAUGUCUTsT 683 AD-14135 55% 6%
    GAAGcucAcAAuGAuuuAATsT 684 UuAAAUcAUUGUGAGCUUCTsT 685 AD-14136 29% 3%
    AcAuGuAucuuuuucucGATsT 686 UCGAGAAAAAGAuAcAUGUTsT 687 AD-14137 40% 3%
    ucGAuucAAAucuuAAcccTsT 688 GGGUuAAGAUUUGAAUCGATsT 689 AD-14138 39% 5%
    ucuuAAcccuuAGGAcucuTsT 690 AGAGUCCuAAGGGUuAAGATsT 691 AD-14139 71% 11%
    GcucAcGAuGAGuuuAGuGTsT 692 cACuAAACUcAUCGUGAGCTsT 693 AD-14140 43% 15%
    cAuAAGcGAuGGAuAAuAcTsT 694 GuAUuAUCcAUCGCUuAUGTsT 695 AD-14141 33% 6%
    AuAAGcGAuGGAuAAuAccTsT 696 GGuAUuAUCcAUCGCUuAUTsT 697 AD-14142 51% 14%
    ccuAAuAAAcuGcccucAGTsT 698 CUGAGGGcAGUUuAUuAGGTsT 699 AD-14143 42% 1%
    ucGGAAAGuuGAAcuuGGuTsT 700 ACcAAGUUcAACUUUCCGATsT 701 AD-14144 4% 4%
    GAAAAcAuuGGccGuucuGTsT 702 cAGAACGGCcAAUGUUUUCTsT 703 AD-14145 92% 5%
    AAGAcuGAucuucuAAGuuTsT 704 AACUuAGAAGAUcAGUCUUTsT 705 AD-14146 13% 2%
    GAGcuuGuuAAAAucGGAATsT 706 UUCCGAUUUuAAcAAGCUCTsT 707 AD-14147 8% 1%
    AcAuuGGccGuucuGGAGcTsT 708 GCUCcAGAACGGCcAAUGUTsT 709 AD-14148 80% 7%
    AAGAAcAucuAuAAuuGcATsT 710 UGcAAUuAuAGAUGUUCUUTsT 711 AD-14149 44% 7%
    AAAuGuGucuAcucAuGuuTsT 712 AAcAUGAGuAGAcAcAUUUTsT 713 AD-14150 32% 29%
    uGucuAcucAuGuuucucATsT 714 UGAGAAAcAUGAGuAGAcATsT 715 AD-14151 75% 11%
    GuAuAcuGuGuAAcAAucuTsT 716 AGAUUGUuAcAcAGuAuACTsT 717 AD-14152 8% 5%
    uAuAcuGuGuAAcAAucuATsT 718 uAGAUUGUuAcAcAGuAuATsT 719 AD-14153 17% 11%
    cuuAGuAGuGuccAGGAAATsT 720 UUUCCUGGAcACuACuAAGTsT 721 AD-14154 16% 4%
    ucAGAuGGAcGuAAGGcAGTsT 722 CUGCCUuACGUCcAUCUGATsT 723 AD-14155 11% 1%
    AGAuAAAuuGAuAGcAcAATsT 724 UUGUGCuAUcAAUUuAUCUTsT 725 AD-14156 10% 1%
    cAAcAGGuAcGAcAccAcATsT 726 UGUGGUGUCGuACCUGUUGTsT 727 AD-14157 29% 3%
    uGcAAuGuAAAuAcGuAuuTsT 728 AAuACGuAUUuAcAUUGcATsT 729 AD-14158 51% 3%
    AGucAGAAuuuuAucuAGATsT 730 UCuAGAuAAAAUUCUGACUTsT 731 AD-14159 53% 5%
    cuAGAAAucuuuuAAcAccTsT 732 GGUGUuAAAAGAUUUCuAGTsT 733 AD-14160 40% 3%
    AAuAAAucuAAcccuAGuuTsT 734 AACuAGGGUuAGAUUuAUUTsT 735 AD-14161 83% 7%
    AAuuuucuGcucAcGAuGATsT 736 UcAUCGUGAGcAGAAAAUUTsT 737 AD-14162 44% 6%
    GcccucAGuAAAuccAuGGTsT 738 CcAUGGAUUuACUGAGGGCTsT 739 AD-14163 57% 3%
    AcGuuuAAAAcGAGAucuuTsT 740 AAGAUCUCGUUUuAAACGUTsT 741 AD-14164 4% 1%
    AGGAGAuAGAAcGuuuAAATsT 742 UUuAAACGUUCuAUCUCCUTsT 743 AD-14165 11% 1%
    GAccGucAuGGcGucGcAGTsT 744 CUGCGACGCcAUGACGGUCTsT 745 AD-14166 90% 5%
    AccGucAuGGcGucGcAGcTsT 746 GCUGCGACGCcAUGACGGUTsT 747 AD-14167 49% 1%
    GAAcGuuuAAAAcGAGAucTsT 748 GAUCUCGUUUuAAACGUUcTsT 749 AD-14168 12% 2%
    uuGAGcuuAAcAuAGGuAATsT 750 UuACCuAUGUuAAGCUcAATsT 751 AD-14169 66% 4%
    AcuAAAuuGAucucGuAGATsT 752 UCuACGAGAUcAAUUuAGUTsT 753 AD-14170 52% 6%
    ucGuAGAAuuAucuuAAuATsT 754 uAUuAAGAuAAUUCuACGATsT 755 AD-14171 42% 4%
    GGAGAuAGAAcGuuuAAAATsT 756 UUUuAAACGUUCuAUCUCCTsT 757 AD-14172 3% 1%
    AcAAcuuAuuGGAGGuuGuTsT 758 AcAACCUCcAAuAAGUUGUTsT 759 AD-14173 29% 2%
    uGAGcuuAAcAuAGGuAAATsT 760 UUuACCuAUGUuAAGCUcATsT 761 AD-14174 69% 2%
    AucucGuAGAAuuAucuuATsT 762 uAAGAuAAUUCuACGAGAUTsT 763 AD-14175 53% 3%
    cuGcGuGcAGucGGuccucTsT 764 GAGGACCGACUGcACGcAGTsT 765 AD-14176 111% 4%
    cAcGcAGcGcccGAGAGuATsT 766 uACUCUCGGGCGCUGCGUGTsT 767 AD-14177 87% 6%
    AGuAccAGGGAGAcuccGGTsT 768 CCGGAGUCUCCCUGGuACUTsT 769 AD-14178 59% 2%
    AcGGAGGAGAuAGAAcGuuTsT 770 AACGUUCuAUCUCCUCCGUTsT 771 AD-14179 9% 2%
    AGAAcGuuuAAAAcGAGAuTsT 772 AUCUCGUUUuAAACGUUCUTsT 773 AD-14180 43% 2%
    AAcGuuuAAAAcGAGAucuTsT 774 AGAUCUCGUUUuAAACGUUTsT 775 AD-14181 70% 10%
    AGcuuGAGcuuAAcAuAGGTsT 776 CCuAUGUuAAGCUcAAGCUTsT 777 AD-14182 100% 7%
    AGcuuAAcAuAGGuAAAuATsT 778 uAUUuACCuAUGUuAAGCUTsT 779 AD-14183 60% 5%
    uAGAGcuAcAAAAccuAucTsT 780 GAuAGGUUUUGuAGCUCuATsT 781 AD-14184 129% 6%
    uAGuuGuAucccuccuuuATsT 782 uAAAGGAGGGAuAcAACuATsT 783 AD-14185 62% 4%
    AccAcccAGAcAucuGAcuTsT 784 AGUcAGAUGUCUGGGUGGUTsT 785 AD-14186 42% 3%
    AGAAAcuAAAuuGAucucGTsT 786 CGAGAUcAAUUuAGUUUCUTsT 787 AD-14187 123% 12%
    ucucGuAGAAuuAucuuAATsT 788 UuAAGAuAAUUCuACGAGATsT 789 AD-14188 38% 2%
    cAAcuuAuuGGAGGuuGuATsT 790 uAcAACCUCcAAuAAGUUGTsT 791 AD-14189 13% 1%
    uuGuAucccuccuuuAAGuTsT 792 ACUuAAAGGAGGGAuAcAATsT 793 AD-14190 59% 3%
    ucAcAAcuuAuuGGAGGuuTsT 794 AACCUCcAAuAAGUUGUGATsT 795 AD-14191 93% 3%
    AGAAcuGuAcucuucucAGTsT 796 CUGAGAAGAGuAcAGUUCUTsT 797 AD-14192 45% 5%
    GAGcuuAAcAuAGGuAAAuTsT 798 AUUuACCuAUGUuAAGCUCTsT 799 AD-14193 57% 3%
    cAccAAcAucuGuccuuAGTsT 800 CuAAGGAcAGAUGUUGGUGTsT 801 AD-14194 51% 4%
    AAAGcccAcuuuAGAGuAuTsT 802 AuACUCuAAAGUGGGCUUUTsT 803 AD-14195 77% 5%
    AAGcccAcuuuAGAGuAuATsT 804 uAuACUCuAAAGUGGGCUUTsT 805 AD-14196 42% 6%
    GAccuuAuuuGGuAAucuGTsT 806 cAGAUuACcAAAuAAGGUCTsT 807 AD-14197 15% 2%
    GAuuAAuGuAcucAAGAcuTsT 808 AGUCUUGAGuAcAUuAAUCTsT 809 AD-14198 12% 2%
    cuuuAAGAGGccuAAcucATsT 810 UGAGUuAGGCCUCUuAAAGTsT 811 AD-14199 18% 2%
    uuAAAccAAAcccuAuuGATsT 812 UcAAuAGGGUUUGGUUuAATsT 813 AD-14200 72% 9%
    ucuGuuGGAGAucuAuAAuTsT 814 AUuAuAGAUCUCcAAcAGATsT 815 AD-14201 9% 3%
    cuGAuGuuucuGAGAGAcuTsT 816 AGUCUCUcAGAAAcAUcAGTsT 817 AD-14202 25% 3%
    GcAuAcucuAGucGuucccTsT 818 GGGAACGACuAGAGuAUGCTsT 819 AD-14203 21% 1%
    GuuccuuAucGAGAAucuATsT 820 uAGAUUCUCGAuAAGGAACTsT 821 AD-14204 4% 2%
    GcAcuuGGAucucucAcAuTsT 822 AUGUGAGAGAUCcAAGUGCTsT 823 AD-14205 5% 1%
    AAAAAAGGAAcuAGAuGGcTsT 824 GCcAUCuAGUUCCUUUUUUTsT 825 AD-14206 79% 6%
    AGAGcAGAuuAccucuGcGTsT 826 CGcAGAGGuAAUCUGCUCUTsT 827 AD-14207 55% 2%
    AGcAGAuuAccucuGcGAGTsT 828 CUCGcAGAGGuAAUCUGCUTsT 829 AD-14208 100% 4%
    cccuGAcAGAGuucAcAAATsT 830 UUUGUGAACUCUGUcAGGGTsT 831 AD-14209 34% 3%
    GuuuAccGAAGuGuuGuuuTsT 832 AAAcAAcACUUCGGuAAACTsT 833 AD-14210 13% 2%
    uuAcAGuAcAcAAcAAGGATsT 834 UCCUUGUUGUGuACUGuAATsT 835 AD-14211 9% 1%
    AcuGGAucGuAAGAAGGcATsT 836 UGCCUUCUuACGAUCcAGUTsT 837 AD-14212 20% 3%
    GAGcAGAuuAccucuGcGATsT 838 UCGcAGAGGuAAUCUGCUCTsT 839 AD-14213 48% 5%
    AAAAGAAGuuAGuGuAcGATsT 840 UCGuAcACuAACUUCUUUUTsT 841 AD-14214 28% 18%
    GAccAuuuAAuuuGGcAGATsT 842 UCUGCcAAAUuAAAUGGUCTsT 843 AD-14215 132% 0%
    GAGAGGAGuGAuAAuuAAATsT 844 UUuAAUuAUcACUCCUCUCTsT 845 AD-14216 3% 0%
