US20160017337A1

US20160017337A1 - Compositions and Methods for Inhibiting Expression of Eg5 Gene

Info

Publication number: US20160017337A1
Application number: US14/720,685
Authority: US
Inventors: David Bumcrot; Pamela Tan; Hans-Peter Vornlocher; Anke Geick
Original assignee: Alnylam Pharmaceuticals Inc
Current assignee: Alnylam Pharmaceuticals Inc
Priority date: 2006-03-31
Filing date: 2015-05-22
Publication date: 2016-01-21
Also published as: ES2544861T3; NZ571568A; HK1126782A1; KR101362681B1; US7718629B2; US20070281899A1; IL194419A; IL231688A0; WO2007115168A3; US20120136145A1; KR20090010174A; WO2007115168A2; EA014886B1; SG170780A1; AU2007233109A1; JP5704741B2; KR20120055740A; US9057069B2; EP2527354A1; AU2007233109B2

Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Eg5 gene (Eg5 gene), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of the Eg5 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by Eg5 expression and the expression of the Eg5 gene using the pharmaceutical composition; and methods for inhibiting the expression of the Eg5 gene in a cell.

Description

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/797,176, filed Mar. 12, 2013 (allowed), which is a continuation of U.S. application Ser. No. 13/165,568, filed Jun. 21, 2011 (abandoned), which is a continuation of U.S. application Ser. No. 12/754,110, filed Apr. 5, 2010 (abandoned), which is a divisional of U.S. application Ser. No. 11/694,215, filed Mar. 30, 2007 (now U.S. Pat. No. 7,718,629, issued May 18, 2010) all which claim the benefit of U.S. Provisional Application No. 60/787,762, filed Mar. 31, 2006, and U.S. Provisional Application No. 60/870,259, filed Dec. 15, 2006. All prior applications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 16, 2015, is named 28983_US_CRF_sequencelisting.txt, and is 361,411 bytes in size.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of the Eg5 gene and the use of the dsRNA to treat pathological processes mediated by Eg5 expression, such as cancer, alone or in combination with a dsRNA targeting vascular endothelian growth factor (VEGF).

BACKGROUND OF THE INVENTION

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.
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.
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 small molecules that more specifically target proteins associated with proliferating cells.
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 a small molecule 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.
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.
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.
Despite significant advances in the field of RNAi and advances in the treatment of pathological processes mediated by Eg5 expression, there remains a need for an agent that can selectively and efficiently silence the Eg5 gene using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target Eg5 gene for use in treating pathological processes mediated by Eg5 expression.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the Eg5 gene in a cell or mammal using such dsRNA, alone or in combination with a dsRNA targeting VEGF. The invention also provides compositions and methods for treating pathological conditions and diseases caused by the expression of the Eg5 gene, such as in cancer. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the Eg5 gene.
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 gene. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding Eg5, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contacting with a cell expressing the Eg5, inhibits the expression of the Eg5 gene by at least 40%.
For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Tables 1-3 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1-3. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may 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 comprising nucleotide. Generally, such modified sequence will be based on a first sequence of said dsRNA selected from the group consisting of the sense sequences of Tables 1-3 and a second sequence selected from the group consisting of the antisense sequences of Tables 1-3.
In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is generally a mammalian cell, such as a human cell.
In another embodiment, the invention provides a pharmaceutical composition for inhibiting the expression of the Eg5 gene in an organism, generally a human subject, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier or delivery vehicle.
In another embodiment, the invention provides a method for inhibiting the expression of the Eg5 gene in a cell, comprising the following steps:

- (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a region of complementarity which is substantially complementary to at least a part of a mRNA encoding Eg5, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein the dsRNA, upon contact with a cell expressing the Eg5, inhibits expression of the Eg5 gene by at least 40%; and
- (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the Eg5 gene, thereby inhibiting expression of the Eg5 gene in the cell.

In another embodiment, the invention provides methods for treating, preventing or managing pathological processes mediated by Eg5 expression, e.g. cancer, comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.
In another embodiment, the invention provides vectors for inhibiting the expression of the Eg5 gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of the Eg5 gene in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
In a further embodiment, the invention provides the Eg5 dsRNA and the uses thereof as described above in combination with a second dsRNA targeting the VEGF mRNA. A combination of a dsRNA targeting Eg5 and a second dsRNA targeting VEGF provides complementary and synergiatic activity for treating hyperproliferative discords, particularly hepatic carcinoma.

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the Eg5 gene in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of the Eg5 gene using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The invention further provides this dsRNA in combination with a second dsRNA that inhibits the expression of the VEGF gene.
The dsRNAs of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of the Eg5 gene. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and or maintenance of cancer cells in mammals. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the Eg5 gene. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by Eg5 expression, e.g. cancer, by targeting a gene involved in mitotic division.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of the Eg5 gene, as well as compositions and methods for treating diseases and disorders caused by the expression of Eg5, such as cancer, alone or in combination with a second dsRNA targeting the VEGF gene. The pharmaceutical compositions of the invention comprise 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. As discussed above, such compositions can further include a second dsRNA targeting VEGF.
Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Eg5 gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Eg5 gene. The invention further provides the above pharmaceutical compositions further containing a second dsRNA designed to inhibit the expression of VEGF.

I. Definitions

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.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. 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. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “Eg5” refers to the human kinesin family member 11, which is also known as KIF11, Eg5, HKSP, KNSL1 or TRIPS. Eg5 sequence can be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeq ID number:NM_—004523.
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 gene, including mRNA that is a product of RNA processing of a primary transcription product.
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.
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, herein incorporated by reference).
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.
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.
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.
“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.
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.
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). 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.
The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having 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.
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.
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 are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
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.
“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.
The terms “silence” and “inhibit the expression of”, in as far as they refer to the Eg5 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 mRNA transcribed from the Eg5 gene which may be isolated from a first cell or group of cells in which the Eg5 gene is transcribed and which has or have been treated such that the expression of the Eg5 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
$\frac{(mRNA in control cells) - (mRNA in treated cells)}{(mRNA in control cells)} \cdot 100 %$
Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Eg5 gene transcription, e.g. the amount of protein encoded by the Eg5 gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g apoptosis. In principle, Eg5 gene silencing may 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.
For example, in certain instances, expression of the Eg5 gene (or VEGF gene) is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, the Eg5 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the Eg5 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. Tables 1-3 provides values for inhibition of expression using various Eg5 dsRNA molecules at various concentrations.
As used herein in the context of Eg5 expression, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by Eg5 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 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.
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 expression or an overt symptom of pathological processes mediated by Eg5 expression (alone or in combination with 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 expression, the patient's history and age, the stage of pathological processes mediated by Eg5 expression, and the administration of other anti-pathological processes mediated by Eg5 expression agents.
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.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. 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.
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)

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 gene (alone or incombinaton with a second dsRNA for inhibiting the expression of VEGF) 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 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 gene, inhibits the expression of said Eg5 gene by at least 40%. The dsRNA comprises two RNA 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 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. 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. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). 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. In a preferred embodiment, the Eg5 gene is the human Eg5 gene. In specific embodiments, the antisense strand of the dsRNA comprises the sense sequences 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 embodiments using a second dsRNA targeting VEGF, such agents are exemplified in the Examples and in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.
The dsRNA 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
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.
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 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.
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.
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. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. 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 may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs 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.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. 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.
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.
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
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.
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.
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.
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.
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 C.sub.1 to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)—O]_mCH₃, O(CH₂)—OCH₃, O(CH₂)—NH₂, O(CH₂)—CH₃, O(CH₂)—ONH₂, and O(CH₂)—ON[(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 C₁₀lower 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)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, 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 hereinbelow.
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.
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 cytosines, 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.
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.
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-S-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).
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.
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:DNAduplex. 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 deoxydsRNAs 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.
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-S-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.
Vector Encoded RNAi Agents
The dsRNA of the invention can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors of the invention comprise sequences encoding the dsRNA of the invention and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA of the invention to cells in vivo is discussed in more detail below.
dsRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
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.
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.
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.
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 comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
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.
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.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of the Eg5 gene, such as pathological processes mediated by Eg5 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery.
In another embodiment, such compositions will further comprise a second dsRNA that inhibits VEGF expression. dsRNA directed to VEGF are described in the Examples and in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080.
The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of the Eg5 gene (and VEGF expression when a second dsRNA is included). In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even 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.
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.
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 expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds of the invention. 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), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
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. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the dsRNAs of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred 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 of 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. Preferred fatty acids and esters include but are not limited 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 C_1-10alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.
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. Preferred oral formulations are those in which dsRNAs of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred 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. Preferred 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). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred 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 of 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. Particularly preferred 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. application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.
Compositions and formulations for parenteral, 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.
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. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
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.
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.
Emulsions
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m 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.
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).
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).
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.
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).
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.
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.
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.
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).
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.
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.
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.
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.
Liposomes
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.
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.
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.
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.
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.
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.
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
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).
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).
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.
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).
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).
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 G.sub.M1, 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).
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 G.sub.M1, 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 G.sub.M1 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).
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, 2C.sub.1215G, 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.
A limited 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 an dsRNA RNA. 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 dsRNA dsRNAs targeted to the raf gene.
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.
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).
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.
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.
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.
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.
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).
Penetration Enhancers
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.
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.
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).
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, C.sub.1-10 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).
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. The bile salts of the invention 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).
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). Chelating agents of the invention 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).
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).
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.
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.
Carriers
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 coadministration 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 extracirculatory 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 coadministered 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.
Excipients
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).
Pharmaceutically acceptable organic or inorganic excipient 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.
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.
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.
Other Components
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.
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.
Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
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 which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of 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 method of 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.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by Eg5 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.
Methods for Treating Diseases Caused by Expression of the Eg5 Gene
The invention relates in particular to the use of a dsRNA or a pharmaceutical composition prepared therefrom for the treatment of cancer, e.g., for inhibiting tumor growth and tumor metastasis. For example, the dsRNA or a pharmaceutical composition prepared therefrom may be used for the treatment of solid tumors, like breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, liver cancer, 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 an dsRNA according to the invention or a pharmaceutical composition prepared therefrom for inhibiting eg5 expression and/or for inhibiting accumulation of ascites fluid and pleural effusion in different types of cancer, e.g., breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, liver cancer, 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 effect on eg5 expression, an dsRNA according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
The invention furthermore relates to the use of an 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. Other embodiments include the use of a second dsRNA used to inhibit the expression of VEGF.
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, or another dsRNA used to inhibit the expression of VEGF. 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.
Methods for Inhibiting Expression of the Eg5 Gene
In yet another aspect, the invention provides a method for inhibiting the expression of the Eg5 gene in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target Eg5 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target Eg5 gene. Compositions and methods for inhibiting the expression of these Eg5 genes using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of the Eg5 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.
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

Gene Walking of the Eg5 Gene

Initial Screening Set
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:NM_—004523, was used.
siRNA duplexes cross-reactive to human and mouse Eg5 were designed. Twenty-four duplexes were synthesized for screening. (Table 1).
Expanded Screening Set
A second screening set was defined with 266 siRNAs targeting human EG5, as well as its rhesus monkey ortholog (Table 2). An expanded screening set was selected with 328 siRNA targeting human EG5, with no necessity to hit any EG5 mRNA of other species (Table 3).
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:NM_—004523.2, 4908 bp, and Rhesus EG5: XM_—001087644.1, 878 bp (only 5′ part of human EG5)
For identification of further rhesus EG5 sequences a mega blast search with the human sequence was conducted at NCBI against rhesus reference genome. The downloaded rhesus sequence and the hit regions in the blast hit were assembled to a rhesus consensus sequence with ˜92% identity to human EG5 over the full-length.
All possible 19mers were extracted from the human mRNA sequence, resulting in the pool of candidate target sites corresponding to 4890 (sense strand) sequences of human-reactive EG5 siRNAs.
Human-rhesus cross-reactivity as prerequisite for in silico selection of siRNAs for an initial screening set out of this candidate pool. To determine rhesus-reactive siRNAs, each candidate siRNA target site was searched for presence in the assembled rhesus sequence. Further, the predicted specificity of the siRNA as criterion for selection of out the pool of human-rhesus cross-reactive siRNAs, manifested by targeting human EG5 mRNA sequences, but not other human mRNAs.
The specificity of an siRNA can be expressed via its potential to target other genes, which are referred to as “off-target genes”.
For predicting the off-target potential of an siRNA, the following assumptions were made:

