COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF A GENE FROM THE EBOLA

Abstract
The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a gene from the Ebola virus.
Description
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 one of the genes of the Ebola virus and the use of the dsRNA to treat pathological processes mediated by Ebola infection, such as systemic hemorrhage and multi-organ failure.


BACKGROUND OF THE INVENTION
Ebola Virus

Minus-strand (−) RNA viruses are major causes of human suffering that cause epidemics of serious human illness. In humans the diseases caused by these viruses include influenza (Orthomyxoviridae), mumps, measles, upper and lower respiratory tract disease (Paramyxoviridae), rabies (Rhabdoviridae), hemorrhagic fever (Filoviridae, Bunyaviridae and Arenaviridae), encephalitis (Bunyaviridae) and neurological illness (Bomaviridae). Virtually the entire human population is thought to be infected by many of these viruses.


The Ebola virus comes from the Filoviridae family, similar to the Marburg virus. It is named after the Ebola River in Zaire, Africa, near where the first outbreak was noted by Dr. Ngoy Mushola in 1976 after a significant outbreaks in both Yambuku, Zaire (now the Democratic Republic of the Congo), and Nzara, in western Sudan. Of 602 identified cases, there were 397 deaths.


The two strains identified in 1976 were named Ebola-Zaire (EBO-Z) and Ebola-Sudan (EBO-S). The outbreak in Sudan showed a lower fatality rate—50%—compared to the 90% mortality rate of the Zaire strain. In 1990, a second, similar virus was identified in Reston, Va. amongst monkeys imported from the Philippines, and was named Ebola-Reston.


Further outbreaks have occurred in Zaire/Congo (1995 and 2003), Gabon (1994, 1995 and 1996), and in Uganda (2000). A new subtype was identified from a single human case in the Côte d'Ivoire in 1994, EBO-CI.


Of around 1500 identified human Ebola infections, two-thirds of the patients have died. The animal (or other) reservoir which sustains the virus between outbreaks has not been identified.


Among humans, the Ebola virus is transmitted by direct contact with infected body fluids such as blood.


The incubation period of Ebola hemorrhagic fever varies from two days to four weeks. Symptoms are variable too, but the onset is usually sudden and characterised by high fever, prostration, myalgia, arthralgia, abdominal pains and headache. These symptoms progress to vomiting, diarrhea, oropharyngeal lesions, conjunctivitis, organ damage (notably the kidney and liver) by co-localized necrosis, proteinuria, and bleeding both internal and external, commonly through the gastrointestinal tract. Death or recovery to convalescence occurs within six to ten days of onset of symptomology.


The development of a successful therapeutic for Ebola virus is a long-sought and seemingly difficult endeavor. Although they cause only a few hundred deaths worldwide each year, filoviruses are considered a significant world health threat and have many of the characteristics commonly associated with biological weapons since they can be grown in large quantities, can be fairly stable, are highly infectious as an aerosol, and are exceptionally deadly. Filoviruses are relatively simple viruses of 19 Kb genomes and consist of seven genes which encode nucleoprotein (NP), glycoprotein (GP), four smaller viral proteins (VP24, VP30, VP35 and VP40), and the RNA-dependent RNA polymerase (L protein) all in a single strand of negative-sensed RNA. Administration of type I interferons, therapeutic vaccines, immune globulins, ribavirin, and other nucleoside analogues have been somewhat successful in rodent Ebola virus models, but not in nonhuman primate infection models.


In view of the severity of the diseases caused by (−) RNA viruses, in particular members of the Filoviridae family of viruses, and the lack of effective prevention or therapies, it is therefore an object of the present invention to provide therapeutic compounds and methods for treating a host infected with a (−) RNA virus.


siRNA


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.


Recent reports have indicated that in vitro, RNAi may show some promising in reducing Ebola replication and providing protection in guinea pigs (Geisbert, et al., The Journal of Infectious Diseases, 193 (2006), 1650-1657). However, the RNAi agents examined were not designed against all known Ebola strains and were not selected for stability and other properties needed for in vivo therapeutic RNAi agents. Accordingly, despite significant advances in the field of RNAi, there remains a need for an agent that can selectively and efficiently silence a gene in the Ebola virus using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit replication of the Ebola virus for use in treating pathological processes mediated by Ebola infection.


SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the Ebola virus in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by Ebola viral infection, such as systemic hemorrhage and multi-organ failure. The dsRNA featured in the invention includes 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 a gene from the Ebola virus.


In one embodiment, the invention provides dsRNA molecules for inhibiting the expression of a gene of the Ebola virus and viral replication. 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 encoded by a gene from the Ebola virus, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contact with a cell infected with the Ebola virus, inhibits the expression of a gene from the Ebola virus by at least 40%.


For example, the dsRNA molecules of the invention can include a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Table 2 and the second sequence selected from the group consisting of the antisense sequences of Table 2. The dsRNA molecules featured in the invention can include naturally occurring nucleotides or can include 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 Table 2 and a second sequence selected from the group consisting of the antisense sequences of Table 2.


In another embodiment, the invention provides a cell having a dsRNA 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 replication of the Ebola virus in an organism, generally a human subject. The composition includes 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 a gene in the Ebola virus in a cell, including the following steps:

    • (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA includes at least two sequences that are complementary to each other. The dsRNA includes a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand includes a region of complementarity which is substantially complementary to at least a part of an mRNA encoded by the Ebola virus, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and optionally, wherein the dsRNA, upon contact with a cell infected with the Ebola virus, inhibits expression of a gene from the Ebola virus by at least 40%, such as in an assay described herein (e.g., a fluorscence-based assay); and
    • (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a Ebola gene, thereby inhibiting expression of a gene from the Ebola virus in the cell.


In another embodiment, the invention provides methods for treating, preventing or managing pathological processes mediated by Ebola infection, such as systemic hemorrhage and multi-organ failure, 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 a gene of the Ebola virus in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a dsRNA of the invention.


In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of a gene of the Ebola virus in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of a dsRNA of the invention.


In one aspect, the invention provides for a method of increasing the life-span of a subject (e.g., a mammal, such as a human or nonhuman primate) infected with an Ebola virus. The method includes administering a dsRNA to the subject, where the dsRNA includes an antisense RNA 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 a gene from the Ebola virus. The dsRNA is administered in an amount sufficient to increase the lifespan of the subject. In one embodiment, the dsRNA includes an antisense RNA strand having a region that is substantially complementary to at least part of an mRNA transcript of a gene selected from the VP30, VP35, NP, L, VP24, VP40 and GP genes. In a preferred embodiment, the dsRNA includes an antisense RNA strand having a region that is substantially complementary to at least part of an mRNA transcript of the VP35 gene. In some embodiments, the subject does not experience a decrease in one or both of lymphocyte or platelet count after administration of the dsRNA. In other embodiments, the subject does not experience an increase in cytokine levels (e.g., IFN-alpha or TNF-alpha levels).


In another aspect, the invention features a method of decreasing viral titre in a subject (e.g., a mammal, such as a human or nonhuman primate) infected with an Ebola virus. The method includes administering a dsRNA to the subject, where the dsRNA includes an antisense RNA 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 a gene from the Ebola virus. In one embodiment, the dsRNA includes an antisense RNA strand having a region that is substantially complementary to at least part of an mRNA transcript of the VP35 gene. In another embodiment, the subject does not experience a decrease in one or both of lymphocyte or platelet count after administration of the dsRNA. In other embodiments, the subject does not experience an increase in cytokine levels (e.g., IFN-alpha or TNF-alpha levels).


In another aspect, the invention features a method of sustaining lymphocyte or platelet count in a mammal (e.g., a human or nonhuman primate) infected with an Ebola virus. The method includes administering a dsRNA to the subject, where the dsRNA includes an antisense RNA 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 a gene from the Ebola virus. In one embodiment, the dsRNA includes an antisense RNA strand having a region that is substantially complementary to at least part of an mRNA transcript of the VP35 gene. In other embodiments, the subject does not experience an increase in cytokine levels (e.g., IFN-alpha or TNF-alpha levels).





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 is a graph showing that siRNAs formulated with lipidoid LNP01 protected mice from a lethal Ebola virus challenge.



FIG. 2 is a graph showing that a single injection of a liposomally formulated siRNA delivered by ip or iv protected mice from a lethal Ebola challenge. VP35 siRNA was AD-11570



FIG. 3 is the structure of NP98 lipid.



FIG. 4 is a graph showing that siRNAs formulated with DODMA protected mice from a lethal Ebola virus challenge.



FIG. 5 is a graph showing that siRNAs formulated with DODMA were effective down to 0.04 mg/kg to protect mice injected with Ebola.



FIG. 6 is a graph showing that siRNAs formulated with DODMA were effective to protect guinea pigs from a lethal Ebola virus challenge.



FIG. 7 is a graph showing the efficacy of siRNAs against different Ebola genes formulated with DODMA in a guinea pig model of Ebola.



FIG. 8 is a graph presenting the observed decrease in viral titers in the serum of mice following administration of LNP01-formulated VP35 siRNA.





DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of a gene from the Ebola virus 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 Ebola infection using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).


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 a gene from the Ebola virus. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and or maintenance of Ebola infection and the occurrence of systemic hemorrhage and multi-organ failure in a subject infected with the Ebola virus. 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 a gene from the Ebola virus. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by Ebolaviral infection by targeting a gene involved in Ebola relication and/or maintenance in a cell.


The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a gene from the Ebola virus, as well as compositions and methods for treating diseases and disorders caused by the infection with the Ebola virus, such as systemic hemorrhage and multi-organ failure. 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 a gene from the Ebola virus, together with a pharmaceutically acceptable carrier.


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 a gene in a gene from the Ebola virus, and methods of using the pharmaceutical compositions to treat diseases caused by infection with the Ebola virus.


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, “Ebola viruses”, are members of the family Filoviridae, are associated with outbreaks of highly lethal hemorrhagic fever in humans and nonhuman primates. Human pathogens include Ebola Zaire, Ebola Sudan, and Ebola Ivory Coast. Ebola Reston is a monkey pathogen and is not considered a significant human pathogen. The natural reservoir of the virus is unknown and there are currently no available vaccines or effective therapeutic treatments for filovirus infections. The genome of Ebola virus consists of a single strand of negative sense RNA that is approximately 19 kb in length. This RNA contains seven sequentially arranged genes that produce 8 mRNAs upon infection. Ebola virions, like virions of other filoviruses, contain seven proteins: a surface glycoprotein (GP), a nucleoprotein (NP), four virion structural proteins (VP40, VP35, VP30, and VP24), and an RNA-dependent RNA polymerase (L) (Feldmann et al. (1992) Virus Res. 24, 1-19; Sanchez et al., (1993) Virus Res. 29, 215-240; reviewed in Peters et al. (1996) In Fields Viroloqy, Third ed. pp. 1161-1176. Fields, B. N., Knipe, D. M., Howley, P. M., et al. eds. Lippincott-Raven Publishers, Philadelphia). The glycoprotein of Ebola virus is unusual in that it is encoded in two open reading frames. Transcriptional editing is needed to express the transmembrane form that is incorporated into the virion (Sanchez et al. (1996) Proc. Natl. Acad. Sci. USA 93, 3602-3607; Volchkov et al, (1995) Virology 214, 421-430).


As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene from the Ebola virus, including mRNA that is a product of RNA processing of a primary transcription product.


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 Ebola). For example, a polynucleotide is complementary to at least a part of a Ebola mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding Ebola.


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. dsRNAs as used herein are also referred to as “siRNAs” (short interfering RNAs).


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 a gene from the Ebola virus, herein refer to the at least partial suppression of the expression of a gene from the Ebola virus, as manifested by a reduction of the amount of mRNA transcribed from a gene from the Ebola virus which may be isolated from a first cell or group of cells in which a gene from the Ebola virus is transcribed and which has or have been treated such that the expression of a gene from the Ebola virus 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










(

mRNA





in





control





cells

)

-

(

mRNA





in





treated





cells

)



(

mRNA





in





control





cells

)


·
100


%




Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Ebola genome transcription, e.g. the amount of protein encoded by a gene from the Ebola virus, or the number of cells displaying a certain phenotype, e.g infection with the Ebola virus. In principle, Ebola genome 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 a gene from the Ebola virus 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 a gene from the Ebola virus is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, a gene from the Ebola virus is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, a gene from the Ebola virus is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention.


As used herein in the context of Ebola expression, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by Ebola infection. 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 Ebola 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, or to reduce the amount of virus present in the infected subject.


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 Ebola infection or an overt symptom of pathological processes mediated by Ebola expression or the amount virus present in the patient. 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 Ebola infection, the patient's history and age, the stage of pathological processes mediated by Ebola infection, and the administration of other anti-pathological processes mediated by Ebola infection.


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. Further, the pharmaceutical composition can be designed to enhance targeting cells involved in Ebola infection such as dendritic cells, macrophages, hepatocytes, and other parenchymal cells.


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 a gene from the Ebola virus 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 a gene from the Ebola virus, 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 the gene from the Ebola virus, inhibits the expression of the Ebola virus 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 a gene from the Ebola virus, 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 one embodiment, a gene from the Ebola virus is the from human Ebola genome. In specific embodiments, the antisense strand of the dsRNA comprises the sense sequences of Table 2 and the second sequence is selected from the group consisting of the antisense sequences of Table 2. Alternative antisense agents that target elsewhere in the target sequence provided in Table 2 can readily be determined using the target sequence and the flanking Ebola sequence.


In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Table 2. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of a gene from the Ebola virus. Generally, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Table 2 and the second oligonucleotide is described as the antisense strand in Table 2


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 Table 2, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Table 2 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 Table 2, and differing in their ability to inhibit the expression of a gene from the Ebola virus 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 Table 2 can readily be made using the Ebola virus sequence and the target sequence provided.


In addition, the RNAi agents provided in Table 2 identify a site in the Ebola virus 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 Table 2 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a gene from the Ebola virus. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target Ebola genome produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Table 2.


The dsRNA of the invention can contain one or more mismatches to the target sequence. In one 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 a gene from the Ebola virus, 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 a gene from the Ebola virus. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a gene from the Ebola virus is important, especially if the particular region of complementarity in a gene from the Ebola virus 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 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 certain 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.


Other embodiments featured in the invention include dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH.sub.2-NH—CH.sub.2-, —CH.sub.2-N(CH.sub.3)-O—CH.sub.2-[known as a methylene (methylimino) or MMI backbone], —CH.sub.2-O—N(CH.sub.3)-CH.sub.2-, —CH.sub.2-N(CH.sub.3)-N(CH.sub.3)-CH.sub.2- and —N(CH.sub.3)-CH.sub.2-CH.sub.2-[wherein the native phosphodiester backbone is represented as —O—P—O—CH.sub.2-] 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. In some embodiment, the dsRNAs include 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 C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, 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—CH.sub.2CH.sub.2OCH.sub.3, 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(CH.sub.2).sub.20N(CH.sub.3).sub.2 group, 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—CH.sub.2-O—CH.sub.2-N(CH.sub.2).sub.2, also described in examples hereinbelow.


Other preferred modifications include 2′-methoxy (2′-OCH.sub.3), 2′-aminopropoxy (2′-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) 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.degree. 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.


Other nucleotide substitutions, such as “Universal” bases can be incorporated into siRNA duplexes to increase the number of target sequences (or in this case, number of different Ebola strains) any particular siRNA might have complementarity to and activity against. Universal bases are non-canonical synthetic molecules that mimic structures of traditional nucleotides (the genetic building blocks of DNA and RNA). However, instead of selectively pairing according to Watson/Crick rules (A with T or U, C with G), universal bases ‘stack’ equally well with all natural bases. Incorporating universal bases into siRNAs may enable the siRNA to tolerate a mutation at that specific site in its target mRNA. Thus, by decreasing the need for absolute complementarity between siRNA and its mRNA target, universal-base containing siRNAs may be an approach to (1) prevent drug resistance caused by site-specific viral mutations and (2) create siRNAs able to be broadly reactive across viral species with similar, but not absolutely conserved, targets. Among the modifications that can be used as universal basaes are: 3-Nitropyrrole, 5-Nitroindole, Imidazole-4-Carboxamide, 2,4-difluorotoluoyl, and Inosine.


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). Preferred conjugates will assist in targeting cells infected by Ebola virus such as dendritic cells and macrophages which are involved in early stages of infection and epatocytes and other parenchymal cells which are involved in later phases of the infection. Such conjugates include, but are not limited to, mannose and folate conjugates.


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:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate 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-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.


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 a gene from the Ebola virus and/or viral infection, such as systemic hemorrhage and multi-organ failure. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery.


The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of a gene from the Ebola virus. A maximum dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or completely suppress expression of a gene from the Ebola virus.


In general, a suitable dose of dsRNA will be in the range of 0.01 to 20.0 milligrams per kilogram body weight of the recipient per day, and optimally in the range of 0.01 to 3 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. 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 animal modeling has generated a number of Ebola infection models (mouse, guinea pig, and non-human primate) that reproduce all the major pathologies associated with human Ebola infection (reviewed in Warfield, K. L. et al. (2006) “Chapter 13: Viral Hemorrhagic Fevers” in Biodefense: Research Methodology and Animal Models, pages 227-258, J. R. Swearengen, Ed., Taylor & Francis, Boca Raton). 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.


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.


Compositions and formulations for parenteral, intrathecal or intraventricular 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.


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.


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. Liposomal delivery systems have been used to effectively deliver siRNA in vivo and silence genes in hepatocytes [Zimmermann et al. (2006) Nature, 441:111-114]. Such siRNA-liposomal formulations have also been used for therapeutic benefit in animal models of dyslipidemias [Zimmermann et al. (2006) Nature, 441:111-114], HBV infection [Morrissey et al. (2005) Nature Biotech 23:1002-1007], Ebola infection [Geisbert, et al. (2006) The Journal of Infectious Diseases, 193:1650-1657], and rheumatoid arthritis [Khoury et al. (2006) Arthritis & Rheumatism, 54:1867-1877]. 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 phagocytosed by macrophages and other cells in vivo.


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.


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.


Liposomes and other nanoparticles have also been designed which contain specific targeting molecules. Targeting molecules used for siRNA delivery in vivo have included integrin-binding RGD peptides [Schiffelers et al. (2004) Nucleic Acids Research 32:e149), anisamide [Li and Huang (2006) Molecular Pharmaceutics 3:579-588] and folate [Hu-Lieskovan et al. (2005) Cancer Research 65:8984-8992]. For delivery to myeloid and dendritic cells which are presumed to be important in early Ebola infection, incorporation of targeting agents such as mannose and folate into liposomes and nanoparticles may improve both siRNA delivery and therapeutic effect. Mannose-conjugated oligonucleotides have been shown to specifically improve delivery to myeloid cells [Rojanasakul et al. (1997) Journal of Biological Chemistry 272:3910-3914; Diebold et al (2002) Somatic Cell and Molecular Genetics 27:65-73] and mannosylated liposomes are effective targeting agents in vivo [Diebold et al. (2002) Somatic Cell and Molecular Genetics 27:65-73; Hattori et al. (2006) Journal Gene Medicine 8:824-834; Hattori et al. (2006) Journal of Pharmacology and Experimental Therapeutics 318:828-834]. Folate conjugation has proven an effective delivery vehicle in a wide variety of contexts [reviewed in Hilgenbrink and Low (2005) Journal Pharmaceutical Sciences 94:2135-2146] and incorporation of folate into liposomes is possible using commercially available reagents such as DSPE-PEG(2000)Folate (Avanti Polar Lipids, Alabaster, Ala.). 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 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).


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.


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-hydroxyperoxycyclophosphoramide, 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 Ebola 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 Infection with the Ebola Virus


The invention relates in particular to the use of a dsRNA or a pharmaceutical composition prepared therefrom for the treatment or prevention of pathological conditions associated with Ebola infection, e.g., systemic hemorrhage and multi-organ failure.


The invention furthermore relates to the use of an dsRNA or a pharmaceutical composition thereof for treating systemic hemorrhage and multi-organ failure 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 viral infection and systemic hemorrhage. Preference is given to a combination with interferon or other antiviral agents.


Methods for Inhibiting Expression of a Gene from the Ebola Virus


In yet another aspect, the invention provides a method for inhibiting the expression of a gene from the Ebola virus in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target Ebola genome is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target Ebola gene. Compositions and methods for inhibiting the expression of these Ebola 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 a gene from the Ebola virus, to 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, intraperitoneal, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, administration. In some 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
Example 1
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 1.









TABLE 1







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
Nucleotide(s)





A
adenosine-5′-phosphate


C
cytidine-5′-phosphate


G
guanosine-5′-phosphate


T
2′-deoxy-thymidine-5′-phosphate


U
uridine-5′-phosphate


N
any nucleotide (G, A, C, or T)


a
2′-O-methyladenosine-5′-phosphate


c
2′-O-methylcytidine-5′-phosphate


g
2′-O-methylguanosine-5′-phosphate


u
2′-O-methyluridine-5′-phosphate


sT
2′-deoxy-thymidine-5′phosphate-phosphorothioate









Example 2
Gene Walking of a Gene from the Ebola Virus

Design and In Silico Selection of siRNAs Targeting Ebola Virus


siRNA design was carried out to identify siRNAs targeting Ebola virus mRNAs for genes VP30, VP35, NP, L, VP24, VP40 and GP with a focus on sequences isolated from the Zaire region (EBOV-Z), as well as sequences from Sudan (EBOV-S). The siRNA in silico selection resulted in 521 siRNAs satisfying our selection criteria (Table 2).


Ebola Zaire sequence AY354458 was downloaded from NCBI Nucleotide database and further on used as reference sequence for EBOV-Z. Ebola Sudan sequence AY729654 was used as reference sequence for EBOV-S, respectively.


Sequence regions encoding target genes VP30, VP35, NP, L, VP24, VP40 and GP according to the information in the Genbank file were extracted from both reference sequences, followed by extraction of all possible 19 mers for each gene, resulting in the candidate siRNA target regions (and siRNA sense sequences) for each distinct gene.


In order to identify siRNAs targeting all available EBOV-Z and EBOV-S sequences, it was necessary to compile available Ebola sequences from all sequenced isolates. For this, each of the 7 target gene sequences extracted from the Ebola Zaire reference sequences was used in a blast search against viruses at NCBI with default parameters and resulting Ebola mRNA hits were downloaded.


Each candidate target region was tested for conservation across the Ebola sequences by searching the relevant target gene for presence of the 19 mer target region. The percentage of conserved sequences across all downloaded sequences for the relevant gene was calculated for each candidate target region by dividing of the number of conserved sequences with the total number of downloaded sequences.


Example 3
Ebola siRNA In Vitro Screening

Table 3 provides a summary of the screening results of the siRNAs described in Table 2. Following initial screening using a GFP-expressing Ebola-Zaire virus and immunofluorescence screening using Ebola-Sudan virus, siRNA showing activity were further tested by plaque assay for anti-viral activity against Ebola-Zaire and Ebola-Sudan strains. Several siRNAs were identified that had significant activity against one or more Ebola strains. At a concentration of 100 nM many of the siRNA identified showed greater than a 1 log reduction (>90% inhibition) in Ebola virus titers. Negative control luciferase and GFP siRNA at the same concentration showed reductions in virus titer of between 10 and 35% (Table 3). Three previously identified Ebola siRNA (LS L#1, LS NP#1, LS VP35#1) were also tested in parallel and these inhibited Ebola virus by roughly 70%. The previously identified Ebola siRNA are 25 nucleotide blunt-ended duplexes with the following composition: LS L#1, sense: 5′ CCAUCUCUGAGACACGACAUAUCUU 3′ anti-sense: 5′ AAGAUAUGUCGUGUCUCAGAGAUGG 3′; LS NP#1, sense: 5′ GGUUCAAAGGCAAAUUCAAGUACAU 3′ anti-sense 5′ AUGUACUUGAAUUUGCCUUUGAACC 3′; LS VP35#1, sense 5′ CCCAAAUGCAACAAACCAAGCCAAA 3′ anti-sense 5′ UUUGGCUUGGUUUGUUGCAUUUGGG 3′. The siRNA sequences for AD-1955 and AD-5179 are as follows: AD-1955, sense: 5′ CUUACGCUGAGUACUUCGAdTsdT 3′ anti-sense: 5′ UCGAAGUACUCAGCGUAAGdTsdT 3′; AD-5179, sense: 5′ CcAcAuGAAGcAGcACGACusU 3′ anti-sense: 5′ AAGUCGUGCUGCUUCAUGUGgsusC 3′.


Lead siRNAs were also screened for immunostimulatory activity (IFNalpha and TNFalpha). Immunostimulatory activity was assayed by transfecting siRNAs into human peripheral blood mononuclear cells and measuring cytokine release by ELISA as outlined in Hornung et al. Nature Medicine 2005. Cytokine levels were normalized to a positive control siRNA included in every assay. The lead candidates had no immunostimulatory activity.


The following procedures were used in generating the screening results.


GFP Ebola-Zaire Assay


VERO cells were transfected at ˜2×10E4 cells per well in a black-walled 96 well plate. Transfection was performed in EMEM with 10% FCS overnight at 100, 10 and 1 nM siRNA complexed with lipofectamine (1.2 ul of lipofectamine per well in 50 ul volume; complexation was performed at room temperature for 15-20 min).


Next day cells were infected with GFP-EBOLA virus ( 1/50 dilution of stock EBOLA-Zaire GFP, stock E6(4) from 11OCT05, USAMRIID) in 50 ul of EMEM with 10% FCS. 2 days later cells were fixed in 10% neutral-buffered formalin for >3 days. Formalin was changed before removing from BSL-4 suite. Next formalin was replaced with PBS.


To quantify infection level of cells in individual wells, cells were stained with 10-20 ul/well of 10 ug/ml Hoescht dye and read on Discovery 1 microscope. GFP signal normalized to Hoescht signal was read as a measure of infection level.


Immunofluorescence Ebola-Sudan Assay


VERO cells were transfected at ˜2×10E4 cell per well in a black-walled 96 well plate. Transfection was performed in EMEM with 10% FCS overnight at 100, 10 and 1 nM siRNA complexed with lipofectamine (1.2 ul of lipofectamine per well in 50 ul volume; complexation was performed at room temperature for 15-20 min).


Next day cells were infected with EBOLA-Sudan virus ( 1/100 dilution of EBOV-Sudan (Boniface), stock GP(1)V(2)E6(2) from 23 May 2006, USAMRIID) in 50 μl of EMEM with 10% FCS. Two days later cells were fixed in 10% neutral-buffered formalin for >3 days. Formalin was changed before removing from BSL-4 suite. Next formalin was replaced with PBS.


To detect infected cells, cells were stained for 4 h at room temperature with mouse anti-Sudan Boniface polyclonal sera (sera was collected from 20 animals and pooled at day 30 post infection of C57BL/6 mice infected with ˜1000 pfu of the EBOLA SUDAN-BONIFACE, stock GP(1) V(2) V(1) E6(2) from 23 May 2006, USAMRIID) at 1:200 dilution in PBS. Then cells were washed with PBS 2× for 5 minutes. Goat anti-mouse IgG-AlexaFluor488 (Molecular Probes) was added at 1:500 dilution in PBS. Cells were washed again with PBS for 5 minutes, 100 ul of PBS was added to each well. To quantify infection level of cells in individual wells, cell were stained with 10-20 ul/well of 10 ug/ml Hoescht dye and read on Discovery 1 microscope. AlexaFluor488 signal normalized to Hoescht signal was read as a measure of infection level.


Plaque Assay for Filoviruses for In Vitro Assay


Vero cells were transfected in 24 well plates at the density of ˜1.5×10E5/well density. Transfection was performed in EMEM with 10% FCS overnight at 100 nM siRNA complexed with lipofectamine (3 ul of lipofectamine per well in 200 ul volume; complexation was performed at room temperature for 15-20 min). Transfection was done in duplicates. 24 hours later duplicate plates were infected in 50 ul/well with either 1/500 diluted Zaire-EBOV [(E6P2) stock from 20 Jun. 2006, USAMRIID] or 1/1000 diluted EBOV-Sudan [(strain Boniface), stock GP(1)V(2)E6(2) from 23 May 2006, USAMRIID]. After 1 hour at 37° C. virus inoculum was replaced with 500 ul of fresh 10% FCS/EMEM.


48-72 h later supernatants were harvested from each well.


Plaque assay was performed with supernatants at 10−1, 10−2, 10−3, 10−4, 10−5 and 10−6 dilutions. Fresh Vero cells in 6-well plates were infected with diluted supernatants for 1 hour at 37° C. with rocking plates every 15 minutes; overlaid with 2 ml/well of 0.5% agarose in EMEM, 5% FCS, Pen/Strep. Six days later plates were overlaid with 2 ml of overlay media+4% neutral red solution and read plates the following day.


siRNA Activity Determination Using the Plasmid System.


Consensus sequences of NP (SEQ ID NO: 1043), GP (SEQ ID NO: 1044), L, VP24 (SEQ ID NO: 1045), VP30 (SEQ ID NO:1046), VP35 (SEQ ID NO:1047), VP40 (SEQ ID NO: 1048) were synthesized by GENEART (Regensburg, Germany) and cloned into GENEART standard vector. The L gene was generated as 2 fragments (SEQ ID NO: 1049 and SEQ ID NO: 1050). All genes were subcloned into individual psiCheck-2 (Promega, Mannheim, Germany) vectors via XhoI and NotI sites, resulting in a construct with the flu gene between the stop-codon and the polyA-signal of Renilla luciferase. Correct cloning was confirmed by end sequencing performed by GATC Biotech (Konstanz, Germany).