    cuGGAGGAuuGGcuGAcAATsT 846 UUGUcAGCcAAUCCUCcAGTsT 847 AD-14217 19% 1%
    cucuAGucGuucccAcucATsT 848 UGAGUGGGAACGACuAGAGTsT 849 AD-14218 67% 8%
    GAuAccAuuAcuAcAGuAGTsT 850 CuACUGuAGuAAUGGuAUCTsT 851 AD-14219 76% 4%
    uucGucuGcGAAGAAGAAATsT 852 UUUCUUCUUCGcAGACGAATsT 853 AD-14220 33% 8%
    GAAAAGAAGuuAGuGuAcGTsT 854 CGuAcACuAACUUCUUUUCTsT 855 AD-14221 25% 2%
    uGAuGuuuAccGAAGuGuuTsT 856 AAcACUUCGGuAAAcAUcATsT 857 AD-14222 7% 2%
    uGuuuGuccAAuucuGGAuTsT 858 AUCcAGAAUUGGAcAAAcATsT 859 AD-14223 19% 2%
    AuGAAGAGuAuAccuGGGATsT 860 UCCcAGGuAuACUCUUcAUTsT 861 AD-14224 13% 1%
    GcuAcucuGAuGAAuGcAuTsT 862 AUGcAUUcAUcAGAGuAGCTsT 863 AD-14225 15% 2%
    GcccuuGuAGAAAGAAcAcTsT 864 GUGUUCUUUCuAcAAGGGCTsT 865 AD-14226 11% 0%
    ucAuGuuccuuAucGAGAATsT 866 UUCUCGAuAAGGAAcAUGATsT 867 AD-14227 5% 1%
    GAAuAGGGuuAcAGAGuuGTsT 868 cAACUCUGuAACCCuAUUCTsT 869 AD-14228 34% 3%
    cAAAcuGGAucGuAAGAAGTsT 870 CUUCUuACGAUCcAGUUUGTsT 871 AD-14229 15% 2%
    cuuAuuuGGuAAucuGcuGTsT 872 cAGcAGAUuACcAAAuAAGTsT 873 AD-14230 20% 1%
    AGcAAuGuGGAAAccuAAcTsT 874 GUuAGGUUUCcAcAUUGCUTsT 875 AD-14231 18% 1%
    AcAAuAAAGcAGAcccAuuTsT 876 AAUGGGUCUGCUUuAUUGUTsT 877 AD-14232 21% 1%
    AAccAcuuAGuAGuGuccATsT 878 UGGAcACuACuAAGUGGUUTsT 879 AD-14233 106% 12%
    AGucAAGAGccAucuGuAGTsT 880 CuAcAGAUGGCUCUUGACUTsT 881 AD-14234 35% 3%
    cucccuAGAcuucccuAuuTsT 882 AAuAGGGAAGUCuAGGGAGTsT 883 AD-14235 48% 4%
    AuAGcuAAAuuAAAccAAATsT 884 UUUGGUUuAAUUuAGCuAUTsT 885 AD-14236 23% 3%
    uGGcuGGuAuAAuuccAcGTsT 886 CGUGGAAUuAuACcAGCcATsT 887 AD-14237 79% 9%
    uuAuuuGGuAAucuGcuGuTsT 888 AcAGcAGAUuACcAAAuAATsT 889 AD-14238 92% 7%
    AAcuAGAuGGcuuucucAGTsT 890 CUGAGAAAGCcAUCuAGUUTsT 891 AD-14239 20% 2%
    ucAuGGcGucGcAGccAAATsT 892 UUUGGCUGCGACGCcAUGATsT 893 AD-14240 71% 6%
    AcuGGAGGAuuGGcuGAcATsT 894 UGUcAGCcAAUCCUCcAGUTsT 895 AD-14241 14% 1%
    cuAuAAuuGcAcuAucuuuTsT 896 AAAGAuAGUGcAAUuAuAGTsT 897 AD-14242 11% 2%
    AAAGGucAccuAAuGAAGATsT 898 UCUUcAUuAGGUGACCUUUTsT 899 AD-14243 11% 1%
    AuGAAuGcAuAcucuAGucTsT 900 GACuAGAGuAUGcAUUcAUTsT 901 AD-14244 15% 2%
    AAcAuAuuGAAuAAGccuGTsT 902 cAGGCUuAUUcAAuAUGUUTsT 903 AD-14245 80% 7%
    AAGAAGGcAGuuGAccAAcTsT 904 GUUGGUcAACUGCCUUCUUTsT 905 AD-14246 57% 5%
    GAuAcuAAAAGAAcAAucATsT 906 UGAUUGUUCUUUuAGuAUCTsT 907 AD-14247 9% 3%
    AuAcuGAAAAucAAuAGucTsT 908 GACuAUUGAUUUUcAGuAUTsT 909 AD-14248 39% 4%
    AAAAAGGAAcuAGAuGGcuTsT 910 AGCcAUCuAGUUCCUUUUUTsT 911 AD-14249 64% 2%
    GAAcuAGAuGGcuuucucATsT 912 UGAGAAAGCcAUCuAGUUCTsT 913 AD-14250 18% 2%
    GAAAccuAAcuGAAGAccuTsT 914 AGGUCUUcAGUuAGGUUUCTsT 915 AD-14251 56% 6%
    uAcccAucAAcAcuGGuAATsT 916 UuACcAGUGUUGAUGGGuATsT 917 AD-14252 48% 6%
    AuuuuGAuAucuAcccAuuTsT 918 AAUGGGuAGAuAUcAAAAUTsT 919 AD-14253 39% 5%
    AucccuAuAGuucAcuuuGTsT 920 cAAAGUGAACuAuAGGGAUTsT 921 AD-14254 44% 8%
    AuGGGcuAuAAuuGcAcuATsT 922 uAGUGcAAUuAuAGCCcAUTsT 923 AD-14255 108% 8%
    AGAuuAccucuGcGAGcccTsT 924 GGGCUCGcAGAGGuAAUCUTsT 925 AD-14256 108% 6%
    uAAuuccAcGuAcccuucATsT 926 UGAAGGGuACGUGGAAUuATsT 927 AD-14257 23% 2%
    GucGuucccAcucAGuuuuTsT 928 AAAACuGAGuGGGAACGACTsT 929 AD-14258 21% 3%
    AAAucAAucccuGuuGAcuTsT 930 AGUcAAcAGGGAUUGAUUUTsT 931 AD-14259 19% 2%
    ucAuAGAGcAAAGAAcAuATsT 932 uAUGUUCUUUGCUCuAUGATsT 933 AD-14260 10% 1%
    uuAcuAcAGuAGcAcuuGGTsT 934 CcAAGUGCuACUGuAGuAATsT 935 AD-14261 76% 3%
    AuGuGGAAAccuAAcuGAATsT 936 UUcAGUuAGGUUUCcAcAUTsT 937 AD-14262 13% 2%
    uGuGGAAAccuAAcuGAAGTsT 938 CUUcAGUuAGGUUUCcAcATsT 939 AD-14263 14% 2%
    ucuuccuuAAAuGAAAGGGTsT 940 CCCUUUcAUUuAAGGAAGATsT 941 AD-14264 65% 3%
    uGAAGAAccucuAAGucAATsT 942 UUGACUuAGAGGUUCUUcATsT 943 AD-14265 13% 1%
    AGAGGucuAAAGuGGAAGATsT 944 UCUUCcACUUuAGACCUCUTsT 945 AD-14266 18% 3%
    AuAucuAcccAuuuuucuGTsT 946 cAGAAAAAUGGGuAGAuAUTsT 947 AD-14267 50% 9%
    uAAGccuGAAGuGAAucAGTsT 948 CUGAUUcACUUcAGGCUuATsT 949 AD-14268 13% 3%
    AGAuGcAGAccAuuuAAuuTsT 950 AAUuAAAUGGUCUGcAUCUTsT 951 AD-14269 19% 4%
    AGuGuuGuuuGuccAAuucTsT 952 GAAUUGGAcAAAcAAcACUTsT 953 AD-14270 11% 2%
    cuAuAAuGAAGAGcuuuuuTsT 954 AAAAAGCUCUUcAUuAuAGTsT 955 AD-14271 11% 1%
    AGAGGAGuGAuAAuuAAAGTsT 956 CUUuAAUuAUcACUCCUCUTsT 957 AD-14272 7% 1%
    uuucucuGuuAcAAuAcAuTsT 958 AUGuAUUGuAAcAGAGAAATsT 959 AD-14273 14% 2%
    AAcAucuAuAAuuGcAAcATsT 960 UGUUGcAAUuAuAGAUGUUTsT 961 AD-14274 73% 4%
    uGcuAGAAGuAcAuAAGAcTsT 962 GUCUuAUGuACUUCuAGcATsT 963 AD-14275 10% 1%
    AAuGuAcucAAGAcuGAucTsT 964 GAUcAGUCUUGAGuAcAUUTsT 965 AD-14276 89% 2%
    GuAcucAAGAcuGAucuucTsT 966 GAAGAUcAGUCUUGAGuACTsT 967 AD-14277 7% 1%
    cAcucuGAuAAAcucAAuGTsT 968 cAUUGAGUUuAUcAGAGUGTsT 969 AD-14278 12% 1%
    AAGAGcAGAuuAccucuGcTsT 970 GcAGAGGuAAUCUGCUCUUTsT 971 AD-14279 104% 3%
    ucuGcGAGcccAGAucAAcTsT 972 GUUGAUCUGGGCUCGcAGATsT 973 AD-14280 21% 2%
    AAcuuGAGccuuGuGuAuATsT 974 uAuAcAcAAGGCUcAAGUUTsT 975 AD-14281 43% 3%
    GAAuAuAuAuAucAGccGGTsT 976 CCGGCUGAuAuAuAuAUUCTsT 977 AD-14282 45% 6%
    uGucAucccuAuAGuucAcTsT 978 GUGAACuAuAGGGAUGAcATsT 979 AD-14283 35% 5%
    GAucuGGcAAccAuAuuucTsT 980 GAAAuAUGGUUGCcAGAUCTsT 981 AD-14284 58% 3%
    uGGcAAccAuAuuucuGGATsT 982 UCcAGAAAuAUGGUUGCcATsT 983 AD-14285 48% 3%
    GAuGuuuAccGAAGuGuuGTsT 984 cAAcACUUCGGuAAAcAUCTsT 985 AD-14286 49% 3%
    uuccuuAucGAGAAucuAATsT 986 UuAGAUUCUCGAuAAGGAATsT 987 AD-14287 6% 1%
    AGcuuAAuuGcuuucuGGATsT 988 UCcAGAAAGcAAUuAAGCUTsT 989 AD-14288 50% 2%
    uuGcuAuuAuGGGAGAccATsT 990 UGGUCUCCcAuAAuAGcAATsT 991 AD-14289 48% 1%
    GucAuGGcGucGcAGccAATsT 992 UUGGCUGCGACGCcAUGACTsT 993 AD-14290 112% 7%
    uAAuuGcAcuAucuuuGcGTsT 994 CGcAAAGAuAGUGcAAUuATsT 995 AD-14291 77% 2%
    cuAucuuuGcGuAuGGccATsT 996 UGGCcAuACGcAAAGAuAGTsT 997 AD-14292 80% 6%
    ucccuAuAGuucAcuuuGuTsT 998 AcAAAGUGAACuAuAGGGATsT 999 AD-14293 58% 2%
    ucAAccuuuAAuucAcuuGTsT 1000 cAAGUGAAUuAAAGGUUGATsT 1001 AD-14294 77% 2%
    GGcAAccAuAuuucuGGAATsT 1002 UUCcAGAAAuAUGGUUGCCTsT 1003 AD-14295 62% 2%
    AuGuAcucAAGAcuGAucuTsT 1004 AGAUcAGUCUUGAGuAcAUTsT 1005 AD-14296 59% 4%
    GcAGAccAuuuAAuuuGGcTsT 1006 GCcAAAUuAAAUGGUCUGCTsT 1007 AD-14297 37% 1%