- 1) off-target potential of a strand can be deduced from the number and distribution of mismatches to an off-target
- 2) the most relevant off-target, that is the gene predicted to have the highest probability to be silenced due to tolerance of mismatches, determines the off-target potential of the strand
- 3) positions 2 to 9 (counting 5′ to 3′) of a strand (seed region) may contribute more to off-target potential than rest of sequence (that is non-seed and cleavage site region)
- 4) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage site region) may contribute more to off-target potential than non-seed region (that is positions 12 to 18, counting 5′ to 3′)
- 5) positions 1 and 19 of each strand are not relevant for off-target interactions
- 6) off-target potential can be expressed by the off-target score of the most relevant off-target, calculated based on number and position of mismatches of the strand to the most homologous region in the off-target gene considering assumptions 3 to 5
- 7) off-target potential of antisense and sense strand will be relevant, whereas potential abortion of sense strand activity by internal modifications introduced is likely

SiRNAs with low off-target potential were defined as preferable and assumed to be more specific.
In order to identify human EG5-specific siRNAs, all other human transcripts, which were all considered potential off-targets, were searched for potential target regions for human-rhesus cross-reactive 19mer sense strand sequences as well as complementary antisense strands. For this, the fastA algorithm was used to determine the most homologues hit region in each sequence of the human RefSeq database, which we assume to represent the comprehensive human transcriptome.
To rank all potential off-targets according to assumptions 3 to 5, and by this identify the most relevant off-target gene and its off-target score, fastA output files were analyzed further by a perl script.
The script extracted the following off-target properties for each 19mer input sequence and each off-target gene to calculate the off-target score:
Number of mismatches in non-seed region
Number of mismatches in seed region
Number of mismatches in cleavage site region
The off-target score was calculated by considering assumptions 3 to 5 as follows:
Off-target score=number of seed mismatches*10
+number of cleavage site mismatches*1.2
+number of non-seed mismatches*1
The most relevant off-target gene for each 19mer sequence was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as representative for the off-target potential of a strand.
For the screening set in Table 2, an off-target score of 3 or more for the antisense strand and 2 or more for the sense strand was chosen as prerequisite for selection of siRNAs, whereas all sequences containing 4 or more consecutive G's (poly-G sequences) were excluded. 266 human-rhesus cross-reactive sequences passing the specificity criterion, were selected based on this cut-off (see Table 2).
For definition of the expanded screening set the cross-reactivity to rhesus was disgarded, re-calculated the predicted specificity based on the newly available human RefSeq database and selected only those 328 non-poly-G siRNAs with off-target score of 2,2 or more for the antisense and sense strand (see Table 3).
For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine: u,c-2′-O-methyl nucleotides: s-phosphorothioate linkage.

TABLE 1

TABLE 1A

position	SEQ	sequence of	SEQ	sense	SEQ	antisense
in human	ID	total 23mer	ID	sequence	ID	sequence	duplex
access. #	NO:	target site	No:	(5′-3′)	No:	(5′-3′)	name

385-407	1244	ACCGAAGUGUUG	1	cGAAGuGuuGu	2	UUGGAcAAAcA	AL-DP-
		UUUGUCCAAUU		uuGuccAATsT		AcACUUCGTsT	6226

347-369	1245	UAUGGUGUUUGG	3	uGGuGuuuGGA	4	GuAGAUGCUCc	AL-DP-
		AGCAUCUACUA		GcAucuAcTsT		AAAcACcATsT	6227

1078-1100	1246	AAUCUAAACUAA	5	ucuAAAcuAAc	6	GGAUUCuAGUu	AL-DP-
		CUAGAAUCCUC		uAGAAuccTsT		AGUUuAGATsT	6228

1067-1089	1247	UCCUUAUCGAGA	7	cuuAucGAGAA	8	AGUUuAGAUUC	AL-DP-
		AUCUAAACUAA		ucuAAAcuTsT		UCGAuAAGTsT	6229

374-396	1248	GAUUGAUGUUUA	9	uuGAuGuuuAc	10	AcACUUCGGuA	AL-DP-
		CCGAAGUGUUG		cGAAGuGuTsT		AAcAUcAATsT	6230

205-227	1249	UGGUGAGAUGCA	11	GuGAGAuGcAG	12	uAAAUGGUCUG	AL-DP-
		GACCAUUUAAU		AccAuuuATsT		cAUCUcACTsT	6231

1176-1198	1250	ACUCUGAGUACA	13	ucuGAGuAcAu	14	AuAUUCcAAUG	AL-DP-
		UUGGAAUAUGC		uGGAAuAuTsT		uACUcAGATsT	6232

386-408	1251	CCGAAGUGUUGU	15	GAAGuGuuGuu	16	AUUGGAcAAAc	AL-DP-
		UUGUCCAAUUC		uGuccAAuTsT		AAcACUUCTsT	6233

416-438	1252	AGUUAUUAUGGG	17	uuAuuAuGGGc	18	cAAUuAuAGCC	AL-DP-
		CUAUAAUUGCA		uAuAAuuGTsT		cAuAAuAATsT	6234

485-507	1253	GGAAGGUGAAAG	19	AAGGuGAAAGG	20	UuAGGUGACCU	AL-DP-
		GUCACCUAAUG		ucAccuAATsT		UUcACCUUTsT	6235

476-498	1254	UUUUACAAUGGA	21	uuAcAAuGGAA	22	CUUUcACCUUC	AL-DP-
		AGGUGAAAGGU		GGuGAAAGTsT		cAUUGuAATsT	6236

486-508	1255	GAAGGUGAAAGG	23	AGGuGAAAGGu	24	AUuAGGUGACC	AL-DP-
		UCACCUAAUGA		cAccuAAuTsT		UUUcACCUTsT	6237

487-509	1256	AAGGUGAAAGGU	25	GGuGAAAGGuc	26	cAUuAGGUGAC	AL-DP-
		CACCUAAUGAA		AccuAAuGTsT		CUUUcACCTsT	6238

1066-1088	1257	UUCCUUAUCGAG	27	ccuuAucGAGA	28	GUUuAGAUUCU	AL-DP-
		AAUCUAAACUA		AucuAAAcTsT		CGAuAAGGTsT	6239

1256-1278	1258	AGCUCUUAUUAA	29	cucuuAuuAAG	30	GuAuACUCCUu	AL-DP-
		GGAGUAUACGG		GAGuAuAcTsT		AAuAAGAGTsT	6240

2329-2351	1259	CAGAGAGAUUCU	31	GAGAGAuucuG	32	CcAAAGcAcAG	AL-DP-
		GUGCUUUGGAG		uGcuuuGGTsT		AAUCUCUCTsT	6241

1077-1099	1260	GAAUCUAAACUA	33	AucuAAAcuAA	34	GAUUCuAGUuA	AL-DP-
		ACUAGAAUCCU		cuAGAAucTsT		GUUuAGAUTsT	6242

1244-1266	1261	ACUCACCAAAAA	35	ucAccAAAAAA	36	AuAAGAGCUUU	AL-DP-
		AGCUCUUAUUA		GcucuuAuTsT		UUUGGUGATsT	6243

637-659	1262	AAGAGCUUUUUG	37	GAGcuuuuuGA	38	uAAGAAGAUcA	AL-DP-
		AUCUUCUUAAU		ucuucuuATsT		AAAAGCUCTsT	6244

1117-1139	1263	GGCGUACAAGAA	39	cGuAcAAGAAc	40	UuAuAGAUGUU	AL-DP-
		CAUCUAUAAUU		AucuAuAATsT		CUUGuACGTsT	6245

373-395	1264	AGAUUGAUGUUU	41	AuuGAuGuuuA	42	cACUUCGGuAA	AL-DP-
		ACCGAAGUGUU		ccGAAGuGTsT		AcAUcAAUTsT	6246

1079-1101	1265	AUCUAAACUAAC	43	cuAAAcuAAcu	44	AGGAUUCuAGU	AL-DP-
		UAGAAUCCUCC		AGAAuccuTsT		uAGUUuAGTsT	6247

383-405	1266	UUACCGAAGUGU	45	AccGAAGuGuu	46	GGAcAAAcAAc	AL-DP-
		UGUUUGUCCAA		GuuuGuccTsT		ACUUCGGUTsT	6248

200-222	1267	GGUGGUGGUGAG	47	uGGuGGuGAGA	48	GGUCUGcAUCU	AL-DP-
		AUGCAGACCAU		uGcAGAccTsT		cACcACcATsT	6249

TABLE 1B

	single
	dose	SDs 2nd
	screen @	screen
	25 nM [%	(among
duplex	residual	quadru-
name	mRNA]	plicates)

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%

TABLE 2

TABLE 2A

position	SEQ	sequence of	SEQ	sense	SEQ	antisense
in human	ID	total 19mer	ID	sequence	ID	sequence	duplex
access. #	NO:	target site	NO:	(5′-3′)	NO:	(5′-3′)	name

829-847	1268	CAUACUCUAG	49	cAuAcucuAGu	50	UGGGAACGACu	AD-
		UCGUUCCCA		cGuucccATsT		AGAGuAUGTsT	12072

246-264	1269	AGCGCCCAUU	51	AGcGcccAuuc	52	CuACuAUUGAA	AD-
		CAAUAGUAG		AAuAGuAGTsT		UGGGCGCUTsT	12073

238-256	1270	GGAAAGCUAG	53	GGAAAGcuAGc	54	GAAUGGGCGCu	AD-
		CGCCCAUUC		GcccAuucTsT		AGCUUUCCTsT	12074

239-257	1271	GAAAGCUAGC	55	GAAAGcuAGcG	56	UGAAUGGGCGC	AD-
		GCCCAUUCA		cccAuucATsT		uAGCUUUCTsT	12075

878-896	1272	AGAAACUACG	57	AGAAAcuAcGA	58	UCcAUcAAUCG	AD-
		AUUGAUGGA		uuGAuGGATsT		uAGUUUCUTsT	12076

1064-1082	1273	UGUUCCUUAU	59	uGuuccuuAuc	60	AGAUUCUCGAu	AD-
		CGAGAAUCU		GAGAAucuTsT		AAGGAAcATsT	12077

3278-3296	1274	CAGAUUACCU	61	cAGAuuAccuc	62	GGCUCGcAGAG	AD-
		CUGCGAGCC		uGcGAGccTsT		GuAAUCUGTsT	12078

247-265	1275	GCGCCCAUUC	63	GcGcccAuucA	64	UCuACuAUUGA	AD-
		AAUAGUAGA		AuAGuAGATsT		AUGGGCGCTsT	12079

434-452	1276	UUGCACUAUC	65	uuGcAcuAucu	66	AuACGcAAAGA	AD-
		UUUGCGUAU		uuGcGuAuTsT		uAGUGcAATsT	12080

232-250	1277	CAGAGCGGAA	67	cAGAGcGGAAA	68	GCGCuAGCUUU	AD-
		AGCUAGCGC		GcuAGcGcTsT		CCGCUCUGTsT	12081

1831-1849	1278	AGACCUUAUU	69	AGAccuuAuuu	70	AGAUuACcAAA	AD-
		UGGUAAUCU		GGuAAucuTsT		uAAGGUCUTsT	12082

1105-1123	1279	AUUCUCUUGG	71	AuucucuuGGA	72	GuACGCCCUCc	AD-
		AGGGCGUAC		GGGcGuAcTsT		AAGAGAAUTsT	12083