Transfections:


Cos-7 cells were seeded at 1.5×104 cells/well on white 96-well plates with clear bottom (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium. Directly after seeding the cells, 50 ng of plasmid/well were transfected with Lipofectamine-2000 (Invitrogen) as described below for the siRNAs, with the plasmid diluted in Opti-MEM to a final volume of 12.5 μl/well, prepared as a mastermix for the whole plate.


siRNA transfections were performed in quadruplicates 4 h after plasmid transfection. For each well 0.5 μl Lipofectamine-2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. For an siRNA concentration of 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 Lipofectamine-2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. During incubation, the growth medium was removed from cells and replaced by 75 μl/well of fresh medium. 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% CO2 in a humidified incubator (Heraeus GmbH, Hanau, Germany).


Cells were harvested by removing growth medium and application of 150 μl of a 1:1 mixture consisting of medium and Dual-Glo Luciferase substrate, from the Dual-Glo Luciferase Assay System (Promega, Mannheim, Germany).The luciferase assay was performed according to the manufacturer's protocol for Dual-Glo Luciferase assay and luminescence was measured in a Victor-Light 1420 Luminescence Counter (Perkin Elmer, Rodgau-Jügesheim, Germany). Values obtained with Renilla luciferase were normalized to the respective values obtained with Firefly luciferase. Values acquired with siRNAs directed against an Ebola gene were normalized to the value obtained with an unspecific siRNA (directed against neomycin resistance gene) set to 100%.


Example 4
In Vivo Filovirus Infection Model

Liposome-formulated siRNAs targeting Ebola genes protected mice from a lethal Ebola virus challenge. Details on the liposome formulation are detailed in the next section. Mice were treated with liposome-formulated siRNA (described below) twice, at 2 hours prior to Ebola infection (5 mg/kg i.v.) and at 3 days after Ebola infection (3 mg/kg i.p.). Mice were infected intraperitoneally with 30,000 LD50 of Ebola-Zaire (LD50 is lethal dose of Ebola infection where 50% of animals die). Mice were monitored for survival with n=10 per treatment group. Negative controls included untreated mice and mice treated with liposome-formulated luciferase siRNA (AD-1955). EK1 is a previously published siRNA sequence targeting the Ebola L gene [Geisbert et al. (2006) The Journal of Infectious Disease 193:1950-1657] that was used as a positive control. The siRNA sequences for EK1 are as follows:











5′ GUACGAAGCUGUAUAUAAAdTdT 3′ (sense)



and







5′ UUUAUAUACAGCUUCGUACdTdT 3′ (antisense).







FIG. 1 provides the results. All the negative control-treated animals (untreated and liposomally-formulated luciferase siRNA-treated) died within 6-8 days following Ebola infection. Several of the liposomally-formulated Ebola siRNAs showed significant increases in survival rates compared to the negative controls. Multiple Ebola siRNA (AD-11691, AD-11710, AD-11588, AD-11599, AD-11570) showed more protection against lethal Ebola infection than the previously published EK1 siRNA (FIG. 1).


A further experiment was conducted utilizing one of the active Ebola siRNA (AD-11570) to investigate different dosing routes and treatment regimens. Mice were treated with liposome-formulated Ebola VP35 siRNA (AD-11570) or negative control luciferase siRNA (AD-1955) 2 hours prior to Ebola infection (5 mg/kg i.p.). Mice were infected intraperitoneally with 30,000 LD50 of Ebola-Zaire (LD50 is lethal dose of Ebola infection where 50% of animals die). Mice were monitored for survival with n=10 per treatment group.



FIG. 2 provides the results. The animals treated with lipsomally-formulated AD-11570 showed near complete protection against lethal Ebola infection as compared to the negative control-treated animals (untreated and liposomally-formulated luciferase siRNA-treated) (FIG. 2). These results indicate that a single siRNA administration either via intravenous or intraperitoneal route is able to have a significant impact on survival.


Formulation Procedure


The lipidoid ND98.4HCl (MW 1487) (FIG. 3), 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%.


Example 5
siRNAs Targeting Ebola Increased the Life-Span of Mice and Guinea Pigs Infected with Ebola

siRNA directed against different Ebola genes were formulated in liposomes. A single dose of siRNA targeting the VP35 gene (AD-11570) protected both mice and guinea pigs against lethal Ebola infection (1000 PFU; 30000×LD50). Protection was associated with reduction in viral titres and was seen when drug was administered either prophylactically or therapeutically. Irrelevant siRNA (targeting luciferase) similarly formulated showed no protective effect or impact on virus titer.


Studies were conducted in mouse, guinea pig, and nonhuman primate lethal Ebola infection models. The studies are summarized below.


Mouse study 1: Demonstrated efficacy in mouse model of Ebola for multiple siRNA sequences formulated in LNP01. siRNAs were administered by intravenous (i.v.) injection on day 0, followed by intraperitoneal (i.p.) injection on day 3. See FIG. 1.


Mouse study 3: Demonstrated efficacy of siRNA in LNP01 formulation in the mouse model of Ebola when given by either IV or IP route. See FIG. 2. Mice were monitored for survival with n=10 per treatment group.


Mouse study #14: Demonstrated efficacy of siRNA in DODMA in the mouse model of Ebola by the IP route. See FIG. 4. siRNAs were formulated in DODMA:DSPC:Chol:PEG-DMG. Mice were monitored for survival with n=10 per treatment group. Treatment with 10 mg/kg DODMA-formulated AD-11570 siRNAs was also effective to protect guinea pigs infected with Ebola.


Mouse study #15: Demonstrated that siRNA in DODMA formulation is effective down to 0.04 mg/kg in the mouse model of Ebola. AD-11570 consistently gave 25-50% protection, but no clear dose response was seen. The control siRNA AD-1955 gave 25-50% protection, but again, no dose response was observed. See FIG. 5.


Guinea pig study #6: Demonstrated efficacy of multiple doses of siRNA in DODMA formulation in the guinea pig model of Ebola. AD-11570 siRNAs formulated in DODMA:DSPC:Chol:PEG-DMG were effective to protect guinea pigs from Ebola. See FIG. 6.


Guinea pig study #11: Efficacy of siRNAs formulated with DODMA and targeting different Ebola genes in a guinea pig model of Ebola. See FIG. 7


A 95% decrease in viral titers was also observed following administration of LNP01 formulated VP35 siRNA to BALB/c mice (n=5 per group) (FIG. 8). Mice were dosed systemically with LNP01 formulated siRNA at 5 mg/kg i.v. at day 0, then 3 mg/kg i.p. at day 3. Two hours post-siRNA injection at day 0, mice were injected with 1,000 pfu Ebola-Zaire virus and monitored for survival. On day 6 post-infection, the mice were sacrificed and their blood viral titers were determined by plaque assay.


Table 3 shows the results of cell-based and plaque assays.


Table 4 shows the results of plaque assays for control siRNAs.


Table 5. shows the sequences of modified duplexes, and Table 6 shows the effect of the modified duplexes on plaque assay activity and IC50 values in the plasmid-based system.


Table 7 shows the effect of siRNAs on cytokine levels (IFN-alpha and TNF-alpha).


Table 8 shows the siRNA silencing in the plasmid system and calculated IC50 values.


Table 9 shows that nonhuman primates administered siRNAs targeting Ebola did not demonstrate a decrease in lymphocyte or platelet count.














TABLE 2









double overhang design





position

sense strand
antisense strand
















in


Seq


Seq

duplex



target
Target
name
ID
sequence (5′-3′)
name
ID
sequence (5′-3′)
name



















 3-21
VP35
A18480
1
GAUGAAGAUUAAAACCUUCTsT
A18481
2
GAAGGUUUUAAUCUUCAUCTsT
AD-11542






 4-22
VP35
A18482
3
AUGAAGAUUAAAACCUUCATsT
A18483
4
UGAAGGUUUUAAUCUUCAUTsT
AD-11543





 5-23
VP35
A18484
5
UGAAGAUUAAAACCUUCAUTsT
A18485
6
AUGAAGGUUUUAAUCUUCATsT
AD-11544





 6-24
VP35
A18486
7
GAAGAUUAAAACCUUCAUCTsT
A18487
8
GAUGAAGGUUUUAAUCUUCTsT
AD-11545





1354-1372
VP35
A18488
9
UGAUGAAGAUUAAGAAAAATsT
A18489
10
UUUUUCUUAAUCUUCAUCATsT
AD-11546





 7-25
VP35
A18490
11
AAGAUUAAAACCUUCAUCATST
A18491
12
UGAUGAAGGUUUUAAUCUUTsT
AD-11547





 8-26
VP35
A18492
13
AGAUUAAAACCUUCAUCAUTsT
A18493
14
AUGAUGAAGGUUUUAAUCUTsT
AD-11548





 9-27
VP35
A18494
15
GAUUAAAACCUUCAUCAUCTsT
A18495
16
GAUGAUGAAGGUUUUAAUCTsT
AD-11549





10-28
VP35
A18496
17
AUUAAAACCUUCAUCAUCCTsT
A18497
18
GGAUGAUGAAGGUUUUAAUTsT
AD-11550





11-29
VP35
A18498
19
UUAAAACCUUCAUCAUCCUTST
A18499
20
AGGAUGAUGAAGGUUUUAATsT
AD-11551





12-30
VP35
A18500
21
UAAAACCUUCAUCAUCCUUTsT
A18501
22
AAGGAUGAUGAAGGUUUUATsT
AD-11552





 1-19
VP30
A18502
23
GAUGAAGAUUAAGAAAAAGTsT
A18503
24
CUUUUUCUUAAUCUUCAUCTsT
AD-11553





 3-21
VP35
A18504
151
GAuGAAGAuuAAAAccuucTsT
A18505
152
GAAGGUUUuAAUCUUcAUCTsT
AD-11554





 4-22
VP35
A18506
153
AuGAAGAuuAAAAccuucATsT
A18507
154
UGAAGGUUUuAAUCUUcAUTsT
AD-11555





 5-23
VP35
A18508
155
uGAAGAuuAAAAccuucAuTsT
A18509
156
AUGAAGGUUUuAAUCUUcATsT
AD-11556





 6-24
VP35
A18510
157
GAAGAuuAAAAccuucAucTsT
A18511
158
GAUGAAGGUUUuAAUCUUCTsT
AD-11557





1354-1372
VP35
A18512
159
uGAuGAAGAuuAAGAAAAATsT
A18513
160
UUUUUCUuAAUCUUcAUcATsT
AD-11558





 7-25
VP35
A18514
161
AAGAuuAAAAccuucAucATsT
A18515
162
UGAUGAAGGUUUuAAUCUUTsT
AD-11559





 8-26
VP35
A18516
163
AGAuuAAAAccuucAucAuTsT
A18517
164
AUGAUGAAGGUUUuAAUCUTsT
AD-11560





 9-27
VP35
A18518
165
GAuuAAAAccuucAucAucTsT
A18519
166
GAUGAUGAAGGUUUuAAUCTsT
AD-11561





10-28
VP35
A18520
167
AuuAAAAccuucAucAuccTsT
A18521
168
GGAUGAUGAAGGUUUuAAUTsT
AD-11562





11-29
VP35
A18522
169
uuAAAAccuucAucAuccuTsT
A18523
170
AGGAUGAUGAAGGUUUuAATsT
AD-11563





12-30
VP35
A18524
171
uAAAAccuucAucAuccuuTsT
A18525
172
AAGGAUGAUGAAGGUUUuATsT
AD-11564





 1-19
VP30
A18526
173
GAUGAAGAUUAAGAAAAAGTST
A18527
174
CUUUUUCUuAAUCUUcAUCTsT
AD-11565





 3-21
VP35
A18528
1019
GAuGAAGAuuAAAAccuucTsT
A18529
1020
GAAGGuuuUAAUCuUcAUCTsT
AD-11566





 4-22
VP35
A18530
1021
AuGAAGAuuAAAAccuucATsT
A18531
1022
uGAAGGuuuUAAUCuUcAUTsT
AD-11567





 5-23
VP35
A18532
1023
uGAAGAuuAAAAccuucAuTsT
A18533
1024
AuGAAGGuuuuAAUCuUcATsT
AD-11568





 6-24
VP35
A18534
1025
GAAGAuuAAAAccuucAucTsT
A18535
1026
GAuGAAGGuuuuAAUCuUCTsT
AD-11569





1354-1372
VP35
A18536
1027
uGAuGAAGAuuAAGAAAAATsT
A18537
1028
uuuuUCuuAAUCuUcAUcATsT
AD-11570





 7-25
VP35
A18538
1029
AAGAuuAAAAccuucAucATsT
A18539
1030
uGAuGAAGGuuuuAAUCuUTsT
AD-11571





 8-26
VP35
A18540
1031
AGAuuAAAAccuucAucAuTsT
A18541
1032
AuGAuGAAGGuuuuAAUCUTsT
AD-11572





 9-27
VP35
A18542
1033
GAuuAAAAccuucAucAucTsT
A18543
1034
GAuGAuGAAGGuuuuAAUCTsT
AD-11573





10-28
VP35
A18544
1035
AuuAAAAccuucAucAuccTsT
A18545
1036
GCAuGAuGAAGGuuuuAAUTsT
AD-11574





11-29
VP35
A18546
1037
uuAAAAccuucAucAuccuTsT
A18547
1038
AGGAuGAuGAAGGuuuuAATsT
AD-11575





12-30
VP35
A18548
1039
uAAAAccuucAucAuccuuTsT
A18549
1040
AAGGAuGAuGAAGGuuuuATsT
AD-11576





 1-19
VP30
A18550
1041
GAuGAAGAuuAAGAAAAAGTsT
A18551
1042
CuuuuUCuuAAUCuUcAUCTsT
AD-11577





 996-1014
NP
A18552
25
UGGACACAUGAUGGUGAUCTsT
A18553
26
GAUCACCAUCAUGUGUCCATsT
AD-11578





1467-1485
NP
A18554
27
AAGCAACUCCAACAAUAUGTsT
A18555
28
CAUAUUGUUGGAGUUGCUUTsT
AD-11579





11-29
VP35
A18556
29
AAAACCUUCAUCAUCCUUUTsT
A18557
30
AAAGGAUGAUGAAGGUUUUTsT
AD-11580





1357-1375
VP35
A18558
31
UUGAUGAAGAUUAAGAAAATsT
A18559
32
UUUUCUUAAUCUUCAUCAATsT
AD-11581





 1-19
VP40
A18560
33
GAUGAAGAUUAAGAAAAAGTsT
A18561
34
CUUUUUCUUAAUCUUCAUCTsT
AD-11582





647-665
VP40
A18562
35
CCCUGCUGCAACAUGGACATsT
A18563
36
UGUCCAUGUUGCAGCAGGGTsT
AD-11583





 2-20
VP30
A18564
37
AUGAAGAUUAAGAAAAAGUTsT
A18565
38
ACUUUUUCUUAAUCUUCAUTsT
AD-11584





 1-19
L
A18566
39
AAGAUUAAGAAAAAGUCCATsT
A18567
40
UGGACUUUUUCUUAAUCUUTsT
AD-11585





 5-23
NP
A18568
41
AAGAUUAAUAAUUUUCCUCTsT
A18569
42
GAGGAAAAUUAUUAAUCUUTsT
AD-11586





823-841
NP
A18570
43
AUGCCGGAAGAGGAGACAATsT
A18571
44
UUGUCUCCUCUUCCGGCAUTsT
AD-11587





824-842
NP
A18572
45
UGCCGGAAGAGGAGACAACTsT
A18573
46
GUUGUCUCCUCUUCCGGCATsT
AD-11588





 987-1005
NP
A18574
47
GCAAUCAGUAGGACACAUGTsT
A18575
48
CAUGUGUCCUACUGUUGCTsT
AD-11589





 988-1006
NP
A18576
49
CAAUCAGUAGGACACAUGATsT
A18577
50
UCAUGUGUCCUACUGAUUGTsT
AD-11590





 989-1007
NP
A18578
51
AAUCAGUAGGACACAUGAUTsT
A18579
52
AUCAUGUGUCCUACUGAUUTsT
AD-11591





 990-1008
NP
A18580
53
AUCAGUAGGACACAUGAUGTsT
A18581
54
CAUCAUGUGUCCUACUGAUTsT
AD-11592





 991-1009
NP
A18582
55
UCAGUAGGACACAUGAUGGTsT
A18583
56
CCAUCAUGUGUCCUACUGATsT
AD-11593





 992-1010
NP
A18584
57
CAGUAGGACACAUGAUGGUTsT
A18585
58
ACCAUCAUGUGUCCUACUGTsT
AD-11594





 993-1011
NP
A18586
59
AGUAGGACACAUGAUGGUGTST
A18587
60
CACCAUCAUGUGUCCUACUTsT
AD-11595





 994-1012
NP
A18588
61
GUAGGACACAUGAUGGUGATsT
A18589
62
UCACCAUCAUGUGUCCUACTsT
AD-11596





 995-1013
NP
A18590
63
UAGGACACAUGAUGGUGAUTsT
A18591
64
AUCACCAUCAUGUGUCCUATsT
AD-11597





1005-1023
NP
A18592
65
GAUGGUGAUUUUCCGUUUGTsT
A18593
66
CAAACGGAAAAUCACCAUCTsT
AD-11598





1006-1024
NP
A18594
67
AUGGUGAUUUUCCGUUUGATsT
A18595
68
UCAAACGGAAAAUCACCAUTsT
AD-11599





1007-1025
NP
A18596
69
UGGUGAUUUUCCGUUUGAUTsT
A18597
70
AUCAAACGGAAAAUCACCATsT
AD-11600





1008-1026
NP
A18598
71
GGUGAUUUUCCGUUUGAUGTsT
A18599
72
CAUCAAACGGAAAAUCACCTsT
AD-11601





1462-1480
NP
A18600
73
GCUGAGAAGCAACUCCAACTsT
A18601
74
GUUGGAGUUGCUUCUCAGCTsT
AD-11602





1463-1481
NP
A18602
75
CUGAGAAGCAACUCCAACATsT
A18603
76
UGUUGGAGUUGCUUCUCAGTsT
AD-11603





1464-1482
NP
A18604
77
UGAGAAGCAACUCCAACAATST
A18605
78
UUGUUGGAGUUGCUUCUCATsT
AD-11604





1465-1483
NP
A18606
79
GAGAAGCAACUCCAACAAUTsT
A18607
80
AUUGUUGGAGUUGCUUCUCTsT
AD-11605





1466-1484
NP
A18608
81
AGAAGCAACUCCAACAAUATsT
A18609
82
UAUUGUUGGAGUUGCUUCUTsT
AD-11606





1353-1371
VP35
A18610
83
AAAAGUGAUGAAGAUUAAGTsT
A18611
84
CUUAAUCUUCAUCACUUUUTsT
AD-11607





1354-1372
VP35
A18612
85
AAAGUGAUGAAGAUUAAGATsT
A18613
86
UCUUAAUCUUCAUCACUUUTsT
AD-11608





1355-1373
VP35
A18614
87
AAGUGAUGAAGAUUAAGAATsT
A18615
88
UUCUUAAUCUUCAUCACUUTsT
AD-11609





1356-1374
VP35
A18616
89
AGUGAUGAAGAUUAAGAAATsT
A18617
90
UUUCUUAAUUUCAUCACUTsT
AD-11610





645-663
VP40
A18618
91
CUGCCUGCUGCAACAUGGATsT
A18619
92
UCCAUGUUGCAGCAGGCAGTsT
AD-11611





646-664
VP40
A18620
93
UGCCUGCUGCAACAUGGACTsT
A18621
94
GUCCAUGUtJGCAGCAGGCATsT
AD-11612





451-469
GP
A18622
95
GGCUGAAAACUGCUACAAUTsT
A18623
96
AUUGUAGCAGUUUUCAGCCTsT
AD-11613





452-470
GP
A18624
97
GCUGAAAACUGCUACAAUCTsT
A18625
98
GAUUGUAGCAGUUUUCAGCTsT
AD-11614





453-471
GP
A18626
99
CUGAAAACUGCUACAAUCUTsT
A18627
100
AGAUUGUAGCAGUUUUCAGTsT
AD-11615





454-472
GP
A18628
101
UGAAAACUGCUACAAUCUUTsT
A18629
102
AAGAUUGUAGCAGUUUUCATsT
AD-11616





455-473
GP
A18630
103
GAAAACUGCUACAAUCUUGTsT
A18631
104
CAAGAUUGUAGCAGUUUUCTsT
AD-11617





456-474
GP
A18632
105
AAAACUGCUACAAUCUUGATsT
A18633
106
UCAAGAUUGUAGCAGUUUUTsT
AD-11618





457-475
GP
A18634
107
AAACUGCUACAAUCUUGAATsT
A18635
108
UUCAAGAUUGUAGCAGUUUTsT
AD-11619





458-476
GP
A18636
109
AACUGCUACAAUCUUGAAATsT
A18637
110
UUUCAAGAUUGUAGCAGUUTsT
AD-11620





459-477
GP
A18638
111
ACUGCUACAAUCUUGAAAUTsT
A18639
112
AUUUCAAGAUUGUAGCAGUTsT
AD-11621





599-617
VP30
A18640
113
AGCAAAUCCAACGGCUGAUTsT
A18641
114
AUCAGCCGUUGGAUUUGCUTsT
AD-11622





600-618
VP30
A18642
115
GCAAAUCCAACGGCUGAUGTsT
A18643
116
CAUCAGCCGUUGGAUUUGCTsT
AD-11623





601-619
VP30
A18644
117
CAAAUCCAACGGCUGAUGATsT
A18645
118
UCAUCAGCCGUUGGAUUUGTsT
AD-11624





135-153
L
A18646
119
UUGGACCAAUGUGACCUAGTsT
A18647
120
CUAGGUCACAUUGGUCCAATsT
AD-11625





136-154
L
A18648
121
UGGACCAAUGUGACCUAGUTsT
A18649
122
ACUAGGUCACAUUGGUCCATsT
AD-11626





2100-2118
L
A18650
123
AUGCAUGUCAGUGAUUAUUTsT
A18651
124
AAUAAUCACUGACAUGCAUTsT
AD-11627





2101-2119
L
A18652
125
UGCAUGUCAGUGAUUAUUATsT
A18653
126
UAAUAAUCACUGACAUGCATsT
AD-11628





2102-2120
L
A18654
127
GCAUGUCAGUGAUUAUUAUTST
A18655
128
AUAAUAAUCACUGACAUGCTsT
AD-11629





2103-2121
L
A18656
129
CAUGUCAGUGAUUAUUAUATsT
A18657
130
UAUAAUAAUCACUGACAUGTsT
AD-11630





2104-2122
L
A18658
131
AUGUCAGUGAUUAUUAUAATsT
A18659
132
UUAUAAUAAUCACUGACAUTsT
AD-11631





2114-2132
L
A18660
133
UUAUUAUAAUCCACCACAUTsT
A18661
134
AUGUGGUGGAUUAUAAUAATsT
AD-11632





2115-2133
L
A18662
135
UAUUAUAAUCCACCACAUATsT
A18663
136
UAUGUGGUGGAUUAUAAUATsT
AD-11633





2116-2134
L
A18664
137
AUUAUAAUCCACCACAUAATsT
A18665
138
UUAUGUGGUGGAUUAUAAUTsT
AD-11634





2412-2430
L
A18666
139
AAAGUUACAAGUGCCUGUGTsT
A18667
140
CACAGGCACUUGUAACUUUTsT
AD-11635





2413-2431
L
A18668
141
AAGUUACAAGUGCCUGUGGTsT
A18669
142
CCACAGGCACUUGUAACUUTsT
AD-11636





2466-2484
L
A18670
143
UCAGGUUUUAUCUAUUUUGTsT
A18671
144
CAAAAUAGAUAAAACCUGATsT
AD-11637





2467-2485
L
A18672
145
CAGGUUUUAUCUAUUUUGGTsT
A18673
146
CCAAAAUAGAUAAAACCUGTsT
AD-11638





2556-2574
L
A18674
147
UCUGAUGCAAUUUUUGAUGTsT
A18675
148
CAUCAAAAAUUGCAUCAGATsT
AD-11639





2557-2557
L
A18676
149
CUGAUGCAAUUUUUGAUGATsT
A18677
150
UCAUCAAAAAUUGCAUCAGTsT
AD-11640





1825-1843
NP
A18678
151
AGUUACUCGGAAAACGGCATST
A18679
152
UGCCGUUUUCCGAGuAACUTsT
AD-11641





1588-1606
NP
A18680
153
AAcGcuAuGGuAAcucuAATsT
A18681
154
UuAGAGUuACcAuAGCGUUTsT
AD-11642





1827-1845
NP
A18682
155
uuAcucGGAAAAcGGcAuGTsT
A18683
156
cAUGCCGUUUUCCGAGuAATsT
AD-11643





1583-1601
NP
A18684
157
AAAcAAAcGcuAuGGuAAcTsT
A18685
158
GUuACcAuAGCGUUUGUUUTsT
AD-11644





1488-1506
NP
A18686
159
AGAGucucGcGAAcuuGAcTsT
A18687
160
GUcAAGUUCGCGAGACUCUTsT
AD-11645





1489-1507
NP
A18688
161
GAGucucGcGAAcuuGAccTsT
A18689
162
GGUcAAGUUCGCGAGACUCTsT
AD-11646





1585-1603
NP
A18690
163
AcAAAcGcuAuGGuAAcucTsT
A18691
164
GAGUuACcAuAGCGUUUGUTsT
AD-11647





1586-1604
NP
A18692
165
cAAAcGcuAuGGuAAcucuTsT
A18693
166
AGAGUuACcAuAGCGUUUGTsT
AD-16648





2231-2249
NP
A18694
167
cAccGGcucccGuAuAcAGTsT
A18695
168
CUGuAuACGGGAGCCGGUGTsT
AD-11649





2873-2891
NP
A18696
169
cuAAcuAGcGAuuuAucuATsT
A18697
170
uAGAuAAAUCGCuAGUuAGTsT
AD-11650





1172-1190
VP35
A18698
171
GCUGAACUAUAGGGUACGUTsT
A18699
172
ACGuACCCuAuAGUUcAGCTsT
AD-11651





1176-1194
VP35
A18700
173
AAcuAuAGGGuAcGuuAcATsT
A18701
174
UGuAACGuACCCuAuAGUUTsT
AD-11652





1174-1192
VP35
A18702
175
uGAAcuAuAGGGuAcGuuATsT
A18703
176
uAACGuACCCuAuAGUUcATsT
AD-11653





1178-1196
VP35
A18706
177
cuAuAGGGuAcGuuAcAuuTsT
A18707
178
AAUGuAACGuACCCuAuAGTsT
AD-11655





251-269
VP35
A18704
179
GGAuuAuGcuAcGcAucccTsT
A18705
180
GGGAUGCGuAGcAuAAUCCTsT
AD-11654





416-434
VP35
A18708
181
uuAGAAcAAcGcAuuAcGATsT
A18709
182
UCGuAAUGCGUUGUUCuAATsT
AD-16656





421-439
VP35
A18710
183
AcAAcGcAuuAcGAGucuuTsT
A18711
184
AAGACUCGuAAUGCGUUGUTsT
AD-11657





1057-1075
VP35
A18712
185
uGAucGAGGuuGGGuAuGuTsT
A18713
186
AcAuACCcAACCUCGAUcATsT
AD-11658





167-185
GP
A18714
187
ccucGuGAucGAuucAAGATsT
A18715
188
UCUUGAAUCGAUcACGAGGTsT
AD-11659





163-181
GP
A18716
189
GuuAccucGuGAucGAuucTsT
A18717
190
GAAUCGAUcACGAGGuAACTsT
AD-11660





658-676
GP
A18720
191
AAcGAcuuucGcuGAAGGuTsT
A18721
192
ACCUUcAGCGAAAGUCGUUTsT
AD-11662





755-773
GP
A18722
193
AcGGAGGAcccGucuAGuGTsT
A18723
194
cACuAGACGGGUCCUCCGUTsT
AD-11663





966-984
GP
A18724
195
AGGucAAccccGAAAuuGATsT
A18725
196
UcAAUUUCGGGGUUGACCUTsT
AD-11664





978-996
GP
A18726
197
AAAuuGAuAcAAcAAucGGTsT
A18727
198
CCGAUUGUUGuAUcAAUUUTsT
AD-11665





 985-1003
GP
A18728
199
uAcAAcAAucGGGGAGuGGTsT
A18729
200
CcACUCCCCGAUUGUUGuATsT
AD-11666





1101-1119
GP
A18730
201
AGAGuccGGcGcGAAcuucTsT
A18731
202
GAAGuUCGCGCCGGACUCUTsT
AD-11667





1730-1748
GP
A18718
203
uGGAuAccAuAuuucGGGcTsT
A18719
204
GCCCGAAAuAUGGuAUCcATsT
AD-11661





1820-1838
GP
A18732
205
cuGGccAAcGAGAcGAcucTsT
A18733
206
GAGUCGUCUCGUUGGCcAGTsT
AD-11668





1298-1316
VP30
A18734
207
uAucGcucGuAAuAuAAccTsT
A18735
208
GGUuAuAUuACGAGCGAuATsT
AD-11669





295-313
VP30
A18736
209
uucGAGcAcGAucAucAucTsT
A18737
210
GAuGAuGAUCGuGCUCGAATsT
AD-11670





590-608
VP30
A18738
211
cucGcGcuuAGcAAAuccATsT
A18739
212
UGGAUUUGCuAAGCGCGAGTsT
AD-11671





519-537
VP30
A18740
213
uuAcuccuAcuAAucGcccTsT
A18741
214
GGGCGAUuAGuAGGAGuAATsT
AD-11672





126-144
VP30
A18742
215
cuGcGAAccGGuAGAGuuuTsT
A18743
216
AAACUCuACCGGUUCGcAGTsT
AD-11673





133-151
VP30
A18744
217
CcGGuAGAGuuuAGuuGcATsT
A18745
218
UGcAACuAAACUCuACCGGTsT
AD-11674