    ucuGAGAGAcuAcAGAuGuTsT 1008 AcAUCUGuAGUCUCUcAGATsT 1009 AD-14298 21% 1%
    uGcucAuAGAGcAAAGAAcTsT 1010 GUUCUUUGCUCuAUGAGcATsT 1011 AD-14299 6% 1%
    AcAuAAGAccuuAuuuGGuTsT 1012 ACcAAAuAAGGUCUuAUGUTsT 1013 AD-14300 17% 2%
    uuuGuGcuGAuucuGAuGGTsT 1014 CcAUcAGAAUcAGcAcAAATsT 1015 AD-14301 97% 6%
    ccAucAAcAcuGGuAAGAATsT 1016 UUCUuACcAGUGUUGAUGGTsT 1017 AD-14302 13% 1%
    AGAcAAuuccGGAuGuGGATsT 1018 UCcAcAUCCGGAAUUGUCUTsT 1019 AD-14303 13% 3%
    GAAcuuGAGccuuGuGuAuTsT 1020 AuAcAcAAGGCUcAAGUUCTsT 1021 AD-14304 38% 2%
    uAAuuuGGcAGAGcGGAAATsT 1022 UUUCCGCUCUGCcAAAUuATsT 1023 AD-14305 14% 2%
    uGGAuGAAGuuAuuAuGGGTsT 1024 CCcAuAAuAACUUcAUCcATsT 1025 AD-14306 22% 4%
    AucuAcAuGAAcuAcAAGATsT 1026 UCUUGuAGUUcAUGuAGAUTsT 1027 AD-14307 26% 6%
    GGuAuuuuuGAucuGGcAATsT 1028 UUGCcAGAUcAAAAAuACCTsT 1029 AD-14308 62% 8%
    cuAAuGAAGAGuAuAccuGTsT 1030 cAGGuAuACUCUUcAUuAGTsT 1031 AD-14309 52% 5%
    uuuGAGAAAcuuAcuGAuATsT 1032 uAUcAGuAAGUUUCUcAAATsT 1033 AD-14310 32% 3%
    cGAuAAGAuAGAAGAucAATsT 1034 UUGAUCUUCuAUCUuAUCGTsT 1035 AD-14311 23% 2%
    cuGGcAAccAuAuuucuGGTsT 1036 CcAGAAAuAUGGUUGCcAGTsT 1037 AD-14312 49% 6%
    uAGAuAccAuuAcuAcAGuTsT 1038 ACUGuAGuAAUGGuAUCuATsT 1039 AD-14313 69% 4%
    GuAuuAAAuuGGGuuucAuTsT 1040 AUGAAACCcAAUUuAAuACTsT 1041 AD-14314 52% 3%
    AAGAccuuAuuuGGuAAucTsT 1042 GAUuACcAAAuAAGGUCUUTsT 1043 AD-14315 66% 4%
    GcuGuuGAuAAGAGAGcucTsT 1044 GAGCUCUCUuAUcAAcAGCTsT 1045 AD-14316 19% 4%
    uAcucAuGuuucucAGAuuTsT 1046 AAUCUGAGAAAcAUGAGuATsT 1047 AD-14317 16% 5%
    cAGAuGGAcGuAAGGcAGcTsT 1048 GCUGCCUuACGUCcAUCUGTsT 1049 AD-14318 52% 11%
    uAucccAAcAGGuAcGAcATsT 1050 UGUCGuACCUGUUGGGAuATsT 1051 AD-14319 28% 11%
    cAuuGcuAuuAuGGGAGAcTsT 1052 GUCUCCcAuAAuAGcAAUGTsT 1053 AD-14320 52% 10%
    cccucAGuAAAuccAuGGuTsT 1054 ACcAUGGAUUuACUGAGGGTsT 1055 AD-14321 53% 6%
    GGucAuuAcuGcccuuGuATsT 1056 uAcAAGGGcAGuAAUGACCTsT 1057 AD-14322 20% 2%
    AAccAcucAAAAAcAuuuGTsT 1058 cAAAUGUUUUUGAGUGGUUTsT 1059 AD-14323 116% 6%
    uuuGcAAGuuAAuGAAucuTsT 1060 AGAUUcAUuAACUUGcAAATsT 1061 AD-14324 14% 2%
    uuAuuuucAGuAGucAGAATsT 1062 UUCUGACuACUGAAAAuAATsT 1063 AD-14325 50% 2%
    uuuucucGAuucAAAucuuTsT 1064 AAGAUUuGAAUCGAGAAAATsT 1065 AD-14326 47% 3%
    GuAcGAAAAGAAGuuAGuGTsT 1066 cACuAACUUCUUUUCGuACTsT 1067 AD-14327 18% 2%
    uuuAAAAcGAGAucuuGcuTsT 1068 AGcAAGAUCUCGUUUuAAATsT 1069 AD-14328 19% 1%
    GAAuuGAuuAAuGuAcucATsT 1070 UGAGuAcAUuAAUcAAUUCTsT 1071 AD-14329 94% 10%
    GAuGGAcGuAAGGcAGcucTsT 1072 GAGCUGCCUuACGUCcAUCTsT 1073 AD-14330 60% 4%
    cAucuGAcuAAuGGcucuGTsT 1074 cAGAGCcAUuAGUcAGAUGTsT 1075 AD-14331 54% 7%
    GuGAuccuGuAcGAAAAGATsT 1076 UCUUUUCGuAcAGGAUcACTsT 1077 AD-14332 22% 4%
    AGcucuuAuuAAGGAGuAuTsT 1078 AuACUCCUuAAuAAGAGCUTsT 1079 AD-14333 70% 10%
    GcucuuAuuAAGGAGuAuATsT 1080 uAuACUCCUuAAuAAGAGCTsT 1081 AD-14334 18% 3%
    ucuuAuuAAGGAGuAuAcGTsT 1082 CGuAuACUCCUuAAuAAGATsT 1083 AD-14335 38% 6%
    uAuuAAGGAGuAuAcGGAGTsT 1084 CUCCGuAuACUCCUuAAuATsT 1085 AD-14336 16% 3%
    cuGcAGcccGuGAGAAAAATsT 1086 UUUUUCUcACGGGCUGcAGTsT 1087 AD-14337 65% 4%
    ucAAGAcuGAucuucuAAGTsT 1088 CUuAGAAGAUcAGUCUUGATsT 1089 AD-14338 18% 0%
    cuucuAAGuucAcuGGAAATsT 1090 UUUCcAGUGAACUuAGAAGTsT 1091 AD-14339 20% 4%
    uGcAAGuuAAuGAAucuuuTsT 1092 AAAGAUUcAUuAACUUGcATsT 1093 AD-14340 24% 1%
    AAucuAAGGAuAuAGucAATsT 1094 UUGACuAuAUCCUuAGAUUTsT 1095 AD-14341 27% 3%
    AucucuGAAcAcAAGAAcATsT 1096 UGUUCUUGUGUUcAGAGAUTsT 1097 AD-14342 13% 1%
    uucuGAAcAGuGGGuAucuTsT 1098 AGAuACCcACUGUUcAGAATsT 1099 AD-14343 19% 1%
    AGuuAuuuAuAcccAucAATsT 1100 UUGAUGGGuAuAAAuAACUTsT 1101 AD-14344 23% 2%
    AuGcuAAAcuGuucAGAAATsT 1102 UUUCUGAAcAGUUuAGcAUTsT 1103 AD-14345 21% 4%
    cuAcAGAGcAcuuGGuuAcTsT 1104 GuAACcAAGUGCUCUGuAGTsT 1105 AD-14346 18% 2%
    uAuAuAucAGccGGGcGcGTsT 1106 CGCGCCCGGCUGAuAuAuATsT 1107 AD-14347 67% 2%
    AuGuAAAuAcGuAuuucuATsT 1108 uAGAAAuACGuAUUuAcAUTsT 1109 AD-14348 39% 3%
    uuuuucucGAuucAAAucuTsT 1110 AGAUUuGAAUCGAGAAAAATsT 1111 AD-14349 83% 6%
    AAucuuAAcccuuAGGAcuTsT 1112 AGUCCuAAGGGUuAAGAUUTsT 1113 AD-14350 54% 2%
    ccuuAGGAcucuGGuAuuuTsT 1114 AAAuACcAGAGUCCuAAGGTsT 1115 AD-14351 57% 8%
    AAuAAAcuGcccucAGuAATsT 1116 UuACUGAGGGcAGUUuAUUTsT 1117 AD-14352 82% 3%
    GAuccuGuAcGAAAAGAAGTsT 1118 CUUCUUUUCGuAcAGGAUCTsT 1119 AD-14353 2% 1%
    AAuGuGAuccuGuAcGAAATsT 1120 UUUCGuAcAGGAUcAcAUUTsT 1121 AD-14354 18% 11%
    GuGAAAAcAuuGGccGuucTsT 1122 GAACGGCcAAUGUUUUcACTsT 1123 AD-14355 2% 1%
    cuuGAGGAAAcucuGAGuATsT 1124 uACUcAGAGUUUCCUcAAGTsT 1125 AD-14356 8% 2%
    cGuuuAAAAcGAGAucuuGTsT 1126 cAAGAUCUCGUUUuAAACGTsT 1127 AD-14357 6% 3%
    uuAAAAcGAGAucuuGcuGTsT 1128 cAGcAAGAUCUCGUUUuAATsT 1129 AD-14358 98% 17%
    AAAGAuGuAucuGGucuccTsT 1130 GGAGACcAGAuAcAUCUUUTsT 1131 AD-14359 10% 1%
    cAGAAAAuGuGucuAcucATsT 1132 UGAGuAGAcAcAUUUUCUGTsT 1133 AD-14360 6% 4%
    cAGGAAuuGAuuAAuGuAcTsT 1134 GuAcAUuAAUcAAUUCCUGTsT 1135 AD-14361 30% 5%
    AGucAAcuAAAGcAuAuuuTsT 1136 AAAuAUGCUUuAGUUGACUTsT 1137 AD-14362 28% 2%
    uGuGuAAcAAucuAcAuGATsT 1138 UcAUGuAGAUUGUuAcAcATsT 1139 AD-14363 60% 6%
    AuAccAuuuGuuccuuGGuTsT 1140 ACcAAGGAAcAAAUGGuAUTsT 1141 AD-14364 12% 9%
    GcAGAAAucuAAGGAuAuATsT 1142 uAuAUCCUuAGAUUUCUGCTsT 1143 AD-14365 5% 2%
    uGGcuucucAcAGGAAcucTsT 1144 GAGUUCCUGUGAGAAGCcATsT 1145 AD-14366 28% 5%
    GAGAuGuGAAucucuGAAcTsT 1146 GUUcAGAGAUUcAcAUCUCTsT 1147 AD-14367 42% 4%
    uGuAAGccAAuGuuGuGAGTsT 1148 CUcAcAAcAUUGGCUuAcATsT 1149 AD-14368 93% 12%
    AGccAAuGuuGuGAGGcuuTsT 1150 AAGCCUcAcAAcAUUGGCUTsT 1151 AD-14369 65% 4%
    uuGuGAGGcuucAAGuucATsT 1152 UGAACUUGAAGCCUcAcAATsT 1153 AD-14370 5% 2%
    AGGcAGcucAuGAGAAAcATsT 1154 UGUUUCUcAUGAGCUGCCUTsT 1155 AD-14371 54% 5%
    AuAAAuuGAuAGcAcAAAATsT 1156 UUUUGUGCuAUcAAUUuAUTsT 1157 AD-14372 4% 1%
    AcAAAAucuAGAAcuuAAuTsT 1158 AUuAAGUUCuAGAUUUUGUTsT 1159 AD-14373 5% 1%
    GAuAucccAAcAGGuAcGATsT 1160 UCGuACCUGUUGGGAuAUCTsT 1161 AD-14374 92% 6%
    AAGuuAuuuAuAcccAucATsT 1162 UGAUGGGuAuAAAuAACUUTsT 1163 AD-14375 76% 4%
    uGuAAAuAcGuAuuucuAGTsT 1164 CuAGAAAuACGuAUUuAcATsT 1165 AD-14376 70% 5%