536-554	1280	GGCUGGUAUA	73	GGcuGGuAuAA	74	ACGUGGAAUuA	AD-
		AUUCCACGU		uuccAcGuTsT		uACcAGCCTsT	12084

236-254	1281	GCGGAAAGCU	75	GcGGAAAGcuA	76	AUGGGCGCuAG	AD-
		AGCGCCCAU		GcGcccAuTsT		CUUUCCGCTsT	12085

435-453	1282	UGCACUAUCU	77	uGcAcuAucuu	78	cAuACGcAAAG	AD-
		UUGCGUAUG		uGcGuAuGTsT		AuAGUGcATsT	12086

541-559	1283	GUAUAAUUCC	79	GuAuAAuuccA	80	AGGGuACGUGG	AD-
		ACGUACCCU		cGuAcccuTsT		AAUuAuACTsT	12087

1076-1094	1284	AGAAUCUAAA	81	AGAAucuAAAc	82	UCuAGUuAGUU	AD-
		CUAACUAGA		uAAcuAGATsT		uAGAUUCUTsT	12088

1432-1450	1285	AGGAGCUGAA	83	AGGAGcuGAAu	84	GuAACCCuAUU	AD-
		UAGGGUUAC		AGGGuuAcTsT		cAGCUCCUTsT	12089

1821-1839	1286	GAAGUACAUA	85	GAAGuAcAuAA	86	AuAAGGUCUuA	AD-
		AGACCUUAU		GAccuuAuTsT		UGuACUUCTsT	12090

2126-2144	1287	GACAGUGGCC	87	GAcAGuGGccG	88	uAUCUuAUCGG	AD-
		GAUAAGAUA		AuAAGAuATsT		CcACUGUCTsT	12091

2373-2391	1288	AAACCACUUA	89	AAAccAcuuAG	90	GGAcACuACuA	AD-
		GUAGUGUCC		uAGuGuccTsT		AGUGGUUUTsT	12092

4026-4044	1289	UCCCUAGACU	91	ucccuAGAcuu	92	AAAuAGGGAAG	AD-
		UCCCUAUUU		cccuAuuuTsT		UCuAGGGATsT	12093

4030-4048	1290	UAGACUUCCC	93	uAGAcuucccu	94	AGCGAAAuAGG	AD-
		UAUUUCGCU		AuuucGcuTsT		GAAGUCuATsT	12094

144-162	1291	GCGUCGCAGC	95	GcGucGcAGcc	96	ACGAAUUUGGC	AD-
		CAAAUUCGU		AAAuucGuTsT		UGCGACGCTsT	12095

242-260	1292	AGCUAGCGCC	97	AGcuAGcGccc	98	uAUUGAAUGGG	AD-
		CAUUCAAUA		AuucAAuATsT		CGCuAGCUTsT	12096

879-897	1293	GAAACUACGA	99	GAAAcuAcGAu	100	CUCcAUcAAUC	AD-
		UUGAUGGAG		uGAuGGAGTsT		GuAGUUUCTsT	12097

2134-2152	1294	CCGAUAAGAU	101	ccGAuAAGAuA	102	UGAUCUUCuAU	AD-
		AGAAGAUCA		GAAGAucATsT		CUuAUCGGTsT	12098

245-263	1295	UAGCGCCCAU	103	uAGcGcccAuu	104	uACuAUUGAAU	AD-
		UCAAUAGUA		cAAuAGuATsT		GGGCGCuATsT	12099

444-462	1296	UUUGCGUAUG	105	uuuGcGuAuGG	106	cAGUUUGGCcA	AD-
		GCCAAACUG		ccAAAcuGTsT		uACGcAAATsT	12100

550-568	1297	CACGUACCCU	107	cAcGuAcccuu	108	AUUUGAUGAAG	AD-
		UCAUCAAAU		cAucAAAuTsT		GGuACGUGTsT	12101

442-460	1298	UCUUUGCGUA	109	ucuuuGcGuAu	110	GUUUGGCcAuA	AD-
		UGGCCAAAC		GGccAAAcTsT		CGcAAAGATsT	12102

386-404	1299	CCGAAGUGUU	111	ccGAAGuGuuG	112	UGGAcAAAcAA	AD-
		GUUUGUCCA		uuuGuccATsT		cACUUCGGTsT	12103

233-251	1300	AGAGCGGAAA	113	AGAGcGGAAAG	114	GGCGCuAGCUU	AD-
		GCUAGCGCC		cuAGcGccTsT		UCCGCUCUTsT	12104

243-261	1301	GCUAGCGCCC	115	GcuAGcGcccA	116	CuAUUGAAUGG	AD-
		AUUCAAUAG		uucAAuAGTsT		GCGCuAGCTsT	12105

286-304	1302	AAGUUAGUGU	117	AAGuuAGuGuA	118	CcAGUUCGuAc	AD-
		ACGAACUGG		cGAAcuGGTsT		ACuAACUUTsT	12106

294-312	1303	GUACGAACUG	119	GuAcGAAcuGG	120	CcAAUCCUCcA	AD-
		GAGGAUUGG		AGGAuuGGTsT		GUUCGuACTsT	12107

296-314	1304	ACGAACUGGA	121	AcGAAcuGGAG	122	AGCcAAUCCUC	AD-
		GGAUUGGCU		GAuuGGcuTsT		cAGUUCGUTsT	12108

373-391	1305	AGAUUGAUGU	123	AGAuuGAuGuu	124	CUUCGGuAAAc	AD-
		UUACCGAAG		uAccGAAGTsT		AUcAAUCUTsT	12109

422-440	1306	UAUGGGCUAU	125	uAuGGGcuAuA	126	AGUGcAAUuAu	AD-
		AAUUGCACU		AuuGcAcuTsT		AGCCcAuATsT	12110

441-459	1307	AUCUUUGCGU	127	AucuuuGcGuA	128	UUUGGCcAuAC	AD-
		AUGGCCAAA		uGGccAAATsT		GcAAAGAUTsT	12111

832-850	1308	ACUCUAGUCG	129	AcucuAGucGu	130	GAGUGGGAACG	AD-
		UUCCCACUC		ucccAcucTsT		ACuAGAGUTsT	12112

881-899	1309	AACUACGAUU	131	AAcuAcGAuuG	132	UUCUCcAUcAA	AD-
		GAUGGAGAA		AuGGAGAATsT		UCGuAGUUTsT	12113

975-993	1310	GAUAAGAGAG	133	GAuAAGAGAGc	134	CUUCCCGAGCU	AD-
		CUCGGGAAG		ucGGGAAGTsT		CUCUuAUCTsT	12114

1073-1091	1311	UCGAGAAUCU	135	ucGAGAAucuA	136	AGUuAGUUuAG	AD-
		AAACUAACU		AAcuAAcuTsT		AUUCUCGATsT	12115

1084-1102	1312	AACUAACUAG	137	AAcuAAcuAGA	138	UGGAGGAUUCu	AD-
		AAUCCUCCA		AuccuccATsT		AGUuAGUUTsT	12116

1691-1709	1313	GGAUCGUAAG	139	GGAucGuAAGA	140	AACUGCCUUCU	AD-
		AAGGCAGUU		AGGcAGuuTsT		uACGAUCCTsT	12117

1693-1711	1314	AUCGUAAGAA	141	AucGuAAGAAG	142	UcAACUGCCUU	AD-
		GGCAGUUGA		GcAGuuGATsT		CUuACGAUTsT	12118

1702-1720	1315	AGGCAGUUGA	143	AGGcAGuuGAc	144	UUGUGUUGGUc	AD-
		CCAACACAA		cAAcAcAATsT		AACUGCCUTsT	12119

2131-2149	1316	UGGCCGAUAA	145	uGGccGAuAAG	146	UCUUCuAUCUu	AD-
		GAUAGAAGA		AuAGAAGATsT		AUCGGCcATsT	12120

2412-2430	1317	UCUAAGGAUA	147	ucuAAGGAuAu	148	UGUUGACuAuA	AD-
		UAGUCAACA		AGucAAcATsT		UCCUuAGATsT	12121

2859-2877	1318	ACUAAGCUUA	149	AcuAAGcuuAA	150	GAAAGcAAUuA	AD-
		AUUGCUUUC		uuGcuuucTsT		AGCUuAGUTsT	12122

3294-3312	1319	GCCCAGAUCA	151	GcccAGAucAA	152	AUuAAAGGUUG	AD-
		ACCUUUAAU		ccuuuAAuTsT		AUCUGGGCTsT	12123

223-241	1320	UUAAUUUGGC	153	uuAAuuuGGcA	154	UUCCGCUCUGC	AD-
		AGAGCGGAA		GAGcGGAATsT		cAAAUuAATsT	12124

1070-1088	1321	UUAUCGAGAA	155	uuAucGAGAAu	156	uAGUUuAGAUU	AD-
		UCUAAACUA		cuAAAcuATsT		CUCGAuAATsT	12125

244-262	1322	CUAGCGCCCA	157	cuAGcGcccAu	158	ACuAUUGAAUG	AD-
		UUCAAUAGU		ucAAuAGuTsT		GGCGCuAGTsT	12126

257-275	1323	AAUAGUAGAA	159	AAuAGuAGAAu	160	AGGAUcAcAUU	AD-
		UGUGAUCCU		GuGAuccuTsT		CuACuAUUTsT	12127

277-295	1324	UACGAAAAGA	161	uAcGAAAAGAA	162	AcACuAACUUC	AD-
		AGUUAGUGU		GuuAGuGuTsT		UUUUCGuATsT	12128

284-302	1325	AGAAGUUAGU	163	AGAAGuuAGuG	164	AGUUCGuAcAC	AD-
		GUACGAACU		uAcGAAcuTsT		uAACUUCUTsT	12129

366-384	1326	ACUAAACAGA	165	AcuAAAcAGAu	166	AAAcAUcAAUC	AD-
		UUGAUGUUU		uGAuGuuuTsT		UGUUuAGUTsT	12130

443-461	1327	CUUUGCGUAU	167	cuuuGcGuAuG	168	AGUUUGGCcAu	AD-
		GGCCAAACU		GccAAAcuTsT		ACGcAAAGTsT	12131

504-522	1328	AAUGAAGAGU	169	AAuGAAGAGuA	170	CCcAGGuAuAC	AD-
		AUACCUGGG		uAccuGGGTsT		UCUUcAUUTsT	12132

543-561	1329	AUAAUUCCAC	171	AuAAuuccAcG	172	GAAGGGuACGU	AD-
		GUACCCUUC		uAcccuucTsT		GGAAUuAUTsT	12133

551-569	1330	ACGUACCCUU	173	AcGuAcccuuc	174	AAUUUGAUGAA	AD-
		CAUCAAAUU		AucAAAuuTsT		GGGuACGUTsT	12134

552-570	1331	CGUACCCUUC	175	cGuAcccuucA	176	AAAUUUGAUGA	AD-
		AUCAAAUUU		ucAAAuuuTsT		AGGGuACGTsT	12135

553-571	1332	GUACCCUUCA	177	GuAcccuucAu	178	AAAAUUUGAUG	AD-
		UCAAAUUUU		cAAAuuuuTsT		AAGGGuACTsT	12136

577-595	1333	AACUUACUGA	179	AAcuuAcuGAu	180	GuACcAUuAUc	AD-
		UAAUGGUAC		AAuGGuAcTsT		AGuAAGUUTsT	12137

602-620	1334	UUCAGUCAAA	181	uucAGucAAAG	182	cAGAGAcACUU	AD-
		GUGUCUCUG		uGucucuGTsT		UGACUGAATsT	12138

652-670	1335	UUCUUAAUCC	183	uucuuAAuccA	184	UcAGAUGAUGG	AD-
		AUCAUCUGA		ucAucuGATsT		AUuAAGAATsT	12139