292-310
VP30
A18746
219
AuGuucGAGcAcGAucAucTsT
A18747
220
GAUGAUCGUGCUCGAAcAUTsT
AD-11675





321-339
VP30
A18748
221
AAuuAucGAGGuGAGuAccTsT
A18749
222
GGuACUcACCUCGAuAAUUTsT
AD-11676





910-928
VP30
A18750
223
GGGAccGAcAAucccuAAuTsT
A18751
224
AUuAGGGAUUGUCGGUCCCTsT
AD-11677





1295-1313
VP30
A18752
225
ucGuAucGcucGuAAuAuATsT
A18753
226
uAuAUuACGAGCGAuACGATsT
AD-11678





331-349
VP30
A18754
227
GuGAGuAccGucAAucAAGTsT
A18755
228
CUUGAUUGACGGuACUcACTsT
AD-11679





123-141
VP30
A18756
229
GAucuGcGAAccGGuAGAGTsT
A18757
230
CUCuACCGGUUCGcAGAUCTsT
AD-11680





124-142
VP30
A18758
231
AucuGcGAAccGGuAGAGuTsT
A18759
232
ACUCuACCGGUUCGcAGAUTsT
AD-11681





1293-1311
VP30
A18760
233
ucucGuAucGcucGuAAuATsT
A18761
234
uAUuACGAGCGAuACGAGATsT
AD-11682





145-163
VP30
A18762
235
AGuuGcAAccuAAcAcAcATsT
A18763
236
UGUGUGUuAGGUUGcAACUTsT
AD-11683





293-311
VP30
A18764
237
uGuucGAGcAcGAucAucATsT
A18765
238
UGAUGAUCGUGCUCGAAcATsT
AD-11684





358-376
VP30
A18766
239
cAcAAGuGcGcGuuccuAcTsT
A18767
240
GuAGGAACGCGcACUUGUGTsT
AD-11685





359-377
VP30
A18768
241
AcAAGuGcGcGuuccuAcuTsT
A18769
242
AGuAGGAACGCGcACUUGUTsT
AD-11686





518-536
VP30
A18770
243
AuuAcuccuAcuAAucGccTsT
A18771
244
GGCGAUuAGuAGGAGuAAUTsT
AD-11687





520-538
VP30
A18772
245
uAcuccuAcuAAucGcccGTsT
A18773
246
CGGGCGAUuAGuAGGAGuATsT
AD-11688





524-542
VP30
A18774
247
ccuAcuAAucGcccGuAAGTsT
A18775
248
CUuACGGGCGAUuAGuAGGTsT
AD-11689





525-543
VP30
A18776
249
cuAcuAAucGcccGuAAGATsT
A18777
250
UCUuACGGGCGAUuAGuAGTsT
AD-11690





584-602
VP30
A18778
251
cAAGGAcucGcGcuuAGcATsT
A18779
252
UGCuAAGCGCGAGUCCUUGTsT
AD-11691





469-487
VP24
A18784
253
AGcuACGGGAcGAuAcAAuTsT
A18785
254
AUUGuAUCGUCCCGuAGCUTsT
AD-11694





910-928
VP24
A18786
255
GucGuuGAuucGAuccAAuTsT
A18787
256
AUUGGAUCGAAUcAACGACTsT
AD-11695





467-485
VP24
A18788
257
AAAGcuAcGGGAcGAuACATsT
A18789
258
UGuAUCGUCCCGuAGCUUUTsT
AD-11696





862-880
VP24
A18792
259
cAAcAuGcGAAcAcAAcGuTsT
A18793
260
ACGUUGUGUUCGcAUGUUGTsT
AD-11698





466-484
VP24
A18796
261
uAAAGcuAcGGGAcGAuAcTsT
A18797
262
GuAUCGUCCCGuAGCUUuATsT
AD-11700





523-541
VP24
A18804
263
uGucuuAAGcGAccucuGuTsT
A18805
264
AcAGAGGUCGCUuAAGAcATsT
AD-11704





958-976
VP24
A18806
265
ucuAcAuGucGuGAAcuAcTsT
A18807
266
GuAGUUcACGAcAUGuAGATsT
AD-11705






959-977
VP24
A18808
267
cuAcAuGucGuGAAcuAcATsT
A18809
268
UGuAGUUcACGACAUGuAGTsT
AD-11706





971-989
VP24
A18810
269
AAcuAcAACGGAuuGuuGATsT
A18811
270
UcAAcAAUCCGUUGuAGUUTsT
AD-11707





1071-1089
VP24
A18812
271
ccGAcAAAucGGcAAuGAATsT
A18813
272
UUcAUUGCCGAUUUGUCGGTsT
AD-11708





5886-5904
L
A18816
273
AGAucGAAAuuGuAcGAAGTsT
A18817
274
CUUCGuAcAAUUUCGAUCUTsT
AD-11710





192-210
L
A18818
275
AAuccGcAAcuAcGcAAcuTsT
A18819
276
AGUUGCGuAGUUGCGGAUUTsT
AD-11711





5395-5413
L
A18820
277
cAcGccAAuuAAcGucAucTsT
A18821
278
GAUGACGUuAAUUGGCGUGTsT
AD-11712





193-211
L
A18822
279
AuccGcAAcuAcGcAAcuGTsT
A18823
280
cAGUUGCGuAGUUGCGGAUTsT
AD-11713





219-237
L
A18824
281
ccGAAAcAuAucuAccGuuTsT
A18825
282
AACGGuAGAuAUGUUUCGGTsT
AD-11714





2840-2858
L
A18826
283
uuucuAccGGAAucuAGGATsT
A18827
284
UCCuAGAUUCCGGuAGAAATsT
AD-11715





4779-4797
L
A18828
285
AuuAAucGcGGAAcAAuuGTsT
A18829
286
cAAUUGUUCCGCGAUuAAUTsT
AD-11716





5275-5293
L
A18830
287
AuuucGAucGAucGAGAcATsT
A18831
288
uGUCUCGAUCGAUCGAAAUTsT
AD-11717





5391-5409
L
A18832
289
GGGAcAcGccAAuuAAcGuTsT
A18833
290
ACGUuAAUUGGCGUGUCCCTsT
AD-11718





191-209
L
A18834
291
uAAuccGcAAcuAcGcAAcTsT
A18835
292
GUUGCGuAGUUGCGGAUuATsT
AD-11719





1614-1632
L
A18836
293
AGuAcuAAAcGuGuAccGGTsT
A18837
294
CCGGuAcACGUUuAGuACUTsT
AD-11720





4588-4606
L
A18838
295
cAcAucGcucAuuGcGAAuTsT
A18839
296
AUUCGcAAUGAGCGAUGUGTsT
AD-11721





4590-4608
L
A18840
297
cAucGcucAuuGcGAAuAcTsT
A18841
298
GuAUUCGcAAUGAGCGAUGTsT
AD-11722





5884-5902
L
A18842
299
AcAGAucGAAAuuGuAcGATsT
A18843
300
UCGuAcAAUUUCGAUCUGUTsT
AD-11723





161-179
L
A18844
301
AGcuuGcGGGuuAuAuucATsT
A18845
302
UGAAuAuAACCCGcAAGCUTsT
AD-11724





778-796
L
A18846
303
cuGccGAcGucuuGAuAAuTsT
A18847
304
AUuAUcAAGACGUCGGcAGTsT
AD-11725





5446-5464
L
A18848
305
AGuAcuuAcGGcAAuuGAGTsT
A18849
306
CUcAAUUGCCGuAAGuACUTsT
AD-11726





6297-6315
L
A18850
307
AAcCucGucGAuucAAAAATsT
A18851
308
uuuuuGAAUCGACGAGGuUTsT
AD-11727





5269-5287
L
A18852
309
AAcuAAAuuucGAucGAucTsT
A18853
310
GAUCGAUCGAAAUUuAGUUTsT
AD-11728





1778-1796
L
A18854
311
GccuuAuccGAcucGcAAuTsT
A18855
312
AUUGCGAGUCGGAuAAGGCTsT
AD-11729





1780-1798
L
A18856
313
cuuAuccGAcucGcAAuGuTsT
A18857
314
AcAUUGCGAGUCGGAuAAGTsT
AD-11730





3163-3181
L
A18858
315
GucGuuuuGcGGccGAuAuTsT
A18859
316
AuAUCGGCCGcAAAACGACTsT
AD-11731





3164-3182
L
A18860
317
ucGuuuuGcGGccGAuAucTsT
A18861
318
GAuAUCGGCCGcAAAACGATsT
AD-11732





5273-5291
L
A18862
319
AAAuuucGAucGAucGAGATsT
A18863
320
UCUCGAUCGAUCGAAAuuUTsT
AD-11733





6295-6313
L
A18864
321
AuAAccucGucGAuucAAATsT
A18865
322
UUUGAAUCGACGAGGUuAUTST
AD-11734





1702-1720
L
A18866
323
UACUACCACAAUAUCGGAATST
A18867
324
UUCCGAuAUUGUGGuAGuATsT
AD-11735





1781-1799
L
A18868
325
uuAuccGAcucGcAAuGuuTsT
A18869
326
AAcAUUGCGAGUCGGAuAATsT
AD-11736





5270-5288
L
A18870
327
AcuAAAuuucGAucGAucGTsT
A18871
328
CGAUCGAUCGAAAUUuAGUTsT
AD-11737





5276-5294
L
A18872
329
uuucGAucGAucGAGAcAcTsT
A18873
330
GuGUCUCGAUCGAUCGAAATsT
AD-11738





5394-5412
L
A18874
331
AcAcGccAAuuAAcGucAuTsT
A18875
332
AUGACGUuAAUUGGCGUGUTsT
AD-11739





6242-6260
L
A18876
333
AAGuuAuAuccGccuuGGuTsT
A18877
334
ACcAAGGCGGAuAuAACUUTsT
AD-11740





182-200
L
A18878
335
AuAcucccuuAAuccGcAATsT
A18879
336
UUGCGGAUuAAGGGAGuAUTsT
AD-11741





194-212
L
A18880
337
uccGcAAcuAcGcAAcuGuTsT
A18881
338
AcAGUUGCGuAGUUGCGGATsT
AD-11742





575-593
L
A18882
339
ucGAGGAAAcuCuAGAucATsT
A18883
340
UGAUCuAGAGUUUCCUCGATsT
AD-11743





1565-1583
L
A18884
341
uGcAGuAuucGAGccuAAuTsT
A18885
342
AUuAGGCUCGAAuACUGcATsT
AD-11744





1566-1584
L
A18886
343
GcAGuAuucGAGccuAAuGTsT
A18887
344
cAUuAGGCUCGAAuACUGCTsT
AD-11745





1567-1585
L
A18888
345
cAGuAuucGAGccuAAuGuTsT
A18889
346
AcAUuAGGCUCGAAuACUGTsT
AD-11746





2779-2797
L
A18890
347
CAUUGGCACUAGCGGUACCTST
A18891
348
GGuACCGCuAGUGCcAAUGTsT
AD-11747





2838-2856
L
A18892
349
uGuuucuAccGGAAucuAGTsT
A18893
350
CuAGAUUCCGGuAGAAAcATsT
AD-11748





2892-2910
L
A18894
351
AcuuAucuccGAAuGAuuGTsT
A18895
352
cAAUcAUUCGGAGAuAAGUTsT
AD-11749





2981-2999
L
A18896
353
AAAuccuAGcGGAuuAAAuTsT
A18897
354
AUUuAAUCCGCuAGGAUUUTsT
AD-11750





2982-3000
L
A18898
355
AAUCCUAGCGGAUUAAAUGTST
A18899
356
cAUUuAAUCCGCuAGGAUUTsT
AD-11751





3038-3056
L
A18900
357
GAuuGuAcGcAGGAccAucTsT
A18901
358
GAUGGUCCUGCGuAcAAUCTsT
AD-11752





3149-3167
L
A18902
359
AAcuccuGuuAuGAGucGuTsT
A18903
360
ACGACUcAuAAcAGGAGUUTsT
AD-11753





3168-3186
L
A18904
361
uuuGcGGccGAuAucuuuuTsT
A18905
362
AAAAGAuAUCGGCCGcAAATsT
AD-11754





3889-3907
L
A18906
363
GGuAcAAcGAucAAuAcAGTsT
A18907
364
CUGuAUUGAUCGUUGuACCTsT
AD-11755





3922-3940
L
A18908
365
uGGccAAucGuAuGAGuAATsT
A18909
366
UuACUcAuACGAUUGGCcATsT
AD-11756





4001-4019
L
A18910
367
GucuGcAcGcGAcAGcAAuTsT
A18911
368
AUUGCUGUCGCGUGcAGACTsT
AD-11757





4584-4602
L
A18912
369
cuAccAcAucGcucAuuGcTsT
A18913
370
GcAAUGAGCGAUGUGGuAGTsT
AD-11758





4593-4611
L
A18914
371
cGcucAuuGcGAAuAcuuATsT
A18915
372
uAAGuAUUCGcAAUGAGCGTsT
AD-11759





4598-4616
L
A18916
373
AuuGcGAAuAcuuAAGccATsT
A18917
374
UGGCUuAAGuAUUCGcAAUTsT
AD-11760





4601-4619
L
A18918
375
GcGAAuAcuuAAGccAAcATsT
A18919
376
UGUUGGCUuAAGuAUUCGCTsT
AD-11761





4638-4656
L
A18920
377
AuGucAcGGuuAAuGAGuATsT
A18921
378
uACUcAUuAACCGUGAcAUTsT
AD-11762





4778-4796
L
A18922
379
AAuuAAucGcGGAAcAAuuTsT
A18923
380
AAUUGUUCCGCGAUuAAUUTsT
AD-11763





5274-5292
L
A18924
381
AAuuucGAucGAucGAGAcTsT
A18925
382
GUCUCGAUCGAUCGAAAuUTsT
AD-11764





5392-5410
L
A18926
383
GGAcAcGccAAuuAAcGucTsT
A18927
384
GACGUuAAUUGGCGUGUCCTsT
AD-11765





5649-5667
L
A18928
385
AcGcuAGcuAcuGAGuccATsT
A18929
386
UGGACUcAGuAGCuAGCGUTsT
AD-11766





5833-5851
L
A18930
387
cuAAGcAAGucGAGGuuAuTsT
A18931
388
AuAACCUCGACUUGCUuAGTsT
AD-11767





6243-6261
L
A18932
389
AGuuAuAuccGccuuGCuuTsT
A18933
390
AACcAAGGCGGAuAuAACUTsT
AD-11768





6290-6308
L
A18934
391
cAGGuAuAAccucGucGAuTsT
A18935
392
AUCGACGAGGUuAuACCUGTsT
AD-11769





6291-6309
L
A18936
393
AGGuAuAAccucGucGAuuTsT
A18937
394
AAUCGACGAGGUuAuACCUTsT
AD-11770





1816-1834
NP
A18938
395
AcuAcGAGGAuucGGcuGATsT
A18939
396
UcAGCCGAAUCCUCGuAGUTsT
AD-11771





875-893
NP
A18940
397
ucuAcccAAAcuuGucGuuTsT
A18941
398
AACGAcAAGUUUGGGuAGATsT
AD-11772





1817-1835
NP
A18942
399
cuAcGAGGAuucGGcuGAATsT
A18943
400
UUcAGCCGAAUCCUCGuAGTsT
AD-11773





1812-1830
NP
A18944
401
ccuGAcuAcGAGGAuucGGTsT
A18945
402
CCGAAUCCUCGuAGUcAGGTsT
AD-11774





1819-1837
NP
A18946
403
AcGAGGAuucGGcuGAAGGTsT
A18947
404
CCUUcAGCCGAAUCCUCGUTsT
AD-11775





2140-2158
NP
A18948
405
AcGAGAGucucAcAucccuTsT
A18949
406
AGGGAuGuGAGACUCUCGUTST
AD-11776





730-748
VP35
A18950
407
AAAuuucGGGcGAccuuAcTsT
A18951
408
GuAAGGUCGCCCGAAAUUUTsT
AD-11777





735-753
VP35
A18952
409
ucGGGcGAccuuAcAuuucTsT
A18953
410
GAAAUGuAAGGUCGCCCGATsT
AD-11778





195-213
VP35
A18954
411
uGAccGGcAAAAuAccGcuTsT
A18955
412
AGCGGuAUUUUGCCGGUcATsT
AD-11779





198-216
VP35
A18956
413
ccGGcAAAAuACcGCuAAcTsT
A18957
414
GUuAGCGGuAUUUUGCCGGTsT
AD-11780





379-397
VP35
A18958
415
AGcuGuGcGucGGcAAAccTsT
A18959
416
GGUUUGCCGACGcACAGCUTsT
AD-11781





646-664
VP35
A18960
417
AuuGAAAGAuccGAAcGGGTsT
A18961
418
CCCGUUCGGAUCUUUcAAUTsT
AD-11782





731-749
VP35
A18962
419
AAuuucGGGcGAccuuAcATsT
A18963
420
UGuAAGGUCGCCCGAAAUUTsT
AD-11783





732-750
VP35
A18964
421
AuuucGGGcGAccuuAcAuTsT
A18965
422
AUGUAAGGUCGCCCGAAAUTsT
AD-11784





1193-1211
VP35
A18966
423
GucuAuuGuGucAuAAGcuTsT
A18967
424
AGCUuAUGAcAcAAuAGACTsT
AD-11785





438-456
VP40
A18968
425
cucGcAucuuAuAcGAucATsT
A18969
426
UGAUCGuAuAAGAUGCGAGTsT
AD-11786





1301-1319
VP40
A18970
427
uGcAuAAGcGAuccAuAcuTsT
A18971
428
AGuAUGGAUCGCUuAUGcATsT
AD-11787





1191-1209
VP40
A18972
429
AAuGuAcuAAucGGGucAATsT
A18973
430
UUGACCCGAUuAGuAcAUUTsT
AD-11788





442-460
VP40
A18974
431
cAucuuAuAcGAucAcccATsT
A18975
432
UGGGUGAUCGuAuAAGAUGTsT
AD-11789





443-461
VP40
A18976
433
AUCUUAUACGAUCACCCAUTsT
A18977
434
AUGGGUGAUCGuAuAAGAUTsT
AD-11790





478-496
VP40
A18978
435
AcccccucGuuAGAGuGAATsT
A18979
436
UUcACUCuAACGAGGGGGUTsT
AD-11791





834-852
VP40
A18980
437
AucGuGccAAuuGAuccAGTsT
A18981
438
CUGGAUcAAUUGGcACGAUTsT
AD-11792





1192-1210
VP40
A18982
439
AuGuAcuAAucGGGucAAGTsT
A18983
440
CUUGACCCGAUuAGuAcAUTsT
AD-11793





1194-1212
VP40
A18984
441
GuAcuAAucGGGucAAGGATsT
A18985
442
UCCUUGACCCGAUuAGuACTsT
AD-11794





1300-1318
VP40
A18986
443
AuGcAuAAGcGAuccAuAcTsT
A18987
444
GuAUGGAUCGCUuAUGcAUTsT
AD-11795





465-483
GP
A18988
445
AcGGGAGcGAAuGcuuAccTsT
A18989
446
GGuAAGcAUUCGCUCCCGUTsT
AD-11796





358-376
VP30
A18990
447
AGuuAGAGucccuAcGGuuTsT
A18991
448
AACCGuAGGGACUCuAACUTsT
AD-11797





331-349
VP30
A18992
449
cuAccGuAGuAGucGAAGuTsT
A18993
450
ACUUCGACuACuACGGuAGTsT
AD-11798





250-268
VP30
A18994
451
GAAUUcAcGuGccGAccAGTsT
A18995
452
CUGGUCGGcACGUGAAUUCTsT
AD-11799





1009-1027
VP30
A18996
453
uGcccccccAAGcGuuAAuTsT
A18997
454
AUuAACGCUUGGGGGGGcATsT
AD-11800





1318-1336
VP30
A18998
455
AGAGuGuuAGGAucGuUAuTsT
A18999
456
AuAACGAUCCuAACACUCUTsT
AD-11801





126-144
VP30
A19000
457
AAucccGAGOcGGcAAuucTsT
A19001
458
GAAuuGCCGCCUCGGGAuUTsT
AD-11802





354-372
VP30
A19002
459
CGCAAGUUAGAGUCCCUACTST
A19003
460
GuAGGGACUCuAACUUGCGTsT
AD-11803





553-571
VP30
A19004
461
uGAuucAucGcuuAAuAuATsT
A19005
462
uAuAUuAAGCGAUGAAUcATsT
AD-11804





583-601
VP30
A19006
463
AGAccuAAGAcuAGcAAAuTsT
A19007
464
AUUUGCuAGUCUuAGGUCUTsT
AD-11805





652-670
VP30
A19008
465
AuuAcuAGucGAGAcuGcuTsT
A19009
466
AGcAGUCUCGACuAGuAAUTsT
AD-11806





 992-1010
VP30
A19010
467
ucAGGccuAcGcuuAcuuGTsT
A19011
468
cAAGuAAGCGuAGGCCUGATsT
AD-11807





1013-1031
VP30
A19012
469
ccccCAAGcGuuAAuGAAGTsT
A19013
470
CUUcAUuAACGCUUGGGGGTsT
AD-11808





404-422
VP24
A19014
471
AuuAuAcGGGuccAuuAAuTsT
A19015
472
AUuAAUGGACCCGuAuAAUTsT
AD-11809





888-906
VP24
A19016
473
cucAAcGAGuAAAGGAccATsT
A19017
474
UGGUCCUUuACUCGUUGAGTsT
AD-11810





1247-1265
VP24
A19018
475
uuGUAcGAuAGGGcuAAcATsT
A19019
476
UGUuAGCCCuAUCGuAcAATsT
AD-11811





536-554
VP24
A19020
477
GuuGuGuuuAGcGAccuAuTsT
A19021
478
AuAGGUCGCuAAAcAcAACTsT
AD-11812





1050-1068
VP24
A19022
479
GGACUAAUAuGGGuuAucuTsT
A19023
480
AGAuAACCcAuAUuAGUCCTsT
AD-11813





1095-1113
VP24
A19024
481
CUGCGAUGGAUAUACGACATsT
A19025
482
UGUCGuAuAUCcAUCGcAGTsT
AD-11814





535-553
VP24
A19026
483
AGuuGuGuuuAGcGAccuATsT
A19027
484
uAGGUCGCuAAAcAcAACUTsT
AD-11815





196-214
VP24
A19028
485
uuGAAcuAGucuAcucGcATsT
A19029
486
UGCGAGuAGACuAGUUcAATsT
AD-11816





215-233
VP24
A19030
487
GAAUCCUACCGGGAAUAGATsT
A19031
488
UCuAUUCCCGGuAGGAUUCTsT
AD-11817





403-421
VP24
A19032
489
uAuuAuAcGGGuccAuuAATsT
A19033
490
UuAAUGGACCCGuAuAAuATsT
AD-11818





406-424
VP24
A19034
491
uAuAcGGGuccAuuAAuuuTsT
A19035
492
AAAUuAAUGGACCCGuAuATsT
AD-11819





1140-1158
VP24
A19036
493
uAcAuGAAuCGAcAcuuAATsT
A19037
494
UuAAGUGUCGAUUcAUGuATsT
AD-11820





1243-1261
VP24
A19038
495
AAAAuuGuAcGAuAGGGcuTsT
A19039
496
AGCCCuAUCGuAcAAUUUUTsT
AD-11821





1249-1267
VP24
A19040
497
GuAcGAuAGGGcuAAcAuuTsT
A19041
498
AAUGUuAGCCCuAUCGuACTsT
AD-11822





1590-1608
VP24
A19042
499
GAGcccAAAuuAAcAcGGuTsT
A19043
500
ACCGUGUuAAUUUGGGCUCTsT
AD-11823





3688-3706
L
A19044
501
ccCGcuAuuAAGccGAGGuTsT
A19045
502
ACCUCGGCUuAAuAGCGGGTsT
AD-11824





3687-3705
L
A19046
503
GcccGcuAuuAAGccGAGGTsT
A19047
504
CCUCGGCUuAAuAGCGGGCTsT
AD-11825





2956-2974
L
A19048
505
AAuuGuAGcGcAAuuGAcuTsT
A19049
506
AGUcAAUUGCGCuAcAAUUTsT
AD-11826





2615-2633
L
A19050
507
AGcGAucAAucuccGAAAcTsT
A19051
508
GuuUCGGAGAuuGAUCGCUTsT
AD-11827





2612-2630
L
A19052
509
uuGAGcGAucAAucuccGATsT
A19053
510
UCGGAGAUUGAUCGCUcAATsT
AD-11828





4595-4613
L
A19054
511
uucGAAucuucAAAccGAcTsT
A19055
512
GUCGGuuuGAAGAuUCGAATsT
AD-11829





2613-2631
L
A19056
513
uGAGcGAucAAucuCCGAATsT
A19057
514
UUCGGAGAUUGAUCGCUcATsT
AD-11830





2614-2632
L
A19058
515
GAGcGAucAAucuccGAAATsT
A19059
516
uuUCGGAGAuuGAUCGCUCTsT
AD-11831





3941-3959
L
A19060
517
CAAcGcGcuuGAuGGuAucTsT
A19061
518
GAuACcAUcAAGCGCGUUGTsT
AD-11832





3942-3960
L
A19062
519
AAcGcGcuuGAuGGuAucuTsT
A19063
520
AGAuACcAUcAAGCGCGUUTsT
AD-11833





1680-1698
L
A19064
521
AuACGcCcAAGAAcuuAGGTsT
A19065
522
CCuAAGUUCUUGGGCGuAUTsT
AD-11834





3686-3704
L
A19066
523
AGcccGcuAuuAAGccGAGTsT
A19067
524
CUCGGCUuAAuAGCGGGCUTsT
AD-11835





4255-4273
L
A19068
525
uuAucGAuuGAcAGuccuuTsT
A19069
526
AAGGACUGUcAAUCGAuAATsT
AD-11836





1374-1392
L
A19070
527
AGAccGAuGuuuAAcGccGTsT
A19071
528
CGGCGUuAAAcAUCGGUCUTsT
AD-11837





5470-5488
L
A19072
529
AccAuAuAuuGucGcuucATsT
A19073
530
UGAAGCGAcAAuAuAUGGUTsT
AD-11838





3872-3890
L
A19074
531
AuAuuGuGcAucGGuAuAATsT
A19075
532
UuAUACCGAUGCAcAAuAUTsT
AD-11839





1384-1402
L
A19076
533
uuAAcGccGGGAuuGAAuuTsT
A19077
534
AAUUcAAUCCCGGCGUuAATsT
AD-11840





4519-4537
L
A19078
535
UGCACGAAAAAGAUCGGACTsT
A19079
536
GUCCGAUCUUUUUCGUGcATsT
AD-11841





3682-3700
L
A19080
537
GGucAGcccGcuAuuAAGcTsT
A19081
538
GCUuAAuAGCGGGCUGACCTsT
AD-11842





2954-2972
L
A19082
539
GGAAuuGuAGcGcAAuuGATsT
A19083
540
UcAAUUGCGCuAcAAUUCCTsT
AD-11843





5467-5485
L
A19084
541
AcuAccAuAuAuuGucGcuTsT
A19085
542
AGCGAcAAuAuAUGGuAGUTsT
AD-11844





1376-1394
L
A19086
543
AccGAuGuuuAAcGccGGGTsT
A19087
544
CCCGGCGUuAAAcAUCGGUTsT
AD-11845





2448-2466
L
A19088
545
uGAuGAGAcuuucGuAcAcTsT
A19089
546
GUGuACGAAAGUCUcAUcATsT
AD-11846





1023-1041
L
A19090
547
AcGAAAAGGGcGGuuuuuATsT
A19091
548
uAAAAACCGCCCUUUUCGUTsT
AD-11847





1377-1395
L
A19092
549
ccGAuGuuuAAcGccGGGATsT
A19093
550
UCCCGGCGUuAAAcAUCGGTsT
AD-11848





2619-2637
L
A19094
551
AucAAuCuCCGAAAcuAGATsT
A19095
552
UCuAGUUUCGGAGAUUGAUTsT
AD-11849





5608-5626
L
A19096
553
AAAuAcGGcGuuAAGAAGuTsT
A19097
554
ACUUCUuAACGCCGuAUUUTsT
AD-11850





5607-5625
L
A19098
555
AAAAuAcGGCGuuAAGAAGTsT
A19099
556
CUUCUuAACGCCGuAUUUUTsT
AD-11851





6396-6414
L
A19100
557
ucGAAcccAGAcuuAucAuTsT
A19101
558
AUGAuAAGUCUGGGUUCGATsT
AD-11852





4165-4183
L
A19102
559
AcAAccAcGcuAAAucUAGTsT
A19103
560
CuAGAUUuAGCGUGGUUGUTsT
AD-11853





4250-4268
L
A19104
561
GcAAcuuAucGAuuGAcAGTsT
A19105
562
CUGUcAAUCGAuAAGUUGCTsT
AD-11854





6434-6452
L
A19106
563
GAcGGAuAAcuAAAcuAGuTsT
A19107
564
ACuAGUUuAGUuAUCCGUCTsT
AD-11855





2959-2977
L
A19108
565
uGuAGcGcAAuuGAcutauGTsT
A19109
566
cAAAGUcAAUUGCGCuAcATsT
AD-11856





6433-6451
L
A19110
567
GGACGGAuAAcuAAAcuAGTsT
A19111
568
CuAGUUuAGUuAUCCGUCCTsT
AD-11857





 83-101
L
A19112
569
uGGcuAcccAAcAuAcAcATsT
A19113
570
UGUGuAUGUUGGGuAGCcATsT
AD-11858





