# Patent

## Title
Lipid formulated compositions and methods for inhibiting expression of a gene from the ebola virus

## Abstract

The invention relates to lipid formulated double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a gene from the Ebola virus.

## Background
RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application 61/234,236, filed Aug. 14, 2009, U.S. provisional application 61/242,732, filed Sep. 15, 2009, and U.S. provisional application 61/259,106, filed Nov. 6, 2009, each of which is hereby incorporated by reference for all purposes.


GOVERNMENT SUPPORT
This invention was made with government support under contract number HHSN266200600012C, ADB N01-AI-60012, awarded by the National Institute of Allergy and Infectious Diseases/National Institutes of Health/Department of Health and Human Services (NIAID/NIH/DHHS). The government has certain rights in the invention.


REFERENCE TO A SEQUENCE LISTING
This application includes a Sequence Listing submitted electronically as a text file named 17150PCT_sequencelisting.txt, created on Aug. 27, 2010, with a size of 386,645 bytes. The sequence listing is incorporated by reference.
FIELD OF THE INVENTION
This invention relates to lipid formulated 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 a pharmaceutical composition including a double-stranded ribonucleic acid (dsRNA) and a lipid formulation, as well as methods for inhibiting the expression of the Ebola virus in a cell, mammal, or organism using such pharmaceutical composition. 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 pharmaceutical composition featured in the invention includes an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 15-30 or 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 a pharmaceutical composition including 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 15-30 or 19-24 nucleotides in length. The pharmaceutical composition, upon contact with a cell infected with the Ebola virus, inhibits the expression of a gene from the Ebola virus by at least 40% compared to a control.
For example, the pharmaceutical compositions 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 other embodiments, the first sequence of the dsRNA consists of the sequence of SEQ ID NO:1027, and the second sequence consists of the sequence of SEQ ID NO:1028.
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 lipid formulation. In a related embodiment, the lipid formulation includes a cationic lipid of formula A:




where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In an embodiment, R1 and R2 of formula A are independently selected from oleoyl, pamitoyl, steroyl, or linoleyl. In another embodiment, R1 and R2 of formula A are both linoleyl. In another embodiment, R3 and R4 of formula A are methyl. In an embodiment, the cationic lipid of formula A is 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane In an embodiment, the pharmaceutical composition can include a neutral lipid, a sterol, a PEG, or a PEG-modified lipid.

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 pharmaceutical composition including a double-stranded ribonucleic acid (dsRNA) and a lipid formulation, 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 15-30 or 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% compared to a control, 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 pharmaceutical compositions of the invention. In certain related embodiments, the pharmaceutical composition is administered to said subject prior to infection with Ebola virus. In other embodiments, the pharmaceutical composition is administered to said subject after infection with Ebola virus. In yet other embodiments, the pharmaceutical composition is administered in a dose of about 1 to 5 mgs/kg, or about 2 to 3 mgs/kg (mgs/kg refers to mgs of siRNA per kg of subject weight). In still other embodiments, the pharmaceutical composition is administered to said subject by parenteral administration, e.g., intraperitoneally. In still other embodiments, the pharmaceutical composition is administered on a daily or semi-daily (e.g., bi-daily) basis for 7 to 10 days. In still other embodiments, the administration of said pharmaceutical composition reduces weight loss in said subject relative to the weight loss that would be observed after administration of a control composition or another treatment that did not include said pharmaceutical composition.
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 pharmaceutical composition including a dsRNA and a lipid formulation to the subject, where the dsRNA includes an antisense RNA strand having a region which is less than 30 nucleotides in length, generally 15-30 or 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 pharmaceutical composition is administered in an amount sufficient to increase the lifespan of the subject. In one embodiment, the pharmaceutical composition 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 one embodiment, the pharmaceutical composition 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 lymphocyte count of the subject is sustained. 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 pharmaceutical composition including a dsRNA and a lipid formulation to the subject, where the dsRNA includes an antisense RNA strand having a region which is less than 30 nucleotides in length, generally 15-30 or 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 lymphocyte count of the subject is sustained. 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 pharmaceutical composition including a dsRNA and a lipid formulation to the subject, where the dsRNA includes an antisense RNA strand having a region which is less than 30 nucleotides in length, generally 15-30 or 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.
FIG. 9 shows the survival of guinea pigs treated with DODMA or LNP09 formulated siRNA or saline control.
FIG. 10 shows the weight loss of guinea pigs treated with DODMA or LNP09 formulated siRNA or saline control as measured against a percentage of the guinea pig's starting weight.
FIG. 11 shows the survival of guinea pigs treated with LNP09 formulated siRNA versus controls, administered according to the indicated dosage regimens.
FIG. 12 shows the weight loss of guinea pigs treated with LNP09 formulated siRNA versus controls, administered according to the indicated dosage regimens.
FIG. 13 shows the survival of guinea pigs treated with Formulation M formulated siRNA versus controls, administered according to the indicated dosage regimen.
FIG. 14 shows the weight loss of guinea pigs treated with Formulation M formulated siRNA versus controls, administered according to the indicated dosage regimen.
FIG. 15 shows the survival of guinea pigs treated with different lipid formulations of siRNA via different administration routes, administered according to the indicated dosage regimen.
FIG. 16 shows the weight loss of guinea pigs treated with different lipid formulations of siRNA via different administration routes, administered according to the indicated dosage regimen. The P/Q treated group includes 3 animals treated with AD-1955 E/F formulation on day 0 and AD-11570 P/Q formulation on days 1, 2, and 4.
FIG. 17 shows the survival of guinea pigs treated with different dosages of siRNA, administered according to the indicated dosage regimen.
FIG. 18 shows the weight change of guinea pigs treated with different dosages of siRNA, administered according to the indicated dosage regimen.
FIG. 19 shows the survival of guinea pigs treated with different formulations of siRNA, administered according to the indicated dosage regimen.
FIG. 20 shows the weight change of guinea pigs treated with different formulations of siRNA, administered according to the indicated dosage regimen.
FIG. 21 shows the survival of guinea pigs treated with siRNA post-Ebola challenge, administered according to the indicated dosage regimen.
FIG. 22 shows the disease index of guinea pigs treated with siRNA post-Ebola challenge, administered according to the indicated dosage regimen. 0=Healthy; no clinical signs of disease, animal active and responsive. 1=Slightly ruffled fur, reduced mobility. 2=Severely reduced mobility, hunched posture, ruffled fur, reduced responsiveness. 3=Moribund; Unresponsive, non-mobile, labored breathing. 4=Dead.
FIG. 23 shows the weight change of guinea pigs treated with siRNA post-Ebola challenge, administered according to the indicated dosage regimen.
FIG. 24 shows the viremia in guinea pigs treated with siRNA or control, administered according to the indicated dose regimen. Serum titer of Ebola virus was quantified by RT-qPCR. Treatment with AD-11570 specific siRNA in a lipid D formulation results in a dramatic suppression of Ebola virus titer.



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. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” or “deoxyribonucleotide” 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 Virology, 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. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.
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) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a 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. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.
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,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like, 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 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




  
    
      
        