747-765	1336	ACAGUACACA	185	AcAGuAcAcAA	186	cAUCCUUGUUG	AD-
		ACAAGGAUG		cAAGGAuGTsT		UGuACUGUTsT	12140

877-895	1337	AAGAAACUAC	187	AAGAAAcuAcG	188	CcAUcAAUCGu	AD-
		GAUUGAUGG		AuuGAuGGTsT		AGUUUCUUTsT	12141

880-898	1338	AAACUACGAU	189	AAAcuAcGAuu	190	UCUCcAUcAAU	AD-
		UGAUGGAGA		GAuGGAGATsT		CGuAGUUUTsT	12142

965-983	1339	UGGAGCUGUU	191	uGGAGcuGuuG	192	UCUCUuAUcAA	AD-
		GAUAAGAGA		AuAAGAGATsT		cAGCUCcATsT	12143

1086-1104	1340	CUAACUAGAA	193	cuAAcuAGAAu	194	CCUGGAGGAUU	AD-
		UCCUCCAGG		ccuccAGGTsT		CuAGUuAGTsT	12144

1191-1209	1341	GAAUAUGCUC	195	GAAuAuGcucA	196	UUGCUCuAUGA	AD-
		AUAGAGCAA		uAGAGcAATsT		GcAuAUUCTsT	12145

1195-1213	1342	AUGCUCAUAG	197	AuGcucAuAGA	198	UUCUUUGCUCu	AD-
		AGCAAAGAA		GcAAAGAATsT		AUGAGcAUTsT	12146

1412-1430	1343	AAAAAUUGGU	199	AAAAAuuGGuG	200	CUcAAcAGcAC	AD-
		GCUGUUGAG		cuGuuGAGTsT		cAAUUUUUTsT	12147

1431-1449	1344	GAGGAGCUGA	201	GAGGAGcuGAA	202	uAACCCuAUUc	AD-
		AUAGGGUUA		uAGGGuuATsT		AGCUCCUCTsT	12148

1433-1451	1345	GGAGCUGAAU	203	GGAGcuGAAuA	204	UGuAACCCuAU	AD-
		AGGGUUACA		GGGuuAcATsT		UcAGCUCCTsT	12149

1434-1452	1346	GAGCUGAAUA	205	GAGcuGAAuAG	206	CUGuAACCCuA	AD-
		GGGUUACAG		GGuuAcAGTsT		UUcAGCUCTsT	12150

1435-1453	1347	AGCUGAAUAG	207	AGcuGAAuAGG	208	UCUGuAACCCu	AD-
		GGUUACAGA		GuuAcAGATsT		AUUcAGCUTsT	12151

1436-1454	1348	GCUGAAUAGG	209	GcuGAAuAGGG	210	CUCUGuAACCC	AD-
		GUUACAGAG		uuAcAGAGTsT		uAUUcAGCTsT	12152

1684-1702	1349	CCAAACUGGA	211	ccAAAcuGGAu	212	UUCUuACGAUC	AD-
		UCGUAAGAA		cGuAAGAATsT		cAGUUUGGTsT	12153

1692-1710	1350	GAUCGUAAGA	213	GAucGuAAGAA	214	cAACUGCCUUC	AD-
		AGGCAGUUG		GGcAGuuGTsT		UuACGAUCTsT	12154

1833-1851	1351	ACCUUAUUUG	215	AccuuAuuuGG	216	GcAGAUuACcA	AD-
		GUAAUCUGC		uAAucuGcTsT		AAuAAGGUTsT	12155

1872-1890	1352	UUAGAUACCA	217	uuAGAuAccAu	218	CUGuAGuAAUG	AD-
		UUACUACAG		uAcuAcAGTsT		GuAUCuAATsT	12156

1876-1894	1353	AUACCAUUAC	219	AuAccAuuAcu	220	GCuACUGuAGu	AD-
		UACAGUAGC		AcAGuAGcTsT		AAUGGuAUTsT	12157

1883-1901	1354	UACUACAGUA	221	uAcuAcAGuAG	222	UCcAAGUGCuA	AD-
		GCACUUGGA		cAcuuGGATsT		CUGuAGuATsT	12158

1987-2005	1355	AAAGUAAAAC	223	AAAGuAAAAcu	224	UGuAGuAcAGU	AD-
		UGUACUACA		GuAcuAcATsT		UUuACUUUTsT	12159

2022-2040	1356	CUCAAGACUG	225	cucAAGAcuGA	226	UuAGAAGAUcA	AD-
		AUCUUCUAA		ucuucuAATsT		GUCUUGAGTsT	12160

2124-2142	1357	UUGACAGUGG	227	uuGAcAGuGGc	228	UCUuAUCGGCc	AD-
		CCGAUAAGA		cGAuAAGATsT		ACUGUcAATsT	12161

2125-2143	1358	UGACAGUGGC	229	uGAcAGuGGcc	230	AUCUuAUCGGC	AD-
		CGAUAAGAU		GAuAAGAuTsT		cACUGUcATsT	12162

2246-2264	1359	GCAAUGUGGA	231	GcAAuGuGGAA	232	AGUuAGGUUUC	AD-
		AACCUAACU		AccuAAcuTsT		cAcAUUGCTsT	12163

2376-2394	1360	CCACUUAGUA	233	ccAcuuAGuAG	234	CCUGGAcACuA	AD-
		GUGUCCAGG		uGuccAGGTsT		CuAAGUGGTsT	12164

2504-2522	1361	AGAAGGUACA	235	AGAAGGuAcAA	236	AACcAAUUUUG	AD-
		AAAUUGGUU		AAuuGGuuTsT		uACCUUCUTsT	12165

2852-2870	1362	UGGUUUGACU	237	uGGuuuGAcuA	238	AUuAAGCUuAG	AD-
		AAGCUUAAU		AGcuuAAuTsT		UcAAACcATsT	12166

2853-2871	1363	GGUUUGACUA	239	GGuuuGAcuAA	240	AAUuAAGCUuA	AD-
		AGCUUAAUU		GcuuAAuuTsT		GUcAAACCTsT	12167

3110-3128	1364	UCUAAGUCAA	241	ucuAAGucAAG	242	AGAUGGCUCUU	AD-
		GAGCCAUCU		AGccAucuTsT		GACUuAGATsT	12168

3764-3782	1365	UCAUCCCUAU	243	ucAucccuAuA	244	AAGUGAACuAu	AD-
		AGUUCACUU		GuucAcuuTsT		AGGGAUGATsT	12169

3765-3783	1366	CAUCCCUAUA	245	cAucccuAuAG	246	AAAGUGAACuA	AD-
		GUUCACUUU		uucAcuuuTsT		uAGGGAUGTsT	12170

4027-4045	1367	CCCUAGACUU	247	cccuAGAcuuc	248	GAAAuAGGGAA	AD-
		CCCUAUUUC		ccuAuuucTsT		GUCuAGGGTsT	12171

4031-4049	1368	AGACUUCCCU	249	AGAcuucccuA	250	AAGCGAAAuAG	AD-
		AUUUCGCUU		uuucGcuuTsT		GGAAGUCUTsT	12172

4082-4100	1369	UCACCAAACC	251	ucAccAAAccA	252	UCuAcAAAUGG	AD-
		AUUUGUAGA		uuuGuAGATsT		UUUGGUGATsT	12173

4272-4290	1370	UCCUUUAAGA	253	uccuuuAAGAG	254	AGUuAGGCCUC	AD-
		GGCCUAACU		GccuAAcuTsT		UuAAAGGATsT	12174

4275-4293	1371	UUUAAGAGGC	255	uuuAAGAGGcc	256	AUGAGUuAGGC	AD-
		CUAACUCAU		uAAcucAuTsT		CUCUuAAATsT	12175

4276-4294	1372	UUAAGAGGCC	257	uuAAGAGGccu	258	AAUGAGUuAGG	AD-
		UAACUCAUU		AAcucAuuTsT		CCUCUuAATsT	12176

4282-4300	1373	GGCCUAACUC	259	GGccuAAcucA	260	AGGGUGAAUGA	AD-
		AUUCACCCU		uucAcccuTsT		GUuAGGCCTsT	12177

4571-4589	1374	UGGUAUUUUU	261	uGGuAuuuuuG	262	UGCcAGAUcAA	AD-
		GAUCUGGCA		AucuGGcATsT		AAAuACcATsT	12178

4677-4695	1375	AGUUUAGUGU	263	AGuuuAGuGuG	264	AAACUUuAcAc	AD-
		GUAAAGUUU		uAAAGuuuTsT		ACuAAACUTsT	12179

152-170	1376	GCCAAAUUCG	265	GccAAAuucGu	266	CUUCGcAGACG	AD-
		UCUGCGAAG		cuGcGAAGTsT		AAUUUGGCTsT	12180

156-174	1377	AAUUCGUCUG	267	AAuucGucuGc	268	UCUUCUUCGcA	AD-
		CGAAGAAGA		GAAGAAGATsT		GACGAAUUTsT	12181

491-509	1378	UGAAAGGUCA	269	uGAAAGGucAc	270	UUcAUuAGGUG	AD-
		CCUAAUGAA		cuAAuGAATsT		ACCUUUcATsT	12182

215-233	1379	CAGACCAUUU	271	cAGAccAuuuA	272	UGCcAAAUuAA	AD-
		AAUUUGGCA		AuuuGGcATsT		AUGGUCUGTsT	12183

216-234	1380	AGACCAUUUA	273	AGAccAuuuAA	274	CUGCcAAAUuA	AD-
		AUUUGGCAG		uuuGGcAGTsT		AAUGGUCUTsT	12184

416-434	1381	AGUUAUUAUG	275	AGuuAuuAuGG	276	AUuAuAGCCcA	AD-
		GGCUAUAAU		GcuAuAAuTsT		uAAuAACUTsT	12185

537-555	1382	GCUGGUAUAA	277	GcuGGuAuAAu	278	uACGUGGAAUu	AD-
		UUCCACGUA		uccAcGuATsT		AuACcAGCTsT	12186

221-239	1383	AUUUAAUUUG	279	AuuuAAuuuGG	280	CCGCUCUGCcA	AD-
		GCAGAGCGG		cAGAGcGGTsT		AAUuAAAUTsT	12187

222-240	1384	UUUAAUUUGG	281	uuuAAuuuGGc	282	UCCGCUCUGCc	AD-
		CAGAGCGGA		AGAGcGGATsT		AAAUuAAATsT	12188

227-245	1385	UUUGGCAGAG	283	uuuGGcAGAGc	284	AGCUUUCCGCU	AD-
		CGGAAAGCU		GGAAAGcuTsT		CUGCcAAATsT	12189

476-494	1386	UUUUACAAUG	285	uuuuAcAAuGG	286	UUcACCUUCcA	AD-
		GAAGGUGAA		AAGGuGAATsT		UUGuAAAATsT	12190

482-500	1387	AAUGGAAGGU	287	AAuGGAAGGuG	288	UGACCUUUcAC	AD-
		GAAAGGUCA		AAAGGucATsT		CUUCcAUUTsT	12191

208-226	1388	UGAGAUGCAG	289	uGAGAuGcAGA	290	UuAAAUGGUCU	AD-
		ACCAUUUAA		ccAuuuAATsT		GcAUCUcATsT	12192

147-165	1389	UCGCAGCCAA	291	ucGcAGccAAA	292	cAGACGAAUUU	AD-
		AUUCGUCUG		uucGucuGTsT		GGCUGCGATsT	12193

426-444	1390	GGCUAUAAUU	293	GGcuAuAAuuG	294	AGAuAGUGcAA	AD-
		GCACUAUCU		cAcuAucuTsT		UuAuAGCCTsT	12194