1382-1400
L
A19114
571
GuuuAAcGccGGGAuuGAATsT
A19115
572
UUcAAUCCCGGCGUuAAACTsT
AD-11859





1014-1032
NP
A19116
573
uuuccGuuuGAuGcGAAcATsT
A19117
574
UGUUCGcAUcAAACGGAAATsT
AD-11860





1805-1823
NP
A19118
575
GAAGcuAcGGcGAAuAccATsT
A19119
576
UGGuAUUCGCCGuAGCUUCTsT
AD-11861





1862-1880
NP
A19120
577
uGGuccuAuucGAucuAGATsT
A19121
578
UCuAGAUCGAAuAGGACcATsT
AD-11862





1016-1034
NP
A19122
579
uccGuuuGAuGcGAAcAAATsT
A19123
580
UUUGUUCGcAUcAAACGGATsT
AD-11863





2230-2248
NP
A19124
581
ccAccGGcucccGuAuAcATsT
A19125
582
UGuAuACGGGAGCCGGUGGTsT
AD-11864





2233-2251
NP
A19126
583
ccGGcucccGuAuAcAGAGTsT
A19127
584
CUCUGuAuACGGGAGCCGGTsT
AD-11865





959-977
NP
A19136
585
AAGGAcuGAuACAAuAuccTsT
A19137
586
GGAuAUUGuAUcAGUCCUUTsT
AD-11870





1017-1035
NP
A19138
587
ccGuuuGAuGcGAAcAAAuTsT
A19139
588
AUUUGUUCGcAUcAAACGGTsT
AD-11871





2124-2142
NP
A19140
589
cccAcuGGAcGAuGccGAcTsT
A19141
590
GUCGGcAUCGUCcAGUGGGTsT
AD-11872





745-763
NP
A19142
591
cGuGAuGGAGuGAAGcGccTsT
A19143
592
GGCGCUUcACUCcAUcACGTsT
AD-11873





2229-2247
NP
A19144
593
cccAccGGcucccGuAuAcTsT
A19145
594
GuAuACGGGAGCCGGUGGGTsT
AD-11874





2119-2137
NP
A20118
595
GcAGAcccAcuGGAcGAuGTsT
A20119
596
cAUCGUCcAGUGGGUCUGCTsT
AD-12462





1587-1605
NP
A20120
597
AAAcGcuAuGGuAAcucuATsT
A20121
598
uAGAGUuACcAuAGCGUUUTsT
AD-12463





1300-1318
NP
A20122
599
uucGcccGAcuuuuGAAccTsT
A20123
600
GGUUcAAAAGUCGGGCGAATsT
AD-12464





1808-1826
NP
A20124
601
GcuAcGGcGAAuAccAGAGTsT
A20125
602
CUCUGGuAUUCGCCGuAGCTsT
AD-12465





1813-1831
NP
A20126
603
GGcGAAuAccAGAGuuAcuTsT
A20127
604
AGuAACUCUGGuAUUCGCCTsT
AD-12466





532-550
VP35
A19146
605
GGuGAuGAcAAccGGucGGTsT
A19147
606
CCGACCGGUUGUcAUcACCTsT
AD-11875





417-435
VP35
A19148
607
uAGAAcAAcGcAuuAcGAGTsT
A19149
608
CUCGuAAUGCGUUGUUCuATsT
AD-11876





741-759
VP35
A19152
609
GGAAAccuGAcAuuucGGcTsT
A19153
610
GCCGAAAUGUcAGGUUUCCTsT
AD-11878





1049-1067
VP35
A19154
611
cccAAGAuuGAucGAGGuuTsT
A19155
612
AACCUCGAUcAAUCUUGGGTsT
AD-11879





206-224
VP35
A19160
613
AGAAuuccuGuAAGCGAcATsT
A19161
614
UGUCGCUuAcAGGAAUUCUTsT
AD-11882





246-264
VP35
A19162
615
AUCCAGGAUUAUGCUACGCTsT
A19163
616
GCGuAGcAuAAUCCUGGAUTsT
AD-11883





247-265
VP35
A19164
617
uCcAGGAuuAuGcuAcGcATsT
A19165
618
UGCGuAGcAuAAUCCUGGATsT
AD-11884





287-305
VP35
A19166
619
CCAAACCCGAAGACGCGCATsT
A19167
620
uGCGCGUCuUCGGGuuuGGTsT
AD-11885





314-332
VP35
A19168
621
AcccAAAcCGAcccAAuuuTsT
A19169
622
AAAuuGGGUCCGuuuGGGUTsT
AD-11886





319-337
VP35
A19170
623
AAcGGAcccAAuuuGcAAuTsT
A19171
624
AUUGcAAAUUGGGUCCGUUTsT
AD-11887





414-432
VP35
A19172
625
cAuuAGAAcAAcGcAuuAcTsT
A19173
626
GuAAUGCGUUGUUCuAAUGTsT
AD-11888





415-433
VP35
A19174
627
AuuAGAAcAAcGcAuuAcGTsT
A19175
628
CGuAAUGCGUUGUUCuAAUTsT
AD-11889





439-457
VP35
A19176
629
uGAGAAuGGucuAAAGccATsT
A19177
630
UGGCUUuAGACcAUUCUcATsT
AD-11890





576-594
VP35
A19178
631
AGGcuUAuUGGGccGAAcATsT
A19179
632
UGUUCGGCCcAAuAAGCCUTsT
AD-11891





413-431
VP35
A20128
633
ucAuuAGAAcAAcGcAuuATsT
A20129
634
uAAUGCGUUGUUCuAAUGATsT
AD-12467





583-601
VP35
A20130
635
UUGGGCCGAACAUGGUCAATsT
A20131
636
UUGACcAUGUUCGGCCcAATsT
AD-12468





 983-1001
VP35
A20132
637
cAcAuccGcucucGAGCuGTsT
A20133
638
cACCUCGAGAGCGGAUGUGTsT
AD-12469





318-336
VP35
A20134
639
AAAcGGAcccAAuuuGcAATsT
A20135
640
UUGcAAAUUGGGUCCGUUUTsT
AD-12470





420-438
VP35
A20136
641
AAcAAcGcAuuAcGAGucuTsT
A20137
642
AGACUCGuAAUGCGUUGUUTsT
AD-12471





419-437
VP35
A20138
643
GAACAACGCAUUACGAGUCTsT
A20139
644
GACUCGuAAUGCGUUGUUCTsT
AD-12472





134-152
VP35
A20140
645
GccAcGAcucAAAAcGAcATsT
A20141
646
UGUCGUUUUGAGUCGUGGCTsT
AD-12473





893-911
VP40
A19180
647
CCACAAGCUGACCGGUAAGTsT
A19181
648
CUuACCGGUcAGCUUGUGGTsT
AD-11892





892-910
VP40
A19182
649
uccAcAAGCuGAccGGuAATsT
A19183
650
UuACCGGUcAGCUUGUGGATsT
AD-11893





325-343
VP40
A19188
651
uGAAuGucAuAucGGGcccTsT
A19189
652
GGGCCCGAuAUGAcAUUcATsT
AD-11896





450-468
VP40
A19190
653
AcuAucAcccAuuucGGcATsT
A19191
654
UGCCGAAAUGGGUGAuAGUTsT
AD-11897





662-680
VP40
A19194
655
GAccGAuGAcAcuccAAcATsT
A19195
656
UGUUGGAGUGUcAUCGGUCTsT
AD-11899





200-218
VP40
A19198
657
GAcAccGGAGucAGucAAuTsT
A19199
658
AuuGACuGACUCCGGuGUCTsT
AD-11901





203-221
VP40
A19200
659
AccGGAGucAGucAAuGGGTsT
A19201
660
CCcAUUGACUGACUCCGGUTsT
AD-11902





204-222
VP40
A19202
661
ccGGAGucAGucAAuGGGGTsT
A19203
662
CCCcAUUGACUGACUCCGGTsT
AD-11903





225-243
VP40
A19204
663
AcuccAucGAAuccAcucATsT
A19205
664
uGAGuGGAuUCGAuGGAGUTsT
AD-11904





386-404
VP40
A19206
665
uGucGcuGAucAAAAGACcTsT
A19207
666
GGUCUUUUGAUcAGCGAcATsT
AD-11905





584-602
VP40
A19208
667
AGuccAAcuAccccAGuAuTsT
A19209
668
AuACUGGGGuAGUUGGACUTsT
AD-11906





631-649
VP40
A19210
669
uGAucAcccAAccAcuGccTsT
A19211
670
GGcAGUGGUUGGGUGAUcATsT
AD-11907





660-678
VP40
A19212
671
uGGAccGAuGAcAcuccAATsT
A19213
672
UUGGAGUGUcAUCGGUCcATsT
AD-11908





663-681
VP40
A19214
673
AccGAuGAcAcucCAAcAGTsT
A19215
674
CUGUUGGAGUGUcAUCGGUTsT
AD-11909





929-947
VP40
A19218
675
uGGAcAAccAAucAucccuTsT
A19219
676
AGGGAUGAUUGGUUGUCcATsT
AD-11911





1019-1037
VP40
A19220
677
uuGuGAcAcGuGucAuucuTsT
A19221
678
AGAAUGAcACGUGUcAcAATsT
AD-11912





243-261
VP40
A19224
679
AGGccAAuuGccGAuGAcATsT
A19225
680
UGUcAUCGGcAAUUGGCCUTsT
AD-11914





140-158
VP40
A20142
681
AuAcccuGucAGGucAAAuTsT
A20143
682
AUUUGACCUGAcAGGGuAUTsT
AD-12474





141-159
VP40
A20144
683
UACCCUGUCAGGUCAAAUUTsT
A20145
684
AAUUUGACCUGAcAGGGuATsT
AD-12475





378-396
VP40
A20146
685
ccucuAGGuGucGcuGAucTsT
A20147
686
GAUCAGCGACACCuAGAGGTsT
AD-12476





427-445
VP40
A20148
687
ccGccAucAuGcuuGcuucTsT
A20149
688
GAAGcAAGCAUGAUGGCGGTsT
AD-12477





898-916
VP40
A20150
689
AGcuGAccGGuAAGAAGGuTsT
A20151
690
ACCUUCUuACCGGUcAGCUTsT
AD-12478





199-217
VP40
A20152
691
uGAcAccGGAGucAGucAATsT
A20153
692
UUGACUGACUCCGGUGUcATsT
AD-12479





568-586
VP40
A20154
693
AGuucGuucuuccGccAGuTsT
A20155
694
ACUGGCGGAAGAACGAACUTsT
AD-12480





569-587
VP40
A20156
695
GuucGuucuuccGccAGucTsT
A20157
696
GACUGGCGGAAGAACGAACTsT
AD-12481





1728-1746
GP
A19232
697
ccuGGAuAccAuAuuucGGTsT
A19233
698
CCGAAAuAUGGuAUCcAGGTsT
AD-11918





1729-1747
GP
A19234
699
cuGGAuAccAuAuuucGGGTsT
A19235
700
CCCGAAAuAUGGuAUCcAGTsT
AD-11919





1818-1836
GP
A19246
701
AGcuGGccAAcGAGAcGAcTsT
A19247
702
GUCGUCUCGUUGGCcAGCUTsT
AD-11925





1821-1839
GP
A19248
703
uGGccAAcGAGAcGAcucATsT
A19249
704
UGAGUCGUCUCGUUGGCcATsT
AD-11926





1732-1750
GP
A19250
705
GAuAccAuAuuucGGGccATsT
A19251
706
UGGCCCGAAAuAUGGuAUCTsT
AD-11927





1956-1974
GP
A20158
707
cGGAcuGcuGuAucGAAccTsT
A20159
708
GGUUCGAuAcAGcAGUCCGTsT
AD-12482





2107-2125
GP
A20160
709
uGGAGuuAcAGGcGuuAuATsT
A20161
710
uAuAACGCCUGuAACUCCATsT
AD-12483





2124-2142
GP
A20162
711
uAAuuGcAGuuAucGcuuuTsT
A20163
712
AAAGCGAuAACUGcAAUuATsT
AD-12484





2109-2127
GP
A20164
713
GAGuuACAGGcGuuAuAAuTsT
A20165
714
AUuAuAACGCCUGuAACUCTsT
AD-12485





1958-1976
GP
A20166
715
GAcuGcuGuAucGAAccAcTsT
A20167
716
GUGGUUCGAuAcAGcAGUCTsT
AD-12486





1890-1908
GP
A20168
717
UCCUCAACCGUAAGGCAAUTsT
A20169
718
AUUGCCUuACGGUUGAGGATsT
AD-12487





1891-1909
GP
A20170
719
ccucAAccGuAAGGcAAuuTsT
A20171
720
AAUUGCCUuACGGUUGAGGTsT
AD-12488





1307-1325
GP
A20172
721
AAuAcAcccGuGuAuAAAcTsT
A20173
722
GUUuAuAcACGGGUGuAUUTsT
AD-12489





1823-1841
GP
A20174
723
GccAAcGAGAcGAcucAAGTsT
A20175
724
CUUGAGUCGUCUCGUUGGCTsT
AD-12490





2110-2128
GP
A20176
725
AGuuAcAGGcGuuAuAAuuTsT
A20177
726
AAUuAuAACGCCUGuAACUTsT
AD-12491





1308-1326
GP
A20178
727
AuAcAcccGuGuAuAAAcuTsT
A20179
728
AGUUuAuAcACGGGUGuAUTsT
AD-12492





2113-2131
GP
A20180
729
uAcAGGcGuuAuAAuuGcATsT
A20181
730
UGCAAUuAuAACGCCUGuATsT
AD-12493





1654-1672
GP
A20182
731
cAAuGcucAAcccAAAuGcTsT
A20183
732
GcAUUUGGGUUGAGcAUUGTsT
AD-12494





1824-1842
GP
A20184
733
ccAAcGAGAcGAcucAAGcTsT
A20185
734
GCUUGAGUCGUCUCGUUGGTsT
AD-12495





1313-1331
GP
A20186
735
cccGuGuAuAAAcuuGAcATsT
A20187
736
UGUcAAGUUuAuAcACGGGTsT
AD-12496





1873-1891
GP
A20188
737
GcuAcGcAccuuuucAAucTsT
A20189
738
GAUUGAAAAGGUGCGuAGCTsT
AD-12497





1953-1971
GP
A20190
739
GAccGGAcuGcuGuAucGATsT
A20191
740
UCGAuAcAGcAGUCCGGUCTsT
AD-12498





1964-1982
GP
A20192
741
uGuAucGAAccAcAuGAuuTsT
A20193
742
AAUcAUGUGGUUCGAuAcATsT
AD-12499





329-347
VP30
A20194
743
AGGuGAGuAccGucAAucATsT
A20195
744
UGAUUGACGGuACUcACCUTsT
AD-12500





426-444
VP30
A20196
745
AAAGAcAuAuGuccGAccuTsT
A20197
746
AGGUCGGAcAuAUGUCUUUTsT
AD-12501





842-860
VP30
A20198
747
UCUCGAAGUAUAUCAACGATsT
A20199
748
UCGUUGAuAuACUUCGAGATsT
AD-12502





909-927
VP30
A20200
749
uGGGAccGAcAAucccuAATsT
A20201
750
UuAGGGAUUGUCGGUCCcATsT
AD-12506





523-541
VP30
A20202
751
uccuAcuAAucGcccGuAATsT
A20203
752
UuACGGGCGAUuAGuAGGATsT
AD-12504





429-447
VP30
A20204
753
GAcAuAuGuccGAccuuGATsT
A20205
754
UcAAGGUCGGAcAuAUGUCTsT
AD-12505





521-539
VP30
A20206
755
ACUCCUACUAAUCGCCCGUTsT
A20207
756
ACGGGCGAUUAGuAGGAGUTsT
AD-12506





903-921
VP30
A20208
757
CAAcAAuGGGAccGAcAAuTsT
A20209
758
AUUGUCGGUCCcAUUGUUGTsT
AD-12507





355-373
VP30
A20210
759
ccucAcAAGuGcGcGuuccTsT
A20211
760
GGAACGCGcACUUGUGAGGTsT
AD-12508





337-355
VP30
A20212
761
AccGucAAucAAGGAGcGcTsT
A20213
762
GCGCUCCUUGAUUGACGGUTsT
AD-12509





908-926
VP24
A19262
763
cuGucGuuGAuucGAuccATsT
A19263
764
UGGAUCGAAUcAACGAcAGTsT
AD-11933





522-540
VP24
A19272
765
uuGucuuAAGcGAccucuGTsT
A19273
766
cAGAGGUCGCUuAAGAcAATsT
AD-11938





790-808
VP24
A19274
767
uuuGAuuGAAcccuuAGcATsT
A19275
768
UGCuAAGGGUUcAAUcAAATsT
AD-11939





863-881
VP24
A20214
769
AAcAuGcGAAcACAAcGuGTsT
A20215
770
cACGUUGUGUUCGcAUGUUTsT
AD-12510





1102-1120
VP24
A20216
771
uGGGccGGcGAAAuuuuccTsT
A20217
772
GGAAAAUUUCGCCGGCCcATsT
AD-12511





912-930
VP24
A20218
773
cGuuGAuucGAuccAAuAuTsT
A20219
774
AuAUUGGAUCGAAUCAACGTsT
AD-12512





954-972
VP24
A20220
775
AuGcucuAcAuGucGuGAATsT
A20221
776
UUcACGAcAUGuAGAGcAUTsT
AD-12513





475-493
VP24
A20222
777
GGGAcGAuAcAAucuAAuATsT
A20223
778
uAUuAGAUUGuAUCGUCCCTsT
AD-12514





1069-1087
VP24
A20224
779
AcccGAcAAAucGGcAAuGTsT
A20225
780
cAUUGCCGAUUUGUCGGGUTsT
AD-12515





486-504
VP24
A20226
781
AucuAAuAucGcccAAAAATsT
A20227
782
UUUUUGGGCGAuAUuAGAUTsT
AD-12516





525-543
VP24
A20228
783
ucuuAAGcGAccucuGuAATsT
A20229
784
UuAcAGAGGUCGCUuAAGATsT
AD-12517





867-885
VP24
A20230
785
uGcGAAcAcAAcGuGucAATsT
A20231
786
UUGAcACGUUGUGUUCGcATsT
AD-12518





1028-1046
VP24
A20232
787
AuAAcucGAAcuAAcAuGGTsT
A20233
788
CcAUGUuAGUUCGAGUuAUTsT
AD-12519





471-489
VP24
A20234
789
CUACGGGACGAUACAAUCUTsT
A20235
790
AGAUUGuAUCGUCCCGuAGTsT
AD-12520





1029-1047
VP24
A20236
791
uAAcucGAAcuAAcAuGGGTsT
A20237
792
CCcAUGUuAGUUCGAGUuATsT
AD-12521





1948-1966
L
A20238
793
cAGuuAGAGGGAGuAGcuuTsT
A20239
794
AAGCuACUCCCUCuAACUGTsT
AD-12522





2003-2021
L
A20240
795
AuAuGAGuuuAcAGcAccuTsT
A20241
796
AGGUGCUGuAAACUcAuAUTsT
AD-12523





2005-2023
L
A20242
797
AuGAGuuuAcAGcAccuuuTsT
A20243
798
AAAGGUGCUGuAAACUcAUTsT
AD-12524





2070-2088
L
A20244
799
uGGAuGcAuuAuAcAAuccTsT
A20245
800
GGAUUGuAuAAUGcAUCcATsT
AD-12525





1959-1977
NP
A19278
801
AccuuGGAcGGAGcGAAAATsT
A19279
802
UUUUCGCUCCGUCcAAGGUTsT
AD-11941





1687-1705
NP
A19280
803
cAuuucccGGGccGAucuATsT
A19281
804
uAGAUCGGCCCGGGAAAUGTsT
AD-11942





1775-1793
NP
A19282
805
uGuuGuuGAcccGuAuGAuTsT
A19283
806
AUCAuACGGGUcAAcAAcATsT
AD-11943





384-402
NP
A19284
807
GuuuAccuGAGAGccuAcATsT
A19285
808
UGuAGGCUCUcAGGuAAACTsT
AD-11944





400-418
NP
A19286
809
AcAAcAuGGAuAAAcGGGuTsT
A19287
810
ACCCGUUuAUCcAUGUUGUTsT
AD-11945





1773-1791
NP
A19288
811
GGuGuuGuuGAcccGuAuGTsT
A19289
812
cAuACGGGUcAAcAAcACCTsT
AD-11946





1964-1982
NP
A19290
813
GGAcGGAGcGAAAAAGGuGTsT
A19291
814
cACCUUUUUCGCUCCGUCCTsT
AD-11947





411-429
NP
A19292
815
AAAcGGGuGAGAGGuucAuTsT
A19293
816
AUGAACCUCUcACCCGUUUTsT
AD-11948





1815-1833
NP
A19294
817
GAcuAcGAGGAuucGGcuGTsT
A19295
818
cAGCCGAAUCCUCGuAGUCTsT
AD-11949





407-425
NP
A19296
819
GGAuAAAcGGGuGAGAGGuTsT
A19297
820
ACCUCUCACCCGUUuAUCCTsT
AD-11950





2405-2423
NP
A19298
821
uuAucAccuAAuGAGuGAuTsT
A19299
822
AUcACUcAUuAGGUGAuAATsT
AD-11951





409-427
NP
A19300
823
AuAAAcGGGuGAGAGGuucTsT
A19301
824
GAACCUCUcACCCGUUuAUTsT
AD-11952





1811-1829
NP
A19302
825
UCCUGACUACGAGCAUUCGTsT
A19303
826
CGAAUCCUCGuAGUcAGGATsT
AD-11953





408-426
NP
A19304
827
GAuAAAcGGGuGAGAGGuuTsT
A19305
828
AACCUCUcACCCGUUuAUCTsT
AD-11954





1958-1976
NP
A19306
829
GAccuuGGACGGAGcGAAATsT
A19307
830
UUUCGCUCCGUCcAAGGUCTsT
AD-11955





1973-1991
NP
A19308
831
GAAAAAGGuGccGGAGuuGTsT
A19309
832
cAACUCCGGcACCUUUUUCTsT
AD-11956





1810-1828
NP
A19310
833
AuccuGAcuAcGAGGAuucTsT
A19311
834
GAAUCCUCGuAGUCAGGAUTsT
AD-11957





1953-1971
NP
A19312
835
GAuccGAccUuGGACGGAGTsT
A19313
836
CUCCGUCCAAGGUCGGAUCTsT
AD-11958





1692-1710
NP
A19314
837
CccGGGccGAucuAuGAuGTsT
A19315
838
cAUcAuAGAUCGGCCCGGGTsT
AD-11959





197-215
VP35
A19316
839
AccGGcAAAAuAccGcuAATsT
A19317
840
UuAGCGGuAUUUUGCCGGUTsT
AD-11960





196-214
VP35
A19318
841
GAcCGGcAAAAuAccGcuATsT
A19319
842
uAGCGGuAUUUUGCCGGUCTsT
AD-11961





409-427
VP35
A19320
843
AucAcuAGAAGGucGAGuATsT
A19321
844
uACUCGACCUUCuAGUGAUTsT
AD-11962





476-494
VP35
A19322
845
AuAucAucccuGAAucGcATsT
A19323
846
UGCGAUUcAGGGAUGAuAUTsT
AD-11963





611-629
VP35
A19324
847
ccAucAuuGuAcGAGGAuGTsT
A19325
848
CAUCCUCGuAcAAUGAUGGTsT
AD-11964





645-663
VP35
A19326
849
AAuuGAAAGAuccGAAcGGTsT
A19327
850
CCGUUCGGAUCUUUcAAUUTsT
AD-11965





726-744
VP35
A19328
851
AGGAAAAuuucGGGcGAccTsT
A19329
852
GGUCGCCCGAAAuuuUCCUTsT
AD-11966





1130-1148
VP35
A19330
853
GuAAGcucAuuuuGcGAuGTsT
A19331
854
cAUCGcAAAAUGAGCUuACTsT
AD-11967





729-747
VP35
A19332
855
AAAAuuucGGGcGAccuuATsT
A19333
856
uAAGGUCGCCCGAAAUUUUTsT
AD-11968





606-624
VP35
A19334
857
cAGGcccAucAuuGuAcGATsT
A19335
858
UCGuAcAAUGAUGGGCCUGTsT
AD-11969





256-274
VP35
A19336
859
cccGAuAAccAuuAuuAGuTsT
A19337
860
ACuAAuAAUGGUuAUCGGGTsT
AD-11970





478-496
VP35
A19338
861
AucAucccuGAAucGcAGcTsT
A19339
862
GCUGCGAUUcAGGGAUGAUTsT
AD-11971





724-742
VP35
A19340
863
uGAGGAAAAuuucGGGcGATsT
A19341
864
UCGCCCGAAAUUUUCCUcATsT
AD-11972





644-662
VP35
A19342
865
AAAuuGAAAGAuccGAAcGTsT
A19343
866
CGUUCGGAUCUUUcAAUUUTsT
AD-11973





1239-1257
VP35
A19344
867
AuccuAAucAAuuGAuAAuTsT
A19345
868
AUuAUcAAUUGAUuAGGAUTsT
AD-11974





1052-1070
VP35
A19346
869
AucGAuAGAGGuuGGGucuTsT
A19347
870
AGACCcAACCUCuAUCGAUTsT
AD-11975





429-447
VP40
A19348
871
GcAAuuAuGcucGcAucuuTsT
A19349
872
AAGAUGCGAGCAuAAUUGCTsT
AD-11976





1189-1207
VP40
A19350
873
AAAAuGuAcuAAucGGGucTsT
A19351
874
GACCCGAUuAGuAcAUUUUTsT
AD-11977





1190-1208
VP40
A19352
875
AAAuGuAcuAAucGGGucATsT
A19353
876
UGACCCGAUuAGuAcAUUUTsT
AD-11978





373-391
VP40
A19354
877
GGuuGccAcucGGAAuuGcTsT
A19355
878
GcAAUUCCGAGUGGcAACCTsT
AD-11979





439-457
VP40
A19356
879
ucGcAucuuAuAcGAucAcTsT
A19357
880
GUGAUCGuAuAAGAUGCGATsT
AD-11980





441-459
VP40
A19358
881
GcAucuuAuAcGAucAcccTsT
A19359
882
GGGUGAUCGuAuAAGAUGCTsT
AD-11981





1121-1139
VP40
A19360
883
AuAGcAAcucAAucGAcuuTsT
A19361
884
AAGUCGAUUGAGUUGCuAUTsT
AD-11982





1127-1145
VP40
A19362
885
AcucAAucGAcuuuuAGGATsT
A19363
886
UCCuAAAAGUCGAUUGAGUTsT
AD-11983





1193-1211
VP40
A19364
887
uGuAcuAAucGGGucAAGGTsT
A19365
888
CCUUGACCCGAUuAGuAcATsT
AD-11984





1298-1316
VP40
A19366
889
ACAuGCAuAAGcGAuccAuTsT
A19367
890
AUGGAUCGCUuAUGcAUGUTsT
AD-11985





1307-1325
VP40
A19368
891
AGcGAuccAuAcuucGcccTsT
A19369
892
GGGCGAAGuAUGGAUCGCUTsT
AD-11986





361-379
VP40
A19370
893
AAAucccuAuuuGGuuGccTsT
A19371
894
GGcAACcAAAuAGGGAUUUTsT
AD-11987





437-455
VP40
A19372
895
GcucGcAucuuAuAcGAucTsT
A19373
896
GAUCGuAuAAGAUGCGAGCTsT
AD-11988





857-875
VP40
A19374
897
GAGuAuCAuuGGGAucGAGTsT
A19375
898
CUCGAUCCcAAUGAuACUCTsT
AD-11989





484-502
VP40
A19376
899
ucGuuAGAGuGAAucGAcuTsT
A19377
900
AGUCGAUUcACUCuAACGATsT
AD-11990





1845-1863
GP
A19378
901
GGAGcuGcGGAcAuAuAccTsT
A19379
902
GGuAuAUGUCCGcAGCUCCTsT
AD-11991





254-272
GP
A19380
903
GAGAuuGAccAGCuAGucuTsT
A19381
904
AGACuAGCUGGUcAAUCUCTsT
AD-11992





461-479
GP
A19382
905
ccGGAcGGGAGcGAAuGcuTsT
A19383
906
AGcAUUCGCUCCCGUCCGGTsT
AD-11993





466-484
GP
A19384
907
cGGGAGcGAAuGcuuAcccTsT
A19385
908
GGGuAAGcAUUCGCUCCCGTsT
AD-11994





933-951
GP
A19386
909
uAAuuuGGAcAcuAGAuGcTsT
A19387
910
GcAUCuAGUGUCcAAAUuATsT
AD-11995





1045-1363
GP
A19388
911
UUAUCGCUCAACGAGACAGTsT
A19389
912
CUGUCUCGUUGAGCGAuAATsT
AD-11996





1100-1118
GP
A19390
913
GAAGAAucuccGAccGGGcTsT
A19391
914
GCCCGGUCGGAGAuUCuUCTsT
AD-11997





1102-1120
GP
A19392
915
AGAAucuccGAccGGGccATsT
A19393
916
UGGCCCGGUCGGAGAUUCUTsT
AD-11998





1191-1209
GP
A19394
917
AAcAACAuuGcCGucucAGTsT
A19395
918
CUGAGACGGcAAUGUUGUUTsT
AD-11999





1203-1221
GP
A19396
919
GucucAGAAuucGAcAGAATsT
A19397
920
uUCuGUCGAAuUCuGAGACTsT
AD-12000





1844-1862
GP
A19398
921
cGGAGcuGcGGAcAuAuAcTsT
A19399
922
GuAuAUGUCCGcAGCUCCGTsT
AD-12001