    ucuAGuuuucAuAuAAAGuTsT 1166 ACUUuAuAUGAAAACuAGATsT 1167 AD-14377 48% 4%
    AuAAAGuAGuucuuuuAuATsT 1168 uAuAAAAGAACuACUUuAUTsT 1169 AD-14378 48% 3%
    ccAuuuGuAGAGcuAcAAATsT 1170 UUUGuAGCUCuAcAAAUGGTsT 1171 AD-14379 44% 5%
    uAuuuucAGuAGucAGAAuTsT 1172 AUUCUGACuACUGAAAAuATsT 1173 AD-14380 35% 16%
    AAAucuAAcccuAGuuGuATsT 1174 uAcAACuAGGGUuAGAUUUTsT 1175 AD-14381 44% 5%
    cuuuAGAGuAuAcAuuGcuTsT 1176 AGcAAUGuAuACUCuAAAGTsT 1177 AD-14382 28% 1%
    AucuGAcuAAuGGcucuGuTsT 1178 AcAGAGCcAUuAGUcAGAUTsT 1179 AD-14383 55% 11%
    cAcAAuGAuuuAAGGAcuGTsT 1180 cAGUCCUuAAAUcAUUGUGTsT 1181 AD-14384 48% 9%
    ucuuuuucucGAuucAAAuTsT 1182 AUUuGAAUCGAGAAAAAGATsT 1183 AD-14385 36% 2%
    cuuuuucucGAuucAAAucTsT 1184 GAUUuGAAUCGAGAAAAAGTsT 1185 AD-14386 41% 7%
    AuuuucuGcucAcGAuGAGTsT 1186 CUcAUCGUGAGcAGAAAAUTsT 1187 AD-14387 38% 3%
    uuucuGcucAcGAuGAGuuTsT 1188 AACUcAUCGUGAGcAGAAATsT 1189 AD-14388 50% 4%
    AGAGcuAcAAAAccuAuccTsT 1190 GGAuAGGUUUUGuAGCUCUTsT 1191 AD-14389 98% 6%
    GAGccAAAGGuAcAccAcuTsT 1192 AGUGGUGuACCUUUGGCUCTsT 1193 AD-14390 43% 8%
    GccAAAGGuAcAccAcuAcTsT 1194 GuAGUGGUGuACCUUUGGCTsT 1195 AD-14391 48% 4%
    GAAcuGuAcucuucucAGcTsT 1196 GCUGAGAAGAGuAcAGUUCTsT 1197 AD-14392 44% 3%
    AGGuAAAuAucAccAAcAuTsT 1198 AUGUUGGUGAuAUUuACCUTsT 1199 AD-14393 37% 2%
    AGcuAcAAAAccuAuccuuTsT 1200 AAGGAuAGGUUUUGuAGCUTsT 1201 AD-14394 114% 7%
    uGuGAAAGcAuuuAAuuccTsT 1202 GGAAUuAAAUGCUUUcAcATsT 1203 AD-14395 55% 4%
    GcccAcuuuAGAGuAuAcATsT 1204 UGuAuACUCuAAAGUGGGCTsT 1205 AD-14396 49% 5%
    uGuGccAcAcuccAAGAccTsT 1206 GGUCUUGGAGUGUGGcAcATsT 1207 AD-14397 71% 6%
    AAAcuAAAuuGAucucGuATsT 1208 uACGAGAUcAAUUuAGUUUTsT 1209 AD-14398 81% 7%
    uGAucucGuAGAAuuAucuTsT 1210 AGAuAAUUCuACGAGAUcATsT 1211 AD-14399 38% 4%
    GcGuGcAGucGGuccuccATsT 1212 UGGAGGACCGACUGcACGCTsT 1213 AD-14400 106% 8%
    AAAGuuuAGAGAcAucuGATsT 1214 UcAGAUGUCUCuAAACUUUTsT 1215 AD-14401 47% 3%
    cAGAAGGAAuAuGuAcAAATsT 1216 UUUGuAcAuAUUCCUUCUGTsT 1217 AD-14402 31% 1%
    cGcccGAGAGuAccAGGGATsT 1218 UCCCUGGuACUCUCGGGCGTsT 1219 AD-14403 105% 4%
    cGGAGGAGAuAGAAcGuuuTsT 1220 AAACGUUCuAUCUCCUCCGTsT 1221 AD-14404 3% 1%
    AGAuAGAAcGuuuAAAAcGTsT 1222 CGUUUuAAACGUUCuAUCUTsT 1223 AD-14405 15% 1%
    GGAAcAGGAAcuucAcAAcTsT 1224 GUuGuGAAGUUCCuGUUCCTsT 1225 AD-14406 44% 5%
    GuGAGccAAAGGuAcAccATsT 1226 UGGUGuACCUUUGGCUcACTsT 1227 AD-14407 41% 4%
    AuccucccuAGAcuucccuTsT 1228 AGGGAAGUCuAGGGAGGAUTsT 1229 AD-14408 104% 3%
    cAcAcuccAAGAccuGuGcTsT 1230 GcAcAGGUCUUGGAGUGUGTsT 1231 AD-14409 67% 4%
    AcAGAAGGAAuAuGuAcAATsT 1232 UUGuAcAuAUUCCUUCUGUTsT 1233 AD-14410 22% 1%
    uuAGAGAcAucuGAcuuuGTsT 1234 cAAAGUcAGAUGUCUCuAATsT 1235 AD-14411 29% 3%
    AAuuGAucucGuAGAAuuATsT 1236 uAAUUCuACGAGAUcAAUUTsT 1237 AD-14412 31% 4%
  • [0268]
    dsRNA Targeting the VEGF Gene
  • [0269]
    Four hundred target sequences were identified within exons 1-5 of the VEGF-A121 mRNA sequence. reference transcript is: NM003376.
  • [0000]
    (SEQ ID NO: 1539)
    1 augaacuuuc ugcugucuug ggugcauugg agccuugccu ugcugcucua ccuccaccau
    61 gccaaguggu cccaggcugc acccauggca gaaggaggag ggcagaauca ucacgaagug
    121 gugaaguuca uggaugucua ucagcgcagc uacugccauc caaucgagac ccugguggac
    181 aucuuccagg aguacccuga ugagaucgag uacaucuuca agccauccug ugugccccug
    241 augcgaugcg ggggcugcug caaugacgag ggccuggagu gugugcccac ugaggagucc
    301 aacaucacca ugcagauuau gcggaucaaa ccucaccaag gccagcacau aggagagaug
    361 agcuuccuac agcacaacaa augugaaugc agaccaaaga aagauagagc aagacaagaa
    421 aaaugugaca agccgaggcg guga
  • [0270]
    Table 4a includes the identified target sequences. Corresponding siRNAs targeting these sequences were subjected to a bioinformatics screen.
  • [0271]
    To ensure that the sequences were specific to VEGF sequence and not to sequences from any other genes, the target sequences were checked against the sequences in Genbank using the BLAST search engine provided by NCBI. The use of the BLAST algorithm is described in Altschul et al., J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth. Enzymol. 266:460, 1996.
  • [0272]
    siRNAs were also prioritized for their ability to cross react with monkey, rat and human VEGF sequences.
  • [0273]
    Of these 400 potential target sequences 80 were selected for analysis by experimental screening in order to identify a small number of lead candidates. A total of 114 siRNA molecules were designed for these 80 target sequences 114 (Table 4b).
  • [0000]
    TABLE 4a
    Target sequences in VEGF-121
    position TARGET SEQUENCE IN
    SEQ ID in VEGF- VEGF121 mRNA
    NO: 121 ORF 5′ to 3′
    1540 1 AUGAACUUUCUGCUGUCUUGGGU
    1541 2 UGAACUUUCUGCUGUCUUGGGUG
    1542 3 GAACUUUCUGCUGUCUUGGGUGC
    1543 4 AACUUUCUGCUGUCUUGGGUGCA
    1544 5 ACUUUCUGCUGUCUUGGGUGCAU
    1545 6 CUUUCUGCUGUCUUGGGUGCAUU
    1546 7 UUUCUGCUGUCUUGGGUGCAUUG
    1547 8 UUCUGCUGUCUUGGGUGCAUUGG
    1548 9 UCUGCUGUCUUGGGUGCAUUGGA
    1549 10 CUGCUGUCUUGGGUGCAUUGGAG
    1550 11 UGCUGUCUUGGGUGCAUUGGAGC
    1551 12 GCUGUCUUGGGUGCAUUGGAGCC
    1552 13 CUGUCUUGGGUGCAUUGGAGCCU
    1553 14 UGUCUUGGGUGCAUUGGAGCCUU
    1554 15 GUCUUGGGUGCAUUGGAGCCUUG
    1555 16 UCUUGGGUGCAUUGGAGCCUUGC
    1556 17 CUUGGGUGCAUUGGAGCCUUGCC
    1557 18 UUGGGUGCAUUGGAGCCUUGCCU
    1558 19 UGGGUGCAUUGGAGCCUUGCCUU
    1559 20 GGGUGCAUUGGAGCCUUGCCUUG
    1560 21 GGUGCAUUGGAGCCUUGCCUUGC
    1561 22 GUGCAUUGGAGCCUUGCCUUGCU
    1562 23 UGCAUUGGAGCCUUGCCUUGCUG
    1563 24 GCAUUGGAGCCUUGCCUUGCUGC
    1564 25 CAUUGGAGCCUUGCCUUGCUGCU
    1565 26 AUUGGAGCCUUGCCUUGCUGCUC
    1566 27 UUGGAGCCUUGCCUUGCUGCUCU
    1567 28 UGGAGCCUUGCCUUGCUGCUCUA
    1568 29 GGAGCCUUGCCUUGCUGCUCUAC
    1569 30 GAGCCUUGCCUUGCUGCUCUACC
    1570 31 AGCCUUGCCUUGCUGCUCUACCU
    1571 32 GCCUUGCCUUGCUGCUCUACCUC
    1572 33 CCUUGCCUUGCUGCUCUACCUCC
    1573 34 CUUGCCUUGCUGCUCUACCUCCA
    1574 35 UUGCCUUGCUGCUCUACCUCCAC
    1575 36 UGCCUUGCUGCUCUACCUCCACC
    1576 37 GCCUUGCUGCUCUACCUCCACCA
    1577 38 CCUUGCUGCUCUACCUCCACCAU
    1578 39 CUUGCUGCUCUACCUCCACCAUG
    1579 40 UUGCUGCUCUACCUCCACCAUGC
    1580 41 UGCUGCUCUACCUCCACCAUGCC