2123-2141	1391	AUUGACAGUG	295	AuuGAcAGuGG	296	CUuAUCGGCcA	AD-
		GCCGAUAAG		ccGAuAAGTsT		CUGUcAAUTsT	12195

4029-4047	1392	CUAGACUUCC	297	cuAGAcuuccc	298	GCGAAAuAGGG	AD-
		CUAUUUCGC		uAuuucGcTsT		AAGUCuAGTsT	12196

438-456	1393	ACUAUCUUUG	299	AcuAucuuuGc	300	GGCcAuACGcA	AD-
		CGUAUGGCC		GuAuGGccTsT		AAGAuAGUTsT	12197

830-848	1394	AUACUCUAGU	301	AuAcucuAGuc	302	GUGGGAACGAC	AD-
		CGUUCCCAC		GuucccAcTsT		uAGAGuAUTsT	12198

876-894	1395	AAAGAAACUA	303	AAAGAAAcuAc	304	cAUcAAUCGuA	AD-
		CGAUUGAUG		GAuuGAuGTsT		GUUUCUUUTsT	12199

115-133	1396	GCCUUGAUUU	305	GccuuGAuuuu	306	CCCGCcAAAAA	AD-
		UUUGGCGGG		uuGGcGGGTsT		AUcAAGGCTsT	12200

248-266	1397	CGCCCAUUCA	307	cGcccAuucAA	308	UUCuACuAUUG	AD-
		AUAGUAGAA		uAGuAGAATsT		AAUGGGCGTsT	12201

1834-1852	1398	CCUUAUUUGG	309	ccuuAuuuGGu	310	AGcAGAUuACc	AD-
		UAAUCUGCU		AAucuGcuTsT		AAAuAAGGTsT	12202

3050-3068	1399	AGAGACAAUU	311	AGAGAcAAuuc	312	cAcAUCCGGAA	AD-
		CCGGAUGUG		cGGAuGuGTsT		UUGUCUCUTsT	12203

4705-4723	1400	UGACUUUGAU	313	uGAcuuuGAuA	314	AAUUuAGCuAU	AD-
		AGCUAAAUU		GcuAAAuuTsT		cAAAGUcATsT	12204

229-247	1401	UGGCAGAGCG	315	uGGcAGAGcGG	316	CuAGCUUUCCG	AD-
		GAAAGCUAG		AAAGcuAGTsT		CUCUGCcATsT	12205

234-252	1402	GAGCGGAAAG	317	GAGcGGAAAGc	318	GGGCGCuAGCU	AD-
		CUAGCGCCC		uAGcGcccTsT		UUCCGCUCTsT	12206

282-300	1403	AAAGAAGUUA	319	AAAGAAGuuAG	320	UUCGuAcACuA	AD-
		GUGUACGAA		uGuAcGAATsT		ACUUCUUUTsT	12207

433-451	1404	AUUGCACUAU	321	AuuGcAcuAuc	322	uACGcAAAGAu	AD-
		CUUUGCGUA		uuuGcGuATsT		AGUGcAAUTsT	12208

540-558	1405	GGUAUAAUUC	323	GGuAuAAuucc	324	GGGuACGUGGA	AD-
		CACGUACCC		AcGuAcccTsT		AUuAuACCTsT	12209

831-849	1406	UACUCUAGUC	325	uAcucuAGucG	326	AGUGGGAACGA	AD-
		GUUCCCACU		uucccAcuTsT		CuAGAGuATsT	12210

872-890	1407	UAUGAAAGAA	327	uAuGAAAGAAA	328	AAUCGuAGUUU	AD-
		ACUACGAUU		cuAcGAuuTsT		CUUUcAuATsT	12211

1815-1833	1408	AUGCUAGAAG	329	AuGcuAGAAGu	330	UCUuAUGuACU	AD-
		UACAUAAGA		AcAuAAGATsT		UCuAGcAUTsT	12212

1822-1840	1409	AAGUACAUAA	331	AAGuAcAuAAG	332	AAuAAGGUCUu	AD-
		GACCUUAUU		AccuuAuuTsT		AUGuACUUTsT	12213

3002-3020	1410	ACAGCCUGAG	333	AcAGccuGAGc	334	cAUuAAcAGCU	AD-
		CUGUUAAUG		uGuuAAuGTsT		cAGGCUGUTsT	12214

3045-3063	1411	AAAGAAGAGA	335	AAAGAAGAGAc	336	CCGGAAUUGUC	AD-
		CAAUUCCGG		AAuuccGGTsT		UCUUCUUUTsT	12215

3224-3242	1412	CACACUGGAG	337	cAcAcuGGAGA	338	UUuAGACCUCU	AD-
		AGGUCUAAA		GGucuAAATsT		CcAGUGUGTsT	12216

3226-3244	1413	CACUGGAGAG	339	cAcuGGAGAGG	340	ACUUuAGACCU	AD-
		GUCUAAAGU		ucuAAAGuTsT		CUCcAGUGTsT	12217

3227-3245	1414	ACUGGAGAGG	341	AcuGGAGAGGu	342	cACUUuAGACC	AD-
		UCUAAAGUG		cuAAAGuGTsT		UCUCcAGUTsT	12218

145-163	1415	CGUCGCAGCC	343	cGucGcAGccA	344	GACGAAUUUGG	AD-
		AAAUUCGUC		AAuucGucTsT		CUGCGACGTsT	12219

1700-1718	1416	GAAGGCAGUU	345	GAAGGcAGuuG	346	GUGUUGGUcAA	AD-
		GACCAACAC		AccAAcAcTsT		CUGCCUUCTsT	12220

4291-4309	1417	CAUUCACCCU	347	cAuucAcccuG	348	AACUCUGUcAG	AD-
		GACAGAGUU		AcAGAGuuTsT		GGUGAAUGTsT	12221

4278-4296	1418	AAGAGGCCUA	349	AAGAGGccuAA	350	UGAAUGAGUuA	AD-
		ACUCAUUCA		cucAuucATsT		GGCCUCUUTsT	12222

3051-3069	1419	GAGACAAUUC	351	GAGAcAAuucc	352	CcAcAUCCGGA	AD-
		CGGAUGUGG		GGAuGuGGTsT		AUUGUCUCTsT	12223

3058-3076	1420	UUCCGGAUGU	353	uuccGGAuGuG	354	UCuAcAUCcAc	AD-
		GGAUGUAGA		GAuGuAGATsT		AUCCGGAATsT	12224

241-259	1421	AAGCUAGCGC	355	AAGcuAGcGcc	356	AUUGAAUGGGC	AD-
		CCAUUCAAU		cAuucAAuTsT		GCuAGCUUTsT	12225

285-303	1422	GAAGUUAGUG	357	GAAGuuAGuGu	358	cAGUUCGuAcA	AD-
		UACGAACUG		AcGAAcuGTsT		CuAACUUCTsT	12226

542-560	1423	UAUAAUUCCA	359	uAuAAuuccAc	360	AAGGGuACGUG	AD-
		CGUACCCUU		GuAcccuuTsT		GAAUuAuATsT	12227

2127-2145	1424	ACAGUGGCCG	361	AcAGuGGccGA	362	CuAUCUuAUCG	AD-
		AUAAGAUAG		uAAGAuAGTsT		GCcACUGUTsT	12228

3760-3778	1425	UCUGUCAUCC	363	ucuGucAuccc	364	GAACuAuAGGG	AD-
		CUAUAGUUC		uAuAGuucTsT		AUGAcAGATsT	12229

3993-4011	1426	UUCUUGCUAU	365	uucuuGcuAuG	366	AcAcAAGUcAu	AD-
		GACUUGUGU		AcuuGuGuTsT		AGcAAGAATsT	12230

1696-1714	1427	GUAAGAAGGC	367	GuAAGAAGGcA	368	UGGUcAACUGC	AD-
		AGUUGACCA		GuuGAccATsT		CUUCUuACTsT	12231

2122-2140	1428	CAUUGACAGU	369	cAuuGAcAGuG	370	UuAUCGGCcAC	AD-
		GGCCGAUAA		GccGAuAATsT		UGUcAAUGTsT	12232

2371-2389	1429	AGAAACCACU	371	AGAAAccAcuu	372	AcACuACuAAG	AD-
		UAGUAGUGU		AGuAGuGuTsT		UGGUUUCUTsT	12233

3143-3161	1430	GGAUUGUUCA	373	GGAuuGuucAu	374	GCcAAUUGAUG	AD-
		UCAAUUGGC		cAAuuGGcTsT		AAcAAUCCTsT	12234

4277-4295	1431	UAAGAGGCCU	375	uAAGAGGccuA	376	GAAUGAGUuAG	AD-
		AACUCAUUC		AcucAuucTsT		GCCUCUuATsT	12235

287-305	1432	AGUUAGUGUA	377	AGuuAGuGuAc	378	UCcAGUUCGuA	AD-
		CGAACUGGA		GAAcuGGATsT		cACuAACUTsT	12236

1823-1841	1433	AGUACAUAAG	379	AGuAcAuAAGA	380	AAAuAAGGUCU	AD-
		ACCUUAUUU		ccuuAuuuTsT		uAUGuACUTsT	12237

3379-3397	1434	UGAGCCUUGU	381	uGAGccuuGuG	382	AAUCuAuAcAc	AD-
		GUAUAGAUU		uAuAGAuuTsT		AAGGCUcATsT	12238

4273-4291	1435	CCUUUAAGAG	383	ccuuuAAGAGG	384	GAGUuAGGCCU	AD-
		GCCUAACUC		ccuAAcucTsT		CUuAAAGGTsT	12239

2375-2393	1436	ACCACUUAGU	385	AccAcuuAGuA	386	CUGGAcACuAC	AD-
		AGUGUCCAG		GuGuccAGTsT		uAAGUGGUTsT	12240

4439-4457	1437	GAAACUUCCA	387	GAAAcuuccAA	388	AGAcAuAAUUG	AD-
		AUUAUGUCU		uuAuGucuTsT		GAAGUUUCTsT	12241

827-845	1438	UGCAUACUCU	389	uGcAuAcucuA	390	GGAACGACuAG	AD-
		AGUCGUUCC		GucGuuccTsT		AGuAUGcATsT	12242

1699-1717	1439	AGAAGGCAGU	391	AGAAGGcAGuu	392	UGUUGGUcAAC	AD-
		UGACCAACA		GAccAAcATsT		UGCCUUCUTsT	12243

1824-1842	1440	GUACAUAAGA	393	GuAcAuAAGAc	394	cAAAuAAGGUC	AD-
		CCUUAUUUG		cuuAuuuGTsT		UuAUGuACTsT	12244

429-447	1441	UAUAAUUGCA	395	uAuAAuuGcAc	396	cAAAGAuAGUG	AD-
		CUAUCUUUG		uAucuuuGTsT		cAAUuAuATsT	12245

856-874	1442	UCUCUGUUAC	397	ucucuGuuAcA	398	AuAUGuAUUGu	AD-
		AAUACAUAU		AuAcAuAuTsT		AAcAGAGATsT	12246

1194-1212	1443	UAUGCUCAUA	399	uAuGcucAuAG	400	UCUUUGCUCuA	AD-
		GAGCAAAGA		AGcAAAGATsT		UGAGcAuATsT	12247

392-410	1444	UGUUGUUUGU	401	uGuuGuuuGuc	402	cAGAAUUGGAc	AD-
		CCAAUUCUG		cAAuucuGTsT		AAAcAAcATsT	12248

1085-1103	1445	ACUAACUAGA	403	AcuAAcuAGAA	404	CUGGAGGAUUC	AD-
		AUCCUCCAG		uccuccAGTsT		uAGUuAGUTsT	12249

2069-2087	1446	UGUGGUGUCU	405	uGuGGuGucuA	406	UUUcAGuAuAG	AD-
		AUACUGAAA		uAcuGAAATsT		AcACcAcATsT	12250

4341-4359	1447	UAUUAUGGGA	407	uAuuAuGGGAG	408	UGGGUGGUCUC	AD-
		GACCACCCA		AccAcccATsT		CcAuAAuATsT	12251