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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 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 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)
As described in more detail herein, 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 of the invention can further include one or more single-stranded nucleotide overhangs.
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. 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, or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ.
Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is 25-30 nucletoides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ.
The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand.
A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In one embodiment, a gene from the Ebola virus is the from human Ebola genome. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences of Table 2 or Table 5 and the antisense strand is one of the antisense sequences of Table 2 or Table 5. Alternative antisense agents that target elsewhere in the target sequences provided in Table 2 or Table 5 can readily be determined using the target sequence and the flanking Ebola sequence.
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 or Table 5, the dsRNAs of the invention can comprise at least one strand of a length of described therein. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Table 2 or Table 5 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, 21, or 22 or more contiguous nucleotides from one of the sequences of Table 2 or Table 5, and differing in their ability to inhibit the expression of a gene from the Ebola virus in an 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 or Table 5 can readily be made using the Ebola virus sequence and the target sequence provided.
In addition, the dsRNA agents provided in Table 2 or Table 5 identify a site in the Ebola virus mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes dsRNA agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second dsRNA agent is said to target within the sequence of a first or Table 5 agent if the second or Table 5 agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first or Table 5 agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 2 or Table 5 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.
Modifications
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 of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
Other preferred modifications include 2′-methoxy(2′-OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N2, 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 bases 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.
Conjugates
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.
Synthesis
Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2′-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.
In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In one embodiment, the oligonucleotides or linked nucleosides featured in the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of an oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group. A summary listing of some of the oligonucleotide modifications known in the art is found at, for example, PCT Publication WO 200370918.
In some embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5′-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5′-position through a linking group. The amino group at the 5′-terminus can be prepared utilizing a 5′-Amino-Modifier C6 reagent. In one embodiment, ligand molecules may be conjugated to oligonucleotides at the 5′-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5′-hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.
Examples of modified internucleoside linkages or 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 United States patents relating to the preparation of the above phosphorus-atom-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 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; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference.
Examples of modified internucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar 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 United States patents relating to 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; 5,264,562; 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; 5,610,289; 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 certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, 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 oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides 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 oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate. The use of a cholesterol conjugate is particularly preferred since such a moiety can increase targeting liver cells, a site of target expression.
Vector Encoded dsRNA
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
In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a target gene, such as pathological processes mediated by target gene expression. Such pharmaceutical compositions are formulated based on the mode of delivery. In an embodiment, the invention provides pharmaceutical compositions having an agent, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same. In an embodiment, the invention provides pharmaceutical compositions having a dsRNA and an agent, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same.
Dosage
The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of target genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.
In another embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg.
In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. For example, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.
The dsRNA can be administered at a dose of 0.03, 0.1, 0.3, or 1.3, or 3.0 mg/kg.
The pharmaceutical composition can be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on target mRNA levels is long lasting, such that subsequent doses are administered at not more than 7 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
In one embodiment the lipid formulated mRNA targeted dsRNA is administered at a first dose of about 3 mg/kg followed by administering at least one subsequent dose once a week, wherein the subsequent dose is lower than the first dose, e.g., the subsequent dose is about 1.0 mg/kg or about 0.3 mg/kg. The subsequent dose can be administered, e.g., once a week for four weeks. In some embodiments the dsRNA is administered using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by target gene expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing a human target gene. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses a human target gene.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately to determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Administration
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, and subdermal, oral or parenteral, e.g., subcutaneous.
For example, when treating a mammal with hyperlipidemia, the dsRNA molecules are administered systemically via parental means. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. For example, dsRNAs, conjugated or unconjugated or formulated with or without liposomes, can be administered intravenously to a patient. For such, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers). Formulations are described in more detail herein.
The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
Formulations
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. In one aspect are formulations that target the liver when treating hepatic disorders such as hyperlipidemia.
In addition, dsRNA that target the target gene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the target gene can contain other therapeutic agents, such as other cancer therapeutics or one or more dsRNA compounds that target other target genes.
Oral, parenteral, topical, and biologic formulations
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. dsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Publication. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). dsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto. Alternatively, dsRNAs may be complexed to lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference. In addition, dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine. Numerous lipophilic agents are commercially available, including LipofectinTM (Invitrogen/Life Technologies, Carlsbad, Calif.) and EffecteneTM (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am. Soc. Nephrol. 7: 1728 (1996)). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat Biotechnol. 23(8):1002-7.
Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpes virus vectors) can be used to deliver dsRNA molecules to liver cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.
Liposomal Formulations
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; and liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al., S.T.P. Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al.).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
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).
Nucleic Acid Lipid Particles
In one embodiment, a dsRNA featured in the invention (e.g., a dsRNA targeting a gene of interest or, e.g., an agent such as a dsRNA targeting an Ebola gene) is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle. Nucleic acid-lipid particles typically contain a cationic lipid, a non-cationic lipid, a sterol, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). Nucleic acid-lipid particles are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
Nucleic acid-lipid particles can further include one or more additional lipids and/or other components such as cholesterol. Other lipids may be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation or to attach ligands onto the liposome surface. Any of a number of lipids may be present, including amphipathic, neutral, cationic, and anionic lipids. Such lipids can be used alone or in combination. Specific examples of additional lipid components that may be present are described herein.
Additional components that may be present in a nucleic acid-lipid particle include bilayer stabilizing components such as polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins, detergents, lipid-derivatives, such as PEG coupled to phosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat. No. 5,885,613).
A nucleic acid-lipid particle can include one or more of a second amino lipid or cationic lipid, a neutral lipid, a sterol, and a lipid selected to reduce aggregation of lipid particles during formation, which may result from steric stabilization of particles which prevents charge-induced aggregation during formation.
Nucleic acid-lipid particles include, e.g., a SPLP, pSPLP, and SNALP. The term“SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. The term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683.
The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1, or about 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, or 33:1.
Cationic Lipids
Cationic lipids can include ionizable cationic lipids and non-ionizable cationic lipids. A cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALNY-100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3), or a mixture thereof.
Other cationic lipids, which carry a net positive charge at about physiological pH, in addition to those specifically described above, may also be included in lipid particles of the invention. Such cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”); 3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”), N-(1-[2,3-d]oleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”), 1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL). In particular embodiments, a cationic lipid is an amino lipid.
As used herein, the term “amino lipid” is meant to include those lipids having one or two fatty acid or fatty alkyl chains and an amino head group (including an alkylamino or dialkylamino group) that may be protonated to form a cationic lipid at physiological pH.
Other amino lipids would include those having alternative fatty acid groups and other dialkylamino groups, including those in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, N-propyl-N-ethylamino- and the like). For those embodiments in which R11 and R12 are both long chain alkyl or acyl groups, they can be the same or different. In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 are preferred. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid. Suitable scaffolds are known to those of skill in the art.
In certain embodiments, amino or cationic lipids of the invention have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwiterrionic, are not excluded from use in the invention.
In certain embodiments, protonatable lipids according to the invention have a pKa of the protonatable group in the range of about 4 to about 11. Most preferred is pKa of about 4 to about 7, because these lipids will be cationic at a lower pH formulation stage, while particles will be largely (though not completely) surface neutralized at physiological pH around pH 7.4. One of the benefits of this pKa is that at least some nucleic acid associated with the outside surface of the particle will lose its electrostatic interaction at physiological pH and be removed by simple dialysis; thus greatly reducing the particle's susceptibility to clearance.
One example of a cationic lipid is 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA). Synthesis and preparation of nucleic acid-lipid particles including DlinDMA is described in International application number PCT/CA2009/00496, filed Apr. 15, 2009.
In one embodiment, the cationic lipid XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) is used to prepare nucleic acid-lipid particles. Synthesis of XTC is described, e.g., in PCT/US10/22614 filed on Jan. 29, 2010, which is hereby incorporated by reference.
In another embodiment, the cationic lipid MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), (e.g., DLin-M-C3-DMA) is used to prepare nucleic acid-lipid particles. Synthesis of MC3 and MC3 comprising formulations are described, e.g., in U.S. Ser. No. 12/813,448, filed Jun. 10, 2010, which is hereby incorporated by reference.
In another embodiment, the cationic lipid ALNY-100 ((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine) is used to prepare nucleic acid-lipid particles. Synthesis of ALNY-100 is described in International patent application number PCT/US09/63933 filed on Nov. 10, 2009, which is herein incorporated by reference.
The cationic lipid may comprise from about 20 mol % to about 70 mol % or about 45-65 mol % or about 40 mol % of the total lipid present in the particle.
Non-Cationic Lipids
The nucleic acid-lipid particles of the invention can include a non-cationic lipid. The non-cationic lipid may be an anionic lipid or a neutral lipid. Examples include but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.
Anionic lipids suitable for use in lipid particles of the invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
Neutral lipids, when present in the lipid particle, can be any of a number of lipid species which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., liposome size and stability of the liposomes in the bloodstream. Preferably, the neutral lipid component is a lipid having two acyl groups, (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In one group of embodiments, lipids containing saturated fatty acids with carbon chain lengths in the range of C14 to C22 are preferred. In another group of embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths in the range of C14 to C22 are used. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. Preferably, the neutral lipids used in the invention are DOPE, DSPC, POPC, or any related phosphatidylcholine. The neutral lipids useful in the invention may also be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.
In one embodiment the non-cationic lipid is distearoylphosphatidylcholine (DSPC). In another embodiment the non-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).
The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 5 mol % to about 10 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
Conjugated Lipids
Conjugated lipids can be used in nucleic acid-lipid particle to prevent aggregation, including polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No. 6,320,017). Other compounds with uncharged, hydrophilic, steric-barrier moieties, which prevent aggregation during formulation, like PEG, Gml or ATTA, can also be coupled to lipids for use as in the methods and compositions of the invention. ATTA-lipids are described, e.g., in U.S. Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Pat. Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, the concentration of the lipid component selected to reduce aggregation is about 1 to 15% (by mole percent of lipids).
Specific examples of PEG-modified lipids (or lipid-polyoxyethylene conjugates) that are useful in the invention can have a variety of “anchoring” lipid portions to secure the PEG portion to the surface of the lipid vesicle. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which are described in co-pending U.S. Ser. No. 08/486,214, incorporated herein by reference, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modified diacylglycerols and dialkylglycerols.