    1581 42 GCUGCUCUACCUCCACCAUGCCA
    1582 43 CUGCUCUACCUCCACCAUGCCAA
    1583 44 UGCUCUACCUCCACCAUGCCAAG
    1584 45 GCUCUACCUCCACCAUGCCAAGU
    1585 46 CUCUACCUCCACCAUGCCAAGUG
    1586 47 UCUACCUCCACCAUGCCAAGUGG
    1587 48 CUACCUCCACCAUGCCAAGUGGU
    1588 49 UACCUCCACCAUGCCAAGUGGUC
    1589 50 ACCUCCACCAUGCCAAGUGGUCC
    1590 51 CCUCCACCAUGCCAAGUGGUCCC
    1591 52 CUCCACCAUGCCAAGUGGUCCCA
    1592 53 UCCACCAUGCCAAGUGGUCCCAG
    1593 54 CCACCAUGCCAAGUGGUCCCAGG
    1594 55 CACCAUGCCAAGUGGUCCCAGGC
    1595 56 ACCAUGCCAAGUGGUCCCAGGCU
    1596 57 CCAUGCCAAGUGGUCCCAGGCUG
    1597 58 CAUGCCAAGUGGUCCCAGGCUGC
    1598 59 AUGCCAAGUGGUCCCAGGCUGCA
    1599 60 UGCCAAGUGGUCCCAGGCUGCAC
    1600 61 GCCAAGUGGUCCCAGGCUGCACC
    1601 62 CCAAGUGGUCCCAGGCUGCACCC
    1602 63 CAAGUGGUCCCAGGCUGCACCCA
    1603 64 AAGUGGUCCCAGGCUGCACCCAU
    1604 65 AGUGGUCCCAGGCUGCACCCAUG
    1605 66 GUGGUCCCAGGCUGCACCCAUGG
    1606 67 UGGUCCCAGGCUGCACCCAUGGC
    1607 68 GGUCCCAGGCUGCACCCAUGGCA
    1608 69 GUCCCAGGCUGCACCCAUGGCAG
    1609 70 UCCCAGGCUGCACCCAUGGCAGA
    1610 71 CCCAGGCUGCACCCAUGGCAGAA
    1611 72 CCAGGCUGCACCCAUGGCAGAAG
    1612 73 CAGGCUGCACCCAUGGCAGAAGG
    1613 74 AGGCUGCACCCAUGGCAGAAGGA
    1614 75 GGCUGCACCCAUGGCAGAAGGAG
    1615 76 GCUGCACCCAUGGCAGAAGGAGG
    1616 77 CUGCACCCAUGGCAGAAGGAGGA
    1617 78 UGCACCCAUGGCAGAAGGAGGAG
    1618 79 GCACCCAUGGCAGAAGGAGGAGG
    1619 80 CACCCAUGGCAGAAGGAGGAGGG
    1620 81 ACCCAUGGCAGAAGGAGGAGGGC
    1621 82 CCCAUGGCAGAAGGAGGAGGGCA
    1622 83 CCAUGGCAGAAGGAGGAGGGCAG
    1623 84 CAUGGCAGAAGGAGGAGGGCAGA
    1624 85 AUGGCAGAAGGAGGAGGGCAGAA
    1625 86 UGGCAGAAGGAGGAGGGCAGAAU
    1626 87 GGCAGAAGGAGGAGGGCAGAAUC
    1627 88 GCAGAAGGAGGAGGGCAGAAUCA
    1628 89 CAGAAGGAGGAGGGCAGAAUCAU
    1629 90 AGAAGGAGGAGGGCAGAAUCAUC
    1630 91 GAAGGAGGAGGGCAGAAUCAUCA
    1631 92 AAGGAGGAGGGCAGAAUCAUCAC
    1632 93 AGGAGGAGGGCAGAAUCAUCACG
    1633 94 GGAGGAGGGCAGAAUCAUCACGA
    1634 95 GAGGAGGGCAGAAUCAUCACGAA
    1635 96 AGGAGGGCAGAAUCAUCACGAAG
    1636 97 GGAGGGCAGAAUCAUCACGAAGU
    1637 98 GAGGGCAGAAUCAUCACGAAGUG
    1638 99 AGGGCAGAAUCAUCACGAAGUGG
    1639 100 GGGCAGAAUCAUCACGAAGUGGU
    1640 101 GGCAGAAUCAUCACGAAGUGGUG
    1641 102 GCAGAAUCAUCACGAAGUGGUGA
    1642 103 CAGAAUCAUCACGAAGUGGUGAA
    1643 104 AGAAUCAUCACGAAGUGGUGAAG
    1644 105 GAAUCAUCACGAAGUGGUGAAGU
    1645 106 AAUCAUCACGAAGUGGUGAAGUU
    1646 107 AUCAUCACGAAGUGGUGAAGUUC
    1647 108 UCAUCACGAAGUGGUGAAGUUCA
    1648 109 CAUCACGAAGUGGUGAAGUUCAU
    1649 110 AUCACGAAGUGGUGAAGUUCAUG
    1650 111 UCACGAAGUGGUGAAGUUCAUGG
    1651 112 CACGAAGUGGUGAAGUUCAUGGA
    1652 113 ACGAAGUGGUGAAGUUCAUGGAU
    1653 114 CGAAGUGGUGAAGUUCAUGGAUG
    1654 115 GAAGUGGUGAAGUUCAUGGAUGU
    1655 116 AAGUGGUGAAGUUCAUGGAUGUC
    1656 117 AGUGGUGAAGUUCAUGGAUGUCU
    1657 118 GUGGUGAAGUUCAUGGAUGUCUA
    1658 119 UGGUGAAGUUCAUGGAUGUCUAU
    1659 120 GGUGAAGUUCAUGGAUGUCUAUC
    1660 121 GUGAAGUUCAUGGAUGUCUAUCA
    1661 122 UGAAGUUCAUGGAUGUCUAUCAG
    1662 123 GAAGUUCAUGGAUGUCUAUCAGC
    1663 124 AAGUUCAUGGAUGUCUAUCAGCG
    1664 125 AGUUCAUGGAUGUCUAUCAGCGC
    1665 126 GUUCAUGGAUGUCUAUCAGCGCA
    1666 127 UUCAUGGAUGUCUAUCAGCGCAG
    1667 128 UCAUGGAUGUCUAUCAGCGCAGC
    1668 129 CAUGGAUGUCUAUCAGCGCAGCU
    1669 130 AUGGAUGUCUAUCAGCGCAGCUA
    1670 131 UGGAUGUCUAUCAGCGCAGCUAC
    1671 132 GGAUGUCUAUCAGCGCAGCUACU
    1672 133 GAUGUCUAUCAGCGCAGCUACUG
    1673 134 AUGUCUAUCAGCGCAGCUACUGC
    1674 135 UGUCUAUCAGCGCAGCUACUGCC
    1675 136 GUCUAUCAGCGCAGCUACUGCCA
    1676 137 UCUAUCAGCGCAGCUACUGCCAU
    1677 138 CUAUCAGCGCAGCUACUGCCAUC
    1678 139 UAUCAGCGCAGCUACUGCCAUCC
    1679 140 AUCAGCGCAGCUACUGCCAUCCA
    1680 141 UCAGCGCAGCUACUGCCAUCCAA
    1681 142 CAGCGCAGCUACUGCCAUCCAAU
    1682 143 AGCGCAGCUACUGCCAUCCAAUC
    1683 144 GCGCAGCUACUGCCAUCCAAUCG
    1684 145 CGCAGCUACUGCCAUCCAAUCGA
    1685 146 GCAGCUACUGCCAUCCAAUCGAG
    1686 147 CAGCUACUGCCAUCCAAUCGAGA
    1687 148 AGCUACUGCCAUCCAAUCGAGAC
    1688 149 GCUACUGCCAUCCAAUCGAGACC
    1689 150 CUACUGCCAUCCAAUCGAGACCC
    1690 151 UACUGCCAUCCAAUCGAGACCCU
    1691 152 ACUGCCAUCCAAUCGAGACCCUG
    1692 153 CUGCCAUCCAAUCGAGACCCUGG
    1693 154 UGCCAUCCAAUCGAGACCCUGGU
    1694 155 GCCAUCCAAUCGAGACCCUGGUG
    1695 156 CCAUCCAAUCGAGACCCUGGUGG
    1696 157 CAUCCAAUCGAGACCCUGGUGGA
    1697 158 AUCCAAUCGAGACCCUGGUGGAC
    1698 159 UCCAAUCGAGACCCUGGUGGACA
    1699 160 CCAAUCGAGACCCUGGUGGACAU
    1700 161 CAAUCGAGACCCUGGUGGACAUC
    1701 162 AAUCGAGACCCUGGUGGACAUCU
    1702 163 AUCGAGACCCUGGUGGACAUCUU
    1703 164 UCGAGACCCUGGUGGACAUCUUC
    1704 165 CGAGACCCUGGUGGACAUCUUCC
    1705 166 GAGACCCUGGUGGACAUCUUCCA
    1706 167 AGACCCUGGUGGACAUCUUCCAG
    1707 168 GACCCUGGUGGACAUCUUCCAGG
    1708 169 ACCCUGGUGGACAUCUUCCAGGA
    1709 170 CCCUGGUGGACAUCUUCCAGGAG
    1710 171 CCUGGUGGACAUCUUCCAGGAGU
    1711 172 CUGGUGGACAUCUUCCAGGAGUA
    1712 173 UGGUGGACAUCUUCCAGGAGUAC
    1713 174 GGUGGACAUCUUCCAGGAGUACC
    1714 175 GUGGACAUCUUCCAGGAGUACCC
    1715 176 UGGACAUCUUCCAGGAGUACCCU
    1716 177 GGACAUCUUCCAGGAGUACCCUG
    1717 178 GACAUCUUCCAGGAGUACCCUGA
    1718 179 ACAUCUUCCAGGAGUACCCUGAU
    1719 180 CAUCUUCCAGGAGUACCCUGAUG
    1720 181 AUCUUCCAGGAGUACCCUGAUGA
    1721 182 UCUUCCAGGAGUACCCUGAUGAG
    1722 183 CUUCCAGGAGUACCCUGAUGAGA
    1723 184 UUCCAGGAGUACCCUGAUGAGAU
    1724 185 UCCAGGAGUACCCUGAUGAGAUC
    1725 186 CCAGGAGUACCCUGAUGAGAUCG
    1726 187 CAGGAGUACCCUGAUGAGAUCGA
    1727 188 AGGAGUACCCUGAUGAGAUCGAG
    1728 189 GGAGUACCCUGAUGAGAUCGAGU
    1729 190 GAGUACCCUGAUGAGAUCGAGUA
    1730 191 AGUACCCUGAUGAGAUCGAGUAC
    1731 192 GUACCCUGAUGAGAUCGAGUACA
    1732 193 UACCCUGAUGAGAUCGAGUACAU
    1733 194 ACCCUGAUGAGAUCGAGUACAUC
    1734 195 CCCUGAUGAGAUCGAGUACAUCU
    1735 196 CCUGAUGAGAUCGAGUACAUCUU
    1736 197 CUGAUGAGAUCGAGUACAUCUUC
    1737 198 UGAUGAGAUCGAGUACAUCUUCA
    1738 199 GAUGAGAUCGAGUACAUCUUCAA
    1739 200 AUGAGAUCGAGUACAUCUUCAAG
    1740 201 UGAGAUCGAGUACAUCUUCAAGC
    1741 202 GAGAUCGAGUACAUCUUCAAGCC
    1742 203 AGAUCGAGUACAUCUUCAAGCCA
    1743 204 GAUCGAGUACAUCUUCAAGCCAU
    1744 205 AUCGAGUACAUCUUCAAGCCAUC