759-777	1448	AAGGAUGAAG	409	AAGGAuGAAGu	410	UUUGAuAGACU	AD-
		UCUAUCAAA		cuAucAAATsT		UcAUCCUUTsT	12252

973-991	1449	UUGAUAAGAG	411	uuGAuAAGAGA	412	UCCCGAGCUCU	AD-
		AGCUCGGGA		GcucGGGATsT		CUuAUcAATsT	12253

1063-1081	1450	AUGUUCCUUA	413	AuGuuccuuAu	414	GAUUCUCGAuA	AD-
		UCGAGAAUC		cGAGAAucTsT		AGGAAcAUTsT	12254

1190-1208	1451	GGAAUAUGCU	415	GGAAuAuGcuc	416	UGCUCuAUGAG	AD-
		CAUAGAGCA		AuAGAGcATsT		cAuAUUCCTsT	12255

1679-1697	1452	CCAUUCCAAA	417	ccAuuccAAAc	418	ACGAUCcAGUU	AD-
		CUGGAUCGU		uGGAucGuTsT		UGGAAUGGTsT	12256

1703-1721	1453	GGCAGUUGAC	419	GGcAGuuGAcc	420	AUUGUGUUGGU	AD-
		CAACACAAU		AAcAcAAuTsT		cAACUGCCTsT	12257

1814-1832	1454	CAUGCUAGAA	421	cAuGcuAGAAG	422	CUuAUGuACUU	AD-
		GUACAUAAG		uAcAuAAGTsT		CuAGcAUGTsT	12258

1818-1836	1455	CUAGAAGUAC	423	cuAGAAGuAcA	424	AGGUCUuAUGu	AD-
		AUAAGACCU		uAAGAccuTsT		ACUUCuAGTsT	12259

1897-1915	1456	UUGGAUCUCU	425	uuGGAucucuc	426	AuAGAUGUGAG	AD-
		CACAUCUAU		AcAucuAuTsT		AGAUCcAATsT	12260

2066-2084	1457	AACUGUGGUG	427	AAcuGuGGuGu	428	cAGuAuAGAcA	AD-
		UCUAUACUG		cuAuAcuGTsT		CcAcAGUUTsT	12261

2121-2139	1458	UCAUUGACAG	429	ucAuuGAcAGu	430	uAUCGGCcACU	AD-
		UGGCCGAUA		GGccGAuATsT		GUcAAUGATsT	12262

2280-2298	1459	AUAAAGCAGA	431	AuAAAGcAGAc	432	GGGAAUGGGUC	AD-
		CCCAUUCCC		ccAuucccTsT		UGCUUuAUTsT	12263

2369-2387	1460	ACAGAAACCA	433	AcAGAAAccAc	434	ACuACuAAGUG	AD-
		CUUAGUAGU		uuAGuAGuTsT		GUUUCUGUTsT	12264

2372-2390	1461	GAAACCACUU	435	GAAAccAcuuA	436	GAcACuACuAA	AD-
		AGUAGUGUC		GuAGuGucTsT		GUGGUUUCTsT	12265

2409-2427	1462	AAAUCUAAGG	437	AAAucuAAGGA	438	UGACuAuAUCC	AD-
		AUAUAGUCA		uAuAGucATsT		UuAGAUUUTsT	12266

2933-2951	1463	UUAUUUAUAC	439	uuAuuuAuAcc	440	UGUUGAUGGGu	AD-
		CCAUCAACA		cAucAAcATsT		AuAAAuAATsT	12267

3211-3229	1464	ACAGAGGCAU	441	AcAGAGGcAuu	442	AGUGUGUuAAU	AD-
		UAACACACU		AAcAcAcuTsT		GCCUCUGUTsT	12268

3223-3241	1465	ACACACUGGA	443	AcAcAcuGGAG	444	UuAGACCUCUC	AD-
		GAGGUCUAA		AGGucuAATsT		cAGUGUGUTsT	12269

3225-3243	1466	ACACUGGAGA	445	AcAcuGGAGAG	446	CUUuAGACCUC	AD-
		GGUCUAAAG		GucuAAAGTsT		UCcAGUGUTsT	12270

3291-3309	1467	CGAGCCCAGA	447	cGAGcccAGAu	448	AAAGGUUGAUC	AD-
		UCAACCUUU		cAAccuuuTsT		UGGGCUCGTsT	12271

4036-4054	1468	UCCCUAUUUC	449	ucccuAuuucG	450	GGAGAAAGCGA	AD-
		GCUUUCUCC		cuuucuccTsT		AAuAGGGATsT	12272

4180-4198	1469	UCUAAAAUCA	451	ucuAAAAucAc	452	UGUUGAcAGUG	AD-
		CUGUCAACA		uGucAAcATsT		AUUUuAGATsT	12273

151-169	1470	AGCCAAAUUC	453	AGccAAAuucG	454	UUCGcAGACGA	AD-
		GUCUGCGAA		ucuGcGAATsT		AUUUGGCUTsT	12274

250-268	1471	CCCAUUCAAU	455	cccAuucAAuA	456	cAUUCuACuAU	AD-
		AGUAGAAUG		GuAGAAuGTsT		UGAAUGGGTsT	12275

821-839	1472	GAUGAAUGCA	457	GAuGAAuGcAu	458	ACuAGAGuAUG	AD-
		UACUCUAGU		AcucuAGuTsT		cAUUcAUCTsT	12276

1060-1078	1473	CUCAUGUUCC	459	cucAuGuuccu	460	UCUCGAuAAGG	AD-
		UUAUCGAGA		uAucGAGATsT		AAcAUGAGTsT	12277

1075-1093	1474	GAGAAUCUAA	461	GAGAAucuAAA	462	CuAGUuAGUUu	AD-
		ACUAACUAG		cuAAcuAGTsT		AGAUUCUCTsT	12278

1819-1837	1475	UAGAAGUACA	463	uAGAAGuAcAu	464	AAGGUCUuAUG	AD-
		UAAGACCUU		AAGAccuuTsT		uACUUCuATsT	12279

3003-3021	1476	CAGCCUGAGC	465	cAGccuGAGcu	466	UcAUuAAcAGC	AD-
		UGUUAAUGA		GuuAAuGATsT		UcAGGCUGTsT	12280

3046-3064	1477	AAGAAGAGAC	467	AAGAAGAGAcA	468	UCCGGAAUUGU	AD-
		AAUUCCGGA		AuuccGGATsT		CUCUUCUUTsT	12281

3134-3152	1478	UGCUGGUGUG	469	uGcuGGuGuGG	470	UGAAcAAUCcA	AD-
		GAUUGUUCA		AuuGuucATsT		cACcAGcATsT	12282

155-173	1479	AAAUUCGUCU	471	AAAuucGucuG	472	CUUCUUCGcAG	AD-
		GCGAAGAAG		cGAAGAAGTsT		ACGAAUUUTsT	12283

4596-4614	1480	UUUCUGGAAG	473	uuucuGGAAGu	474	AcAUCUcAACU	AD-
		UUGAGAUGU		uGAGAuGuTsT		UCcAGAAATsT	12284

365-383	1481	UACUAAACAG	475	uAcuAAAcAGA	476	AAcAUcAAUCU	AD-
		AUUGAUGUU		uuGAuGuuTsT		GUUuAGuATsT	12285

374-392	1482	GAUUGAUGUU	477	GAuuGAuGuuu	478	ACUUCGGuAAA	AD-
		UACCGAAGU		AccGAAGuTsT		cAUcAAUCTsT	12286

436-454	1483	GCACUAUCUU	479	GcAcuAucuuu	480	CcAuACGcAAA	AD-
		UGCGUAUGG		GcGuAuGGTsT		GAuAGUGCTsT	12287

539-557	1484	UGGUAUAAUU	481	uGGuAuAAuuc	482	GGuACGUGGAA	AD-
		CCACGUACC		cAcGuAccTsT		UuAuACcATsT	12288

1629-1647	1485	AGCAAGCUGC	483	AGcAAGcuGcu	484	CUGUGUuAAGc	AD-
		UUAACACAG		uAAcAcAGTsT		AGCUUGCUTsT	12289

2370-2388	1486	CAGAAACCAC	485	cAGAAAccAcu	486	cACuACuAAGU	AD-
		UUAGUAGUG		uAGuAGuGTsT		GGUUUCUGTsT	12290

2676-2694	1487	AACUUAUUGG	487	AAcuuAuuGGA	488	UuAcAACCUCc	AD-
		AGGUUGUAA		GGuuGuAATsT		AAuAAGUUTsT	12291

3228-3246	1488	CUGGAGAGGU	489	cuGGAGAGGuc	490	CcACUUuAGAC	AD-
		CUAAAGUGG		uAAAGuGGTsT		CUCUCcAGTsT	12292

3703-3721	1489	AAAAAAGAUA	491	AAAAAAGAuAu	492	ACUGCCUuAuA	AD-
		UAAGGCAGU		AAGGcAGuTsT		UCUUUUUUTsT	12293

3737-3755	1490	GAAUUUUGAU	493	GAAuuuuGAuA	494	UGGGuAGAuAU	AD-
		AUCUACCCA		ucuAcccATsT		cAAAAUUCTsT	12294

4573-4591	1491	GUAUUUUUGA	495	GuAuuuuuGAu	496	GUUGCcAGAUc	AD-
		UCUGGCAAC		cuGGcAAcTsT		AAAAAuACTsT	12295

526-544	1492	AGGAUCCCUU	497	AGGAucccuuG	498	AuACcAGCcAA	AD-
		GGCUGGUAU		GcuGGuAuTsT		GGGAUCCUTsT	12296

527-545	1493	GGAUCCCUUG	499	GGAucccuuGG	500	uAuACcAGCcA	AD-
		GCUGGUAUA		cuGGuAuATsT		AGGGAUCCTsT	12297

256-274	1494	CAAUAGUAGA	501	cAAuAGuAGAA	502	GGAUcAcAUUC	AD-
		AUGUGAUCC		uGuGAuccTsT		uACuAUUGTsT	12298

427-445	1495	GCUAUAAUUG	503	GcuAuAAuuGc	504	AAGAuAGUGcA	AD-
		CACUAUCUU		AcuAucuuTsT		AUuAuAGCTsT	12299

554-572	1496	UACCCUUCAU	505	uAcccuucAuc	506	AAAAAUUUGAU	AD-
		CAAAUUUUU		AAAuuuuuTsT		GAAGGGuATsT	12300

1210-1228	1497	AGAACAUAUU	507	AGAAcAuAuuG	508	GGCUuAUUcAA	AD-
		GAAUAAGCC		AAuAAGccTsT		uAUGUUCUTsT	12301

1414-1432	1498	AAAUUGGUGC	509	AAAuuGGuGcu	510	UCCUcAAcAGc	AD-
		UGUUGAGGA		GuuGAGGATsT		ACcAAUUUTsT	12302

1438-1456	1499	UGAAUAGGGU	511	uGAAuAGGGuu	512	AACUCUGuAAC	AD-
		UACAGAGUU		AcAGAGuuTsT		CCuAUUcATsT	12303

1516-1534	1500	AAGAACUUGA	513	AAGAAcuuGAA	514	UGAGUGGUUUc	AD-
		AACCACUCA		AccAcucATsT		AAGUUCUUTsT	12304

2279-2297	1501	AAUAAAGCAG	515	AAuAAAGcAGA	516	GGAAUGGGUCU	AD-
		ACCCAUUCC		cccAuuccTsT		GCUUuAUUTsT	12305

2939-2957	1502	AUACCCAUCA	517	AuAcccAucAA	518	uACcAGUGUUG	AD-
		ACACUGGUA		cAcuGGuATsT		AUGGGuAUTsT	12306