In embodiments where a sterically-large moiety such as PEG or ATTA are conjugated to a lipid anchor, the selection of the lipid anchor depends on what type of association the conjugate is to have with the lipid particle. It is well known that mePEG (mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remain associated with a liposome until the particle is cleared from the circulation, possibly a matter of days. Other conjugates, such as PEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidly exchanges out of the formulation upon exposure to serum, with a T1/2 less than 60 mins. in some assays. As illustrated in U.S. patent application Ser. No. 08/486,214, at least three characteristics influence the rate of exchange: length of acyl chain, saturation of acyl chain, and size of the steric-barrier head group. Compounds having suitable variations of these features may be useful for the invention. For some therapeutic applications, it may be preferable for the PEG-modified lipid to be rapidly lost from the nucleic acid-lipid particle in vivo and hence the PEG-modified lipid will possess relatively short lipid anchors. In other therapeutic applications, it may be preferable for the nucleic acid-lipid particle to exhibit a longer plasma circulation lifetime and hence the PEG-modified lipid will possess relatively longer lipid anchors. Exemplary lipid anchors include those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
It should be noted that aggregation preventing compounds do not necessarily require lipid conjugation to function properly. Free PEG or free ATTA in solution may be sufficient to prevent aggregation. If the particles are stable after formulation, the PEG or ATTA can be dialyzed away before administration to a subject.
The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). Additional conjugated lipids include polyethylene glycol-didimyristoyl glycerol (C14-PEG or PEG-C14, where PEG has an average molecular weight of 2000 Da) (PEG-DMG); (R)-2,3-bis(octadecyloxy)propyl1-(methoxy poly(ethylene glycol)2000)propylcarbamate) (PEG-DSG); PEG-carbamoyl-1,2-dimyristyloxypropylamine, in which PEG has an average molecular weight of 2000 Da (PEG-cDMA); N-Acetylgalactosamine-((R)-2,3-bis(octadecyloxy)propyl1-(methoxy poly(ethylene glycol)2000)propylcarbamate)) (GalNAc-PEG-DSG); and polyethylene glycol-dipalmitoylglycerol (PEG-DPG).
In one embodiment the conjugated lipid is PEG-DMG. In another embodiment the conjugated lipid is PEG-cDMA. In still another embodiment the conjugated lipid is PEG-DPG. Alternatively the conjugated lipid is GalNAc-PEG-DSG.
The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 0.5 to about 5.0 mol % or about 2 mol % of the total lipid present in the particle.
The sterol component of the lipid mixture, when present, can be any of those sterols conventionally used in the field of liposome, lipid vesicle or lipid particle preparation. A preferred sterol is cholesterol.
In some embodiments, the nucleic acid-lipid particle further includes a sterol, e.g., a cholesterol at, e.g., about 10 mol % to about 60 mol % or about 25 to about 40 mol % or about 48 mol % of the total lipid present in the particle.
Lipoproteins
In one embodiment, the formulations of the invention further comprise an apolipoprotein. As used herein, the term “apolipoprotein” or “lipoprotein” refers to apolipoproteins known to those of skill in the art and variants and fragments thereof and to apolipoprotein agonists, analogues or fragments thereof described below.
Suitable apolipoproteins include, but are not limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V and ApoE, and active polymorphic forms, isoforms, variants and mutants as well as fragments or truncated forms thereof. In certain embodiments, the apolipoprotein is a thiol containing apolipoprotein. “Thiol containing apolipoprotein” refers to an apolipoprotein, variant, fragment or isoform that contains at least one cysteine residue. The most common thiol containing apolipoproteins are ApoA-I Milano (ApoA-IM) and ApoA-I Paris (ApoA-IP) which contain one cysteine residue (Jia et al., 2002, Biochem. Biophys. Res. Comm. 297: 206-13; Bielicki and Oda, 2002, Biochemistry 41: 2089-96). ApoA-II, ApoE2 and ApoE3 are also thiol containing apolipoproteins. Isolated ApoE and/or active fragments and polypeptide analogues thereof, including recombinantly produced forms thereof, are described in U.S. Pat. Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189; 5,168,045; 5,116,739; the disclosures of which are herein incorporated by reference. ApoE3 is disclosed in Weisgraber, et al., “Human E apoprotein heterogeneity: cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms,” J. Biol. Chem. (1981) 256: 9077-9083; and Rall, et al., “Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects,” Proc. Nat. Acad. Sci. (1982) 79: 4696-4700. (See also GenBank accession number K00396.)
In certain embodiments, the apolipoprotein can be in its mature form, in its preproapolipoprotein form or in its proapolipoprotein form. Homo- and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-I Milano (Klon et al., 2000, Biophys. J. 79:(3)1679-87; Franceschini et al., 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al., 1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem. 259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem. 201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem. 258(14):8993-9000) can also be utilized within the scope of the invention.
In certain embodiments, the apolipoprotein can be a fragment, variant or isoform of the apolipoprotein. The term “fragment” refers to any apolipoprotein having an amino acid sequence shorter than that of a native apolipoprotein and which fragment retains the activity of native apolipoprotein, including lipid binding properties. By “variant” is meant substitutions or alterations in the amino acid sequences of the apolipoprotein, which substitutions or alterations, e.g., additions and deletions of amino acid residues, do not abolish the activity of native apolipoprotein, including lipid binding properties. Thus, a variant can comprise a protein or peptide having a substantially identical amino acid sequence to a native apolipoprotein provided herein in which one or more amino acid residues have been conservatively substituted with chemically similar amino acids. Examples of conservative substitutions include the substitution of at least one hydrophobic residue such as isoleucine, valine, leucine or methionine for another. Likewise, the present invention contemplates, for example, the substitution of at least one hydrophilic residue such as, for example, between arginine and lysine, between glutamine and asparagine, and between glycine and serine (see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term “isoform” refers to a protein having the same, greater or partial function and similar, identical or partial sequence, and may or may not be the product of the same gene and usually tissue specific (see Weisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol. 18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sacre et al., 2003, FEBS Lett. 540 (1-3):181-7; Weers, et al., 2003, Biophys. Chem. 100 (1-3):481-92; Gong et al., 2002, J. Biol. Chem. 277(33):29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23):14888-93 and U.S. Pat. No. 6,372,886).
In certain embodiments, the methods and compositions of the present invention include the use of a chimeric construction of an apolipoprotein. For example, a chimeric construction of an apolipoprotein can be comprised of an apolipoprotein domain with high lipid binding capacity associated with an apolipoprotein domain containing ischemia reperfusion protective properties. A chimeric construction of an apolipoprotein can be a construction that includes separate regions within an apolipoprotein (i.e., homologous construction) or a chimeric construction can be a construction that includes separate regions between different apolipoproteins (i.e., heterologous constructions). Compositions comprising a chimeric construction can also include segments that are apolipoprotein variants or segments designed to have a specific character (e.g., lipid binding, receptor binding, enzymatic, enzyme activating, antioxidant or reduction-oxidation property) (see Weisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sorenson et al., 1999, Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996, Arterioscler. Throb. Vasc. Biol. 16(2):328-38: Thurberg et al., J. Biol. Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill 1998, J. Biol. Chem. 273(47):30979-84).
Apolipoproteins utilized in the invention also include recombinant, synthetic, semi-synthetic or purified apolipoproteins. Methods for obtaining apolipoproteins or equivalents thereof, utilized by the invention are well-known in the art. For example, apolipoproteins can be separated from plasma or natural products by, for example, density gradient centrifugation or immunoaffinity chromatography, or produced synthetically, semi-synthetically or using recombinant DNA techniques known to those of the art (see, e.g., Mulugeta et al., 1998, J. Chromatogr. 798 (1-2): 83-90; Chung et al., 1980, J. Lipid Res. 21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson, et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528, 5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 and WO 87/02062).
Apolipoproteins utilized in the invention further include apolipoprotein agonists such as peptides and peptide analogues that mimic the activity of ApoA-I, ApoA-I Milano (ApoA-IM), ApoA-I Paris (ApoA-IP), ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be any of those described in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166, and 5,840,688, the contents of which are incorporated herein by reference in their entireties.
Apolipoprotein agonist peptides or peptide analogues can be synthesized or manufactured using any technique for peptide synthesis known in the art including, e.g., the techniques described in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166. For example, the peptides may be prepared using the solid-phase synthetic technique initially described by Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptide synthesis techniques may be found in Bodanszky et al., Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) and other references readily available to those skilled in the art. A summary of polypeptide synthesis techniques can be found in Stuart and Young, Solid Phase Peptide. Synthesis, Pierce Chemical Company, Rockford, Ill., (1984). Peptides may also be synthesized by solution methods as described in The Proteins, Vol. II, 3d Ed., Neurath et al., Eds., p. 105-237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in different peptide syntheses are described in the above-mentioned texts as well as in McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptides of the present invention might also be prepared by chemical or enzymatic cleavage from larger portions of, for example, apolipoprotein A-I.
In certain embodiments, the apolipoprotein can be a mixture of apolipoproteins. In one embodiment, the apolipoprotein can be a homogeneous mixture, that is, a single type of apolipoprotein. In another embodiment, the apolipoprotein can be a heterogeneous mixture of apolipoproteins, that is, a mixture of two or more different apolipoproteins. Embodiments of heterogenous mixtures of apolipoproteins can comprise, for example, a mixture of an apolipoprotein from an animal source and an apolipoprotein from a semi-synthetic source. In certain embodiments, a heterogenous mixture can comprise, for example, a mixture of ApoA-I and ApoA-I Milano. In certain embodiments, a heterogeneous mixture can comprise, for example, a mixture of ApoA-I Milano and ApoA-I Paris. Suitable mixtures for use in the methods and compositions of the invention will be apparent to one of skill in the art.
If the apolipoprotein is obtained from natural sources, it can be obtained from a plant or animal source. If the apolipoprotein is obtained from an animal source, the apolipoprotein can be from any species. In certain embodiments, the apolipoprotien can be obtained from an animal source. In certain embodiments, the apolipoprotein can be obtained from a human source. In preferred embodiments of the invention, the apolipoprotein is derived from the same species as the individual to which the apolipoprotein is administered.
Other Components
In numerous embodiments, amphipathic lipids are included in lipid particles of the invention. “Amphipathic lipids” refer to any suitable material, wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, can also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
Also suitable for inclusion in the lipid particles of the invention are programmable fusion lipids. Such lipid particles have little tendency to fuse with cell membranes and deliver their payload until a given signal event occurs. This allows the lipid particle to distribute more evenly after injection into an organism or disease site before it starts fusing with cells. The signal event can be, for example, a change in pH, temperature, ionic environment, or time. In the latter case, a fusion delaying or “cloaking” component, such as an ATTA-lipid conjugate or a PEG-lipid conjugate, can simply exchange out of the lipid particle membrane over time. Exemplary lipid anchors include those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
A lipid particle conjugated to a nucleic acid agent can also include a targeting moiety, e.g., a targeting moiety that is specific to a cell type or tissue. Targeting of lipid particles using a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies, has been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044). The targeting moieties can include the entire protein or fragments thereof. Targeting mechanisms generally require that the targeting agents be positioned on the surface of the lipid particle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting agents and methods are known and available in the art, including those described, e.g., in Sapra, P. and Allen, T M, Prog. Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res. 12:1-3, (2002).
The use of lipid particles, i.e., liposomes, with a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains, for targeting has been proposed (Allen, et al., Biochimica et Biophysica Acta 1237: 99-108 (1995); DeFrees, et al., Journal of the American Chemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica et Biophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal of Liposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky, Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353: 71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press, Boca Raton Fla. (1995). In one approach, a ligand, such as an antibody, for targeting the lipid particle is linked to the polar head group of lipids forming the lipid particle. In another approach, the targeting ligand is attached to the distal ends of the PEG chains forming the hydrophilic polymer coating (Klibanov, et al., Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBS Letters 388: 115-118 (1996)).
Standard methods for coupling the target agents can be used. For example, phosphatidylethanolamine, which can be activated for attachment of target agents, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used. Antibody-targeted liposomes can be constructed using, for instance, liposomes that incorporate protein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990) and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990). Other examples of antibody conjugation are disclosed in U.S. Pat. No. 6,027,726, the teachings of which are incorporated herein by reference. Examples of targeting moieties can also include other proteins, specific to cellular components, including antigens associated with neoplasms or tumors. Proteins used as targeting moieties can be attached to the liposomes via covalent bonds (see, Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)). Other targeting methods include the biotin-avidin system.
Production of Nucleic Acid-Lipid Particles
In one embodiment, the nucleic acid-lipid particle formulations of the invention are produced via an extrusion method or an in-line mixing method.
The extrusion method (also refer to as preformed method or batch process) is a method where the empty liposomes (i.e. no nucleic acid) are prepared first, followed by the addition of nucleic acid to the empty liposome. Extrusion of liposome compositions through a small-pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome complex size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size. In some instances, the lipid-nucleic acid compositions which are formed can be used without any sizing. These methods are disclosed in the U.S. Pat. No. 5,008,050; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,737,323; Biochim Biophys Acta. 1979 Oct. 19; 557(1):9-23; Biochim Biophys Acta. 1980 Oct. 2; 601(3):559-7; Biochim Biophys Acta. 1986 Jun. 13; 858(1):161-8; and Biochim. Biophys. Acta 1985 812, 55-65, which are hereby incorporated by reference in their entirety.
The in-line mixing method is a method wherein both the lipids and the nucleic acid are added in parallel into a mixing chamber. The mixing chamber can be a simple T-connector or any other mixing chamber that is known to one skill in the art. These methods are disclosed in U.S. Pat. No. 6,534,018 and U.S. Pat. No. 6,855,277; US publication 2007/0042031 and Pharmaceuticals Research, Vol. 22, No. 3, March 2005, p. 362-372, which are hereby incorporated by reference in their entirety.
It is further understood that the formulations of the invention can be prepared by any methods known to one of ordinary skill in the art.
Characterization of Nucleic Acid-Lipid Particles
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. In one embodiment, the formulations of the invention are entrapped by at least 75%, at least 80% or at least 90%.
For nucleic acid-lipid particle formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Formulations of Nucleic Acid-Lipid Particles
LNP01
One example of synthesis of a nucleic acid-lipid particle is as follows. Nucleic acid-lipid particles are synthesized using the lipidoid ND98.4HCl (MW 1487) (Formula I), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids). This nucleic acid-lipid particle is sometimes referred to as a LNP01 particles. Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.




LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Additional exemplary nucleic acid-lipid particle formulations are described in the following table. It is to be understood that the name of the nucleic acid-lipid particle in the table is not meant to be limiting. For example, as used herein, the term SNALP refers to formulations that include the cationic lipid DLinDMA.















cationic lipid/non-cationic lipid/cholesterol/




PEG-lipid conjugate mol % ratio



Name
Lipid:siRNA ratio









SNALP
DLinDMA/DPPC/Cholesterol/PEG-cDMA




(57.1/7.1/34.4/1.4)




lipid:siRNA ~7:1



LNP-S-X
XTC/DPPC/Cholesterol/PEG-cDMA




57.1/7.1/34.4/1.4




lipid:siRNA ~7:1



LNP05
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA ~6:1



LNP06
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA ~11:1



LNP07
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA ~6:1



LNP08
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA ~11:1



LNP09
XTC/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




lipid:siRNA ~10:1



LNP10
ALNY-100/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




lipid:siRNA ~10:1



LNP11
MC3/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




lipid:siRNA ~10:1



LNP13
XTC/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




lipid:siRNA ~33:1



LNP14
MC3/DSPC/Cholesterol/PEG-DMG




40/15/40/5




lipid:siRNA ~11:1



LNP15
MC3/DSPC/Cholesterol/PEG-DSG/GalNAc-PEG-DSG




50/10/35/4.5/0.5




lipid:siRNA ~11:1



LNP16
MC3/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




lipid:siRNA ~7:1



LNP17
MC3/DSPC/Cholesterol/PEG-DSG




50/10/38.5/1.5




lipid:siRNA ~10:1



LNP18
MC3/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




lipid:siRNA ~12:1



LNP19
MC3/DSPC/Cholesterol/PEG-DMG




50/10/35/5




lipid:siRNA ~8:1



LNP20
MC3/DSPC/Cholesterol/PEG-DPG




50/10/38.5/1.5




lipid:siRNA ~10:1



LNP22
XTC/DSPC/Cholesterol/PEG-DSG




50/10/38.5/1.5




lipid:siRNA ~10:1










XTC comprising formulations are described, e.g., in PCT/US10/22614 filed on Jan. 29, 2010, which is hereby incorporated by reference.
MC3 comprising formulations are described, e.g., in U.S. Ser. No. 12/813,448, filed Jun. 10, 2010, and PCT/US10/38224, filed Jun. 10, 2010, which is hereby incorporated by reference.
ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
TechG1 (also known as C12-200) comprising formulations are described, e.g., International patent application number PCT/US10/33777, filed on May 15, 2010, which is hereby incorporated by reference. An embodiment of TechG1 is shown below.