    1745 206 UCGAGUACAUCUUCAAGCCAUCC
    1746 207 CGAGUACAUCUUCAAGCCAUCCU
    1747 208 GAGUACAUCUUCAAGCCAUCCUG
    1748 209 AGUACAUCUUCAAGCCAUCCUGU
    1749 210 GUACAUCUUCAAGCCAUCCUGUG
    1750 211 UACAUCUUCAAGCCAUCCUGUGU
    1751 212 ACAUCUUCAAGCCAUCCUGUGUG
    1752 213 CAUCUUCAAGCCAUCCUGUGUGC
    1753 214 AUCUUCAAGCCAUCCUGUGUGCC
    1754 215 UCUUCAAGCCAUCCUGUGUGCCC
    1755 216 CUUCAAGCCAUCCUGUGUGCCCC
    1756 217 UUCAAGCCAUCCUGUGUGCCCCU
    1757 218 UCAAGCCAUCCUGUGUGCCCCUG
    1758 219 CAAGCCAUCCUGUGUGCCCCUGA
    1759 220 AAGCCAUCCUGUGUGCCCCUGAU
    1760 221 AGCCAUCCUGUGUGCCCCUGAUG
    1761 222 GCCAUCCUGUGUGCCCCUGAUGC
    1762 223 CCAUCCUGUGUGCCCCUGAUGCG
    1763 224 CAUCCUGUGUGCCCCUGAUGCGA
    1764 225 AUCCUGUGUGCCCCUGAUGCGAU
    1765 226 UCCUGUGUGCCCCUGAUGCGAUG
    1766 227 CCUGUGUGCCCCUGAUGCGAUGC
    1767 228 CUGUGUGCCCCUGAUGCGAUGCG
    1768 229 UGUGUGCCCCUGAUGCGAUGCGG
    1769 230 GUGUGCCCCUGAUGCGAUGCGGG
    1770 231 UGUGCCCCUGAUGCGAUGCGGGG
    1771 232 GUGCCCCUGAUGCGAUGCGGGGG
    1772 233 UGCCCCUGAUGCGAUGCGGGGGC
    1773 234 GCCCCUGAUGCGAUGCGGGGGCU
    1774 235 CCCCUGAUGCGAUGCGGGGGCUG
    1775 236 CCCUGAUGCGAUGCGGGGGCUGC
    1776 237 CCUGAUGCGAUGCGGGGGCUGCU
    1777 238 CUGAUGCGAUGCGGGGGCUGCUG
    1778 239 UGAUGCGAUGCGGGGGCUGCUGC
    1779 240 GAUGCGAUGCGGGGGCUGCUGCA
    1780 241 AUGCGAUGCGGGGGCUGCUGCAA
    1781 242 UGCGAUGCGGGGGCUGCUGCAAU
    1782 243 GCGAUGCGGGGGCUGCUGCAAUG
    1783 244 CGAUGCGGGGGCUGCUGCAAUGA
    1784 245 GAUGCGGGGGCUGCUGCAAUGAC
    1785 246 AUGCGGGGGCUGCUGCAAUGACG
    1786 247 UGCGGGGGCUGCUGCAAUGACGA
    1787 248 GCGGGGGCUGCUGCAAUGACGAG
    1788 249 CGGGGGCUGCUGCAAUGACGAGG
    1789 250 GGGGGCUGCUGCAAUGACGAGGG
    1790 251 GGGGCUGCUGCAAUGACGAGGGC
    1791 252 GGGCUGCUGCAAUGACGAGGGCC
    1792 253 GGCUGCUGCAAUGACGAGGGCCU
    1793 254 GCUGCUGCAAUGACGAGGGCCUG
    1794 255 CUGCUGCAAUGACGAGGGCCUGG
    1795 256 UGCUGCAAUGACGAGGGCCUGGA
    1796 257 GCUGCAAUGACGAGGGCCUGGAG
    1797 258 CUGCAAUGACGAGGGCCUGGAGU
    1798 259 UGCAAUGACGAGGGCCUGGAGUG
    1799 260 GCAAUGACGAGGGCCUGGAGUGU
    1800 261 CAAUGACGAGGGCCUGGAGUGUG
    1801 262 AAUGACGAGGGCCUGGAGUGUGU
    1802 263 AUGACGAGGGCCUGGAGUGUGUG
    1803 264 UGACGAGGGCCUGGAGUGUGUGC
    1804 265 GACGAGGGCCUGGAGUGUGUGCC
    1805 266 ACGAGGGCCUGGAGUGUGUGCCC
    1806 267 CGAGGGCCUGGAGUGUGUGCCCA
    1807 268 GAGGGCCUGGAGUGUGUGCCCAC
    1808 269 AGGGCCUGGAGUGUGUGCCCACU
    1809 270 GGGCCUGGAGUGUGUGCCCACUG
    1810 271 GGCCUGGAGUGUGUGCCCACUGA
    1811 272 GCCUGGAGUGUGUGCCCACUGAG
    1812 273 CCUGGAGUGUGUGCCCACUGAGG
    1813 274 CUGGAGUGUGUGCCCACUGAGGA
    1814 275 UGGAGUGUGUGCCCACUGAGGAG
    1815 276 GGAGUGUGUGCCCACUGAGGAGU
    1816 277 GAGUGUGUGCCCACUGAGGAGUC
    1817 278 AGUGUGUGCCCACUGAGGAGUCC
    1818 279 GUGUGUGCCCACUGAGGAGUCCA
    1819 280 UGUGUGCCCACUGAGGAGUCCAA
    1820 281 GUGUGCCCACUGAGGAGUCCAAC
    1821 282 UGUGCCCACUGAGGAGUCCAACA
    1822 283 GUGCCCACUGAGGAGUCCAACAU
    1823 284 UGCCCACUGAGGAGUCCAACAUC
    1824 285 GCCCACUGAGGAGUCCAACAUCA
    1825 286 CCCACUGAGGAGUCCAACAUCAC
    1826 287 CCACUGAGGAGUCCAACAUCACC
    1827 288 CACUGAGGAGUCCAACAUCACCA
    1828 289 ACUGAGGAGUCCAACAUCACCAU
    1829 290 CUGAGGAGUCCAACAUCACCAUG
    1830 291 UGAGGAGUCCAACAUCACCAUGC
    1831 292 GAGGAGUCCAACAUCACCAUGCA
    1832 293 AGGAGUCCAACAUCACCAUGCAG
    1833 294 GGAGUCCAACAUCACCAUGCAGA
    1834 295 GAGUCCAACAUCACCAUGCAGAU
    1835 296 AGUCCAACAUCACCAUGCAGAUU
    1836 297 GUCCAACAUCACCAUGCAGAUUA
    1837 298 UCCAACAUCACCAUGCAGAUUAU
    1838 299 CCAACAUCACCAUGCAGAUUAUG
    1839 300 CAACAUCACCAUGCAGAUUAUGC
    1840 301 AACAUCACCAUGCAGAUUAUGCG
    1841 302 ACAUCACCAUGCAGAUUAUGCGG
    1842 303 CAUCACCAUGCAGAUUAUGCGGA
    1843 304 AUCACCAUGCAGAUUAUGCGGAU
    1844 305 UCACCAUGCAGAUUAUGCGGAUC
    1845 306 CACCAUGCAGAUUAUGCGGAUCA
    1846 307 ACCAUGCAGAUUAUGCGGAUCAA
    1847 308 CCAUGCAGAUUAUGCGGAUCAAA
    1848 309 CAUGCAGAUUAUGCGGAUCAAAC
    1849 310 AUGCAGAUUAUGCGGAUCAAACC
    1850 311 UGCAGAUUAUGCGGAUCAAACCU
    1851 312 GCAGAUUAUGCGGAUCAAACCUC
    1852 313 CAGAUUAUGCGGAUCAAACCUCA
    1853 314 AGAUUAUGCGGAUCAAACCUCAC
    1854 315 GAUUAUGCGGAUCAAACCUCACC
    1855 316 AUUAUGCGGAUCAAACCUCACCA
    1856 317 UUAUGCGGAUCAAACCUCACCAA
    1857 318 UAUGCGGAUCAAACCUCACCAAG
    1858 319 AUGCGGAUCAAACCUCACCAAGG
    1859 320 UGCGGAUCAAACCUCACCAAGGC
    1860 321 GCGGAUCAAACCUCACCAAGGCC
    1861 322 CGGAUCAAACCUCACCAAGGCCA
    1862 323 GGAUCAAACCUCACCAAGGCCAG
    1863 324 GAUCAAACCUCACCAAGGCCAGC
    1864 325 AUCAAACCUCACCAAGGCCAGCA
    1865 326 UCAAACCUCACCAAGGCCAGCAC
    1866 327 CAAACCUCACCAAGGCCAGCACA
    1867 328 AAACCUCACCAAGGCCAGCACAU
    1868 329 AACCUCACCAAGGCCAGCACAUA
    1869 330 ACCUCACCAAGGCCAGCACAUAG
    1870 331 CCUCACCAAGGCCAGCACAUAGG
    1871 332 CUCACCAAGGCCAGCACAUAGGA
    1872 333 UCACCAAGGCCAGCACAUAGGAG
    1873 334 CACCAAGGCCAGCACAUAGGAGA
    1874 335 ACCAAGGCCAGCACAUAGGAGAG
    1875 336 CCAAGGCCAGCACAUAGGAGAGA
    1876 337 CAAGGCCAGCACAUAGGAGAGAU
    1877 338 AAGGCCAGCACAUAGGAGAGAUG
    1878 339 AGGCCAGCACAUAGGAGAGAUGA
    1879 340 GGCCAGCACAUAGGAGAGAUGAG
    1880 341 GCCAGCACAUAGGAGAGAUGAGC
    1881 342 CCAGCACAUAGGAGAGAUGAGCU
    1882 343 CAGCACAUAGGAGAGAUGAGCUU
    1883 344 AGCACAUAGGAGAGAUGAGCUUC
    1884 345 GCACAUAGGAGAGAUGAGCUUCC
    1885 346 CACAUAGGAGAGAUGAGCUUCCU
    1886 347 ACAUAGGAGAGAUGAGCUUCCUA
    1887 348 CAUAGGAGAGAUGAGCUUCCUAC
    1888 349 AUAGGAGAGAUGAGCUUCCUACA
    1889 350 UAGGAGAGAUGAGCUUCCUACAG
    1890 351 AGGAGAGAUGAGCUUCCUACAGC
    1891 352 GGAGAGAUGAGCUUCCUACAGCA
    1892 353 GAGAGAUGAGCUUCCUACAGCAC
    1893 354 AGAGAUGAGCUUCCUACAGCACA
    1894 355 GAGAUGAGCUUCCUACAGCACAA
    1895 356 AGAUGAGCUUCCUACAGCACAAC
    1896 357 GAUGAGCUUCCUACAGCACAACA
    1897 358 AUGAGCUUCCUACAGCACAACAA
    1898 359 UGAGCUUCCUACAGCACAACAAA
    1899 360 GAGCUUCCUACAGCACAACAAAU
    1900 361 AGCUUCCUACAGCACAACAAAUG
    1901 362 GCUUCCUACAGCACAACAAAUGU
    1902 363 CUUCCUACAGCACAACAAAUGUG
    1903 364 UUCCUACAGCACAACAAAUGUGA
    1904 365 UCCUACAGCACAACAAAUGUGAA
    1905 366 CCUACAGCACAACAAAUGUGAAU
    1906 367 CUACAGCACAACAAAUGUGAAUG
    1907 368 UACAGCACAACAAAUGUGAAUGC