255-273
GP
A19400
923
AGAuuGAccAGcuAGucuGTsT
A19401
924
cAGACuAGCUGGUcAAUCUTsT
AD-12002





1212-1230
GP
A19402
925
uucGAcAGAAGGucGAAGATsT
A19403
926
UCUUCGACCUUCUGUCGAATsT
AD-12003





1706-1724
GP
A19404
927
GGAucccGuAcuuuGGAccTsT
A19405
928
GGUCcAAAGuACGGGAUCCTsT
AD-12004





125-143
GP
A19406
929
cuuAGccuAcuccAAuuGcTsT
A19407
930
GcAAUUGGAGuAGGCuAAGTsT
AD-12005





264-282
GP
A19408
931
AGcuAGucuGcAAGGAucATsT
A19409
932
UGAUCCUUGcAGACuAGCUTsT
AD-12006





332-350
GP
A19410
933
AGcGGAGuAucuACuGAuATsT
A19411
934
uAUcAGuAGAuACUCCGCUTsT
AD-12006





464-482
GP
A19412
935
GAcGGGAGcGAAuGcuuAcTsT
A19413
936
GuAAGcAUUCGCUCCCGUCTsT
AD-12008





1210-1228
GP
A19414
937
AAuucGAcAGAAGGucGAATsT
A19415
938
uUCGACCuUCuGUCGAAUUTsT
AD-12009





1213-1231
GP
A19416
939
ucGAcAGAAGGucGAAGAGTsT
A19417
940
CUCuUCGACCuUCuGUCGATsT
AD-12010





1850-1868
GP
A19418
941
uGcGGAcAuAuAccAuAcuTsT
A19419
942
AGuAUGGuAuAUGUCCGcATsT
AD-12011





124-142
GP
A19420
943
ucuuAGccuAcuccAAuuGTsT
A19421
944
cAAUUGGAGuAGGCuAAGATsT
AD-12012





1044-1062
GP
A19422
945
uuuAucGcucAAcGAGAcATsT
A19423
946
UGUCUCGUUGAGCGAuAAATsT
AD-12013





265-283
GP
A19424
947
GcuAGucuGcAAGGAucAuTsT
A19425
948
AUGAUCCUUGcAGACuAGCTsT
AD-12014





361-379
VP30
A19426
949
uAGAGucccuAcGGuuuucTsT
A19427
950
GAAAACCGuAGGGACUCuATsT
AD-12015





324-342
VP30
A19428
951
cAAcAGAcuAccGuAGuAGTsT
A19429
952
CuACuACGGuAGUCUGUUGTsT
AD-12016





 994-1012
VP30
A19430
953
AGGccuAcGcuuAcuuGccTsT
A19431
954
GGcAAGuAAGCGuAGGCCUTsT
AD-12017





248-266
VP30
A19432
955
AGGAAuucAcGuGccGAccTsT
A19433
956
GGUCGGcACGUGAAUUCCUTsT
AD-12018





491-509
VP30
A19434
957
cuuGAAAGCcuAAccGAccTsT
A19435
958
GGUCGGUuAGGCUUUcAAGTsT
AD-12019





322-340
VP30
A19436
959
AAcAAcAGAcuAccGuAGuTsT
A19437
960
ACuACGGuAGUCUGUUGUUTsT
AD-12020





323-341
VP30
A19438
961
AcAAcAGAcuAccGuAGuATsT
A19439
962
uACuACGGuAGUCUGUUGUTsT
AD-12021





517-535
VP30
A19440
963
CCUACUUCUUAUAGCACGGTsT
A19441
964
CCGUGCuAuAAGAAGuAGGTsT
AD-12022





295-313
VP30
A19442
965
GAcAAGAuccAuuucccGGTsT
A19443
966
CCGGGAAAuGGAUCuuGUCTsT
AD-12023





229-247
VP30
A19444
967
ucGuGAGcGCGGGAGAucATsT
A19445
968
UGAUCUCCCGCGCUcACGATsT
AD-12024





251-269
VP30
A19446
969
AAuucAcGuGccGAccAGcTsT
A19447
970
GCUGGUCGGcACGUGAAUUTsT
AD-12025





340-358
VP30
A19448
971
uAGucGAAGuAcuucGcAATsT
A19449
972
UUGCGAAGuACUUCGACuATsT
AD-12026





1350-1368
VP30
A19450
973
ucccuAGAAGcGuuGAAucTsT
A19451
974
GAUUcAACGCUUCuAGGGATsT
AD-12027





1057-1075
VP24
A19452
975
uAuGGGuuAucuuGucGAGTsT
A19453
976
CUCGAcAAGAuAACCcAuATsT
AD-12028





878-896
VP24
A19454
977
AAcAuGAGAAcucAAcGAGTsT
A19455
978
CUCGUUGAGUUCUCAUGUUTsT
AD-12029





1056-1074
VP24
A19456
979
AuAuGGGuuAucuuGucGATsT
A19457
980
UCGAcAAGAuAACCcAuAUTsT
AD-12030





1137-1155
VP24
A19458
981
uAcuAcAuGAAucGAcAcuTsT
A19459
982
AGUGUCGAUUcAUGuAGuATsT
AD-12031





1099-1117
VP24
A19460
983
GAuGGAuAuAcGAcAcCcuTsT
A19461
984
AGGGUGUCGuAuAUCcAUCTsT
AD-12032





1591-1609
VP24
A19462
985
AGCcCAAAuuAAcAcGGuATsT
A19463
986
uACCGUGUuAAUUUGGGCUTsT
AD-12033





1094-1112
VP24
A19464
987
ucuGcGAuGGAuAuAcGAcTsT
A19465
988
GUCGuAuAUCcAUCGcAGATsT
AD-12034





1135-1153
VP24
A19466
989
cuuAcuAcAuGAAucGAcATsT
A19467
990
UGUCGAUUcAUGuAGuAAGTsT
AD-12035





152-170
VP24
A19468
991
cuAGGcuAGGGuuuAuAGuTsT
A19469
992
ACuAuAAACCCuAGCCuAGTsT
AD-12036





624-642
VP24
A19470
993
AccAAAAGGGuAuuAcccuTsT
A19471
994
AGGGuAAuACCCUUUUGGUTsT
AD-12037




