Additional representative formulations delineated in Tables A and B. Lipid refers to a cationic lipid.







TABLE A







Composition of exemplary nucleic acid-lipid particle


(mole %) prepared via extrusion methods.











Lipid
DSPC
Chol
PEG
Lipid/


(mol %)
(mol %)
(mol %)
(mol %)
siRNA














20
30
40
10
2.13


20
30
40
10
2.35


20
30
40
10
2.37


20
30
40
10
3.23


20
30
40
10
3.91


30
20
40
10
2.89


30
20
40
10
3.34


30
20
40
10
3.34


30
20
40
10
4.10


30
20
40
10
5.64


40
10
40
10
3.02


40
10
40
10
3.35


40
10
40
10
3.74


40
10
40
10
5.80


40
10
40
10
8.00


45
5
40
10
3.27


45
5
40
10
3.30


45
5
40
10
4.45


45
5
40
10
7.00


45
5
40
10
9.80


50
0
40
10
27.03


20
35
40
5
3.00


20
35
40
5
3.32


20
35
40
5
3.05


20
35
40
5
3.67


20
35
40
5
4.71


30
25
40
5
2.47


30
25
40
5
2.98


30
25
40
5
3.29


30
25
40
5
4.99


30
25
40
5
7.15


40
15
40
5
2.79


40
15
40
5
3.29


40
15
40
5
4.33


40
15
40
5
7.05


40
15
40
5
9.63


45
10
40
5
2.44


45
10
40
5
3.21


45
10
40
5
4.29


45
10
40
5
6.50


45
10
40
5
8.67


20
35
40
5
4.10


20
35
40
5
4.83


30
25
40
5
3.86


30
25
40
5
5.38


30
25
40
5
7.07


40
15
40
5
3.85


40
15
40
5
4.88


40
15
40
5
7.22


40
15
40
5
9.75


45
10
40
5
2.83


45
10
40
5
3.85


45
10
40
5
4.88


45
10
40
5
7.05


45
10
40
5
9.29


45
20
30
5
4.01


45
20
30
5
3.70


50
15
30
5
4.75


50
15
30
5
3.80


55
10
30
5
3.85


55
10
30
5
4.13


60
5
30
5
5.09


60
5
30
5
4.67


65
0
30
5
4.75


65
0
30
5
6.06


56.5
10
30
3.5
3.70


56.5
10
30
3.5
3.56


57.5
10
30
2.5
3.48


57.5
10
30
2.5
3.20


58.5
10
30
1.5
3.24


58.5
10
30
1.5
3.13


59.5
10
30
0.5
3.24


59.5
10
30
0.5
3.03


45
10
40
5
7.57


45
10
40
5
7.24


45
10
40
5
7.48


45
10
40
5
7.84


65
0
30
5
4.01


60
5
30
5
3.70


55
10
30
5
3.65


50
10
35
5
3.43


50
15
30
5
3.80


45
15
35
5
3.70


45
20
30
5
3.75


45
25
25
5
3.85


55
10
32.5
2.5
3.61


60
10
27.5
2.5
3.65


60
10
25
5
4.07


55
5
38.5
1.5
3.75


60
10
28.5
1.5
3.43


55
10
33.5
1.5
3.48


60
5
33.5
1.5
3.43


55
5
37.5
2.5
3.75


60
5
32.5
2.5
4.52


60
5
32.5
2.5
3.52


45
 15 (DMPC)
35
5
3.20


45
15 (DPPC)
35
5
3.43


45
 15 (DOPC)
35
5
4.52


45
15 (POPC)
35
5
3.85


55
5
37.5
2.5
3.96


55
10
32.5
2.5
3.56


60
5
32.5
2.5
3.80


60
10
27.5
2.5
3.75


60
5
30
5
4.19


60
5
33.5
1.5
3.48


60
5
33.5
1.5
6.64


60
5
30
5
3.90


60
5
30
5
4.65


60
5
30
5
5.88


60
5
30
5
7.51


60
5
30
5
9.51


60
5
30
5
11.06


62.5
2.5
50
5
6.63


45
15
35
5
3.31


45
15
35
5
6.80


60
5
25
10
6.48


60
5
32.5
2.5
3.43


60
5
30
5
3.90


60
5
30
5
7.61


45
15
35
5
3.13


45
15
35
5
6.42


60
5
25
10
6.48


60
5
32.5
2.5
3.03


60
5
30
5
3.43


60
5
30
5
6.72


60
5
30
5
4.13


70
5
20
5
5.48


80
5
10
5
5.94


90
5
0
5
9.50


60
5
30
5 C12PEG
3.85


60
5
30
5
3.70


60
5
30
5 C16PEG
3.80


60
5
30
5
4.19


60
5
29
5
4.07


60
5
30
5
3.56


60
5
30
5
3.39


60
5
30
5
3.96


60
5
30
5
4.01


60
5
30
5
4.07


60
5
30
5
4.25


60
5
30
5
3.80


60
5
30
5
3.31


60
5
30
5
4.83


60
5
30
5
4.67


60
5
30
5
3.96


57.5
7.5
33.5
1.5
3.39


57.5
7.5
32.5
2.5
3.39


57.5
7.5
31.5
3.5
3.52


57.5
7.5
30
5
4.19


60
5
30
5
3.96


60
5
30
5
3.96


60
5
30
5
3.56


60
5
33.5
1.5
3.52


60
5
25
10
5.18


60
 5 (DPPC)
30
5
4.25


60
5
32.5
2.5
3.70


57.5
7.5
31.5
3.5
3.06


57.5
7.5
31.5
3.5
3.65


57.5
7.5
31.5
3.5
4.70


57.5
7.5
31.5
3.5
6.56
















TABLE B







Composition of exemplary nucleic acid-lipid


particles prepared via in-line mixing











Lipid
DSPC
Chol
PEG
Lipid


(mol %)
(mol %)
(mol %)
(mol %)
A/siRNA














55
5
37.5
2.5
3.96


55
10
32.5
2.5
3.56


60
5
32.5
2.5
3.80


60
10
27.5
2.5
3.75


60
5
30
5
4.19


60
5
33.5
1.5
3.48


60
5
33.5
1.5
6.64


60
5
25
10
6.79


60
5
32.5
2.5
3.96


60
5
34
1
3.75


60
5
34.5
0.5
3.28


50
5
40
5
3.96


60
5
30
5
4.75


70
5
20
5
5.00


80
5
10
5
5.18


60
5
30
5
13.60


60
5
30
5
14.51


60
5
30
5
6.20


60
5
30
5
4.60


60
5
30
5
6.20


60
5
30
5
5.82


40
5
54
1
3.39


40
7.5
51.5
1
3.39


40
10
49
1
3.39


50
5
44
1
3.39


50
7.5
41.5
1
3.43


50
10
39
1
3.35


60
5
34
1
3.52


60
7.5
31.5
1
3.56


60
10
29
1
3.80


70
5
24
1
3.70


70
7.5
21.5
1
4.13


70
10
19
1
3.85


60
5
34
1
3.52


60
5
34
1
3.70


60
5
34
1
3.52


60
7.5
27.5
5
5.18


60
7.5
29
3.5
4.45


60
5
31.5
3.5
4.83


60
7.5
31
1.5
3.48


57.5
7.5
30
5
4.75


57.5
7.5
31.5
3.5
4.83


57.5
5
34
3.5
4.67


57.5
7.5
33.5
1.5
3.43


55
7.5
32.5
5
4.38


55
7.5
34
3.5
4.13


55
5
36.5
3.5
4.38


55
7.5
36
1.5
3.35









Synthesis of Cationic Lipids.
Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.
“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.
“Halogen” means fluoro, chloro, bromo and iodo.
In some embodiments, the methods of the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
Synthesis of Formula A
In one embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:




where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.





Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.




Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
Synthesis of MC3
Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).
Synthesis of ALNY-100
Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:




Synthesis of 515:
To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
Synthesis of 516:
To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]-232.3 (96.94%).
Synthesis of 517A and 517B:
The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction ( ̃3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude
517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]−266.3, [M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
Synthesis of 518:
Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.
General Procedure for the Synthesis of Compound 519:
A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+Calc. 654.6. Found 654.6.
Therapeutic Agent-Lipid Particle Compositions and Formulations
The invention includes compositions comprising a lipid particle of the invention and an active agent, wherein the active agent is associated with the lipid particle. In particular embodiments, the active agent is a therapeutic agent. In particular embodiments, the active agent is encapsulated within an aqueous interior of the lipid particle. In other embodiments, the active agent is present within one or more lipid layers of the lipid particle. In other embodiments, the active agent is bound to the exterior or interior lipid surface of a lipid particle.
“Fully encapsulated” as used herein indicates that the nucleic acid in the particles is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free DNA. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded. Alternatively, full encapsulation may be determined by an Oligreen® assay. Oligreen® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA in solution (available from Invitrogen Corporation, Carlsbad, Calif.). Fully encapsulated also suggests that the particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.
Active agents, as used herein, include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such effects may be biological, physiological, or cosmetic, for example. Active agents may be any type of molecule or compound, including e.g., nucleic acids, peptides and polypeptides, including, e.g., antibodies, such as, e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and PrimatizedTM antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.
In one embodiment, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives may be therapeutically active themselves or they may be prodrugs, which become active upon further modification. Thus, in one embodiment, a therapeutic agent derivative retains some or all of the therapeutic activity as compared to the unmodified agent, while in another embodiment, a therapeutic agent derivative lacks therapeutic activity.
In various embodiments, therapeutic agents include any therapeutically effective agent or drug, such as anti-inflammatory compounds, anti-depressants, stimulants, analgesics, antibiotics, birth control medication, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs, e.g., anti-arrhythmic agents, vasoconstrictors, hormones, and steroids. In certain embodiments, the therapeutic agent is an oncology drug, which may also be referred to as an anti-tumor drug, an anti-cancer drug, a tumor drug, an antineoplastic agent, or the like.
Additional Formulations
Emulsions
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252). Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.
Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
The agent and dsRNAs targeting a gene of interest of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extra-circulatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
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 excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Methods of Preparing Lipid Particles
The methods and compositions of the invention make use of certain cationic lipids, the synthesis, preparation and characterization of which is described below and in the accompanying Examples. In addition, the present invention provides methods of preparing lipid particles, including those associated with a therapeutic agent, e.g., a nucleic acid. In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated in lipid particles wherein the encapsulated nucleic acids are present in a nucleic acid/lipid ratio of about 3 wt % to about 25 wt %, preferably 5 to 15 wt %. The intermediate mixture may optionally be sized to obtain lipid-encapsulated nucleic acid particles wherein the lipid portions are unilamellar vesicles, preferably having a diameter of 30 to 150 nm, more preferably about 40 to 90 nm. The pH is then raised to neutralize at least a portion of the surface charges on the lipid-nucleic acid particles, thus providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.
As described above, several of these cationic lipids are amino lipids that are charged at a pH below the pKa of the amino group and substantially neutral at a pH above the pKa. These cationic lipids are termed titratable cationic lipids and can be used in the formulations of the invention using a two-step process. First, lipid vesicles can be formed at the lower pH with titratable cationic lipids and other vesicle components in the presence of nucleic acids. In this manner, the vesicles will encapsulate and entrap the nucleic acids. Second, the surface charge of the newly formed vesicles can be neutralized by increasing the pH of the medium to a level above the pKa of the titratable cationic lipids present, i.e., to physiological pH or higher. Particularly advantageous aspects of this process include both the facile removal of any surface adsorbed nucleic acid and a resultant nucleic acid delivery vehicle which has a neutral surface. Liposomes or lipid particles having a neutral surface are expected to avoid rapid clearance from circulation and to avoid certain toxicities which are associated with cationic liposome preparations. Additional details concerning these uses of such titratable cationic lipids in the formulation of nucleic acid-lipid particles are provided in U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein by reference.
It is further noted that the vesicles formed in this manner provide formulations of uniform vesicle size with high content of nucleic acids. Additionally, the vesicles have a size range of from about 30 to about 150 nm, more preferably about 30 to about 90 nm.
Without intending to be bound by any particular theory, it is believed that the very high efficiency of nucleic acid encapsulation is a result of electrostatic interaction at low pH. At acidic pH (e.g. pH 4.0) the vesicle surface is charged and binds a portion of the nucleic acids through electrostatic interactions. When the external acidic buffer is exchanged for a more neutral buffer (e.g. pH 7.5) the surface of the lipid particle or liposome is neutralized, allowing any external nucleic acid to be removed. More detailed information on the formulation process is provided in various publications (e.g., U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225).
In view of the above, the present invention provides methods of preparing lipid/nucleic acid formulations. In the methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated in lipid particles, e.g., wherein the encapsulated nucleic acids are present in a nucleic acid/lipid ratio of about 10 wt % to about 20 wt %. The intermediate mixture may optionally be sized to obtain lipid-encapsulated nucleic acid particles wherein the lipid portions are unilamellar vesicles, preferably having a diameter of 30 to 150 nm, more preferably about 40 to 90 nm. The pH is then raised to neutralize at least a portion of the surface charges on the lipid-nucleic acid particles, thus providing an at least partially surface-neutralized lipid-encapsulated nucleic acid composition.
In certain embodiments, the mixture of lipids includes at least two lipid components: a first amino lipid component of the present invention that is selected from among lipids which have a pKa such that the lipid is cationic at pH below the pKa and neutral at pH above the pKa, and a second lipid component that is selected from among lipids that prevent particle aggregation during lipid-nucleic acid particle formation. In particular embodiments, the amino lipid is a novel cationic lipid of the present invention.
In preparing the nucleic acid-lipid particles of the invention, the mixture of lipids is typically a solution of lipids in an organic solvent. This mixture of lipids can then be dried to form a thin film or lyophilized to form a powder before being hydrated with an aqueous buffer to form liposomes. Alternatively, in a preferred method, the lipid mixture can be solubilized in a water miscible alcohol, such as ethanol, and this ethanolic solution added to an aqueous buffer resulting in spontaneous liposome formation. In most embodiments, the alcohol is used in the form in which it is commercially available. For example, ethanol can be used as absolute ethanol (100%), or as 95% ethanol, the remainder being water. This method is described in more detail in U.S. Pat. No. 5,976,567).
In accordance with the invention, the lipid mixture is combined with a buffered aqueous solution that may contain the nucleic acids. The buffered aqueous solution of is typically a solution in which the buffer has a pH of less than the pKa of the protonatable lipid in the lipid mixture. Examples of suitable buffers include citrate, phosphate, acetate, and MES. A particularly preferred buffer is citrate buffer. Preferred buffers will be in the range of 1-1000 mM of the anion, depending on the chemistry of the nucleic acid being encapsulated, and optimization of buffer concentration may be significant to achieving high loading levels (see, e.g., U.S. Pat. No. 6,287,591 and U.S. Pat. No. 6,858,225). Alternatively, pure water acidified to pH 5-6 with chloride, sulfate or the like may be useful. In this case, it may be suitable to add 5% glucose, or another non-ionic solute which will balance the osmotic potential across the particle membrane when the particles are dialyzed to remove ethanol, increase the pH, or mixed with a pharmaceutically acceptable carrier such as normal saline. The amount of nucleic acid in buffer can vary, but will typically be from about 0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL to about 50 mg/mL.
The mixture of lipids and the buffered aqueous solution of therapeutic nucleic acids is combined to provide an intermediate mixture. The intermediate mixture is typically a mixture of lipid particles having encapsulated nucleic acids. Additionally, the intermediate mixture may also contain some portion of nucleic acids which are attached to the surface of the lipid particles (liposomes or lipid vesicles) due to the ionic attraction of the negatively-charged nucleic acids and positively-charged lipids on the lipid particle surface (the amino lipids or other lipid making up the protonatable first lipid component are positively charged in a buffer having a pH of less than the pKa of the protonatable group on the lipid). In one group of preferred embodiments, the mixture of lipids is an alcohol solution of lipids and the volumes of each of the solutions is adjusted so that upon combination, the resulting alcohol content is from about 20% by volume to about 45% by volume. The method of combining the mixtures can include any of a variety of processes, often depending upon the scale of formulation produced. For example, when the total volume is about 10-20 mL or less, the solutions can be combined in a test tube and stirred together using a vortex mixer. Large-scale processes can be carried out in suitable production scale glassware.
Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleic acid) complexes which are produced by combining the lipid mixture and the buffered aqueous solution of therapeutic agents (nucleic acids) can be sized to achieve a desired size range and relatively narrow distribution of lipid particle sizes. Preferably, the compositions provided herein will be sized to a mean diameter of from about 70 to about 200 nm, more preferably about 90 to about 130 nm. Several techniques are available for sizing liposomes to a desired size. One sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUVs) less than about 0.05 microns in size. Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size determination. For certain methods herein, extrusion is used to obtain a uniform vesicle size.
Extrusion of liposome compositions through a small-pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome complex size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size. In some instances, the lipid-nucleic acid compositions which are formed can be used without any sizing.