    1908 369 ACAGCACAACAAAUGUGAAUGCA
    1909 370 CAGCACAACAAAUGUGAAUGCAG
    1910 371 AGCACAACAAAUGUGAAUGCAGA
    1911 372 GCACAACAAAUGUGAAUGCAGAC
    1912 373 CACAACAAAUGUGAAUGCAGACC
    1913 374 ACAACAAAUGUGAAUGCAGACCA
    1914 375 CAACAAAUGUGAAUGCAGACCAA
    1915 376 AACAAAUGUGAAUGCAGACCAAA
    1916 377 ACAAAUGUGAAUGCAGACCAAAG
    1917 378 CAAAUGUGAAUGCAGACCAAAGA
    1918 379 AAAUGUGAAUGCAGACCAAAGAA
    1919 380 AAUGUGAAUGCAGACCAAAGAAA
    1920 381 AUGUGAAUGCAGACCAAAGAAAG
    1921 382 UGUGAAUGCAGACCAAAGAAAGA
    1922 383 GUGAAUGCAGACCAAAGAAAGAU
    1923 384 UGAAUGCAGACCAAAGAAAGAUA
    1924 385 GAAUGCAGACCAAAGAAAGAUAG
    1925 386 AAUGCAGACCAAAGAAAGAUAGA
    1926 387 AUGCAGACCAAAGAAAGAUAGAG
    1927 388 UGCAGACCAAAGAAAGAUAGAGC
    1928 389 GCAGACCAAAGAAAGAUAGAGCA
    1929 390 CAGACCAAAGAAAGAUAGAGCAA
    1930 391 AGACCAAAGAAAGAUAGAGCAAG
    1931 392 GACCAAAGAAAGAUAGAGCAAGA
    1932 393 ACCAAAGAAAGAUAGAGCAAGAC
    1933 394 CCAAAGAAAGAUAGAGCAAGACA
    1934 395 CAAAGAAAGAUAGAGCAAGACAA
    1935 396 AAAGAAAGAUAGAGCAAGACAAG
    1936 397 AAGAAAGAUAGAGCAAGACAAGA
    1937 398 AGAAAGAUAGAGCAAGACAAGAA
    1938 399 GAAAGAUAGAGCAAGACAAGAAA
    1939 400 AAAGAUAGAGCAAGACAAGAAAA
  • [0000]
    TABLE 4b
    VEGF targeted duplexes
    position SEQ SEQ
    in ID Target sequence ID
    ORF NO: (5′-3′) Duplex ID Strand NO: Strand Sequences
    1 2184 AUGAACUUUCUGCUGUCUUGGGU AL-DP-4043 S 1940 5 GAACUUUCUGCUGUCUUGGGU 3
    AS 1941 3 UACUUGAAAGACGACAGAACCCA 5
    22 2185 GUGCAUUGGAGCCUUGCCUUGCU AL-DP-4077 S 1942 5 GCAUUGGAGCCUUGCCUUGCU 3
    AS 1943 3 CACGUAACCUCGGAACGGAACGA 5
    47 2186 UCUACCUCCACCAUGCCAAGUGG AL-DP-4021 S 1944 5 UACCUCCACCAUGCCAAGUTT 3
    AS 1945 3 TTAUGGAGGUGGUACGGUUCA 5
    48 2187 CUACCUCCACCAUGCCAAGUGGU AL-DP-4109 S 1946 5 ACCUCCACCAUGCCAAGUGTT 3
    AS 1947 3 TTUGGAGGUGGUACGGUUCAC 5
    50 2188 ACCUCCACCAUGCCAAGUGGUCC AL-DP-4006 S 1948 5 CUCCACCAUGCCAAGUGGUCC 3
    AS 1949 3 UGGAGGUGGUACGGUUCACCAGG 5
    AL-DP-4083 S 1950 5 CUCCACCAUGCCAAGUGGUTT 3
    AS 1951 3 TTGAGGUGGUACGGUUCACCA 5
    51 2189 CCUCCACCAUGCCAAGUGGUCCC AL-DP-4047 S 1952 5 UCCACCAUGCCAAGUGGUCCC 3
    AS 1953 3 GGAGGUGGUACGGUUCACCAGGG 5
    AL-DP-4017 S 1954 5 UCCACCAUGCCAAGUGGUCTT 3
    AS 1955 3 TTAGGUGGUACGGUUCACCAG 5
    52 2190 CUCCACCAUGCCAAGUGGUCCCA AL-DP-4048 S 1956 5 CCACCAUGCCAAGUGGUCCCA 3
    AS 1957 3 GAGGUGGUACGGUUCACCAGGGU 5
    AL-DP-4103 S 1958 5 CCACCAUGCCAAGUGGUCCTT 3
    AS 1959 3 TTGGUGGUACGGUUCACCAGG 5
    53 2191 UCCACCAUGCCAAGUGGUCCCAG AL-DP-4035 S 1960 5 CACCAUGCCAAGUGGUCCCAG 3
    AS 1961 3 AGGUGGUACGGUUCACCAGGGUC 5
    AL-DP-4018 S 1962 5 CACCAUGCCAAGUGGUCCCTT 3
    AS 1963 3 TTGUGGUACGGUUCACCAGGG 5
    54 2192 CCACCAUGCCAAGUGGUCCCAGG AL-DP-4036 S 1964 5 ACCAUGCCAAGUGGUCCCAGG 3
    AS 1965 3 GGUGGUACGGUUCACCAGGGUCC 5
    AL-DP-4084 S 1966 5 ACCAUGCCAAGUGGUCCCATT 3
    AS 1967 3 TTUGGUACGGUUCACCAGGGU 5
    55 2193 CACCAUGCCAAGUGGUCCCAGGC AL-DP-4093 S 1968 5 CCAUGCCAAGUGGUCCCAGGC 3
    AS 1969 3 GUGGUACGGUUCACCAGGGUCCG 5
    AL-DP-4085 S 1970 5 CCAUGCCAAGUGGUCCCAGTT 3
    AS 1971 3 TTGGUACGGUUCACCAGGGUC 5
    56 2194 ACCAUGCCAAGUGGUCCCAGGCU AL-DP-4037 S 1972 5 CAUGCCAAGUGGUCCCAGGCU 3
    AS 1973 3 UGGUACGGUUCACCAGGGUCCGA 5
    AL-DP-4054 S 1974 5 CAUGCCAAGUGGUCCCAGGTT 3
    AS 1975 3 TTGUACGGUUCACCAGGGUCC 5
    57 2195 CCAUGCCAAGUGGUCCCAGGCUG AL-DP-4038 S 1976 5 AUGCCAAGUGGUCCCAGGCUG 3
    AS 1977 3 GGUACGGUUCACCAGGGUCCGAC 5
    AL-DP-4086 S 1978 5 AUGCCAAGUGGUCCCAGGCTT 3
    AS 1979 3 TTUACGGUUCACCAGGGUCCG 5
    58 2196 CAUGCCAAGUGGUCCCAGGCUGC AL-DP-4049 S 1980 5 UGCCAAGUGGUCCCAGGCUGC 3
    AS 1981 3 GUACGGUUCACCAGGGUCCGACG 5
    AL-DP-4087 S 1982 5 UGCCAAGUGGUCCCAGGCUTT 3
    AS 1983 3 TTACGGUUCACCAGGGUCCGA 5
    59 2197 AUGCCAAGUGGUCCCAGGCUGCA AL-DP-4001 S 1984 5 GCCAAGUGGUCCCAGGCUGCA 3
    AS 1985 3 UACGGUUCACCAGGGUCCGACGU 5
    AL-DP-4052 A 1986 5 GCCAAGUGGUCCCAGGCUGTT 3
    AS 1987 3 TTCGGUUCACCAGGGUCCGAC 5
    60 2198 UGCCAAGUGGUCCCAGGCUGCAC AL-DP-4007 S 1988 5 CCAAGUGGUCCCAGGCUGCAC 3
    AS 1989 3 ACGGUUCACCAGGGUCCGACGUG 5
    AL-DP-4088 S 1990 5 CCAAGUGGUCCCAGGCUGCTT 3
    AS 1991 3 TTGGUUCACCAGGGUCCGACG 5
    61 2199 GCCAAGUGGUCCCAGGCUGCACC AL-DP-4070 S 1992 5 CAAGUGGUCCCAGGCUGCACC 3
    AS 1993 3 CGGUUCACCAGGGUCCGACGUGG 5
    AL-DP-4055 S 1994 5 CAAGUGGUCCCAGGCUGCATT 3
    AS 1995 3 TTGUUCACCAGGGUCCGACGU 5
    62 2200 CCAAGUGGUCCCAGGCUGCACCC AL-DP-4071 S 1996 5 AAGUGGUCCCAGGCUGCACCC 3
    AS 1997 3 GGUUCACCAGGGUCCGACGUGGG 5
    AL-DP-4056 S 1998 5 AAGUGGUCCCAGGCUGCACTT 3
    AS 1999 3 TTUUCACCAGGGUCCGACGUG 5
    63 2201 CAAGUGGUCCCAGGCUGCACCCA AL-DP-4072 S 2000 5 AGUGGUCCCAGGCUGCACCCA 3
    AS 2001 3 GUUCACCAGGGUCCGACGUGGGU 5
    AL-DP-4057 S 2002 5 AGUGGUCCCAGGCUGCACCTT 3
    AS 2003 3 TTUCACCAGGGUCCGACGUGG 5
    64 2202 AAGUGGUCCCAGGCUGCACCCAU AL-DP-4066 S 2004 5 GUGGUCCCAGGCUGCACCCTT 3
    AS 2005 3 TTCACCAGGGUCCGACGUGGG 5
    99 2203 AGGGCAGAAUCAUCACGAAGUGG AL-DP-4022 S 2006 5 GGCAGAAUCAUCACGAAGUTT 3
    AS 2007 3 TTCCGUCUUAGUAGUGCUUCA 5
    100 2204 GGGCAGAAUCAUCACGAAGUGGU AL-DP-4023 S 2008 5 GCAGAAUCAUCACGAAGUGTT 3
    AS 2009 3 TTCGUCUUAGUAGUGCUUCAC 5
    101 2205 GGCAGAAUCAUCACGAAGUGGUG AL-DP-4024 S 2010 5 CAGAAUCAUCACGAAGUGGTT 3
    AS 2011 3 TTGUCUUAGUAGUGCUUCACC 5
    102 2206 GCAGAAUCAUCACGAAGUGGUGA AL-DP-4076 S 2012 5 AGAAUCAUCACGAAGUGGUGA 3
    AS 2013 3 CGUCUUAGUAGUGCUUCACCACU 5
    AL-DP-4019 S 2014 5 AGAAUCAUCACGAAGUGGUTT 3
    AS 2015 3 TTUCUUAGUAGUGCUUCACCA 5
    103 2207 CAGAAUCAUCACGAAGUGGUGAA AL-DP-4025 S 2016 5 GAAUCAUCACGAAGUGGUGTT 3
    AS 2017 3 TTCUUAGUAGUGCUUCACCAC 5
    104 2208 AGAAUCAUCACGAAGUGGUGAAG AL-DP-4110 S 2018 5 AAUCAUCACGAAGUGGUGATT 3
    AS 2019 3 TTUUAGUAGUGCUUCACCACU 5
    105 2209 GAAUCAUCACGAAGUGGUGAAGU AL-DP-4068 S 2020 5 AUCAUCACGAAGUGGUGAATT 3
    AS 2021 3 TTUAGUAGUGCUUCACCACUU 5
    113 2210 ACGAAGUGGUGAAGUUCAUGGAU AL-DP-4078 S 2022 5 GAAGUGGUGAAGUUCAUGGAU 3
    AS 2023 3 UGCUUCACCACUUCAAGUACCUA 5
    121 2211 GUGAAGUUCAUGGAUGUCUAUCA AL-DP-4080 S 2024 5 GAAGUUCAUGGAUGUCUAUCA 3
    AS 2025 3 CACUUCAAGUACCUACAGAUAGU 5