TABLE 3









Ebola

Ebola



Ebola Zaire GFP assay
Zaire plaque
Ebola Sudan IF assay
Sudan plaque


duplex
% reduction
% reduction vs
% reduction
% reduction vs















name
100 nM
10 nM
1 nM
no siRNA
100 nM
10 nM
1 nM
no siRNA


















AD-11542
90.7
80.1
55.2
55.24
−5.3
−1.5
−11.8
−103.39


AD-11543
62.7
55.9
47.8
55.83
−11.9
10.2
−9.0
−7.12


AD-11544
63.5
32.8
41.0
42.06
22.5
34.6
61.1
−16.95


AD-11545
73.0
71.8
61.4
47.23
61.5
71.9
71.8
39.41


AD-11546
94.5
88.3
72.5
86.12
−8.8
−73.8
5.5
55.93


AD-11547
79.8
62.8
48.6
36.44
−20.7
15.8
−4.0
18.64


AD-11548
−15.4
−13.0
−15.6

19.3
29.2
49.3


AD-11549
3.8
13.8
16.1
44.12
63.7
51.5
90.4
94.38


AD-11550
17.5
−1.2
−10.5

−41.1
−39.8
−29.4


AD-11551
−14.2
−20.4
−21.3

−32.6
−23.9
−30.6


AD-11552
−14.7
−11.9
−6.5

−22.7
−9.5
11.9


AD-11553
4.7
7.4
13.9
27.67
44.5
40.9
44.6
80.33


AD-11554
−12.6
−20.5
−32.1

−12.6
−17.7
−8.0


AD-11555
−20.8
−22.2
−36.7

−9.8
−8.9
0.8


AD-11556
6.4
−11.6
−21.7
29.03
11.1
37.1
34.7
−17.37


AD-11557
30.6
22.0
−8.7
−175.73
41.5
48.3
78.3
82.20


AD-11558
3.0
1.2
−33.7
72.97
−23.5
−18.2
−30.8
82.49


AD-11559
−29.1
−38.0
−42.9

−2.4
−37.3
−26.5


AD-11560
−42.0
−46.1
−54.6

19.8
0.7
19.1


AD-11561
−16.4
−16.6
−15.9
37.38
50.5
61.2
57.4
−129.83


AD-11562
12.0
−13.9
−63.1
61.33
−35.3
−46.2
−42.4
63.56


AD-11563
−45.3
−51.6
−61.7

−45.3
−54.7
−42.9


AD-11564
−19.0
−31.3
−44.0

−38.5
−32.3
−0.1


AD-11565
−27.0
−4.4
−10.7
50.32
6.7
47.4
52.9
26.95


AD-11566
6.8
6.0
−0.5

32.0
18.2
0.7


AD-11567
−1.1
−5.7
0.3

3.1
−4.6
14.4


AD-11568
10.3
7.9
11.4

−0.7
27.6
25.6


AD-11569
17.6
22.0
15.4
33.01
47.1
51.3
63.7
−85.01


AD-11570
23.8
5.9
−15.4
98.14
9.3
−5.5
−15.3
−217.80


AD-11571
−27.5
−23.9
−32.4

−8.5
−13.0
10.8


AD-11572
−35.2
−25.7
−26.1

4.3
8.8
18.6


AD-11573
−8.3
16.7
15.7
46.60
55.9
76.5
37.8
−22.05


AD-11574
1.1
−12.0
−27.6

12.5
−2.9
−11.3


AD-11575
−29.9
−30.0
−35.9

1.6
27.3
−11.4


AD-11576
−9.4
−8.2
−20.8

0.1
34.6
3.1


AD-11577
−8.5
−0.1
13.7

45.2
31.5
23.2


AD-11578
9.8
17.0
4.1

−40.1
−56.3
−53.3


AD-11579
15.1
12.2
−7.8

−47.6
−37.3
−18.5


AD-11580
−4.4
2.3
4.1
76.38
14.2
4.9
33.6
12.97


AD-11581
10.6
2.5
3.4
−26.97
57.4
65.1
81.7
−109.94


AD-11582
10.6
4.3
−33.2
−1.17
−99.7
−93.3
−90.8
−533.33


AD-11583
−16.6
−18.4
−28.3

−85.8
−72.4
−55.3


AD-11584
−24.1
−12.0
−18.7
59.87
−1.0
54.0
16.5
1.61


AD-11585
−7.2
−9.6
9.1
51.46
37.5
19.1
45.7
82.71


AD-11586
−6.8
−0.7
−9.1

−81.1
−87.9
−86.9


AD-11587
6.1
−0.4
0.9

−80.7
−6.2
−51.8


AD-11588
20.9
10.4
−9.5
99.40
−12.9
−0.1
20.5
−117.23


AD-11589
28.7
24.3
16.1
35.75
18.2
38.7
54.2
88.14


AD-11590
20.7
28.8
3.0
98.81
−38.5
−55.2
20.9
44.92


AD-11591
−18.3
−29.6
−27.0

−3.1
−40.1
−30.1


AD-11592
−11.3
−14.7
−12.3

−36.3
−38.7
−46.1


AD-11593
3.8
5.0
−12.3

1.8
−15.8
4.6


AD-11594
33.4
4.9
−3.2
99.07
−25.2
−31.3
−21.7
7.63


AD-11595
−30.9
−37.7
−54.2

−11.6
−2.7
−2.7


AD-11596
−8.2
−14.8
−3.3

−7.8
26.8
3.5


AD-11597
13.0
13.0
11.7

21.7
28.2
25.2
83.00


AD-11598
40.0
−1.6
−10.4
98.91
−30.2
−33.7
−17.7
0.00


AD-11599
13.0
24.6
−18.3
98.79
−20.9
−22.8
−50.8
34.32


AD-11600
16.7
2.8
−8.1
98.52
−4.3
−28.5
6.8
−120.34


AD-11601
10.4
25.3
2.0
71.20
23.0
6.3
7.9
−652.16


AD-11602
39.7
45.0
24.4
38.73
47.4
37.7
54.8
48.13


AD-11603
41.2
42.4
36.0
54.85
38.2
35.7
43.8
73.04


AD-11604
39.6
36.3
31.6
−1.51
37.0
56.6
52.8
84.65


AD-11605
50.2
38.8
21.2
60.19
48.1
58.6
77.6
66.95


AD-11606
41.5
36.8
1.4
89.23
53.2
33.2
37.0
79.32


AD-11607
−12.8
2.7
−9.9
38.83
29.0
8.3
21.4
−13.98


AD-11608
10.9
−6.7
6.1
14.24
39.5
49.3
42.9
−75.07


AD-11609
27.3
32.9
29.0
51.46
36.8
40.7
50.2
20.81


AD-11610
23.9
19.7
16.0
23.95
37.7
70.3
49.6
97.93


AD-11611
5.6
11.9
16.1

30.7
8.5
15.6


AD-11612
−2.9
−4.0
−14.4

9.9
21.9
10.0


AD-11613
13.4
32.7
23.7

−2.6
17.5
44.4


AD-11614
9.0
14.3
10.3

21.9
9.9
−29.6


AD-11615
−5.9
22.3
−0.9

−24.8
−16.9
−21.2


AD-11616
2.3
5.5
11.0

−10.4
−7.6
26.4


AD-11617
14.3
5.3
−1.9
−617.15
39.8
62.5
91.5
14.55


AD-11618
−2.5
−4.3
−1.2

−11.0
−26.8
−28.6


AD-11619
−8.3
−37.8
−30.2

−33.9
−43.8
−23.9


AD-11620
11.0
−19.2
−37.5

−30.5
−12.0
0.7


AD-11621
−29.1
−4.5
4.7
−255.66
60.9
64.7
78.8
74.06


AD-11622
11.9
15.0
4.2

9.8
−29.1
−15.8


AD-11623
7.7
−17.8
−24.6

−27.3
−48.1
−19.4


AD-11624
−16.3
−36.6
−45.8

−36.1
−17.2
−8.3


AD-11625
45.0
11.0
0.3
37.98
19.5
48.2
43.1
−23.73


AD-11626
−3.6
13.2
11.9

−25.4
−81.4
−80.3


AD-11627
34.1
24.7
21.1
48.69
−46.8
−73.0
−54.8
12.71


AD-11628
−8.9
−1.4
−13.8
9.60
−9.5
6.1
35.2
59.80


AD-11629
22.9
23.0
11.8
−73.11
65.3
71.2
75.1
3.39


AD-11630
12.2
19.0
−2.0

−99.9
−123.8
−85.0


AD-11631
−10.4
−15.2
−18.3

−127.0
−87.2
−59.2


AD-11632
−50.7
−49.9
−49.4
−128.91
−32.4
1.3
29.8
51.76


AD-11633
−39.4
−10.2
1.8
−19.31
20.6
42.9
57.4
49.57


AD-11634
2.2
12.3
−14.1

−55.8
−129.3
−136.9


AD-11635
−44.3
−46.0
−29.5

−60.2
−89.0
−66.2


AD-11636
−48.8
−44.7
−41.1

−35.4
−17.4
7.3


AD-11637
−36.8
−27.2
−17.3
59.55
34.3
33.8
52.8
80.40


AD-11638
−8.4
8.5
−3.9

−9.4
12.4
28.9


AD-11639
−47.6
−1.7
36.0
23.31
2.9
20.2
12.6
−97.46


AD-11640
69.4
39.4
34.6
75.14
66.0
71.5
82.5
49.15


AD-11641
42.6
15.2
15.9
65.14
73.1
81.9
87.7
96.50


AD-11642
24.8
21.4
32.7
−42.93
89.8
87.4
87.4
4.03


AD-11643
37.7
53.8
52.7
89.14
94.3
94.2
97.5
−22.03


AD-11644
54.1
17.7
5.5
94.29
26.7
38.5
51.2
31.78


AD-11645
5.9
2.5
−2.5
51.97
64.7
68.1
80.1
93.34


AD-11646
−1.7
2.1
7.9
59.55
72.6
76.8
79.0
92.72


AD-11647
8.4
8.8
37.5
57.28
75.6
81.7
93.7
74.71


AD-11648
56.9
19.2
8.1
66.10
−15.4
−9.9
13.0
71.19


AD-11649
−3.3
−6.9
−3.5
65.47
36.0
54.2
64.9
82.84


AD-11650
1.9
−2.5
−1.5
17.28
58.7
65.3
59.6
2.31


AD-11651
6.7
9.4
30.9
65.70
70.3
87.1
91.2
83.96


AD-11652
55.8
27.7
10.3

−56.8
59.6
68.4
33.90


AD-11653
12.4
13.8
10.9
86.84
66.4
70.4
75.8
2.02


AD-11654
13.8
7.2
10.7

76.1
73.8
73.8
−123.92


AD-11655
9.1
14.6
40.5
−7.01
82.4
83.9
92.6
−146.11


AD-11656
39.1
9.6
−1.0
81.71
−21.9
30.7
19.0
20.34


AD-11657
−1.1
1.1
0.2

−3.1
20.7
48.9


AD-11658
7.3
0.9
1.5
86.84
55.5
50.8
41.3
93.52


AD-11659
2.9
4.1
28.0
78.79
46.4
51.1
66.6
79.47


AD-11660
23.8
13.0
0.9
80.57
11.0
20.8
−57.0
90.51


AD-11661
−6.4
−8.0
−6.1

−37.1
−16.9
19.1


AD-11662
−1.6
−7.9
−7.9

10.9
10.7
27.7


AD-11663
1.6
5.6
28.1

25.4
20.4
30.8


AD-11664
11.9
7.6
3.9
50.16
54.4
63.1
66.8
77.95


AD-11665
5.1
1.2
0.2
15.10
52.7
65.2
62.3
−13.26


AD-11666
0.0
−3.5
5.3
5.72
67.9
80.3
80.3
79.54


AD-11667
3.7
2.9
8.0
47.73
83.6
80.4
90.6
84.70


AD-11668
16.3
−0.3
−1.3

−91.6
−4.4
2.8


AD-11669
−1.0
3.8
0.3

3.4
33.5
43.3


AD-11670
−0.1
−4.6
−2.6
35.28
42.7
43.6
62.6
−5.48


AD-11671
0.1
1.1
16.0
80.58
61.0
65.1
72.7
13.54


AD-11672
8.2
−0.1
−2.2

−69.9
25.3
−57.8


AD-11673
1.7
0.9
3.2

−46.1
−22.7
1.5


AD-11674
−4.2
−3.0
−6.0

34.4
14.8
32.2


AD-11675
−0.6
0.6
18.4

11.1
45.1
59.7


AD-11676
22.1
11.5
7.7
78.86
75.7
86.4
78.6
16.95


AD-11677
5.6
−3.1
3.2

86.1
78.0
87.1
1.27


AD-11678
−2.3
−7.8
5.0
87.06
86.4
86.6
86.6
−141.35


AD-11679
5.6
7.3
34.4
28.26
88.3
88.6
90.9
−79.25


AD-11680
30.4
7.9
−0.5
75.62
−38.9
13.4
−88.3
72.03


AD-11681
−3.7
−10.1
−0.2

14.4
25.0
24.2


AD-11682
−10.0
−11.0
−7.7

−39.9
53.8
57.2


AD-11683
−6.4
6.6
32.3
92.99
59.6
53.3
48.2
57.85


AD-11684
31.5
14.4
2.9
33.26
−156.0
−119.6
−198.2
87.80


AD-11685
0.0
−6.5
−9.5
−6.15
−43.0
−107.3
−93.7
−20.61


AD-11686
−12.0
−7.9
−4.7
88.12
−134.6
−69.2
−151.8
43.11


AD-11687
−4.8
6.3
29.0

−77.4
−41.4
−79.0


AD-11688
40.0
28.5
26.7
−15.66
73.4
79.8
88.0
66.95


AD-11689
34.4
27.1
32.4
57.33
83.4
74.8
86.5
85.59


AD-11690
24.0
30.2
42.1
71.71
82.5
89.4
89.4
83.05


AD-11691
47.9
44.1
55.3
87.43
92.0
93.9
97.0
70.96


AD-11694
44.3
8.5
5.7

2.1
4.8
13.6


AD-11695
1.5
0.5
−5.3

42.1
36.7
46.0


AD-11696
5.1
4.8
10.8

49.2
56.9
76.3


AD-11698
18.4
20.4
45.5
59.09
69.5
79.5
82.0
54.66


AD-11700
30.4
8.5
−2.1
84.00
−151.2
−108.0
−67.5
4.52


AD-11704
−7.2
−6.1
−9.9

−37.2
−56.5
−32.4


AD-11705
−2.9
−2.4
−4.7

−7.3
0.9
−14.8


AD-11706
8.5
21.1
99.9

21.5
−8.9
33.9


AD-11707
41.8
25.9
21.9
90.69
79.3
84.2
85.1
63.14


AD-11708
27.2
27.4
28.1
83.92
80.0
81.1
83.7
24.58


AD-11710
31.0
37.7
43.0
77.54
86.9
89.6
89.6
99.70


AD-11711
33.8
36.5
48.2
73.43
91.1
89.8
93.9
−34.75


AD-11712
81.1
20.2
8.0
79.71
22.6
−4.3
27.5
−13.56


AD-11713
−8.6
−4.0
−2.1

39.1
39.1
51.8


AD-11714
−8.7
−8.3
−4.3
63.27
45.0
66.1
74.5
39.48


AD-11715
0.8
14.9
40.7
−92.23
79.0
82.0
86.9
1.15


AD-11716
32.4
8.3
1.8
83.05
−46.0
−51.4
−11.3
−577.97


AD-11717
2.2
−0.5
−0.6

−1.5
−13.0
−14.3


AD-11718
−8.1
−11.9
−19.8

2.4
18.8
29.2


AD-11719
4.2
11.6
33.8
57.93
55.3
56.6
74.0
−2.02


AD-11720
29.8
17.3
10.4
77.05
86.4
91.8
95.6
−373.73


AD-11721
14.6
13.7
12.4
82.56
90.8
91.1
95.1
−31.70


AD-11722
17.0
15.1
23.7
82.96
94.1
91.7
91.7
−112.39


AD-11723
16.0
13.5
30.5
90.99
94.1
95.5
95.7
−55.62


AD-11724
33.9
10.4
5.1
99.98
63.2
73.2
73.7
−1.27


AD-11725
6.4
−2.0
0.0
93.62
85.5
92.4
89.5
−172.62


AD-11726
2.4
0.2
0.2
58.58
90.3
91.9
94.4
−2.54


AD-11727
−1.0
2.8
20.2
4.72
93.9
94.5
98.3
49.86


AD-11728
30.7
12.9
4.0
73.43
26.9
27.5
41.0
30.51


AD-11729
5.1
1.5
−4.3
26.86
51.9
65.4
58.9
−20.46


AD-11730
2.6
1.3
0.8
24.70
60.2
67.7
71.6
52.02


AD-11731
4.9
3.3
24.1
79.94
66.8
67.3
89.0
96.16


AD-11732
33.6
9.3
6.0
63.43
63.0
79.9
87.5
70.34


AD-11733
2.2
2.8
−0.7
75.57
84.2
89.7
85.1
−9.51


AD-11734
8.6
6.7
10.8
90.52
90.8
90.6
90.6
−119.16


AD-11735
6.9
9.3
31.4
−22.87
90.8
88.5
94.5
−77.23


AD-11736
31.5
9.9
3.0
21.14
29.9
39.5
45.3
−611.86


AD-11737
−8.0
−3.4
−1.7
−183.17
78.3
88.4
82.5
14.41


AD-11738
−0.2
1.5
−4.7
−102.05
85.3
86.4
80.4
10.52


AD-11739
7.4
3.8
21.5
52.75
53.8
55.2
83.5
89.27


AD-11740
30.0
11.7
−0.4
53.43
−87.3
−64.8
12.1
38.98


AD-11741
−3.5
−6.1
−2.9
42.39
27.2
47.7
36.1
−32.56


AD-11742
−1.0
−1.9
−6.0
40.35
49.9
56.9
20.5
12.25


AD-11743
0.1
6.6
26.9
37.35
46.3
24.7
71.2
26.08


AD-11744
40.5
24.2
21.0
75.24
42.9
52.0
90.3
32.20


AD-11745
26.3
23.1
23.3
55.43
64.3
74.1
84.5
54.24


AD-11746
7.2
0.9
−4.8
89.82
42.3
74.8
8.6
33.49


AD-11747
1.6
−3.5
−5.4

54.9
5.8
25.4


AD-11748
−4.3
0.5
0.9

30.3
10.9
10.9


AD-11749
8.4
3.9
1.0

41.4
−3.7
54.3


AD-11750
4.8
2.1
0.0

44.9
30.4
71.5


AD-11751
3.3
0.6
−2.2

34.3
−18.6
29.4


AD-11752
17.7
6.5
3.2
−27.63
19.8
19.6
−0.1
61.08


AD-11753
1.7
1.2
0.9

−62.5
−40.7
28.2


AD-11754
3.7
1.8
0.7

69.9
91.1
44.9


AD-11755
37.7
26.6
10.8
−58.93
10.1
0.6
28.8
47.05


AD-11756
−1.0
−0.6
0.3

−39.8
−16.7
−5.7


AD-11757
0.2
0.0
1.8

−46.4
−12.7
−2.7


AD-11758
18.9
11.7
7.5
21.83
27.3
12.4
−9.8
70.02


AD-11759
26.1
15.1
18.8
−595.93
−6.9
−3.8
−22.6
55.98


AD-11760
21.9
19.5
6.0
11.60
−61.2
6.6
6.6
22.49


AD-11761
18.1
8.6
6.4
−67.30
39.7
43.4
58.0
71.77


AD-11762
15.4
9.7
4.2
−335.24
38.7
−86.0
12.4
73.68


AD-11763
6.8
7.3
3.7

−3.6
−0.7
8.7


AD-11764
5.1
5.7
0.9

−34.9
28.7
18.2


AD-11765
14.8
8.0
4.3
5.98
32.1
27.3
58.4
76.08


AD-11766
2.9
3.5
3.9

13.4
−6.1
−68.1


AD-11767
24.1
15.1
5.5
7.18
32.9
−27.7
0.2
9.57


AD-11768
19.2
16.5
4.3
30.34
−92.5
−3.0
4.0
55.98


AD-11769
5.6
2.3
2.6

6.7
25.7
43.9


AD-11770
14.0
7.2
2.2
−16.41
19.1
34.2
35.3
49.60


AD-11771
2.8
2.1
1.3

37.7
19.1
15.6


AD-11772
4.0
2.2
2.6

70.0
52.3
52.3


AD-11773
1.3
−0.6
−0.7

52.0
65.8
65.4


AD-11774
2.0
1.5
0.5

73.1
−18.3
6.5


AD-11775
2.3
0.3
−0.2

57.8
49.1
5.5


AD-11776
2.7
0.4
1.6

59.6
48.4
41.0


AD-11777
3.6
1.3
0.0

10.4
47.2
53.8


AD-11778
0.9
−0.9
−0.8

40.4
−13.5
−17.7


AD-11779
6.3
1.7
−0.5

42.1
2.9
−6.9


AD-11780
5.8
0.1
0.1

29.5
−17.6
20.3


AD-11781
2.3
1.0
−0.2

8.8
33.5
39.1


AD-11782
−1.7
−4.0
−4.1

48.1
44.5
56.1


AD-11783
−3.5
−2.1
−1.5

34.8
16.7
44.2


AD-11784
5.7
3.7
−1.0

50.4
59.1
59.1


AD-11785
2.8
1.0
1.5

75.3
88.0
81.7


AD-11786
14.9
6.5
−2.9
97.46
−7.0
−34.8
−6.5
66.19


AD-11787
−0.3
0.0
−2.9

9.0
−8.6
18.2


AD-11788
−0.1
0.5
1.3

23.5
11.8
55.0


AD-11789
5.1
5.7
3.1

57.8
45.6
62.5


AD-11790
5.7
0.0
−4.0

18.8
−12.3
−1.9


AD-11791
−1.5
−2.6
−4.6

42.1
−27.4
10.4


AD-11792
−0.9
1.8
1.5

27.1
30.8
24.6


AD-11793
2.2
3.7
4.4

6.7
34.8
44.3


AD-11794
0.4
−0.5
−1.6

59.5
81.9
69.3


AD-11795
−1.9
−0.8
0.1

78.7
94.4
87.2


AD-11796
5.0
1.8
0.8

89.6
71.8
71.8


AD-11797
0.6
0.8
1.3

92.1
87.0
83.7


AD-11798
−0.2
−0.2
−1.8

67.4
69.7
45.4


AD-11799
−2.1
−1.9
−1.3

−49.1
−5.6
72.9


AD-11800
1.3
−1.0
−0.4

64.1
58.5
60.6


AD-11801
0.1
0.1
0.8

23.7
35.3
54.5


AD-11802
4.8
0.8
−0.4

−115.9
−81.0
−113.4


AD-11803
3.7
1.3
−2.1

10.5
22.6
16.3


AD-11804
−2.0
−1.1
0.0

16.0
19.8
36.1


AD-11805
−0.5
0.2
−0.4

−6.9
6.5
51.6


AD-11806
−11.1
−16.4
−13.0

79.5
70.3
29.3


AD-11807
13.3
−13.2
−11.2
−78.63
65.7
20.0
50.5
−58.37


AD-11808
−6.3
−9.0
9.4

27.5
30.6
30.6


AD-11809
−0.2
−11.7
−11.7

44.7
61.3
73.0


AD-11810
−15.2
−17.1
−17.8

80.1
81.6
81.7


AD-11811
−9.7
−12.1
−11.2

40.4
38.0
23.7


AD-11812
−8.1
−12.5
−15.0

21.4
12.4
22.7


AD-11813
29.3
−14.5
−6.8
−258.78
31.4
32.0
43.9
−423.13


AD-11814
−19.9
−18.8
−8.1

49.2
15.1
55.1


AD-11815
−12.8
−13.4
−14.9

62.5
29.3
36.0


AD-11816
−14.5
−15.2
−14.6

13.0
−11.9
46.2


AD-11817
−15.4
−15.2
−11.8

43.3
38.2
42.0


AD-11818
9.5
−1.9
−6.6
−176.59
69.0
43.6
35.2
39.23


AD-11819
−3.5
−1.4
−1.9

75.0
50.7
31.2


AD-11820
7.6
4.4
−2.0
−327.48
58.5
50.6
50.6
29.19


AD-11821
1.3
−0.5
1.6

42.4
26.4
49.4


AD-11822
7.7
−4.1
−4.2

82.6
70.2
71.9


AD-11823
−1.5
−2.3
−0.4

63.9
58.8
37.4


AD-11824
3.2
1.7
−2.7

33.5
13.3
37.3


AD-11825
0.1
−0.9
0.7

53.6
42.2
44.8


AD-11826
3.4
−6.1
−3.8

52.8
35.7
39.9


AD-11827
−4.2
−2.6
−1.1

54.5
26.3
15.2


AD-11828
0.8
−1.3
−1.4

9.3
−4.3
19.5


AD-11829
2.0
−1.6
1.1

32.2
16.0
32.0


AD-11830
−4.3
−7.5
−8.0

44.5
48.3
75.6


AD-11831
−6.4
−5.2
−4.1

39.3
56.5
67.3


AD-11832
−3.2
−2.5
−3.0

61.0
76.2
76.2


AD-11833
−1.4
−2.5
−1.2

80.6
87.2
91.0


AD-11834
18.8
2.8
−7.7
−260.69
7.3
−11.5
24.5
25.84


AD-11835
−2.0
−0.2
−4.6

52.7
59.4
61.0


AD-11836
3.3
0.7
−1.9

70.7
76.0
80.0


AD-11837
0.3
−2.3
−0.7

84.6
70.1
80.8


AD-11838
−7.4
−7.1
−8.8

−13.5
9.5
8.9


AD-11839
−5.6
−2.9
−2.2

−0.7
45.3
48.4


AD-11840
−1.7
1.3
−0.2

85.7
61.6
59.6


AD-11841
−3.8
−1.8
−2.3

60.7
52.5
50.8


AD-11842
14.4
7.7
0.8
35.57
−145.5
−117.2
−46.4
42.11


AD-11843
ND
ND
ND

−68.1
−87.6
−36.1


AD-11844
0.0
1.4
−0.5

−72.1
−80.2
−80.2


AD-11845
3.9
2.0
1.1

−34.1
−60.1
−13.9


AD-11846
27.5
20.0
−7.1
25.19
−69.7
−62.0
−63.7
13.40


AD-11847
−5.9
−4.8
−6.2

−93.2
−61.3
−57.4


AD-11848
−1.3
0.6
−1.7

−89.3
−76.1
−18.3


AD-11849
−1.0
−1.4
50.6

−0.9
12.6
18.7


AD-11850
−4.1
2.9
−6.3

−15.1
−3.7
−34.5


AD-11851
−5.0
−4.6
−2.2

−13.3
−10.3
−15.2


AD-11852
−0.4
−0.6
−0.6

7.7
11.6
10.2


AD-11853
1.6
−0.6
−0.8

24.9
28.1
36.7


AD-11854
−0.6
−1.3
−1.8

50.9
35.6
21.2


AD-11855
5.0
3.4
1.6

47.8
24.7
28.6


AD-11856
2.3
0.9
−0.6

60.4
36.7
36.7


AD-11857
3.4
0.7
0.1

23.1
37.0
57.6


AD-11858
−2.3
−2.5
−2.7

59.8
78.3
41.7


AD-11859
0.6
2.0
−1.0

64.0
33.3
40.7


AD-11860
11.4
6.4
0.1
32.44
40.2
−5.8
34.3
54.70


AD-11861
3.2
1.7
−0.4

−6.3
−30.9
26.8


AD-11862
15.3
3.2
−3.1
−55.53
37.3
6.8
51.0
51.20


AD-11863
8.7
8.8
4.1
−77.56
26.0
17.7
37.8
80.86


AD-11864
8.9
3.5
−1.4
−179.90
40.7
27.9
−8.9
69.38


AD-11865
4.3
0.2
0.1

0.3
8.6
14.9


AD-11870
4.5
5.7
1.9

60.1
43.2
68.8


AD-11871
13.9
4.6
−0.4
86.72
59.8
62.3
72.1


AD-11872
12.4
9.9
−0.2
81.93
75.1
77.9
77.9
79.43


AD-11873
16.2
8.7
2.9
51.91
81.9
90.3
92.0


AD-11874
14.0
3.6
−0.6
63.74
36.6
41.1
21.2


AD-11875
37.1
22.5
9.9
92.11
54.4
52.0
47.5
76.56


AD-11876
11.8
4.2
1.3
5.85
35.7
43.8
50.3


AD-11878
3.5
−0.5
−1.7

65.6
62.4
60.0


AD-11879
30.5
21.5
3.8
93.89
−4.3
−19.3
11.4
80.10


AD-11882
16.0
12.4
5.1
80.92
9.1
4.8
19.5
77.03


AD-11883
13.2
6.8
1.4
−37.40
−29.3
12.5
26.9
−1048.33


AD-11884
23.7
11.4
5.5
25.83
41.4
25.2
40.9
−42.26


AD-11885
1.5
−0.8
−2.9

55.9
53.3
53.8


AD-11886
−1.2
−3.4
−4.7

38.7
47.2
67.9


AD-11887
16.6
4.6
−2.2
−85.11
69.8
54.1
54.1
85.65


AD-11888
12.9
3.8
2.3

62.2
66.5
65.7


AD-11889
12.2
5.9
−1.6

4.6
10.5
40.1


AD-11890
8.5
6.2
5.0

−41.7
−12.9
20.9


AD-11891
20.7
9.8
1.0
−69.47
19.3
23.9
32.0


AD-11892
−0.1
−2.5
−2.0

38.4
32.1
38.0


AD-11893
0.1
−2.1
−1.5

27.1
21.1
3.1


AD-11896
3.5
1.3
−1.6

23.3
−6.7
10.0


AD-11897
1.6
−1.3
−2.6

26.4
13.7
13.1


AD-11899
8.3
1.9
0.6

16.4
20.9
28.9


AD-11901
2.5
3.1
1.4

−89.3
71.9
78.6


AD-11902
5.9
0.6
−2.6

−15.2
62.9
79.8


AD-11903
1.1
−1.5
−3.6

64.0
45.9
45.9


AD-11904
0.7
0.4
−0.2

46.4
16.9
70.6


AD-11905
6.9
2.1
−0.1

−42.4
42.5
27.9


AD-11906
−0.4
0.3
−2.5

98.5
93.5
54.3


AD-11907
4.5
0.5
−1.1

95.8
45.3
95.1


AD-11908
−0.9
−1.7
0.2

39.0
86.6
84.5


AD-11909
7.0
2.9
1.1

79.1
−27.1
83.9


AD-11911
5.4
2.6
−1.9

97.4
98.6
94.0


AD-11912
−3.4
−4.5
−3.7

99.4
96.2
65.9


AD-11914
−0.6
−0.2
1.0

91.7
69.5
91.5


AD-11918
−6.3
−8.2
−6.7

−12.4
9.7
8.0


AD-11919
−1.0
−0.1
3.2

18.2
−9.2
32.5


AD-11925
3.5
1.9
−2.7

11.9
13.7
13.7


AD-11926
11.3
4.9
−3.2
46.95
16.9
37.0
66.5
93.16


AD-11927
−6.8
−8.4
−5.7

45.5
−12.2
−17.2


AD-11933
−0.2
1.4
3.3

−6.7
32.7
3.5


AD-11938
0.0
−2.4
−4.1

19.2
−45.8
1.4


AD-11939
−2.3
−2.0
−3.1

3.0
23.1
24.9


AD-11941
−8.3
−9.6
−7.5

42.1
4.0
−25.0


AD-11942
−4.4
−3.5
−2.5

31.6
2.5
1.4


AD-11943
1.9
0.7
−2.7

−5.6
20.5
15.4


AD-11944
−1.1
−2.1
−2.0

51.0
46.7
50.6


AD-11945
−7.7
−8.6
−5.9

77.2
30.5
55.8


AD-11946
0.6
0.8
−2.4

66.9
65.2
65.4


AD-11947
−0.6
−2.2
−4.1

66.6
68.5
68.5


AD-11948
−2.3
−4.0
−4.5

77.1
77.6
88.6


AD-11949
−6.6
−7.9
−6.4

67.5
33.7
2.9


AD-11950
−3.9
0.8
7.9

56.0
51.7
44.8


AD-11951
7.7
0.4
6.0

35.6
45.6
46.1


AD-11952
1.7
−4.5
−4.7

54.8
65.7
66.0


AD-11953
−9.1
−7.7
−6.8

51.4
22.8
30.1


AD-11954
−2.7
8.2
8.2

42.1
26.4
32.1


AD-11955
9.0
7.5
5.9

54.6
43.4
44.2


AD-11956
6.3
−6.5
−4.0

28.1
40.0
55.3


AD-11957
−1.9
−6.4
−2.8

78.2
95.9
69.2


AD-11958
2.0
1.2
−1.0

99.7
89.5
86.2


AD-11959
8.7
3.4
−1.2

81.2
74.5
74.5


AD-11960
2.8
1.7
0.2

71.9
61.4
82.7


AD-11961
−3.4
−5.5
−4.5

84.3
50.6
68.7


AD-11962
−1.4
0.0
−0.2

74.1
89.2
63.9


AD-11963
2.1
−0.3
−0.2

78.8
20.1
50.9


AD-11964
4.5
3.9
−0.1

61.5
51.0
58.4


AD-11965
−3.8
−6.3
−4.4

64.6
14.2
89.4


AD-11966
−0.5
−1.0
−0.5

49.5
−30.0
21.4


AD-11967
5.0
3.8
1.2

24.7
28.2
24.2


AD-11968
−0.8
−0.2
−0.3

9.5
6.5
26.5


AD-11969
3.9
0.6
−2.2

63.5
31.6
56.5


AD-11970
9.2
5.5
−1.6

47.6
36.9
57.1


AD-11971
−1.2
0.1
−1.7

63.8
55.3
55.3


AD-11972
2.7
−0.9
−0.8

66.7
82.1
78.1


AD-11973
3.2
5.3
−0.3

−137.3
−57.5
−69.7


AD-11974
1.7
2.3
2.6

−15.1
−41.3
−55.7


AD-11975
1.8
1.4
1.9

−63.6
−63.1
−54.6


AD-11976
2.3
2.7
2.7

−8.3
10.1
29.0


AD-11977
4.5
3.7
2.7

−168.8
−142.8
−116.4


AD-11978
2.1
1.2
0.8

−66.5
−91.0
−120.2


AD-11979
−1.7
−2.0
−2.2

−140.3
−128.4
−107.4


AD-11980
−1.3
−0.8
−0.6

−21.5
−10.2
−14.2


AD-11981
9.1
6.7
4.6

−181.1
−162.1
−164.3


AD-11982
−0.2
−0.4
−1.4

−189.5
−148.6
−159.6


AD-11983
−2.7
−3.4
−3.7

−131.3
−111.7
−115.3


AD-11984
−1.9
−0.5
0.1

−36.1
−12.9
−10.8


AD-11985
5.5
6.4
2.8

−139.4
−160.3
−216.6


AD-11986
6.1
6.0
5.8

−111.7
−143.8
−134.7


AD-11987
0.9
1.5
2.0

−162.1
−148.5
−132.2


AD-11988
7.4
8.5
8.9

−196.2
−201.7
−178.2


AD-11989
1.9
2.0
1.6

−62.4
−57.6
−61.2


AD-11990
0.8
−0.2
−0.8

−51.3
−69.3
−76.8


AD-11991
−1.9
−2.4
−2.3

−84.5
−65.2
−51.0


AD-11992
0.6
1.3
1.6

−83.1
−60.4
−57.1


AD-11993
6.6
5.6
3.7

−29.9
−33.8
−35.4


AD-11994
0.3
0.1
−0.6

−60.1
−59.5
−59.4


AD-11995
−2.3
−2.1
−1.9

−73.2
−61.7
−58.5


AD-11996
−0.7
0.3
0.4

−4.6
−14.0
−21.4


AD-11997
4.1
4.3
−0.1

64.7
26.4
52.9


AD-11998
0.3
0.0
−0.7

69.1
61.0
52.1


AD-11999
−0.5
−0.1
0.1

31.9
37.3
39.5


AD-12000
−0.8
0.2
0.8

51.7
54.7
60.6


AD-12001
3.7
3.3
2.9

54.7
49.4
47.3


AD-12002
3.8
2.9
2.2

34.0
49.3
62.0


AD-12003
−0.3
−0.4
−0.2

26.3
29.4
32.9


AD-12004
0.6
0.8
1.4

37.7
36.6
36.9


AD-12005
4.9
4.7
3.9

19.6
9.3
7.5


AD-12006
0.8
0.5
0.5

−22.4
−23.6
−19.4


AD-12007
−1.3
−0.7
−0.3

−10.4
−13.6
−21.7


AD-12008
0.7
1.4
1.4

31.6
31.4
33.4


AD-12009
0.8
2.5
−2.2

36.9
25.4
31.0


AD-12010
−1.2
−1.5
−1.6

35.6
23.9
15.8


AD-12011
−1.2
−0.3
0.2

50.4
46.0
41.7


AD-12012
0.7
0.7
0.5

47.1
49.2
51.5


AD-12013
1.3
1.1
−0.5

31.5
25.2
21.8


AD-12014
−1.8
−3.0
−2.7

21.5
26.1
26.3


AD-12015
−4.2
−4.1
−3.8

36.5
35.7
34.2


AD-12016
−1.9
−1.5
−1.2

38.7
41.0
41.9


AD-12017
−0.4
−1.0
−2.2

6.4
12.0
17.5


AD-12018
−1.0
−2.6
−2.9

−19.0
−8.5
−7.1


AD-12019
−3.1
−2.7
−2.3

−3.4
−3.2
0.0


AD-12020
−2.1
−1.4
−1.0

2.2
10.2
12.7


AD-12021
4.9
3.3
2.1

−34.2
−9.2
−30.6


AD-12022
3.9
3.1
2.1

−139.2
−184.9
−191.4


AD-12023
0.4
0.5
0.8

10.7
11.7
−2.8


AD-12024
0.2
1.4
2.3

−8.4
6.0
18.1


AD-12025
6.9
4.8
3.2

18.0
−1.4
−25.4


AD-12026
−0.3
−0.1
0.2

−79.8
−64.2
−44.8


AD-12027
−0.8
−1.0
−0.5

−28.2
−43.7
−57.1


AD-12028
−1.2
−0.2
0.8

−8.1
−0.6
6.2


AD-12029
6.9
5.6
3.8

−65.2
−79.3
−106.3


AD-12030
−0.1
−0.8
−2.0

−70.4
−93.2
−119.7


AD-12031
0.2
0.1
−0.1

−32.4
−34.4
34.9


AD-12032
0.0
0.1
0.6

−86.6
−68.5
−43.4


AD-12033
−4.7
−8.2
−7.6

−115.7
−149.7
−58.5


AD-12034
−6.0
−5.7
−5.7

−217.6
−210.8
−215.6


AD-12035
−7.7
−7.4
−7.6

−99.6
−102.4
−107.1


AD-12036
−6.6
−6.1
−5.1

−111.1
−91.0
−89.8


AD-12037
−7.8
−7.8
−7.7

45.1
−5.3
−44.2


AD-12462
−8.4
−8.6
−9.4

−150.2
−135.8
−130.8


AD-12463
−8.3
−8.4
−8.6

−70.1
−70.9
−75.8


AD-12464
−6.3
−6.6
−6.5

−38.9
−19.7
−2.6


AD-12465
−2.8
−3.8
−5.3

−52.8
−75.2
−92.9


AD-12466
−1.9
−3.1
−3.5

−68.5
−103.9
−113.9


AD-12467
−5.9
−5.8
−5.6

−118.6
−121.8
−117.7


AD-12468
−4.9
−4.2
−3.3

−14.2
−8.4
−6.8


AD-12469
−4.8
−6.0
−6.6

−29.1
−6.1
−10.2


AD-12470
−0.9
−1.4
−1.9

−39.5
−19.2
−1.8


AD-12471
−4.0
−4.0
−4.0

−15.4
−16.3
−17.3


AD-12472
−3.2
−3.4
−2.6

25.9
24.0
27.4


AD-12473
−3.5
−3.8
−4.1

−42.5
−31.5
−26.4


AD-12474
−5.0
−5.3
−5.8

−7.1
−3.0
−1.8


AD-12475
−7.1
−7.6
−7.4

−10.2
−4.3
−0.7


AD-12476
−5.2
−4.7
−3.5

24.3
26.9
32.2


AD-12477
−2.8
−3.8
−4.4

−41.4
−45.4
−37.6


AD-12478
−7.3
−7.6
−7.8

−59.7
−63.8
−67.5


AD-12479
−7.9
−8.3
−8.1

−54.9
−39.3
−8.6


AD-12480
−2.7
−2.4
−1.4

9.3
18.0
12.4


AD-12481
−5.7
−5.5
−7.5

−131.4
−152.8
−48.2


AD-12482
−4.4
−4.5
−4.1

−159.1
−127.8
−140.7


AD-12483
−4.8
−4.8
−4.5

−66.0
−64.8
−60.3


AD-12484
−4.2
−3.9
−3.8

17.9
27.6
29.1


AD-12485
−2.7
−4.0
−5.4

−170.2
−176.6
−186.0


AD-12486
−8.5
−8.6
−8.6

−200.1
−194.6
−169.0


AD-12487
−8.1
−8.0
−7.8

−117.5
−106.8
−86.5


AD-12488
−8.2
−7.9
−7.8

−17.3
−13.1
0.8


AD-12489
−4.2
−5.6
−7.1

−128.0
−152.3
−185.8


AD-12490
−8.6
−8.9
−8.8

−241.7
−258.1
−251.4


AD-12491
−8.9
−9.0
−8.4

−181.6
−184.9
−188.0


AD-12492
−8.6
−7.7
−6.7

−23.3
−22.4
−27.3


AD-12493
−8.2
−6.7
−4.5

−93.8
−54.8
6.7


AD-12494
−7.6
−8.0
−8.2

−62.2
−63.5
−54.7


AD-12495
−6.6
−5.5
−4.9

16.6
0.4
−10.0


AD-12496
−4.1
−4.2
−3.8

40.2
41.7
39.5


AD-12497
−5.1
−5.2
−6.3

−67.9
−65.2
−67.3


AD-12498
−5.8
−6.0
−6.4

−68.6
−84.1
−94.1


AD-12499
−6.6
−6.3
−6.3

−93.4
−80.2
−61.0


AD-12500
−6.0
−5.4
−4.7

9.7
10.2
11.3


AD-12501
−4.8
−5.0
−5.8

−87.1
−108.5
−110.6


AD-12502
−6.8
−7.2
−6.7

−126.5
−125.5
−119.1


AD-12503
−10.4
−9.6
−9.9

−85.2
−76.4
−86.7


AD-12504
−4.0
−3.6
−2.5

−59.7
−52.7
−34.7


AD-12505
18.1
−0.9
−5.5
42.18
3.5
−116.5
−34.2
−282.78


AD-12506
−2.1
−2.7
−4.2

−29.8
−22.3
−11.9


AD-12507
−1.7
7.2
15.0
44.47
29.1
33.0
32.3


AD-12508
−4.2
−3.2
−1.9

40.7
33.9
30.5


AD-12509
−9.2
−8.9
−8.2

−54.3
−67.7
−80.3


AD-12510
−7.9
−7.6
−7.6

−127.6
−117.5
−124.2


AD-12511
−7.1
−6.9
−6.8

−28.4
−18.6
−6.7


AD-12512
−5.2
−4.1
−3.3

−0.4
−1.7
−1.5


AD-12513
−6.9
−7.4
−8.8

−65.5
−80.0
−100.6


AD-12514
−10.8
−11.0
−10.6

−83.8
−85.0
−92.0


AD-12515
−10.7
−9.6
−9.3

−19.3
−41.2
−32.7


AD-12516
−5.8
−5.3
−4.2

−5.9
0.9
15.9


AD-12517
−8.6
−8.7
−7.1

29.2
53.0
49.4


AD-12518
−8.3
−8.0
−8.0

65.7
57.8
53.6


AD-12519
−7.2
−7.2
−7.6

51.2
51.3
53.9


AD-12520
−7.0
−6.3
−5.4

67.7
73.2
78.8


AD-12521
−6.4
−8.0
−9.1

19.8
27.6
35.5


AD-12522
−9.9
−9.0
−9.1

50.3
43.0
34.3


AD-12523
−9.0
−8.7
−8.9

28.3
31.9
38.1


AD-12524
−7.0
−7.4
−7.0

63.7
67.1
65.9


AD-12525
−1.6
−3.8
−6.0

16.1
21.8
21.2
















TABLE 4





In vitro Plaque assay controls (all values average of 3-4 experiments


and expressed as % inhibition relative to no siRNA treatment)



















AD-1955 (luc)
31.37
10.97



AD-5179 (GFP)
15.15
18.65



LS L#1
77.23
N/A



LS NP#1
73.94
N/A



LS VP35#1
67.27
N/A


























TABLE 5






Exo + Endo



seq

seq




parent
light



id

id


duplex
duplex
Target
sense
antisense
no
sense 5′-3′
no
antisense 5′-3′
























AD-11594
AD-3542
NP
A-30769
A-30769
995
cAGuAGGAcAcAuGAuGGucTsdT
996
ACcAUcAUGUGUCCuACUGdTsdT






AD-11596
AD-3543
NP
A-30770
A-30771
997
GuAGGAcAcAuGAuGGuGAdTsdT
998
UcACcAUcAUGUGUCCuACdTsdT





AD-11597
AD-3544
NP
A-30772
A-30773
999
uAGGAcAcAuGAuGGuGAucTsdT
1000
AUcACcAUcAUGUGUCCuAdTsdT





AD-11598
AD-3545
NP
A-30774
A-30775
1001
GAuGGuGAuuuuccGuuuGdTsdT
1002
cAAACGGAAAAUcACcAUCdTscT





AD-11600
AD-3546
NP
A-30776
A-30777
1003
uGGuGAuuuuccGuuuGAudTsdT
1004
AUcAAACGGAAAAUcACcAdTsdT





AD-11603
AD-3547
NP
A-30778
A-30779
1005
cuGAGAAGcAAcuccAAcAcTsdT
1006
UGUUGGAGUUGCUUCUcAGcTsdT





AD-11604
AD-3548
NP
A-30780
A-30781
1007
uGAGAAGcAAcuccAAcAAdTsdT
1008
UUGUUGGAGUUGCUUCUcAdTsdT





AD-11606
AD-3549
NP
A-30782
A-30783
1009
AGAAGcAAcuccAAcAAuAdTsdT
1010
uAUUGUUGGAGUUGCUUCUdTsdT





AD-11609
AD-3550
VP35
A-30784
A-30785
1011
AAGuGAuGAAGAuuAAGAAdTsdT
1012
UUCUuAAUCUUcAUcACUUdTsdT





AD-11610
AD-3551
VP35
A-30786
A-30787
1013
AGuGAuGAAGAuuAAGAAAdTsdT
1014
UUUCUuAAUCUUcAUcACUdTsdT





AD-11611
AD-3552
VP40
A-30788
A-30789
1015
cuGccuGcuGcAAcAuGGAdTsdT
1016
UCcAUGUUGcAGcAGGcAGdTsdT





AD-11613
AD-3553
GP
A-307901
A-30791
1017
GGcuGAAAAcuGcuAcAAudTsdT
1018
AUUGuAGcAGUUUUcAGCCdTsdT





AD-11614
AD-3554
GP
A-30792
A-30793
1019
GcuGAAAAcuGcuAcAAucdTscT
1020
GAUUGuAGcAGUUUUcAGCdTsdT





AD-11615
AD-3555
GP
A-30794
A-30795
1021
cuGAAAAcuGcuAcAAucudTsdT
1022
AGAUUGuAGcAGUUUUcAGdTsdT





AD-11618
AD-3556
GP
A-30796
A-30797
1023
AAAAcuGcuAcAAucuuGAdTsdT
1024
UcAAGAUUGuAGcAGUUUUcTsdT





AD-11621
AD-3557
GP
A-30798
A-30799
1025
AcuGcuAcAAucuuGAAAudTsdT
1026
AUUUcAAGAUUGuAGcAGUdTscT





AD-11622
AD-3558
VP30
A-30800
A-30801
1027
AGcAAAuccAAcGGcuGAudTsdT
1028
AUcAGCCGUUGGAUUUGCUdTsdT





AD-11624
AD-3559
VP30
A-30802
A-30803
1029
cAAAuccAAcGGcuGAuGAdTsdT
1030
UcAUcAGCCGUUGGAUUUGdTsdT





AD-11627
AD-3560
L
A-30804
A-30805
1031
AuGcAuGucAGuGAuuAuudTsdT
1032
AAuAAUcACUGAcAUGcAUdTsdT





AD-11628
AD-3561
L
A-30806
A-30807
1033
uGcAuGucAGuGAuuAuuAdTsdT
1034
uAAuAAUcACUGAcAUGcAdTsdT





AD-11629
AD-3562
L
A-30808
A-30809
1035
GcAuGucAGuGAuuAuuAudTsdT
1036
AuAAuAAUcACUGAcAUGCdTsdT





AD-11630
AD-3563
L
A-30810
A-30811
1037
cAuGucAGuGAuuAuuAuAdTsdT
1038
uAuAAuAAUcACUGAcAUGdTsdT





AD-11631
AD-3564
L
A-30812
A-30813
1039
AuGucAGuGAuuAuuAuAAdTsdT
1040
UuAuAAuAAUcACUGAcAUdTsdT



















TABLE 6






In vitro
In vitro plaque assay
In vitro plaque assay



plasmid
against Ebola-Zaire
against Ebola-Sudan


modified
screen IC50
(% inhibition relative
(% inhibition relative


duplex
(nM)
to no siRNA)
to no siRNA)


















AD-3542
1.60
84%
74%


AD-3543
0.00
74%
77%


AD-3544
0.61
65%
13%


AD-3545
0.00
82%
61%


AD-3546
1.35
88%
71%


AD-3547
0.00
81%
26%


AD-3548
7.82
68%
73%


AD-3549
0.00
68%
74%


AD-3550
0.17
77%
22%


AD-3551
0.08
2%
75%


AD-3552
0.00
78%
70%


AD-3553
0.19
85%
26%


AD-3554
0.77
84%
15%


AD-3555
0.88
−5%
19%


AD-3556
0.00
73%
74%


AD-3557
0.21
100%
28%


AD-3558
0.00
−7%
68%


AD-3559
0.00
−252%
−7%


AD-3560
0.50
16%
−35%


AD-3561
0.00
−105%
19%


AD-3562
0.00
−75%
63%


AD-3563
5.42
73%
−17%


AD-3564
0.97
−86%
19%


AD-3621
0.00
80%
48%


AD-3622
16.00
61%
47%


AD-3623
0.00
98%
37%


AD-3624
0.00
84%
24%


AD-3625
0.00
−7%
−5%


AD-3626
0.00
53%
31%





















TABLE 7









IFN induction normalized

TNF induction normalized




to positive control siRNA

to positive control siRNA












duplex
% of control
duplex
% of control
















AD-11546
131.6
AD-11546
30.1



AD-11558
98.8
AD-11558
57.2



AD-11570
0
AD-11570
0



AD-11588
147
AD-11588
0.0



AD-11590
0.0
AD-11590
130.5



AD-11594
17.8
AD-11594
124.8



AD-11597
22.0
AD-11597
242.9



AD-11598
25.7
AD-11598
180.3



AD-11599
136
AD-11599
183.9



AD-11600
13.5
AD-11600
141.0



AD-11603
69.0
AD-11603
81.4



AD-11606
33.2
AD-11606
79.8



AD-11609
126.9
AD-11609
46.3



AD-11610
138.2
AD-11610
48.5



AD-11611
43.2
AD-11611
41.0



AD-11613
167.0
AD-11613
52.6



AD-11614
162.1
AD-11614
48.1



AD-11615
171.2
AD-11615
60.0



AD-11618
137.3
AD-11618
54.6



AD-11621
0.0
AD-11621
325.8



AD-11622
37.2
AD-11622
28.5



AD-11623
58.0
AD-11623
26.0



AD-11624
63.2
AD-11624
29.2



AD-11627
6.4
AD-11627
125.0



AD-11628
0.0
AD-11628
101.1



AD-11630
0.0
AD-11630
170.3



AD-11631
0.0
AD-11631
156.0



AD-11644
0
AD-11644
0.0



AD-11650
0.0
AD-11650
0.0



AD-11659
0.0
AD-11659
69.0



AD-11673
9.2
AD-11673
0.0



AD-11678
0.0
AD-11678
0.0



AD-11683
0.0
AD-11683
19.6



AD-11684
0.0
AD-11684
24.3



AD-11691
0
AD-11691
0.0



AD-11695
0.0
AD-11695
5.2



AD-11698
5.3
AD-11698
14.0



AD-11706
0.0
AD-11706
0.0



AD-11707
0
AD-11707
0.0



AD-11710
0
AD-11710
0.0



AD-11721
0.0
AD-11721
0.0



AD-11725
0.0
AD-11725
0.0



AD-11732
0.0
AD-11732
0.0



AD-11743
16.1
AD-11743
0.0



AD-11756
0.0
AD-11756
0.0



AD-11757
0.0
AD-11757
0.0



AD-11758
0.0
AD-11758
0.0



AD-11759
0.0
AD-11759
0.0



AD-11773
0.0
AD-11773
0.0



AD-11780
0.0
AD-11780
7.9



AD-11789
0.0
AD-11789
5.7



AD-11804
0.0
AD-11804
0.0



AD-11811
0.0
AD-11811
0.0



AD-11814
0.0
AD-11814
0.0



AD-11816
0.0
AD-11816
0.0



AD-11822
9.8
AD-11822
0.0



AD-11823
0.0
AD-11823
0.0



AD-11832
0.0
AD-11832
0.0



AD-11836
0.0
AD-11836
0.0



AD-11939
0.0
AD-11939
0.0



AD-11976
0.0
AD-11976
0.0



AD-11982
6.9
AD-11982
15.1



AD-11990
12.2
AD-11990
0.0



AD-11992
7.3
AD-11992
0.0



AD-12007
0.0
AD-12007
0.0



AD-12013
0.0
AD-12013
0.0



AD-12019
24.6
AD-12019
5.0



AD-12024
0.0
AD-12024
19.8



AD-12035
0.0
AD-12035
8.5



AD-12475
0.0
AD-12475
0.0



AD-12484
21.0
AD-12484
9.5



AD-12491
0.0
AD-12491
13.1



AD-12500
12.6
AD-12500
53.3



AD-12502
101.6
AD-12502
55.5



AD-3542
0.0
AD-3542
0.0



AD-3543
0.0
AD-3543
0.0



AD-3544
10.1
AD-3544
0.0



AD-3545
11.8
AD-3545
0.0



AD-3546
0.0
AD-3546
0.0



AD-3547
0.0
AD-3547
31.0



AD-3548
0.0
AD-3548
5.9



AD-3549
0.0
AD-3549
10.8



AD-3550
7.4
AD-3550
0.0



AD-3551
0.0
AD-3551
0.0



AD-3552
0.0
AD-3552
0.0



AD-3553
0.0
AD-3553
0.0



AD-3554
0.0
AD-3554
11.5



AD-3555
0.0
AD-3555
6.2



AD-3556
0.0
AD-3556
9.1



AD-3557
0.0
AD-3557
0.0



AD-3558
0.0
AD-3558
5.8



AD-3559
0.0
AD-3559
5.3



AD-3560
0.0
AD-3560
0.0



AD-3561
0.0
AD-3561
0.0



AD-3562
0.0
AD-3562
0.0



AD-3563
0.0
AD-3563
0.0



AD-3564
0.0
AD-3564
0.0



AD-3621
0.0
AD-3621
0.0



AD-3622
0.0
AD-3622
0.0



AD-3623
5.7
AD-3623
0.0



AD-3624
10.0
AD-3624
0.0



AD-3625
0.0
AD-3625
0.0



AD-3626
0.0
AD-3626
0.0





















TABLE 8








In vitro





plasmid
In vitro




screen
plasmid




single dose
screen



duplex
(%
IC50



name
silencing)
(nM)




