In particular embodiments, methods of the present invention further comprise a step of neutralizing at least some of the surface charges on the lipid portions of the lipid-nucleic acid compositions. By at least partially neutralizing the surface charges, unencapsulated nucleic acid is freed from the lipid particle surface and can be removed from the composition using conventional techniques. Preferably, unencapsulated and surface adsorbed nucleic acids are removed from the resulting compositions through exchange of buffer solutions. For example, replacement of a citrate buffer (pH about 4.0, used for forming the compositions) with a HEPES-buffered saline (HBS pH about 7.5) solution, results in the neutralization of liposome surface and nucleic acid release from the surface. The released nucleic acid can then be removed via chromatography using standard methods, and then switched into a buffer with a pH above the pKa of the lipid used.
Optionally the lipid vesicles (i.e., lipid particles) can be formed by hydration in an aqueous buffer and sized using any of the methods described above prior to addition of the nucleic acid. As described above, the aqueous buffer should be of a pH below the pKa of the amino lipid. A solution of the nucleic acids can then be added to these sized, preformed vesicles. To allow encapsulation of nucleic acids into such “pre-formed” vesicles the mixture should contain an alcohol, such as ethanol. In the case of ethanol, it should be present at a concentration of about 20% (w/w) to about 45% (w/w). In addition, it may be necessary to warm the mixture of pre-formed vesicles and nucleic acid in the aqueous buffer-ethanol mixture to a temperature of about 25° C. to about 50° C. depending on the composition of the lipid vesicles and the nature of the nucleic acid. It will be apparent to one of ordinary skill in the art that optimization of the encapsulation process to achieve a desired level of nucleic acid in the lipid vesicles will require manipulation of variable such as ethanol concentration and temperature. Examples of suitable conditions for nucleic acid encapsulation are provided in the Examples. Once the nucleic acids are encapsulated within the preformed vesicles, the external pH can be increased to at least partially neutralize the surface charge. Unencapsulated and surface adsorbed nucleic acids can then be removed as described above.
Methods of Use
The lipid particles of the invention may be used to deliver a therapeutic agent to a cell, in vitro or in vivo. In particular embodiments, the therapeutic agent is a nucleic acid, which is delivered to a cell using a nucleic acid-lipid particles of the invention. While the following description of various methods of using the lipid particles and related pharmaceutical compositions of the invention are exemplified by description related to nucleic acid-lipid particles, it is understood that these methods and compositions may be readily adapted for the delivery of any therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.
In certain embodiments, the invention provides methods for introducing a nucleic acid into a cell. Preferred nucleic acids for introduction into cells are siRNA, immune-stimulating oligonucleotides, plasmids, antisense and ribozymes. These methods may be carried out by contacting the particles or compositions of the invention with the cells for a period of time sufficient for intracellular delivery to occur.
The compositions of the invention can be adsorbed to almost any cell type. Once adsorbed, the nucleic acid-lipid particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. Transfer or incorporation of the nucleic acid portion of the complex can take place via any one of these pathways. Without intending to be limited with respect to the scope of the invention, it is believed that in the case of particles taken up into the cell by endocytosis the particles then interact with the endosomal membrane, resulting in destabilization of the endosomal membrane, possibly by the formation of non-bilayer phases, resulting in introduction of the encapsulated nucleic acid into the cell cytoplasm. Similarly in the case of direct fusion of the particles with the cell plasma membrane, when fusion takes place, the liposome membrane is integrated into the cell membrane and the contents of the liposome combine with the intracellular fluid. Contact between the cells and the lipid-nucleic acid compositions, when carried out in vitro, will take place in a biologically compatible medium. The concentration of compositions can vary widely depending on the particular application, but is generally between about 1 μmol and about 10 mmol. In certain embodiments, treatment of the cells with the lipid-nucleic acid compositions will generally be carried out at physiological temperatures (about 37° C.) for periods of time from about 1 to 24 hours, preferably from about 2 to 8 hours. For in vitro applications, the delivery of nucleic acids can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type. In preferred embodiments, the cells will be animal cells, more preferably mammalian cells, and most preferably human cells.
In one group of embodiments, a lipid-nucleic acid particle suspension is added to 60-80% confluent plated cells having a cell density of from about 103 to about 105 cells/mL, more preferably about 2×104 cells/mL. The concentration of the suspension added to the cells is preferably of from about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.
Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets. Alternatively applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides. In this manner, therapy is provided for genetic diseases by supplying deficient or absent gene products (i.e., for Duchenne's dystrophy, see Kunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cystic fibrosis, see Goodfellow, Nature 341:102-103 (1989)). Other uses for the compositions of the invention include introduction of antisense oligonucleotides in cells (see, Bennett, et al., Mol. Pharm. 41:1023-1033 (1992)).
Alternatively, the compositions of the invention can also be used for deliver of nucleic acids to cells in vivo, using methods which are known to those of skill in the art. With respect to application of the invention for delivery of DNA or mRNA sequences, Zhu, et al., Science 261:209-211 (1993), incorporated herein by reference, describes the intravenous delivery of cytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) expression plasmid using DOTMA-DOPE complexes. Hyde, et al., Nature 362:250-256 (1993), incorporated herein by reference, describes the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to epithelia of the airway and to alveoli in the lung of mice, using liposomes. Brigham, et al., Am. J. Med. Sci. 298:278-281 (1989), incorporated herein by reference, describes the in vivo transfection of lungs of mice with a functioning prokaryotic gene encoding the intracellular enzyme, chloramphenicol acetyltransferase (CAT). Thus, the compositions of the invention can be used in the treatment of infectious diseases.
For in vivo administration, the pharmaceutical compositions are preferably administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In particular embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. For one example, see Stadler, et al., U.S. Pat. No. 5,286,634, which is incorporated herein by reference. Intracellular nucleic acid delivery has also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY, Academic Press, New York. 101:512-527 (1983); Mannino, et al., Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. Drug Carrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993). Still other methods of administering lipid-based therapeutics are described in, for example, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
In other methods, the pharmaceutical preparations may be contacted with the target tissue by direct application of the preparation to the tissue. The application may be made by topical, “open” or “closed” procedures. By “topical,” it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like. “Open” procedures are those procedures which include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue. “Closed” procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin. For example, the preparations may be administered to the peritoneum by needle lavage. Likewise, the pharmaceutical preparations may be administered to the meninges or spinal cord by infusion during a lumbar puncture followed by appropriate positioning of the patient as commonly practiced for spinal anesthesia or metrazamide imaging of the spinal cord. Alternatively, the preparations may be administered through endoscopic devices.
The lipid-nucleic acid compositions can also be administered in an aerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci. 298(4):278-281 (1989)) or by direct injection at the site of disease (Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, New York. pp. 70-71 (1994)).
The methods of the invention may be practiced in a variety of hosts. Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like.
Dosages for the lipid-therapeutic agent particles of the invention will depend on the ratio of therapeutic agent to lipid and the administrating physician's opinion based on age, weight, and condition of the patient.
In one embodiment, the invention provides a method of modulating the expression of a target polynucleotide or polypeptide. These methods generally comprise contacting a cell with a lipid particle of the invention that is associated with a nucleic acid capable of modulating the expression of a target polynucleotide or polypeptide. As used herein, the term “modulating” refers to altering the expression of a target polynucleotide or polypeptide. In different embodiments, modulating can mean increasing or enhancing, or it can mean decreasing or reducing. Methods of measuring the level of expression of a target polynucleotide or polypeptide are known and available in the arts and include, e.g., methods employing reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical techniques. In particular embodiments, the level of expression of a target polynucleotide or polypeptide is increased or reduced by at least 10%, 20%, 30%, 40%, 50%, or greater than 50% as compared to an appropriate control value. For example, if increased expression of a polypeptide desired, the nucleic acid may be an expression vector that includes a polynucleotide that encodes the desired polypeptide. On the other hand, if reduced expression of a polynucleotide or polypeptide is desired, then the nucleic acid may be, e.g., an antisense oligonucleotide, siRNA, or microRNA that comprises a polynucleotide sequence that specifically hybridizes to a polynucleotide that encodes the target polypeptide, thereby disrupting expression of the target polynucleotide or polypeptide. Alternatively, the nucleic acid may be a plasmid that expresses such an antisense oligonucleotide, siRNA, or microRNA.
In one particular embodiment, the invention provides a method of modulating the expression of a polypeptide by a cell, comprising providing to a cell a lipid particle that consists of or consists essentially of a cationic lipid of formula A, a neutral lipid, a sterol, a PEG of PEG-modified lipid, e.g., in a molar ratio of about 35-65% of cationic lipid of formula A, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid, wherein the lipid particle is associated with a nucleic acid capable of modulating the expression of the polypeptide. In particular embodiments, the molar lipid ratio is approximately 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol % LIPID A/DSPC/Chol/PEG-DMG). In another group of embodiments, the neutral lipid in these compositions is replaced with DPPC (dipalmitoylphosphatidylcholine), POPC, DOPE or SM.
In particular embodiments, the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof, such that the expression of the polypeptide is reduced.
In other embodiments, the nucleic acid is a plasmid that encodes the polypeptide or a functional variant or fragment thereof, such that expression of the polypeptide or the functional variant or fragment thereof is increased.
In related embodiments, the invention provides a method of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.
In one embodiment, the pharmaceutical composition comprises a lipid particle that consists of or consists essentially of Lipid A, DSPC, Chol and PEG-DMG, PEG-C-DOMG or PEG-DMA, e.g., in a molar ratio of about 35-65% of cationic lipid of formula A, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid PEG-DMG, PEG-C-DOMG or PEG-DMA, wherein the lipid particle is associated with the therapeutic nucleic acid. In particular embodiments, the molar lipid ratio is approximately 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol % LIPID A/DSPC/Chol/PEG-DMG). In another group of embodiments, the neutral lipid in these compositions is replaced with DPPC, POPC, DOPE or SM.
In another related embodiment, the invention includes a method of treating a disease or disorder characterized by underexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the invention, wherein the therapeutic agent is a plasmid that encodes the polypeptide or a functional variant or fragment thereof.
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.
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.
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, Unterschleitβheim, 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