3142-3160	1503	UGGAUUGUUC	519	uGGAuuGuucA	520	CcAAUUGAUGA	AD-
		AUCAAUUGG		ucAAuuGGTsT		AcAAUCcATsT	12307

3229-3247	1504	UGGAGAGGUC	521	uGGAGAGGucu	522	UCcACUUuAGA	AD-
		UAAAGUGGA		AAAGuGGATsT		CCUCUCcATsT	12308

3763-3781	1505	GUCAUCCCUA	523	GucAucccuAu	524	AGUGAACuAuA	AD-
		UAGUUCACU		AGuucAcuTsT		GGGAUGACTsT	12309

4801-4819	1506	AUAAUGGCUA	525	AuAAuGGcuAu	526	GAGAAAUuAuA	AD-
		UAAUUUCUC		AAuuucucTsT		GCcAUuAUTsT	12310

529-547	1507	AUCCCUUGGC	527	AucccuuGGcu	528	AUuAuACcAGC	AD-
		UGGUAUAAU		GGuAuAAuTsT		cAAGGGAUTsT	12311

425-443	1508	GGGCUAUAAU	529	GGGcuAuAAuu	530	GAuAGUGcAAU	AD-
		UGCACUAUC		GcAcuAucTsT		uAuAGCCCTsT	12312

1104-1122	1509	GAUUCUCUUG	531	GAuucucuuGG	532	uACGCCCUCcA	AD-
		GAGGGCGUA		AGGGcGuATsT		AGAGAAUCTsT	12313

1155-1173	1510	GCAUCUCUCA	533	GcAucucucAA	534	CCUcAAGAUUG	AD-
		AUCUUGAGG		ucuuGAGGTsT		AGAGAUGCTsT	12314

2403-2421	1511	CAGCAGAAAU	535	cAGcAGAAAuc	536	uAUCCUuAGAU	AD-
		CUAAGGAUA		uAAGGAuATsT		UUCUGCUGTsT	12315

3115-3133	1512	GUCAAGAGCC	537	GucAAGAGccA	538	UCuAcAGAUGG	AD-
		AUCUGUAGA		ucuGuAGATsT		CUCUUGACTsT	12316

3209-3227	1513	AAACAGAGGC	539	AAAcAGAGGcA	540	UGUGUuAAUGC	AD-
		AUUAACACA		uuAAcAcATsT		CUCUGUUUTsT	12317

3293-3311	1514	AGCCCAGAUC	541	AGcccAGAucA	542	UuAAAGGUUGA	AD-
		AACCUUUAA		AccuuuAATsT		UCUGGGCUTsT	12318

4574-4592	1515	UAUUUUUGAU	543	uAuuuuuGAuc	544	GGUUGCcAGAU	AD-
		CUGGCAACC		uGGcAAccTsT		cAAAAAuATsT	12319

352-370	1516	UGUUUGGAGC	545	uGuuuGGAGcA	546	UuAGuAGAUGC	AD-
		AUCUACUAA		ucuAcuAATsT		UCcAAAcATsT	12320

741-759	1517	GAAAUUACAG	547	GAAAuuAcAGu	548	UGUUGUGuACU	AD-
		UACACAACA		AcAcAAcATsT		GuAAUUUCTsT	12321

1478-1496	1518	ACUUGACCAG	549	AcuuGAccAGu	550	AGAUUuAcACU	AD-
		UGUAAAUCU		GuAAAucuTsT		GGUcAAGUTsT	12322

1483-1501	1519	ACCAGUGUAA	551	AccAGuGuAAA	552	AGGUcAGAUUu	AD-
		AUCUGACCU		ucuGAccuTsT		AcACUGGUTsT	12323

1967-1985	1520	AGAACAAUCA	553	AGAAcAAucAu	554	UGCUGCuAAUG	AD-
		UUAGCAGCA		uAGcAGcATsT		AUUGUUCUTsT	12324

2247-2265	1521	CAAUGUGGAA	555	cAAuGuGGAAA	556	cAGUuAGGUUU	AD-
		ACCUAACUG		ccuAAcuGTsT		CcAcAUUGTsT	12325

2500-2518	1522	ACCAAGAAGG	557	AccAAGAAGGu	558	AAUUUUGuACC	AD-
		UACAAAAUU		AcAAAAuuTsT		UUCUUGGUTsT	12326

2508-2526	1523	GGUACAAAAU	559	GGuAcAAAAuu	560	CUUcAACcAAU	AD-
		UGGUUGAAG		GGuuGAAGTsT		UUUGuACCTsT	12327

3138-3156	1524	GGUGUGGAUU	561	GGuGuGGAuuG	562	UUGAUGAAcAA	AD-
		GUUCAUCAA		uucAucAATsT		UCcAcACCTsT	12328

4304-4322	1525	AGAGUUCACA	563	AGAGuucAcAA	564	UGGGCUUUUUG	AD-
		AAAAGCCCA		AAAGcccATsT		UGAACUCUTsT	12329

4711-4729	1526	UGAUAGCUAA	565	uGAuAGcuAAA	566	UGGUUuAAUUu	AD-
		AUUAAACCA		uuAAAccATsT		AGCuAUcATsT	12330

1221-1239	1527	AAUAAGCCUG	567	AAuAAGccuGA	568	GAUUcACUUcA	AD-
		AAGUGAAUC		AGuGAAucTsT		GGCUuAUUTsT	12331

1705-1723	1528	CAGUUGACCA	569	cAGuuGAccAA	570	GcAUUGUGUUG	AD-
		ACACAAUGC		cAcAAuGcTsT		GUcAACUGTsT	12332

3137-3155	1529	UGGUGUGGAU	571	uGGuGuGGAuu	572	UGAUGAAcAAU	AD-
		UGUUCAUCA		GuucAucATsT		CcAcACcATsT	12333

4292-4310	1530	AUUCACCCUG	573	AuucAcccuGA	574	GAACUCUGUcA	AD-
		ACAGAGUUC		cAGAGuucTsT		GGGUGAAUTsT	12334

1829-1847	1531	UAAGACCUUA	575	uAAGAccuuAu	576	AUUACcAAAuA	AD-
		UUUGGUAAU		uuGGuAAuTsT		AGGUCUuATsT	12335

2244-2262	1532	AAGCAAUGUG	577	AAGcAAuGuGG	578	UuAGGUUUCcA	AD-
		GAAACCUAA		AAAccuAATsT		cAUUGCUUTsT	12336

2888-2906	1533	UCUGAAACUG	579	ucuGAAAcuGG	580	UGGGAuAUCcA	AD-
		GAUAUCCCA		AuAucccATsT		GUUUcAGATsT	12337

TABLE 2B

	1st single	SDs 1st	2nd single	SDs 2nd		SDs 3^rd
	dose screen	screen	dose screen	screen		screen
	@ 50 nM	(among	@ 25 nM	(among	3rd single	(among
duplex	[% resudual	quadru-	[% resudual	quadru-	dose screen	quadru-
name	mRNA]	plicates)	mRNA]	plicates)	@ 25 nM	plicates)

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%

TABLE 3

					single	SDs
					dose	2nd
					screen @	screen
	SEQ		SEQ		25 nM [%	(among
	ID		ID	duplex	residual	quadru-
sequence (5′-3′)	NO.	sequence (5′-3′)	NO.	name	mRNA]	plicates)

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%

dsRNA Synthesis

Source of Reagents
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.
siRNA Synthesis
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).
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.
For 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

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

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

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-[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}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

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

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

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

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

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 stirred 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.
Cholesterol Derivatised CPG AI
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).
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.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 4.

TABLE 4

Abbreviations of nucleotide monomers used in nucleic
acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are
mutually linked by 5′-3′-phosphodiester bonds.

Abbreviation^a	Nucleotide(s)

A, a	2′-deoxy-adenosine-5′-phosphate, adenosine-5′-
	phosphate
C, c	2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate
G, g	2′-deoxy-guanosine-5′-phosphate, guanosine-5′-
	phosphate
T, t	2′-deoxy-thymidine-5′-phosphate, thymidine-5′-
	phosphate
U, u	2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate
N, n	any 2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g,
	a, c or u)
Am	2′-O-methyladenosine-5′-phosphate
Cm	2′-O-methylcytidine-5′-phosphate
Gm	2′-O-methylguanosine-5′-phosphate
Tm	2′-O-methyl-thymidine-5′-phosphate
Um	2′-O-methyluridine-5′-phosphate
Af	2′-fluoro-2′-deoxy-adenosine-5′-phosphate
Cf	2′-fluoro-2′-deoxy-cytidine-5′-phosphate
Gf	2′-fluoro-2′-deoxy-guanosine-5′-phosphate
Tf	2′-fluoro-2′-deoxy-thymidine-5′-phosphate
Uf	2′-fluoro-2′-deoxy-uridine-5′-phosphate
A, C, G, T, U, a,	underlined: nucleoside-5′-phosphorothioate
c, g, t, u
am, cm, gm, tm,	underlined: 2-O-methyl-nucleoside-5′-phosphorothioate
um

^acapital letters represent 2′-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)

dsRNA Expression Vectors

In another aspect of the invention, Eg5 specific dsRNA molecules that modulate Eg5 gene expression activity 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).
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.
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. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. 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.
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)).
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-D 1-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.
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 multiple Eg5 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.
The Eg5 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 comprise 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.

Eg5 siRNA In Vitro Screening Via Cell Proliferation

As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, et al[2002] Biotechniques 33: 1244-8), a cell viability assay was used for siRNA activity screening. HeLa cells (14000 per well [Screens 1 and 3] or 10000 per well [Screen2])) were seeded in 96-well plates and simultaneously transfected with Lipofectamine 2000 (Invitrogen) at a final siRNA concentration in the well of 30 nM and at final concentrations of 50 nM (1^stscreen) and 25 nM (2^ndscreen). A subset of duplexes was tested at 25 nM in a third screen (Table 5).
Seventy-two hours post-transfection, cell proliferation was assayed the addition of WST-1 reagent (Roche) to the culture medium, and subsequent absorbance measurement at 450 nm. The absorbance value for control (non-transfected) cells was considered 100 percent, and absorbances for the siRNA transfected wells were compared to the control value. Assays were performed in sextuplicate for each of three screens. A subset of the siRNAs was further tested at a range of siRNA concentrations. Assays were performed in HeLa cells (14000 per well; method same as above, Table 5).

	TABLE 5

	Relative absorbance at 450 nm

Screen I

Screen II

Screen III

	Duplex	mean	sd	Mean	sd	mean	Sd

AL-DP-6226	20	10	28	11	43	9
AL-DP-6227	66	27	96	41	108	33
AL-DP-6228	56	28	76	22	78	18
AL-DP-6229	17	3	31	9	48	13
AL-DP-6230	48	8	75	11	73	7
AL-DP-6231	8	1	21	4	41	10
AL-DP-6232	16	2	37	7	52	14
AL-DP-6233	31	9	37	6	49	12
AL-DP-6234	103	40	141	29	164	45
AL-DP-6235	107	34	140	27	195	75
AL-DP-6236	48	12	54	12	56	12
AL-DP-6237	73	14	108	18	154	37
AL-DP-6238	64	9	103	10	105	24
AL-DP-6239	9	1	20	4	31	11
AL-DP-6240	99	7	139	16	194	43
AL-DP-6241	43	9	54	12	66	19
AL-DP-6242	6	1	15	7	36	8
AL-DP-6243	7	2	19	5	33	13
AL-DP-6244	7	2	19	3	37	13
AL-DP-6245	25	4	45	10	58	9
AL-DP-6246	34	8	65	10	66	13
AL-DP-6247	53	6	78	14	105	20
AL-DP-6248	7	0	22	7	39	12
AL-DP-6249	36	8	48	13	61	7

The nine siRNA duplexes that showed the greatest growth inhibition in Table 5 were re-tested at a range of siRNA concentrations in HeLa cells. The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.046 nM. Assays were performed in sextuplicate, and the concentration of each siRNA resulting in fifty percent inhibition of cell proliferation (IC₅₀) was calculated. This dose-response analysis was performed between two and four times for each duplex. Mean IC₅₀values (nM) are given in Table 6.