AD-11542
9%




AD-11543
−3%



AD-11544
−1%



AD-11545
13%



AD-11546
68%
0.72



AD-11547
28%



AD-11548
48%



AD-11549
43%



AD-11550
−5%



AD-11551
8%



AD-11552
−17%



AD-11553
−6%



AD-11554
15%



AD-11555
2%



AD-11556
6%



AD-11557
−6%



AD-11558
70%
0.46



AD-11559
28%



AD-11560
5%



AD-11561
30%



AD-11562
24%



AD-11563
6%



AD-11564
0%



AD-11565
−4%



AD-11566
4%



AD-11567
−2%



AD-11568
0%



AD-11569
12%



AD-11570
73%
0.95



AD-11571
−2%



AD-11572
−3%



AD-11573
2%



AD-11574
15%



AD-11575
−2%



AD-11576
−9%



AD-11577
−9%



AD-11578
77%



AD-11579
37%



AD-11580
35%



AD-11581
60%



AD-11582
21%



AD-11583
47%



AD-11584
0%



AD-11585
−1%



AD-11586
36%



AD-11587
66%



AD-11588
47%



AD-11589
77%



AD-11590
83%
0.57



AD-11591
65%



AD-11592
62%



AD-11593
55%



AD-11594
85%
0.35



AD-11595
72%



AD-11596
84%
0.24



AD-11597
85%
0.33



AD-11598
87%
0.21



AD-11599
91%
0.81



AD-11600
89%
0.29



AD-11601
84%
1.07



AD-11602
71%



AD-11603
80%
1.3



AD-11604
81%
1.44



AD-11605
75%



AD-11606
78%
6.38



AD-11607
53%



AD-11608
60%



AD-11609
75%
0.3



AD-11610
74%
0.15



AD-11611
61%
0.28



AD-11612
−5%



AD-11613
84%
0.077



AD-11614
85%
0.102



AD-11615
79%
0.211



AD-11616
66%



AD-11617
59%



AD-11618
78%
0.24



AD-11619
57%



AD-11620
64%



AD-11621
74%
0.15



AD-11622
70%
0.41



AD-11623
67%
0.54



AD-11624
75%
0.15



AD-11625
11%



AD-11626
51%



AD-11627
71%
0.28



AD-11628
68%
0.33



AD-11629
75%
0.18



AD-11630
73%
0.24



AD-11631
69%
0.31



AD-11632
53%



AD-11633
63%
1.78



AD-11634
65%
0.76



AD-11635
29%



AD-11636
43%



AD-11637
−5%



AD-11638
6%



AD-11639
2%



AD-11640
38%



AD-11641
35%



AD-11642
55%



AD-11643
33%



AD-11644
36%



AD-11645
45%



AD-11646
37%



AD-11647
41%



AD-11648
61%



AD-11649
35%



AD-11650
84%
0.7



AD-11651
13%



AD-11652
64%



AD-11653
61%



AD-11654
6%



AD-11655
59%



AD-11656
38%



AD-11657
39%



AD-11658
59%



AD-11659
82%
0.038



AD-11660
39%



AD-11661
−5%



AD-11662
−1%



AD-11663
14%



AD-11664
19%



AD-11665
7%



AD-11666
−4%



AD-11667
−14%



AD-11668
63%



AD-11669
28%



AD-11670
23%



AD-11671
23%



AD-11672
15%



AD-11673
79%
0.117



AD-11674
67%



AD-11675
46%



AD-11676
20%



AD-11677
34%



AD-11678
79%
0.149



AD-11679
51%



AD-11680
24%



AD-11681
72%



AD-11682
73%



AD-11683
88%
0.056



AD-11684
80%
0.184



AD-11685
33%



AD-11686
72%



AD-11687
32%



AD-11688
15%



AD-11689
58%



AD-11690
26%



AD-11691
60%



AD-11694
54%



AD-11695
81%
0.46



AD-11696
32%



AD-11698
73%
0.2



AD-11700
−9%



AD-11704
35%



AD-11705
39%



AD-11706
67%
0.56



AD-11707
2%



AD-11708
−10%



AD-11710
65%
4.57



AD-11711
−3%



AD-11712
17%



AD-11713
0%



AD-11714
42%



AD-11715
41%



AD-11716
18%



AD-11717
32%



AD-11718
−3%



AD-11719
36%



AD-11720
41%



AD-11721
68%
1.35



AD-11722
31%



AD-11723
49%



AD-11724
27%



AD-11725
67%
0.34



AD-11726
12%



AD-11727
3%



AD-11728
5%



AD-11729
12%



AD-11730
6%



AD-11731
63%
49.4



AD-11732
76%
2.88



AD-11733
60%
8.76



AD-11734
44%



AD-11735
17%



AD-11736
44%



AD-11737
14%



AD-11738
−9%



AD-11739
23%



AD-11740
1%



AD-11741
9%



AD-11742
40%



AD-11743
77%
0.11



AD-11744
24%



AD-11745
27%



AD-11746
−9%



AD-11747
16%



AD-11748
8%



AD-11749



AD-11750
33%



AD-11751
19%



AD-11752
61%
6.92



AD-11753
13%



AD-11754
53%



AD-11755



AD-11756
61%
1.21



AD-11757
63%
1.57



AD-11758
28%



AD-11759
66%
1.61



AD-11760
64%
3.4



AD-11761
59%



AD-11762
54%



AD-11763
47%



AD-11764
21%



AD-11765
−1%



AD-11766
−1%



AD-11767
67%
4.4



AD-11768
52%



AD-11769
21%



AD-11770
55%



AD-11771
36%



AD-11772
41%



AD-11773
76%
0.37



AD-11774
35%



AD-11775
49%



AD-11776
50%



AD-11777
−5%



AD-11778
18%



AD-11779
15%



AD-11780
62%
0.76



AD-11781
14%



AD-11782
38%



AD-11783
46%



AD-11784
23%



AD-11785
−11%



AD-11786
42%



AD-11787
48%



AD-11788
19%



AD-11789
64%
0.38



AD-11790
26%



AD-11791
22%



AD-11792
−8%



AD-11793
26%



AD-11794
57%



AD-11795



AD-11796
59%



AD-11797
11%



AD-11798
11%



AD-11799
35%



AD-11800
2%



AD-11801
−6%



AD-11802
0%



AD-11803
5%



AD-11804
88%
0.281



AD-11805
5%



AD-11806
9%



AD-11807
6%



AD-11808
50%



AD-11809
24%



AD-11810
−1%



AD-11811
66%
1.56



AD-11812
1%



AD-11813
17%



AD-11814
65%
0.43



AD-11815
−1%



AD-11816
65%
0.99



AD-11817
67%
2.98



AD-11818
44%



AD-11819
64%



AD-11820
36%



AD-11821
64%



AD-11822
62%
1.44



AD-11823
69%
0.32



AD-11824
38%



AD-11825
18%



AD-11826
23%



AD-11827
2%



AD-11828
51%



AD-11829



AD-11830
46%



AD-11831
20%



AD-11832
71%
0.94



AD-11833
3%



AD-11834
23%



AD-11835
−8%



AD-11836
65%
1.46



AD-11837
26%



AD-11838
−16%



AD-11839
22%



AD-11840
−5%



AD-11841
60%



AD-11842
19%



AD-11843
57%



AD-11844
18%



AD-11845
8%



AD-11846
48%



AD-11847
57%



AD-11848
2%



AD-11849
66%
0.32



AD-11850
25%



AD-11851
−12%



AD-11852
27%



AD-11853
28%



AD-11854
55%



AD-11855
43%



AD-11856
−20%



AD-11857
61%
3.28



AD-11858
6%



AD-11859
4%



AD-11860
79%



AD-11861
31%



AD-11862
48%



AD-11863
85%
0.39



AD-11864
42%



AD-11865
37%



AD-11870
34%



AD-11871
79%
0.63



AD-11872
70%



AD-11873
70%



AD-11874
39%



AD-11875
39%



AD-11876
34%



AD-11878
−1%



AD-11879
50%



AD-11882
−6%



AD-11883
11%



AD-11884
7%



AD-11885
−3%



AD-11886
−5%



AD-11887
18%



AD-11888
41%



AD-11889
1%



AD-11890
44%



AD-11891
20%



AD-11892
37%



AD-11893
29%



AD-11896
1%



AD-11897
41%



AD-11899
12%



AD-11901
−2%



AD-11902
40%



AD-11903
14%



AD-11904
1%



AD-11905
33%



AD-11906
2%



AD-11907
9%



AD-11908
5%



AD-11909
16%



AD-11911
37%



AD-11912
19%



AD-11914
19%



AD-11918
1%



AD-11919
−1%



AD-11925
−5%



AD-11926
60%



AD-11927
−11%



AD-11933
30%



AD-11938
6%



AD-11939
71%
0.48



AD-11941
47%



AD-11942
33%



AD-11943
48%



AD-11944
51%



AD-11945
69%



AD-11946
39%



AD-11947
39%



AD-11948
83%
0.68



AD-11949
41%



AD-11950
73%



AD-11951
55%



AD-11952
81%
1.42



AD-11953
52%



AD-11954
55%



AD-11955
79%
0.63



AD-11956
37%



AD-11957
39%



AD-11958
34%



AD-11959
36%



AD-11960
19%



AD-11961
−4%



AD-11962
−6%



AD-11963
3%



AD-11964
13%



AD-11965
28%



AD-11966
−4%



AD-11967
15%



AD-11968
4%



AD-11969
0%



AD-11970
−7%



AD-11971
3%



AD-11972
58%



AD-11973
4%



AD-11974
38%



AD-11975
−11%



AD-11976
63%
0.21



AD-11977
2%



AD-11978
56%



AD-11979
5%



AD-11980
5%



AD-11981
19%



AD-11982
65%
0.14



AD-11983
52%



AD-11984
50%



AD-11985
50%



AD-11986
6%



AD-11987
−7%



AD-11988
58%



AD-11989
27%



AD-11990
72%
0.24



AD-11991
29%



AD-11992
76%
0.94



AD-11993
46%



AD-11994
21%



AD-11995
−3%



AD-11996
53%



AD-11997
0%



AD-11998
3%



AD-11999
19%



AD-12000
41%



AD-12001
3%



AD-12002
37%



AD-12003
17%



AD-12004
5%



AD-12005
6%



AD-12006
69%



AD-12007
81%
0.268



AD-12008
35%



AD-12009
22%



AD-12010
34%



AD-12011
10%



AD-12012
25%



AD-12013
75%
0.60



AD-12014
29%



AD-12015
4%



AD-12016
2%



AD-12017
5%



AD-12018
7%



AD-12019
79%
0.904



AD-12020
6%



AD-12021
0%



AD-12022
2%



AD-12023
16%



AD-12024
87%
0.075



AD-12025
4%



AD-12026
5%



AD-12027
66%



AD-12028
24%



AD-12029
−8%



AD-12030
46%



AD-12031
48%



AD-12032
−10%



AD-12033
64%
6.74



AD-12034
45%



AD-12035
70%
0.11



AD-12036
43%



AD-12037



AD-12462
35%



AD-12463
39%



AD-12464
47%



AD-12465
34%



AD-12466
35%



AD-12467
25%



AD-12468
−9%



AD-12469
−3%



AD-12470
50%



AD-12471
12%



AD-12472
9%



AD-12473
52%



AD-12474
1%



AD-12475
62%
0.13



AD-12476
19%



AD-12477
−12%



AD-12478
5%



AD-12479
21%



AD-12480
13%



AD-12481
22%



AD-12482
65%



AD-12483
78%



AD-12484
90%
0.023



AD-12485
76%



AD-12486
13%



AD-12487
60%



AD-12488
54%



AD-12489
11%



AD-12490
72%



AD-12491
86%
0.047



AD-12492
41%



AD-12493
26%



AD-12494
12%



AD-12495
69%



AD-12496
44%



AD-12497
2%



AD-12498
14%



AD-12499
63%



AD-12500
86%
0.057



AD-12501
57%



AD-12502
88%
0.048



AD-12503
−2%



AD-12504
8%



AD-12505
29%



AD-12506
31%



AD-12507
48%



AD-12508
47%



AD-12509
2%



AD-12510
−21%



AD-12511
28%



AD-12512
43%



AD-12513
−22%



AD-12514
38%



AD-12515
−9%



AD-12516
58%



AD-12517
18%



AD-12518
−8%



AD-12519
−5%



AD-12520
62%
1.12



AD-12521
−12%



AD-12522
53%



AD-12523
55%



AD-12524
60%



AD-12525
32%






















TABLE 9








WBC
Platelets
Lymphocyte #






















Animal #1
day 0
7.1
328
2



(AD-11570
day 3
6.9
308
1.9



treatment)
day 5
6.2
394
3.4



Animal #2
day 0
3.6
299
1.5



(AD-11570
day 3
10.9
254
1.5



treatment)
day 5
12.9
281
1.8




day 8
21.2
444
4.3



Animal #3
day 0
3.2
218
2.2



(AD-11570
day 3
10.9
202
1.9



treatment)
day 5
6.4
266
2.4




day 8
18.5
306
3.6



Animal #4
day 0
9.7
398
7.3



(untreated)
day 3
8.4
448
4.4




day 5
6.2
263
1.9




day 8
2.8
143
1.5


















NP Chimeric sequence (SEQ ID NO:1043):



GGTACCCTCGAGGAGGAAGATTAATAATTTTCCTCTCATTGAAATTTATA





TCGGAATTTAAATTGAAATTGTTACTGTAATCACACCTGGTTTGTTTCAG





AGCCACATCACAAAGATAGAGAACAACCTAGGTCTCCGAAGGGAGCAAGG





GCATCAGTGTGCTCAGTTGAAAATCCCTTGTCAACACCTAGGTCTTATCA





CATCACAAGTTCCACCTCAGACTCTGCAGGGTGATCCAACAACCTTAATA





GAAACATTATTGTTAAAGGACAGCATTAGTTCACAGTCAAACAAGCAAGA





TTGAGAATTAACCTTGGTTTTGAACTTGAACACTTAGGGGATTGAAGATT





CAACAACCCTAAAGCTTGGGGTAAAACATTGGAAATAGTTAAAAGACAAA





TTGCTCGGGTTTACCTGAGAGCCTACAACATGGATAAACGGGTGAGAGGT





TCATTGGCGCCGAGTCTCACTGAATCTGACATGGATTACCACAAGATCTT





GACAGCAGGTCTGTCCGTTCAACAGGGGATTGTTCGGCAAAGAGTCATCC





CAGTGTATCAAGTAAACAATCTTGAAGAAATTTGCCAACTTATCATACAG





GCCTTTGAAGCAGGTGTTGATTTTCAAGAGAGTGCGGACAGTTTCCTTCT





CATGCTTTGTCTTCATCATGCGTACCAGGGAGATTACAAACTTTTCTTGG





AAAGTGGCGCAGTCAAGTATTTGGAAGGGCACGGGTTCCGTTTTGAAGTC





AAGAAGCGTGATGGAGTGAAGCGCCTTGAGGAATTGCTGCCAGCAGTATC





TAGTGGAAAAAACATTAAGAGAACACTTGCTGCCATGCCGGAAGAGGAGA





CAACTGAAGCTAATGCCGGTCAGTTTCTCTCCTTTGCAAGTCTATTTCTA





CCCAAACTTGTCGTTGGAGAAAAGGCTTGCCTTGAGAAGGTTCAAAGGCA





AATTCAAGTACATGCAGAGCAAGGACTGATACAATATCCAACAGCTTGGC





AATCAGTAGGACACATGATGGTGATTTTCCGTTTGATGCGAACAAATTTT





CTGATCAAATTTCTCCTAATACACCAAGGGATGCACATGGTTGCCGGGCA





TGATGCCAACGATGCTGTGATTTCAAATTCAGTGGCTCAAGCTCGTTTTT





CAGGCTTATTGATTGTCAAAACAGTACTTGATCATATCCTACAAAAGACA





GAACGAGGAGTTCGTCTCCATCCTCTTGCAAGGACCGCCAAGGTAAAAAA





TGAGGTGAACTCCTTTAAGGCTGCACTCAGCTCCCTGGCCAAGCATGGAG





AGTATGCTCCTTTCGCCCGACTTTTGAACCTTTCTGGAGTAAATAATCTT





GAGCATGGTCTTTTCCCTCAACTATCGGCAATTGCACTCGGAGTCGCCAC





AGCACACGGGAGTACCCTCGCAGGAGTAAATGTTGGAGAACAGTATCAAC





AACTCAGAGAGGCTGCCACTGAGGCTGAGAAGCAACTCCAACAATACGCA





GAGTCTCGCGAACTTGACCATCTTGGACTTGATGATCAGGAAAAGAAAAT





TCTTATGAACTTCCATCAGAAAAAGAACGAAATCAGCTTCCAGCAAACAA





ACGCTATGGTAACTCTAAGAAAAGAGCGCCTGGCCAAGCTGACAGAAGCT





ATCACTGCTGCGTCACTGCCCAAAACAAGTGGACATTACGATGATGATGA





CGACATTCCATTTGCCGGGCCGATCTATGATGACGACAATCCTGGCCATC





AAGATGATGATCCGACTGACTCACAGGATACGACCATTCCCGATGGTGTT





GTTGACCCGTATGATGGAAGCTACGGCGAATATCCTGACTACGAGGATTC





GGCTGAAGGTGCACCAGATGACTTGGTCCTATTCGATCTAGACGAGGACG





ACGAGGACACTAAGCCAGTGCCTAATAGATCGACCAAGGGTGGACAACAG





AAGAACAGTCAAAAGGGCCAGCATATAGAGGGCAGACAGATCCGACCTTG





GACGGAGCGAAAAAGGTGCCGGAGTTGCAGAACAATCCACCACGCCAGTG





CGCCACTCACGGACAATGACAGAAGAAATGAACCCTCCGGCTCAACCAGC





CCTCGCATGCTGACACCAATTAACGAAGAGGCAGACCCACTGGACGATGC





CGACGACGAGAGTCTCACATCCCTGCCCTTGGAGTCAGATGATGAAGAGC





AGGACAGGGACGGAACTTCCAACCGCACACCCACTGTCGCCCCACCGGCT





CCCGTATACAGAGATCACTCTGAAAAGAAAGAACTCCCGCAAGACGAGCA





ACAAGATCAGCACCACACTCAAGAGGCCAGGAACCAGGACAGTGACAACA





CCCAGTCAGAACACTCTTTTGAGGAGATGTATCGCCACATTCTAAGATCA





CAGGGGCCATTTGATGCTGTTTTGTATTATCACCTAATGAGTGATGAGCC





TGTAGTTTTCAGTACCAGTGATGGCAAAGAGTACACGTATCCAGACTCCC





TTGAAGAGGAATATCCACCATGGCTCACTGAAAAAGAGGCTATGAATGAA





GAGAATAGATTTGTTACATTGGATGGTCAACAATTTTATTGGCCGGTGAT





GAATCACAAGAATAAATTCATGGCAATCCTGCAACATCATCAGTGAATGA





GCATGGAACAATGGGATGATTCAACCGACAAATAGCTAACATTAAGTAGT





CAAGGAACGAAAACAGGAAGAATTTTTGATGTCTAAGGTGTGAATTATTA





TCACAATAAAAGTGATTCTTATTTTTGAATTTAAAGCTAGCTTATTATTA





CTAGCCGTTTTTCAAAGTTCAATTTGAGTCTTAATGCAAATAGGCGTTAA





GCCACAGTTATAGCCATAATTGTAACTCAATATTCTAACTAGCGATTTAT





CTAAATTAAATTACATTATGCTTTTATAACTTACCTACTAGCCTGCCCAA





CATTTACACGATCGTTTTATAATTAAGAAAAAAGCGGCCGCAGAGCTC





GP Chimeric sequence (SEQ ID NO:1044):


GGTACCCTCGAGGATGAAGATTAAGCCGACAGTGAGCGTAATCTTCATCT





CTCTTAGATTATTTGTTTTCCAGAGTAGGGGTCGTCAGGTCCTTTTCAAT





CGTGTAACCAAAATAAACTCCACTAGAAGGATATTGTGGGGCAACAACAC





AATGGGCGTTCTTAGCCTACTCCAATTGCCTCGTGATCGATTCAAGAGGA





CATCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATC





CCACTTGGAGTCATCCACAATAGCACATTACAGGTTAGTGAGATTGACCA





GCTAGTCTGCAAGGATCATACTGATATGCCATCTGCAACTAAAAGATGGG





GCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGTGAA





TGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCGGACGGGAGCGA





ATGCTTACCCGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGT





ATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTC





CATAAAGAGGGTGCTTTCTTCCTGTATGATCGACTTGCTTCCACAGTTAT





CTACCGAGGAACGACTTTCGCTGAAGGTGTGCTTGCATTTCTGATACTGC





CCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTC





AATGCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATA





TCAGGCTACCGGTTTTGGAACCAATGAGACAGAGTACTTGTTCGAGGTTG





ACAATTTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTG





CTCCAGCTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCAC





GGGAAAACTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGG





AGTGGGCCTTCTGGGAAACTAAAAAAACCTCACTAGAAAAATTCGCAGTG





AAGAGTTGTCTTTCACAGTTTTATCGCTCAACGAGACAGACATCAGTGGT





CAGAGTCCGGCGCGAACTTCTTCCGGAAGAATCTCCGACCGGGCCACTGA





AGACCACAAAATCATGGCTTCAGAAAATTCCTCTGCAATGGTTCAAGTGC





ACAGTCAAGGAAGGGAAACAACATTGCCGTCTCAGAATTCGACAGAAGGT





CGAAGAGCGAGTCCCCAATCCCTCACAACCAAACCAGGTCCGGACAACAG





CACCCATAATACACCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAG





TTGAACAACATCACCGCAGAACAGACAACGACAGCACAGCCTCCGACACT





CCCTCTGCCACGACCGCAGCCGGACCCCCAAAAGCAGAGAACACCAACAC





GAGCAAGAGCACTGACTTCCTGGACCCCGCCACCACAACAAGTCCCCAAA





ACCACAGCGAGACCGCTGGCAACAACAACACTCATCACCAAGATACCGGA





GAAGAGAGTGCCAGCAGCGGGAAGCTAGGCTTAATTACCAATACTATTGC





TGGAGTCGCAGGACTGATCACAGGCGGGAGAAGAACTCGAAGAGAAGCAA





TTGTCAATGCTCAACCCAAATGCAACCCTAATTTACATTACTGGACTACT





CAGGATGAAGGTGCTGCAATCGGACTGGCCTGGATACCATATTTCGGGCC





AGCAGCCGAGGGAATTTACATAGAGGGGCTAATGCACAATCAAGATGGTT





TAATCTGTGGGTTGAGACAGCTGGCCAACGAGACGACTCAAGCTCTTCAA





CTGTTCCTGAGAGCCACAACGGAGCTGCGGACATATACCATACTCAACCG





TAAGGCAATTGATTTCTTGCTGCAGCGATGGGGCGGCACATGCCACATTC





TGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACA





GACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGA





CCAGGGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGG





CAGGTATTGGAGTTACAGGCGTTATAATTGCAGTTATCGCTTTATTCTGT





ATATGCAAATTTGTCTTTTAGTTTTTCTTCAGATTGCTTCATGGAAAAGC





TCAGCCTCAAATCAATGAAACCAGGATTTAATTATATGGATTACTTGAAT





CTAAGATTACTTGACAAATGATAATATAATACACTGGAGCTTTAAACATA





GCCAATGTGATTCTAACTCCTTTAAACTCACAGTTAATCATAAACAAGGT





TTGACATCAATCTAGTTATCTCTTTGAGAATGATAAACTTGATGAAGATT





AAGAAAAAGCGGCCGCAGAGCTC







The L gene was generated as 2 fragments (L-ABC, SEQ ID NO:1049; and L-DEFG, SEQ ID NO:1050).