    129 2212 CAUGGAUGUCUAUCAGCGCAGCU AL-DP-4111 S 2026 5 UGGAUGUCUAUCAGCGCAGTT 3
    AS 2027 3 TTACCUACAGAUAGUCGCGUC 5
    130 2213 AUGGAUGUCUAUCAGCGCAGCUA AL-DP-4041 S 2028 5 GGAUGUCUAUCAGCGCAGCUA 3
    AS 2029 3 UACCUACAGAUAGUCGCGUCGAU 5
    AL-DP-4062 S 2030 5 GGAUGUCUAUCAGCGCAGCTT 3
    AS 2031 3 TTCCUACAGAUAGUCGCGUCG 5
    131 2214 UGGAUGUCUAUCAGCGCAGCUAC AL-DP-4069 S 2032 5 GAUGUCUAUCAGCGCAGCUTT 3
    AS 2033 3 TTCUACAGAUAGUCGCGUCGA 5
    132 2215 GGAUGUCUAUCAGCGCAGCUACU AL-DP-4112 S 2034 5 AUGUCUAUCAGCGCAGCUATT 3
    AS 2035 3 TTUACAGAUAGUCGCGUCGAU 5
    133 2216 GAUGUCUAUCAGCGCAGCUACUG AL-DP-4026 S 2036 5 UGUCUAUCAGCGCAGCUACTT 3
    AS 2037 3 TTACAGAUAGUCGCGUCGAUG 5
    134 2217 AUGUCUAUCAGCGCAGCUACUGC AL-DP-4095 S 2038 5 GUCUAUCAGCGCAGCUACUGC 3
    AS 2039 3 UACAGAUAGUCGCGUCGAUGACG 5
    AL-DP-4020 S 2040 5 GUCUAUCAGCGCAGCUACUTT 3
    AS 2041 3 TTCAGAUAGUCGCGUCGAUGA 5
    135 2218 UGUCUAUCAGCGCAGCUACUGCC AL-DP-4027 S 2042 5 UCUAUCAGCGCAGCUACUGTT 3
    AS 2043 3 TTAGAUAGUCGCGUCGAUGAC 5
    144 2219 GCGCAGCUACUGCCAUCCAAUCG AL-DP-4081 S 2044 5 GCAGCUACUGCCAUCCAAUCG 3
    AS 2045 3 CGCGUCGAUGACGGUAGGUUAGC 5
    146 2220 GCAGCUACUGCCAUCCAAUCGAG AL-DP-4098 S 2046 5 AGCUACUGCCAUCCAAUCGAG 3
    AS 2047 3 CGUCGAUGACGGUAGGUUAGCUC 5
    149 2221 GCUACUGCCAUCCAAUCGAGACC AL-DP-4028 S 2048 5 UACUGCCAUCCAAUCGAGATT 3
    AS 2049 3 TTAUGACGGUAGGUUAGCUCU 5
    150 2222 CUACUGCCAUCCAAUCGAGACCC AL-DP-4029 S 2050 5 ACUGCCAUCCAAUCGAGACTT 3
    AS 2051 3 TTUGACGGUAGGUUAGCUCUG 5
    151 2223 UACUGCCAUCCAAUCGAGACCCU AL-DP-4030 S 2052 5 CUGCCAUCCAAUCGAGACCTT 3
    AS 2053 3 TTGACGGUAGGUUAGCUCUGG 5
    152 2224 ACUGCCAUCCAAUCGAGACCCUG AL-DP-4031 S 2054 5 UGCCAUCCAAUCGAGACCCTT 3
    AS 2055 3 TTACGGUAGGUUAGCUCUGGG 5
    166 2225 GAGACCCUGGUGGACAUCUUCCA AL-DP-4008 S 2056 5 GACCCUGGUGGACAUCUUCCA 3
    AS 2057 3 CUCUGGGACCACCUGUAGAAGGU 5
    AL-DP-4058 S 2058 5 GACCCUGGUGGACAUCUUCTT 3
    AS 2059 3 TTCUGGGACCACCUGUAGAAG 5
    167 2226 AGACCCUGGUGGACAUCUUCCAG AL-DP-4009 S 2060 5 ACCCUGGUGGACAUCUUCCAG 3
    AS 2061 3 UCUGGGACCACCUGUAGAAGGUC 5
    AL-DP-4059 S 2062 5 ACCCUGGUGGACAUCUUCCTT 3
    AS 2063 3 TTUGGGACCACCUGUAGAAGG 5
    168 2227 GACCCUGGUGGACAUCUUCCAGG AL-DP-4010 S 2064 5 CCCUGGUGGACAUCUUCCAGG 3
    AS 2065 3 CUGGGACCACCUGUAGAAGGUCC 5
    AL-DP-4060 S 2066 5 CCCUGGUGGACAUCUUCCATT 3
    AS 2067 3 TTGGGACCACCUGUAGAAGGU 5
    169 2228 ACCCUGGUGGACAUCUUCCAGGA AL-DP-4073 S 2068 5 CCUGGUGGACAUCUUCCAGGA 3
    AS 2069 3 UGGGACCACCUGUAGAAGGUCCU 5
    AL-DP-4104 S 2070 5 CCUGGUGGACAUCUUCCAGTT 3
    AS 2071 3 TTGGACCACCUGUAGAAGGUC 5
    170 2229 CCCUGGUGGACAUCUUCCAGGAG AL-DP-4011 S 2072 5 CUGGUGGACAUCUUCCAGGAG 3
    AS 2073 3 GGGACCACCUGUAGAAGGUCCUC 5
    AL-DP-4089 S 2074 5 CUGGUGGACAUCUUCCAGGTT 3
    AS 2075 3 TTGACCACCUGUAGAAGGUCC 5
    171 2230 CCUGGUGGACAUCUUCCAGGAGU AL-DP-4074 S 2076 5 UGGUGGACAUCUUCCAGGAGU 3
    AS 2077 3 GGACCACCUGUAGAAGGUCCUCA 5
    AL-DP-4090 S 2078 5 UGGUGGACAUCUUCCAGGATT 3
    AS 2079 3 TTACCACCUGUAGAAGGUCCU 5
    172 2231 CUGGUGGACAUCUUCCAGGAGUA AL-DP-4039 S 2080 5 GGUGGACAUCUUCCAGGAGUA 3
    AS 2081 3 GACCACCUGUAGAAGGUCCUCAU 5
    AL-DP-4091 S 2082 5 GGUGGACAUCUUCCAGGAGTT 3
    AS 2083 3 TTCCACCUGUAGAAGGUCCUC 5
    175 2232 GUGGACAUCUUCCAGGAGUACCC AL-DP-4003 S 2084 5 GGACAUCUUCCAGGAGUACCC 3
    AS 2085 3 CCUGUAGAAGGUCCUCAUGGG 5
    AL-DP-4116 S 2086 5 GGACAUCUUCCAGGAGUACCC 3
    AS 2087 3 CCUGUAGAAGGUCCUCAUGGG 5
    AL-DP-4015 S 2088 5 GGACAUCUUCCAGGAGUACTT 3
    AS 2089 3 TTCCUGUAGAAGGUCCUCAUG 5
    AL-DP-4120 S 2090 5 GGACAUCUUCCAGGAGUAC 3
    AS 2091 3 CCUGUAGAAGGUCCUCAUG 5
    179 2233 ACAUCUUCCAGGAGUACCCUGAU AL-DP-4099 S 2092 5 AUCUUCCAGGAGUACCCUGAU 3
    AS 2093 3 UGUAGAAGGUCCUCAUGGGACUA 5
    191 2234 AGUACCCUGAUGAGAUCGAGUAC AL-DP-4032 S 2094 5 UACCCUGAUGAGAUCGAGUTT 3
    AS 2095 3 TTAUGGGACUACUCUAGCUCA 5
    192 2235 GUACCCUGAUGAGAUCGAGUACA AL-DP-4042 S 2096 5 ACCCUGAUGAGAUCGAGUACA 3
    AS 2097 3 CAUGGGACUACUCUAGCUCAUGU 5
    AL-DP-4063 S 2098 5 ACCCUGAUGAGAUCGAGUATT 3
    AS 2099 3 TTUGGGACUACUCUAGCUCAU 5
    209 2236 AGUACAUCUUCAAGCCAUCCUGU AL-DP-4064 S 2100 5 UACAUCUUCAAGCCAUCCUTT 3
    AS 2101 3 TTAUGUAGAAGUUCGGUAGGA 5
    260 2237 GCAAUGACGAGGGCCUGGAGUGU AL-DP-4044 S 2102 5 AAUGACGAGGGCCUGGAGUGU 3
    AS 2103 3 CGUUACUGCUCCCGGACCUCACA 5
    263 2238 AUGACGAGGGCCUGGAGUGUGUG AL-DP-4045 S 2104 5 GACGAGGGCCUGGAGUGUGUG 3
    AS 2105 3 UACUGCUCCCGGACCUCACACAC 5
    279 2239 GUGUGUGCCCACUGAGGAGUCCA AL-DP-4046 S 2106 5 GUGUGCCCACUGAGGAGUCCA 3
    AS 2107 3 CACACACGGGUGACUCCUCAGGU 5
    281 2240 GUGUGCCCACUGAGGAGUCCAAC AL-DP-4096 S 2108 5 GUGCCCACUGAGGAGUCCAAC 3
    AS 2109 3 CACACGGGUGACUCCUCAGGUUG 5
    283 2241 GUGCCCACUGAGGAGUCCAACAU AL-DP-4040 S 2110 5 GCCCACUGAGGAGUCCAACAU 3
    AS 2111 3 CACGGGUGACUCCUCAGGUUGUA 5
    289 2242 ACUGAGGAGUCCAACAUCACCAU AL-DP-4065 S 2112 5 UGAGGAGUCCAACAUCACCTT 3
    AS 2113 3 TTACUCCUCAGGUUGUAGUGG 5
    302 2243 ACAUCACCAUGCAGAUUAUGCGG AL-DP-4100 S 2114 5 AUCACCAUGCAGAUUAUGCGG 3
    AS 2115 3 UGUAGUGGUACGUCUAAUACGCC 5
    305 2244 UCACCAUGCAGAUUAUGCGGAUC AL-DP-4033 S 2116 5 ACCAUGCAGAUUAUGCGGATT 3
    AS 2117 3 TTUGGUACGUCUAAUACGCCU 5
    310 2245 AUGCAGAUUAUGCGGAUCAAACC AL-DP-4101 S 2118 5 GCAGAUUAUGCGGAUCAAACC 3
    AS 2119 3 UACGUCUAAUACGCCUAGUUUGG 5
    312 2246 GCAGAUUAUGCGGAUCAAACCUC AL-DP-4102 S 2120 5 AGAUUAUGCGGAUCAAACCUC 3
    AS 2121 3 CGUCUAAUACGCCUAGUUUGGAG 5
    315 2247 GAUUAUGCGGAUCAAACCUCACC AL-DP-4034 S 2122 5 UUAUGCGGAUCAAACCUCATT 3
    AS 2123 3 TTAAUACGCCUAGUUUGGAGU 5
    316 2248 AUUAUGCGGAUCAAACCUCACCA AL-DP-4113 S 2124 5 UAUGCGGAUCAAACCUCACTT 3
    AS 2125 3 TTAUACGCCUAGUUUGGAGUG 5
    317 2249 UUAUGCGGAUCAAACCUCACCAA AL-DP-4114 S 2126 5 AUGCGGAUCAAACCUCACCTT 3
    AS 2127 3 TTUACGCCUAGUUUGGAGUGG 5
    319 2250 AUGCGGAUCAAACCUCACCAAGG AL-DP-4002 S 2128 5 GCGGAUCAAACCUCACCAAGG 3
    AS 2129 3 UACGCCUAGUUUGGAGUGGUUCC 5
    AL-DP-4115 S 2130 5 GCGGAUCAAACCUCACCAA 3
    AS 2131 3 CGCCUAGUUUGGAGUGGUU 5
    AL-DP-4014 S 2132 5 GCGGAUCAAACCUCACCAATT 3
    AS 2133 3 TTCGCCUAGUUUGGAGUGGUU 5
    AL-DP-4119 S 2134 5 GCGGAUCAAACCUCACCAA 3
    AS 2135 3 CGCCUAGUUUGGAGUGGUU