	TABLE 6

	Duplex	Mean IC50

	AL-DP-6226	15.5
	AL-DP-6229	3.4
	AL-DP-6231	4.2
	AL-DP-6232	17.5
	AL-DP-6239	4.4
	AL-DP-6242	5.2
	AL-DP-6243	2.6
	AL-DP-6244	8.3
	AL-DP-6248	1.9

Eg5 siRNA In Vitro Screening Via Cell Proliferation
Directly before transfection, Hela S3 (ATCC-Number: CCL-2.2, LCG Promochem GmbH, Wesel, Germany) cells were seeded at 1.5×10⁴cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium (Ham's F12, 10% fetal calf serum, 100 u penicillin/100 μg/ml streptomycin, all from Biochrom AG, Berlin, Germany). Transfections were performed in quadruplicates. For each well 0.5 μl Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. For the siRNA concentration being 50 nM in the 100 μl transfection volume, 1 μl of a 5 μM siRNA were mixed with 11.5 μl Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. siRNA-Lipofectamine2000-complexes were applied completely (25 μl each per well) to the cells and cells were incubated for 24 h at 37° C. and 5% CO₂in a humidified incubator (Heraeus GmbH, Hanau). The single dose screen was done once at 50 nM and at 25 nM, respectively.
Cells were harvested by applying 50 μl of lysis mixture (content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well containing 100 μl of growth medium and were lysed at 53° C. for 30 min. Afterwards, 50 μl of the lysates were incubated with probesets specific to human Eg5 and human GAPDH and proceeded according to the manufacturer's protocol for QuantiGene. In the end chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the hEg5 probeset were normalized to the respective GAPDH values for each well. Values obtained with siRNAs directed against Eg5 were related to the value obtained with an unspecific siRNA (directed against HCV) which was set to 100% (Tables 1, 2 and 3).
Effective siRNAs from the screen were further characterized by dose response curves. Transfections of dose response curves were performed at the following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77 picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9 fM and mock (no siRNA) and diluted with Opti-MEM to a final concentration of 12.5 μl according to the above protocol. Data analysis was performed by using the Microsoft Excel add-in software XL-fit 4.2 (IDBS, Guildford, Surrey, UK) and applying the dose response model number 205 (Tables 1, 2 and 3).
The lead siRNA AD12115 was additionally analyzed by applying the WST-proliferation assay from Roche (as previously described).
A subset of 34 duplexes from Table 2 that showed greatest activity was assayed by transfection in HeLa cells at final concentrations ranging from 100 nM to 10 fM. Transfections were performed in quadruplicate. Two dose-response assays were performed for each duplex. The concentration giving 20% (IC20), 50% (IC50) and 80% (IC80) reduction of KSP mRNA was calculated for each duplex. (Table 7).

TABLE 7

Concentrations given in pM

	IC20s		IC50s		IC80s
Duplex	1^st	2^nd	1st	2nd	1st	2nd
name	screen	screen	screen	screen	screen	screen

AD12077	1.19	0.80	6.14	10.16	38.63	76.16
AD12078	25.43	25.43	156.18	156.18	ND	ND
AD12085	9.08	1.24	40.57	8.52	257.68	81.26
AD12095	1.03	0.97	9.84	4.94	90.31	60.47
AD12113	4.00	5.94	17.18	28.14	490.83	441.30
AD12115	0.60	0.41	3.79	3.39	23.45	23.45
AD12125	31.21	22.02	184.28	166.15	896.85	1008.11
AD12134	2.59	5.51	17.87	22.00	116.36	107.03
AD12149	0.72	0.50	4.51	3.91	30.29	40.89
AD12151	0.53	6.84	4.27	10.72	22.88	43.01
AD12152	155.45	7.56	867.36	66.69	13165.27	ND
AD12157	0.30	26.23	14.60	92.08	14399.22	693.31
AD12166	0.20	0.93	3.71	3.86	46.28	20.59
AD12180	28.85	28.85	101.06	101.06	847.21	847.21
AD12185	2.60	0.42	15.55	13.91	109.80	120.63
AD12194	2.08	1.11	5.37	5.09	53.03	30.92
AD12211	5.27	4.52	11.73	18.93	26.74	191.07
AD12257	4.56	5.20	21.68	22.75	124.69	135.82
AD12280	2.37	4.53	6.89	20.23	64.80	104.82
AD12281	8.81	8.65	19.68	42.89	119.01	356.08
AD12282	7.71	456.42	20.09	558.00	ND	ND
AD12285	ND	1.28	57.30	7.31	261.79	42.53
AD12292	40.23	12.00	929.11	109.10	ND	ND
AD12252	0.02	18.63	6.35	68.24	138.09	404.91
AD12275	25.76	25.04	123.89	133.10	1054.54	776.25
AD12266	4.85	7.80	10.00	32.94	41.67	162.65
AD12267	1.39	1.21	12.00	4.67	283.03	51.12
AD12264	0.92	2.07	8.56	15.12	56.36	196.78
AD12268	2.29	3.67	22.16	25.64	258.27	150.84
AD12279	1.11	28.54	23.19	96.87	327.28	607.27
AD12256	7.20	33.52	46.49	138.04	775.54	1076.76
AD12259	2.16	8.31	8.96	40.12	50.05	219.42
AD12276	19.49	6.14	89.60	59.60	672.51	736.72
AD12321	4.67	4.91	24.88	19.43	139.50	89.49

(ND—not determined)

Silencing of Liver Eg5/KSP in Juvenile Rats Following Single-Bolus Administration of LNP01 Formulated siRNA

From birth until approximately 23 days of age, Eg5/KSP expression can be detected in the growing rat liver. Target silencing with a formulated Eg5/KSP siRNA was evaluated in juvenile rats.

KSP Duplex Tested

Duplex
ID	Target	Sense	Antisense

AD6248	Eg5/	AccGAAGuGuu	GGAcAAAcAAc
	KSP	GuuuGuccTsT	ACUUCGGUTsT
		(SEQ ID	(SEQ ID
		NO: 1238)	NO: 1239)

Methods
Dosing of Animals.
Male, juvenile Sprague-Dawley rats (19 days old) were administered single doses of lipidoid (“LNP01”) formulated siRNA via tail vein injection. Groups of ten animals received doses of 10 milligrams per kilogram (mg/kg) bodyweight of either AD6248 or an unspecific siRNA. Dose level refers to the amount of siRNA duplex administered in the formulation. A third group received phosphate-buffered saline. Animals were sacrificed two days after siRNA administration. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.
mRNA Measurements.
Levels of Eg5/KSP mRNA were measured in livers from all treatment groups. Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of Eg5/KSP and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for Eg5/KSP were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.
Statistical Analysis.
Significance was determined by ANOVA followed by the Tukey post-hoc test
Results
Data Summary
Mean values (±standard deviation) for Eg5/KSP mRNA are given. Statistical significance (p value) versus the PBS group is shown (ns, not significant [p>0.05]).

Experiment 1


	VEGF/GAPDH	p value

PBS		1.0 ± 0.47
AD6248	10 mg/kg	0.47 ± 0.12	<0.001
unspec	10 mg/kg	1.0 ± 0.26	ns

A statistically significant reduction in liver Eg5/KSP mRNA was obtained following treatment with formulated AD6248 at a dose of 10 mg/kg.

Silencing of Rat Liver VEGF Following Intravenous Infusion of LNP01 Formulated siRNA Duplexes

A “lipidoid” formulation comprising an equimolar mixture of two siRNAs was administered to rats. One siRNA (AD3133) was directed towards VEGF. The other (AD12115) was directed towards Eg5/KSP. Since Eg5/KSP expression is nearly undetectable in the adult rat liver, only VEGF levels were measured following siRNA treatment.
siRNA Duplexes Administered


	Duplex
	ID	Target	Sense	Antisense

	AD12115	Eg5/KSP	ucGAGAAucuA	AGUuAGUUuAG
			AAcuAAcuTsT	AUUCUCGATsT
			(SEQ ID	(SEQ ID
			NO: 1240)	NO: 1241)

	AD3133	VEGF	GcAcAuAGGAG	AAGCUcAUCUCU
			AGAuGAGCUsU	CCuAuGuGCusG
			(SEQ ID	(SEQ ID
			NO: 1242)	NO: 1243)

Key:
A,G,C,U-ribonucleotides;
c,u-2′-O-Me ribonucleotides;
s-phorphorothioate.

Methods
Dosing of Animals.
Adult, female Sprague-Dawley rats were administered lipidoid (“LNP01”) formulated siRNA by a two-hour infusion into the femoral vein. Groups of four animals received doses of 5, 10 and 15 milligrams per kilogram (mg/kg) bodyweight of formulated siRNA. Dose level refers to the total amount of siRNA duplex administered in the formulation. A fourth group received phosphate-buffered saline. Animals were sacrificed 72 hours after the end of the siRNA infusion. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.
Formulation Procedure
The lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.
Characterization of Formulations
Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 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 is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is 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%.
mRNA Measurements.
Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of VEGF and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for VEGF were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.
Protein Measurements.
Samples of each liver powder (approximately 60 milligrams) were homogenized in 1 ml RIPA buffer. Total protein concentrations were determined using the Micro BCA protein assay kit (Pierce). Samples of total protein from each animal was used to determine VEGF protein levels using a VEGF ELISA assay (R&D systems). Group means were determined and normalized to the PBS group for each experiment.
Statistical Analysis.
Significance was determined by ANOVA followed by the Tukey post-hoc test
Results
Data Summary
Mean values (±standard deviation) for mRNA (VEGF/GAPDH) and protein (rel. VEGF) are shown for each treatment group. Statistical significance (p value) versus the PBS group for each experiment is shown.


VEGF/GAPDH	p value	rel VEGF	p value

PBS	1.0 ± 0.17		1.0 ± 0.17
5 mg/kg	0.74 ± 0.12	<0.05	0.23 ± 0.03	<0.001
10 mg/kg	0.65 ± 0.12	<0.005	0.22 ± 0.03	<0.001
15 mg/kg	0.49 ± 0.17	<0.001	0.20 ± 0.04	<0.001

Statistically significant reductions in liver VEGF mRNA and protein were measured at all three siRNA dose levels.

Claims

1. A composition comprising a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a human kinesin family member 11 (Eg5) gene in a cell, wherein the dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence complementary to the sequence provided in an even SEQ ID NO: 2-580 or an odd SEQ ID NO: 583-1257, wherein the first sequence is complementary to the second sequence and wherein the dsRNA is between 15 and 30 base pairs in length.

2. The composition of claim 1, wherein the dsRNA comprises at least one modified nucleotide.

3. The composition of claim 2, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

4. The composition of claim 2, wherein the modified nucleotide is 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 comprising nucleotide.

5. The composition of claim 2, wherein the first dsRNA comprises at least one 2′-O-methyl modified ribonucleotide and at least one phosphorothioate.

6. The composition of claim 1, wherein the composition, upon contact with a cell expressing Eg5, inhibits expression of Eg5 gene by at least 40%.

7. The composition of claim 1, wherein the dsRNA is 19-21 base pairs in length.

8. An isolated cell comprising the composition of claim 1.

9. A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of the dsRNA of the composition of claim 1.

10. An isolated cell comprising the vector of claim 9.

11. A pharmaceutical composition for inhibiting Eg5 gene expression comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

12. A method for inhibiting Eg5 gene expression in a cell, the method comprising:

introducing into the cell the composition of claim 1; and

maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the Eg5 gene, thereby inhibiting expression of the Eg5 gene in the cell.

13. A method of treating pathological processes mediated by human Eg5 expression comprising administering to a patient a therapeutically effective amount of the composition of claim 1.