L fragment 1: SEQ ID NO:1049 (L-ABC):



GGCGCGCCTCGAGGAGGAAGATTAAGAAAAACTGCTTATTGGGTCTTTCC





GTGTTTTAGATGAAGCAGTTGAAATTCTTCCTCTTGATATTAAATGGCTA





CCCAACATACACAATACCCAGACGCTAGTTATCATCACCAATTGTATTGG





ACCAATGTGACCTAGTCACTAGAGCTTGCGGGTTATATTCATCATACTCC





CTTAATCCGCAACTACGCAACTGTAAACTCCCGAAACATATCTACCGTTT





GAAATACGTGTAACTGTTACCAAGTTCTTGAGTGATGTACCAGTGGCGAC





ATTGCCCATAGATTTCATAGTCCCAGTTCTTCTCAAGGCAATGTCAGGCA





ATGGATTCTGTCCTGTTGAGCCGCGGTGCCAACAGTTCTAGATGAAATCA





TTAAGTACACAATGCAAGATGCTCTCTTCTTGAAATATTATCTCAAAAAT





GTGGGTGCTCAAGAAGACTGTGTTGATGAACACTTTCAAGAGAAAATCTT





ATCTTCAATTCAGGGCAATGATTTTTACATCAAATGTTTTTCTGGTATGA





TCTGGCTATTTTAACTCGAAGGGGTAGATTAAATCGAGGAAACTCTAGAT





CAACATGGTTTGTTCATGATGATTTAATAGACATCTTAGGCTATGGGGAC





TTGTTTTTTGGAAGATCCCAATTTCAATGTTACCACTGAACACACAAGGA





ATCCCCCATGCTGCTATGGACTGGTATCAGGCATCAGTATTCAAAGAAGC





GGTTCAAGGGCATACACACATTGTTTCTGTTTTACTGCCGACGTCTTGAT





AATGTGCAAAGATTTAATTACATGTCGATTCAACACAACTCTAATCTCAA





AAATAGCAGAGATTGAGGATCCAGTTTGTTCTGATTATCCCAATTTTAAG





ATTGTGTCTATGCTTACCAGAGCGGAGATTACTTACTCTCCATATTAGGG





TCTGATGGGTATAAAATTATTAAGTTCCTCGAACCATTGTGCTTGGCCAA





AATTCAATTATGCTCAAAGTACACTGAACGAAAAGGGCGGTTTTAACACA





AATGCATTTAGCTGTAAATCACACCCTAGAAGAAATTACAGAAATGCGTG





CACTAAAGCCTTCACAGGCTCAAAAGATCCGTGAATTCCATAGAACATTG





ATAAGGCTGGAGATGACGCCACAACACTTTGTGAGCTATTTTCCATTCAA





AAACACTGGGGGCATCCTGTGCTACATAGTGAAACAGCAATCCAAAAAGT





TAAAAAACATGCTACGGTGCTAAAAGCATTACGCCCTATAGTGATTTTCG





AGACATCTGTGTTTTTAAATATAGTATTGCCAAACATTATTTTGATAGTC





AAGGATCTTGGTACAGTGTTACTTCAGACCGATGTTTAACGCCGGGATTG





AATTCTTATATCAAAAGAAATCAATTCCCTCCGTTGCAATGATTAAAGAA





CTACTATGGGAATTTTACCACCTTGACCACCCTCCACTTTTCTCAACCAA





AATTATTAGTGACTTAAGTATTTTTATAAAAGACAGAGCTACCGCAGTAG





AAAGGACATGCTGGGATGAGTATTCGAGCCTAATGTTCTAGGATATAATC





CACCTCACAAATTTAGTACTAAACGTGTACCGGAACAATTTTTAGAGCAA





GAAAACTTTTCTATTGAGAATGTTCTTTCATACGCCCAAGAACTTAGGTT





CTACTACCACAATATCGGAACTTTTCTTTCTCATTGAAAGAGAAAGAGTT





GAATGTAGGTAGAACCTTCGGAAAATTGCCTTATCCGACTCGCAATGTTC





AAACACTTTGTGAAGCTCTGTTAGCTGATGTCTTGCTAAAGCATTTCCTA





GCAATATGATGGTAGTTACGGAACGTGAGCAAAAAGAAAGCTTATTGCAT





CAAGCATCATGGCACCACACAAGTGATGATTTTGGTGAACATGCCACAGT





TAGAGGGAGTACTTTGTAACTGATTTAGAGAAATACAATCTTGCATTTAG





ATATGAGTTTACAGCACCTTTTATAGAATATTGCAACCGTTCCTATGGTG





TTAAGAATGTTTTTAATTGGATGCATTATACAATCCCACAGTTTATATGC





ATGTCAGTGATTATTATAATCCACCACATAACCTCACACTGGAGAATCGA





GACAACCCCCCCGAAGGGCCTAGTTCATACAGGGGTCATATGGGAGGGAT





TGAAGGACTGCAACAAAAACTCTGACAAGTATTTCATGTGCTCAAATTTC





TTTAGTTGAAATTAAGACTGGTTTTAAGTTACGCTCAGCTGTGATGGGTG





ACAATCAGTGCATTACTGTTTTATCAGTCTTCCCCTTAGAGACTGACGCA





GACGGCAGGAACAGAGCGCCGAAGACAATGCAGCGAGGGTGGCCGCCAGC





CTAGCAAAAGTTACAAGTGCCTGTGGAATCTTTTTAAAACCTGATGAGAC





TTTCGTACACTCAGGTTTTATCTATTTTGGAAAAAACAATATTTGAATGG





GGTCCAATTGCCTCAGTCCCTTAAAACGGCTACAAGAATGGCACCATTGT





CTGATGCAATTTTTGATGATCTTCAAGGGACCCTGGCTAGTATAGGCACT





GCTTTTGAGCGATCAACTCCGAAACTAGACATATCTTTCCTTGCAGGATA





ACCGCAGCTTTCCATACGTTTTTTTCGGTGAGAATCTTGCAATATCATCA





TCTCGGGTTCAATAAAGGTTTTGACCTTGGACAGTTAACACTCGGCAACC





TCTGGATTTCGGAACAATATCATTGGCACTAGCGGTACCGCAGGTGCTTG





GAGGGTTATCCTTCTTGAATCCTGAGAAATGTTTCTACCGGAATCTAGGA





GATCCAGTTACCTCAGGCTTATTCCAGTAAAAACTTATCTCCGAATAGAG





ACCTATTGAGCTCCACCGCGGTGGCGGCCGCTCTAGCCCGGGCGGATCCC





CCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAG





GGGGGGCCCGTACCTTACATCGCGTTAATTAACTAGTGGATCGATCCCCA





ATTCG





L fragment 2: SEQ ID NO:1050 (L-DEFG):


CGCGACGTAATACGACTCACTATAGGGCGAATTGGGCGCGCCCCGTCTCA





GAATGATTGAGATGGATGATTTATTCTTACCTTTAATTGCGAAGAACCCT





GGGAATTGTAGCGCAATTGACTTTGTGTAAATCCTAGCGGATTAAATGTC





CCTGGGTCGCAAGACTTAACTTCATTTCTGCGCCAGATTGTACGCAGGAC





CATCACCCTAAGTGCGAAAAACAAACTTATTAATACCTTATTTCATGCGT





CAGCTGACTCGAAGACGAAATGGTTTGTAAATGGCTATTATCATCAACTC





CTGTTATGAGTCGTTTTGCGGCCGATATCTTTTCACGCACGCCGAGCGGG





AAGCGATTGCAAATTCTAGGATACCTGGAAGGAACACGCCATTATTAGCC





TCTAAGATCATCAACAATAATACAGAGACACCGGTTTTGCACAGACTGAG





GAAAATAACATTGCAAAGGTGGAGCCTATGGTTTAGTTATCTTGATCATT





GTGATAATATCCTGGCGGAGCTTTAACCCAAATAACTTGCACAGTTGATT





TAGCACAGATTCTGAGGGAATATTCATGGGCTCATATTTTAGAGGGAAGA





CCTCTTATTGGAGCCACACTCCCATGTATGATTGAGCAATTCAAAGTGTT





TGGCTGAAACCCTACGAACAATGTCCGCAGTGTTCAAATGCAAAGCAACC





AGGTGGGAAACCATTCGTGTCAGTGGCAGTCAAGAAACATATTGTTAGTG





CATGGCCGAACGCATCCCGAATAAGCTGGACTTCGGGGATGGAATCCCAT





ACATTGGATCAAGGACAGAAGATAAGATAGGTCAGCCCGCTATTAAGCCG





AGGTGTCCTTCCGCAGCCTTAAGAGAGGCCATTGAATTGGCGTCCCGTTT





AACATGGGTAACTAAGGCAGTTCGAACAGTGACTTGCTAATAAAACCATT





TTTGGAAGCACGAGTAAATTTAAGTGTTCAAGAAATACTTCAAATGACCC





CTTCACATTACTCAGGAAATATTGTGCATCGGTATAACGATCAAACAGTC





CTCATTCTTTCATGGCCAATCGTATGAGTAATTCAGCAACGCGCTTGATG





GTATCTACAAACACTTTAGGTGAGTTTTCAGGAGGTGGCCAGTCTGCACG





CGACAGCAATATTATTTTCCAGAATTTATAAATTATGCAGTTGCACTGTT





CGATATTAAATTTAGAAACACTGAGGCTACAGATATCCAATATAATCGTG





CTCACCTTCATCTAACTAAGTGTTGCACCCGGGAAGTACCAGCTCAGTAT





TTAACAACACAACCACGCTAAATCTAGATTTAACAAGATACCGAGAAAAC





GAATTGATTTATGACAGTAATCCTCTAAAAGGAGGACTCAATTGCAACTT





ATCGATTGACAGTCCTTTTTTCCAAGGTAAACGGCTGACATTATAGAAGA





TGATCTTATTCGACTGCCTCACTTATCTGGATGGGAGCTAGCCAAGACCA





TCATGCAATCAATTATTTCAGATAGCAACAATTCATCTACAGACCCAATT





AGCAGTGGAGAAACAAGACATTCACTACCCATTTCTTAACTTATCCCAAG





ATAGGACTTCTGTACAGTTTTGGGGCCTTTGTAAGTTATTATCTTGGCAA





TACAATTCTTTGCACGAAAAAGATCGGACTTGACAATTTTTTATATTACT





AACTACTCAAATTCATAATCTACCACATCGCTCATTGCGAATACTTAAGC





CAACATTCAAACATGCAAGCGTTATGTCACGGTTAATGAGTATTGATCCT





CATTTTTCTATTTACATAGGCGGTGCTGCAGTGACAGAGGACTCTCAGAT





GCGGCCAGGTTATTTTTGAGAACGTCCATTTCATCTTTTCTTACATTTGT





AAAAGAATGGATAATTAATCGCGGAACAATTGTCCCTTTATGGATAGTAT





ATCCGCTAGAGGTCAAAACCCAACACCTGTGAATAATTTTCTCTATCAGA





TCGTAGAACTGCTGGTGCATGATTCATCAAGACAACAGGCTTTTAAAACT





ACCATAAGTGATCATGTACATCCTCACGACAATCTTGTTTACCATGTAAG





AGTACAGCCAGCAATTTCTTCCATGCATCATTGGCGTACTGGAGGAGCAG





ACACAGAAACAGCAACCGAAAATACTTGGCAAGAGACTCTTCAACTGGAT





CAAGCACAAACAACAGTGATGGTATATTGAGAGAAGTCAAGAACAAACCA





CCAGAGATCCACATGATGGCACTGAACGGAATCTAGTCCTACAAATGAGC





CATGAAATAAAAAGAACGACAATTCCACAAGAAAACACGCACCAGGGTCC





GTCGTCCAGTCCTTTGTAAGTGACTCTGCTTGTGGTACAGCAAATCCAAA





ACTAAATTTCGATCGATCGAGACACAATGTGAAATTTCAGGATCATAACT





CGGCATCCAAGAGGGAAGGTCATCAAATAATCTCAACCGTCTAGTCCTAC





CTTTCTTTACATTATCTCAAGGGACACGCCAATTAACGTCATCCAATGAG





TCACAAACCCAAGACGAGATATCAAAGTACTTACGGCAATTGAGATCCGT





CATTGATACTACCATAATTGTCGCTTCACCGGTATAGTCTCGTCCATGCA





TTACAAACTTGATGAGGTCCTTTGGGAAATAGAGAGTTTCAAGTCGGCTG





TGACGCTAGCAGAGGGAGAAGGTGCTGGTGCCTTACTATTGATTCAAAAT





ACGGCGTTAAGAAGTTATTTTTCAACACGCTAGCTACTGAGTCCAGTATA





GAGTCAGAAATAGTATCAGGAATGACTACTCCTAGGATGCTTCTACCTGT





TATGTCAAAATTCCATAATGACCAAATTAGATTATTCTTAACAACTCAGC





AAGCCAAATAACAGACATAACAAATCCTACTTGGTTTAAAGACCAAAGAG





CAAGGCTACCTAAGCAAGTCGAGGTTATAACCATGGATGCAGAGACAACA





GAGAATATAACAGATCGAAATTGTACGAAGCTGTATATAAATTGATCTTA





CACCATATTGATCCTAGCGTATTGAAAGCAGTGGTCCTTAAAGTCTTTCT





AAGTGATACTGAGGGTATGTTATGGCTAAATGATAATTTACCCCGTTTTT





TGCCACTGGTTATTTAATTAAGCCAATAACGTCAAGTGCTAGATCTAGTG





AGTGGTATCTTTGTCTGACGAACTTCTTATCAACTACACGTAAGATGCCA





CACCAAAACCATCTCAGTTGTAACAGGTAATACTTACGGCATTGCAACTG





CAAATTCAACCAAGCCCATACTGGCTAAGTCATTTAACTCAGTATGCTGA





CTGTGAGTTACATTTAAGTTATATCCGCCTTGGTTTTCCATCATTAGAGA





AATACTATACCACAGGTATAACCTCGTCGATTCAAAAAGAGGTCCACTAG





TCTCTATCACTCAGCACTTAGCACATCTTAGAGCAGAGATTCGAGAATTA





ACTAATGATTATAATCAACAGCGACAAAGTCGACCCAGACTTATCATTTT





ATTCGTACTGCAAAAGGACGGATAACTAAACTAGTCAATGATTATTTAAA





ATTCTTTCTTATTGTGCAAGCATTAAAACATAATGGGACATGGCAAGCTG





AGTTTAAGAAATTACAGAGTTGATTAGTGTGTGCAATAGGTTCTACCATA





TTAGAGATTGCAATTGTGAAGAACGTTTCTTAGTTCAAACCTTATATTTA





CATAGAATGCAGGATTCTGAAGTTAAGCTTATCGAAAGGCTGACAGGCTT





CTGAGTTTATTTCCGGATGGTCTCTACAGGTTTGATTGAATTACCGTGCA





TAGTATCCTGATACTTGCAAAGGTTGGTTATTAACATACAGATTATAAAA





AAGCGGCCGCAGAGCTCCAGCGGTGGGGCCGCCGGCGTCTAGCCCGGGCG





GATCCCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAG





GGCCCATGCAGGCCGGCCAGGTACCTTAGTTAATTAACAGCTTTTGTTCC





CTTTAGTAGGGTTAATTGACGCGCTC





VP24 (SEQ ID NO:1045):


GGCGCGCCTCGAGGATGAAGATTAATGCGGAGGTCTGATAAGAATAAACC





TTATTATTCAGATTAGGCCCCAAGAGGCATTCTTCATCTCCTTTTAGCAA





AGTACTATTTCAGGGTAGTCCAATTAGTGGCACGTCTTTTAGCTGTATAT





CAGTCGCCCCTAGGCTAGGGTTTATAGTTGTCTCTAAGCTAAATTGGTCT





TGAACTAGTCTACTCGCAGAATCCTACCGGGAATAGACTAATTGAACTTA





GCCGTTTAAAATTTAGTGCATAAATCTGGGCTAACACCACCAGGTCAACT





CCATTGGCTGAAAAGAAGCTTACCTACAACGAACATCACTTTGAGCGCCC





TCACAATTAAAAAATAGGAACGTCGTTCCAACAATCGAGCGCAAGGTTTC





AATATTATACGGGTCCATTAATTTCAACAAAATATTGATACTCCAGACAC





CAAGCAAGACCTGAGAAAAAACCATGGCTAAAGCTACGGGACGATACAAT





CTAATATCGCCCAAAAAGGACCTGGAGAAAGGGGTTGTCTTAAGCGACCT





CTGTAACTTCTTAGTTAGCCAAACTATTCAGGGGTGGAAGGTTTATTGGG





CTGGTATTGAGTTTGATGTGAACCAAAAGGGTATTACCCTATTGCATAGA





CTGAAAACTAATGACTTTGCCCCTGCATGGTCAATGACAAGGAATCTCTT





TCCTCATTTATTTCAAAATCCGAATTCCACAATTGAATCACCGCTGTGGG





CATTGAGAGTCATCCTTGCAGCAGGGATACAGGACCAGCTGATTGACCAG





TCTTTGATTGAACCCTTAGCAGGAGCCCTTGGTCTGATCTCTGATTGGCT





GCTAACAACCAACACTAACCATTTCAACATGCGAACACAACGTGTCAAGG





AACAATTGAGCCTAAAAATGCTGTCGTTGATTCGATCCAATATTCTCAAG





TTTATTAACAAATTGGATGCTCTACATGTCGTGAACTACAACGGATTGTT





GAGCAGTATTGAAATTGGAACTCAAAATCATACAATCATCATAACTCGGA





CTAATATGGGTTATCTTGTCGAGCTCCAAGAACCCGACAAATCTGCGATG





GATATACGACACCCTGGGCCGGCGAAATTTTCCTTACTACATGAATCGAC





ACTTAAAGCATTTACACAAGGATCCTCGACACGAATGCAAAGTTTGATTC





TTGAATTTAATAGCTCTCTTGCTATCTAACTAAGGTAGAAAAAATTGTAC





GATAGGGCTAACATTATGCTGACTCAATAGTTATCTTGACATCTCTGCTT





TCATAATCAGATATATAAGCATAATAAATAAATACTCATATTTCTTGATA





ATTTGTTTAACCACAGATAAATCCTCACTGTAAGCCAGCTTCCAAGTTGA





CACCCTTACAAAAACCAGGACTCAGAATCCCTCAAACAAGAGATTCCAAG





ACAACATCATAGAATTGCTTTATTATATGAATAAGCATTTTATCACCAGA





AATCCTATATACTAAATGGTTAATTGTAACTGAACCCGCAGGTCACATGT





GTTAGGTTTCACAGATTCTATATATTACTAACTCTAGAGCCCAAATTAAC





ACGGTATAAGTAGATTAAGAAAAAAGCCTGAGGAAGATTAAGAAAAAGCG





GCCGCATTAATTAA





VP30 (SEQ ID NO:1046):


GGCGCGCCTCGAGGATGAAGATTAAGAAAAAGGTAATCTTTCGATTATCT





TTAATCTTCATCCTTGATTCTACAATCATGACAGTTGTCTTTAGTGACAA





GGGAAAGAAGCCTTTTTATTAAGTTGTAATAATCAGATCTGCGAACCGGT





AGAGTTTAGTTGCAACCTAACACACATAAAGCATTGGTCAAAAAGTCAAT





AGAAATTTAAACAGTGAGTGGAGACAACTTTTAAATGGAAGCTTCGTGAG





CGCGGGAGATCAAGGAATTCACGTGCCGACCAGCAAGGGATGGACACGAC





CACCATGTTCGAGCACGATCATCATCCAGAGAGAATTATCGAGGTGAGTA





CCGTCAATCAAGGAGCGCCTCACAAGTGCGCGTTCCTACTGTATTTCATA





AGAAGAGAGTTGAACCATTAACAGTTCCTCCAGCACCTAAAGACATATGT





CCGACCTTGAAAAAAGGATTTTTGTGTGACAGTAGTTTTTGCAAAAAAGA





TCACCAGCTTGAAAGCCTAACCGACCGGGAATTACTCCTACTAATCGCCC





GTAAGACTTGTGGATCAGTTGATTCATCGCTTAATATAACTGCACCCAAG





GACTCGCGCTTAGCAAATCCAACGGCTGATGATTTCCAGCAAGAGGAAGG





TCCAAAAAATTACTAGTCGAGACTGCTCAAGACGGCAGAACACTGGGCGA





GACAAGACATCAGAACCATAGAGGATTCAAAATTAAGAGCATTGTTGACT





CTATGTGCTGTGATGACGAGGAAATTCTCAAAATCCCAGCTGAGTCTTTT





ATGTGAGACACACCTAAGGCGCGAGGGGCTTGGGCAAGATCAGGCAGAAC





CCGTTCTCGAAGTATATCAACGATTACACAGTGATAAAGGAGGCAGTTTT





GAAGCTGCACTATGGCAACAATGGGACCGACAATCCCTAATTATGTTTAT





CACTGCATTCTTGAATATTGCTCTCCAGTTACCGTGTGAAAGTTCTGCTG





TCGTTGTTTCAGGCCTACGCTTACTTGCCCCCCCAAGCGTTAATGAAGAA





GCTTCAACCAACCCGGGGACATGCTCATGGTCTGATGAGGGTACCCCTTA





ATAAGGCTGACTAAAACACTATATAACCTTCTACTTGATCACAATACTCC





GTATACCTATCATCATATATTTAATCAAGACGATATCCTTTAAAACTTAT





TCAGTACTATAATCACTCTCGTTTCAAATTAATAAGATGTGCATGATTGC





CCTAATATATGAAGAGGTATGATACAACCCTAACAGTGATCAAAGAAAAT





CATAATCTCGTATCGCTCGTAATATAACCTGCCAAGCATACTCCCTAGAA





GCGTTGAATCTTGTACACAAATAATGTTTTACTCTACAGGAGGTAGCAAC





GATCCATCCCATCAAAAAATAAGTATTTCATGACTTACTAATGATCTCTT





AAAATATTAAGAAAAAGCGGCCGCATTAATTAA





VP35 (SEQ ID NO:1047):


GGTACCGCGATCGCGATGAAGATTAAAACCTTCATCATCCTTACGTCAAT





TGAATTCTCTAGCACTCGAAGCTTATTGTCTTCAATGTAAAAGAAAAGCT





GGTCTAACAAGATGACAACTAGAACAAAGGGCAGGGGCCATACTGCGGCC





ACGACTCAAAACGACAGAATGCCAGGCCCTGAGCTTTCGGGCTGGATCTC





TGAGCAGCTAATGACCGGCAAAATACCGCTAACCGACATCTTCTGTGATA





TTGAGAACAATCCAGGATTATGCTACGCATCCCAAATGCAACAAACGAAG





CCAAACCCGAAGACGCGCAACAGTCAAACCCAAACGGACCCAATTTGCAA





TCATAGTTTTGAGGAGGTAGTACAAACATTGGCTTCATTGGCTACAGCTG





TGCGTCGGCAAACCATCGCATCAGAATCATTAGAACAACGCATTACGAGT





CTTGAGAATGGTCTAAAGCCAGTTTATGATATGGCAAAAACAATATCATC





CCTGAATCGCAGCTGTGCTGAGATGGTTGCAAAATATGATCTTCTGGTGA





TGACAACCGGTCGGGCAACAGCAACCGCTGCGGCAACTGAGGCTTATTGG





GCCGAACATGGTCAACCACCACCAGGCCCATCATTGTACGAGGATGGTGC





GATTCGGGGTAAATTGAAAGATCCGAACGGGACCGTCCCTCAAAGTGTTA





GGGAGGCATTCAACAATCTAAACAGTACCACTTCACTAACTGAGGAAAAT





TTCGGGCGACCTTACATTTCGGCAAAGGATTTGAGAAACATTATGTATGA





TCACTTGCCTGGTTTTGGAACTGCTTTCCACCAATTAGTACAAGTGATTT





GTAAATTGGGAAAAGATAGCAACTCATTGGACATCATTCATGCTGAGTTC





CAGGCCAGCCTGGCTGAAGGAGACTCTCCTCAATGTGCCCTAATTCAAAT





TACAAAAAGAGTTCCAATCTTCCAAGATGCTGCTCCACCTGTCATCCACA





TCCGCTCTCGAGGTGACATTCCCCGAGCTTGCCAGAAAAGCTTGCGTCCA





GTCCCACCATCGCCCAAGATTGATCGAGGTTGGGTATGTGTTTTTCAGCT





TCAAGATGGTAAAACACTTGGACTCAAAATTTGAGCCAATGTAAGCTCAT





TTTGCGATGGGCGAATAATAGCAGAGGCTTCAACTGCTGAACTATAGGGT





ACGTTACATTAATGATACACTTGTGAGTATCAGCCCTGGATAATATAAGT





CAATCCTAATCAATTGATAATATTGTTCATATCTCGCTAGCAGCTTAAAA





TATAAATGTAATAGGAGCTATATCTCTGACAGTATTATAATCAATTGTTA





TTAAGTAACCCAAACCAAAAGTGATGAAGATTAAGAAAAAGCGGCCGCAG





AGCTC





VP40 (SEO ID NO:1048):


GGTACCTCGAGGATGAAGATTAAGAAAAACCTACCTCGGCTGAGAGAGTG





TTTTTTCATTAACCTTCATCTTGTAAACGTTGAGCAAAATTGTTAAAAAT





ATGAGGCGGGTTATATTGCCTACTGCTCCTCCTGAATATATGGAGGCCAT





ATACCCTGTCAGGTCAAATTCAACAATTGCTAGAGGTGGCAACAGCAATA





CAGGCTTCCTGACACCGGAGTCAGTCAATGGGGACACTCCATCGAATCCA





CTCAGGCCAATTGCCGATGACACCATCGACCATGCCAGCCACACACCAGG





CAGTGTGTCATCAGCATTCATCCTTGAAGCTATGGTGAATGTCATATCGG





GCCCCAAAGTGCTAATGAAGCAAATCCCTATTTGGTTGCCTCTAGGTGTC





GCTGATCAAAAGACCTACAGCTTTGACTCAACTACGGCCGCAATTATGCT





CGCATCTTATACGATCACCCATTTCGGCAAGGCAACCAACCCCCTCGTTA





GAGTGAATCGACTGGGTCCTGGAATCCCGGATCATCCCCTCAGGCTCCTG





CGAATTGGAAACCAGGCTTTCCTCCAGGAGTTCGTTCTTCCGCCAGTCCA





ACTACCCCAGTATTTCACCTTTGATTTGACAGCACTCAAACTGATCACCC





AACCACTGCCTGCTGCAACATGGACCGATGACACTCCAACAGGATCAAAT





GGAGCGTTGCGTCCAGGAATTTCATTTCATCCAAAACTTCGCCCCATTCT





TTTACCCAACAAAAGTGGGAAGAAGGGGAACAGTGCCGATCTAACATCTC





CGGAGAAAATCCAAGCAATAATGACTTCACTCCAGGACTTTAAGATCGTG





CCAATTGATCCAGCCAAGAGTATCATTGGGATCGAGGTGCCAGAAACTCT





GGTCCACAAGCTGACCGGTAAGAAGGTGACTTCTAAAAATGGACAACCAA





TCATCCCTGTTCTTTTGCCAAAGTACATTGGGTTGGACCCGGTGGCTCCA





GGAGACCTCACCATGGTAATCACACAGGATTGTGACACGTGTCATTCTCC





TGCAAGTCTTCCAGCTGTGATTGAGAAGTAATTGCAATAATTGACTCAGA





TCCAGTTTTATAGAATCTTCTCAGGGATAGCAACTCAATCGACTTTTAGG





ACCGTCCATTAGAGGAGACACTTTTAATTGAAAAATGTACTAATCGGGTC





AAGGACCATTGTCTTTTTTCTCTCCTAAATGTAGAACTTAACAAAAGACT





CATAATATACTTGTTTTTAAAGGATTGATTGATGAAAGAACATGCATAAG





CGATCCATACTTCGCCCTACTATAATCAATACGGTGATTCAAATGTTAAT





CTTTCTCATTGCACATACTTTTTGCCCTTATCCTCAAATTGCCTGCATGC





TTACATCTGAGGATAGCCAGTGTGACTTGGATTGGAAATGTGGAGAAAAA





ATCGGGACCCATTTCTAGGTTGTTCACAATCCAAGTACAGACATTGCCCT





TCTAATTAAGAAAAAAGCGGCCGCAGAGCTC






Other embodiments are in the claims.

Claims
  • 1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human Ebola genome in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding Ebola, and wherein said region of complementarity is less than 30 nucleotides in length.
  • 2. The dsRNA of claim 1, wherein said first sequence is selected from the group consisting of the sense sequences of Table 2 and said second sequence is selected from the group consisting of the antisense sequences of Table 2.
  • 3. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide.
  • 4. The dsRNA of claim 2, wherein said dsRNA comprises at least one modified nucleotide.
  • 5. The dsRNA of claims 3, wherein said 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.
  • 6. The dsRNA of claim 3, wherein said 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.
  • 7. The dsRNA of claims 3, wherein said first sequence is selected from the group consisting of the sense sequences of Table 2 and said second sequence is selected from the group consisting of the antisense sequences of Table 2.
  • 8. The dsRNA of claim 6, wherein said first sequence is selected from the group consisting of the sense sequences of Table 2 and said second sequence is selected from the group consisting of the antisense sequences of Table 2.
  • 9. A cell comprising the dsRNA of claim 1.
  • 10. A pharmaceutical composition for inhibiting the expression of a gene from an Ebola virus in an organism, comprising a dsRNA of claim 1 and a pharmaceutically acceptable carrier.
  • 11. The pharmaceutical composition of claim 10, wherein said first sequence of said dsRNA is selected from the group consisting of the sense sequences of Table 2 and said second sequence is selected from the group consisting of the antisense sequences of Table 2.
  • 12. The-pharmaceutical composition of claim 10, wherein said first sequence of said dsRNA consists of the sequence of SEQ ID NO: 1029, and said second sequence consists of the sequence of SEQ ID NO: 1030.
  • 13. A method for inhibiting the expression of a gene from an Ebola virus in a cell, the method comprising: (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA) of claim 1; and(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a gene from the Ebola virus, thereby inhibiting expression of a gene from the Ebola virus in the cell.
  • 14. A method of treating, preventing or managing pathological processes mediated by Ebola expression comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA of claim 1.
  • 15. A vector for inhibiting the expression of a gene from the Ebola virus in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes the dsRNA of claim 1.
  • 16. A cell comprising the vector of claim 15.
  • 17. The dsRNA of claim 1, wherein the dsRNA targets the VP35 of Ebola.
  • 18. The dsRNA of claim 1, wherein the dsRNA has a sense strand consisting of the sequence of SEQ ID NO:1029, and antisense strand consisting of the sequence of SEQ ID NO:1030.
  • 19. The dsRNA of claim 1, wherein said dsRNA, upon contact with a cell infected with Ebola virus, inhibits expression of a gene from the virus by at least 40%.
  • 20. The dsRNA of claim 1, wherein said region of complementarity is 15-30 nucleotides in length.
  • 21. The dsRNA of claim 1, wherein said region of complementarity is 19 and 24 nucleotides in length.
  • 22. The vector of claim 15, wherein said dsRNA, upon contact with a cell infected with Ebola virus, inhibits expression of a gene from the virus by at least 40%.
  • 23. The vector of claim 15, wherein said dsRNA is 15-30 base pairs in length.
  • 24. The vector of claim 15, wherein said dsRNA is 19-24 base pairs in length.
  • 25. A method of increasing life-span of a subject infected with an Ebola virus, comprising administering to the subject a dsRNA of claim 1 in an amount sufficient to increase the life-span of the subject.
  • 26. A method of decreasing viral titre in a subject infected with an Ebola virus, comprising administering to the subject a dsRNA of claim 1 in an amount sufficient to decrease viral titre in the subject.
  • 27. A method of sustaining platelet count in a subject infected with an Ebola virus, comprising administering to the subject a dsRNA of claim 1 in an amount sufficient to sustain platelet count.
  • 28. The method of claim 27, wherein the lymphocyte count of the subject is also sustained.
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser. No. 60/908,793, filed Mar. 29, 2007. The entire contents of this provisional application are hereby incorporated by reference in the present application.

GOVERNMENT SUPPORT

The work described herein was carried out, at least in part, using funds from the United States government under contract number HHSN266200600012C, ADB N01-AI-60012, from the National Institute of Allergy and Infectious Diseases/National Institutes of Health/Department of Health and Human Services (NIAID/NIH/DHHS). The government may therefore have certain rights in the invention.

Provisional Applications (1)
Number Date Country
60908793 Mar 2007 US