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




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




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




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




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




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




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




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.










Abbreviation
Nucleotide(s)







A
adenosine-5′-phosphate



C
cytidine-5′-phosphate



G
guanosine-5′-phosphate



T, dT
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, sdT
2′-deoxy-thymidine-5′phosphate-phosphorothioate







It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds.






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 19mers 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 19mer 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 11 Oct. 2005, 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. 6, 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-Lipofectamine-2000-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 titers 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.
Example 6
Inhibition of Ebola Gene Expression in Humans
A human subject is treated with a pharmaceutical composition comprising a pharmaceutical formulation of an siRNA targeting a Ebola virus gene.
A subject infected with an Ebola virus is selected or identified. The subject can be in need of treatment for diseases caused by Ebola viral infection, such as systemic hemorrhage and multi-organ failure.
At time zero, a suitable first dose of the composition is administered to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is evaluated, e.g., by decrease in symptoms and the like. This measurement can be accompanied by a measurement of Ebola gene expression in said subject, and/or the products of the successful siRNA-targeting of Ebola gene expression. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
After treatment, the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.
Example 7
Protection Against Ebola in Guinea Pigs with LNP09 Formulated AD-11570 Administration
To test of efficacy of siRNAs targeting Ebola genes in protecting guinea pigs from a lethal Ebola virus challenge, the AD-11570 and control siRNA (AD-1955) were formulated in lipid particles containing either DODMA or 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane as follows: AD-11570 DODMA, AD-1955 DODMA control, AD-11570 in LNP09, and AD-1955 LNP09 control.
The LNP09 formulation is described above. The DODMA formulation comprises: DODMA (1,2-dioleyloxy-N,N-dimethyl-3-aminopropane) 25.1 mol %; DSPC 20.8 mol %; Cholesterol 44.5 mol %; and PEG-C-DOMG 9.7 mol %.
Forty Hartley guinea pigs, approximately 6 months in age, were used in the study, each with a size of approximately 0.5 kg. Guinea pigs were infected subcutaneously (s.c.) with 100 plaque forming units (PFU) of guinea pig-adapted Ebola-Zaire. Guinea pigs were treated with DODMA or LNP09 formulated siRNA or saline control three times: at 2 hours post infection, 3 days post infection, and 6 days post infection (between 2-3 mg/kg intraperitoneal (i.p.)) (see Table 10 for summary). Guinea pigs were used at n=8 per treatment group. Negative controls included guinea pigs treated with saline and guinea pigs treated with formulated luciferase siRNA (AD-1955). Guinea pigs were monitored for survival (FIG. 9), weight loss (FIG. 10), and general health status.
Results
There were no tolerability issues with LNP09 formulated siRNAs in guinea pigs. In addition, no non-specific virus protection was observed with the LNP09 formulated AD-1955 siRNA control as measured by survival and weight loss of the guinea pigs. Partial virus protection was observed with the LNP09 formulated AD-11570 siRNA compared to controls, as measured by survival and weight loss (See, for example, FIGS. 9 and 10). Less protection was seen with DODMA formulated AD-11570 in this study when compared with previous studies, possibly due to the use of older animals.
Example 8
Dosing Regimen: Protection Against Ebola in Guinea Pigs with LNP09 Formulated AD-11570 Administration
To test the efficacy of siRNAs targeting Ebola genes in protecting guinea pigs from a lethal Ebola virus challenge, the AD-11570 and control siRNA (AD-1955) were formulated in lipid particles containing 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane as follows: AD-11570 LNP09 (132 mg) and AD-1955 LNP09 control (96 mg).
The LNP09 formulation is described above.
Forty-eight Hartley guinea pigs were used in the study, each with a size of approximately 0.5 kg and an age of approximately 3 months. Guinea pigs were infected subcutaneously (s.c.) with 100 plaque forming units (PFU) of guinea pig-adapted Ebola-Zaire. Control and drug were administered similar to the previous Example, except for a modified dosing regimen. In this study, some animals (e.g., Group 1) received 3 mg/kg every other day, while others (Groups 2 and 3) received 1 mg/kg daily. Groups 2 and 3 also differed in the timing of the first dose, e.g., Group 1 received a first dose of 3 mg/kg at 2 hours prior to infection, Group 2 received the first dose of 1 mg/kg at 2 hrs prior to infection, and group 3 received the first dose at 30 mins post-infection (Table 11 summarizes the dosing regimen for each group of guinea pigs). Guinea pigs were used at n=8 per treatment group. Negative controls included saline treated guinea pigs and guinea pigs treated with formulated luciferase siRNA (AD-1955). Guinea pigs were monitored for weight loss, general health status, and survival.
The effectiveness of AD-11570 siRNA with respect to the targeting of Ebola genes and the various dosing regimens is demonstrated by the results shown in FIG. 11 and FIG. 12. FIG. 11 shows that AD-11570 siRNA increases survival after two weeks post-infection by roughly 3-7 fold, regardless of the dosage regimen used. At least a 6-fold increase in survival or 75% protection from Ebola virus is observed with the treated animals. FIG. 12 similarly demonstrates that the LNP09/AD-11570 formulation prevents the severe weight loss observed in the absence of treatment. In addition, the siRNA does not appear to be immunostimulatory.
Example 9
Protection Against Ebola in Guinea Pigs with AD-11570 siRNA Administered as Formulation M
To test the efficacy of siRNAs targeting Ebola genes in protecting guinea pigs from a lethal Ebola virus challenge, the AD-11570 and control siRNA (AD-1955) were formulated in lipid particles containing 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane as Formulation M, described above. The experimental design was similar to that described in the previous example, except here both the control and test guinea pigs (3 groups, with 6 animals per group) were dosed with 3 mgs/kg siRNA on days 0, 2, 4, and 6 at 3 mg/kg, with the 1st dose given 2 hours prior to infection.
The results are shown in FIG. 13 and FIG. 14. As was observed in the previous Example (using a different formulation), 75% protection against Ebola was observed with the AD-11570 siRNA.
Example 10
Comparison of dsRNA Administration Route and Formulation in Guinea Pigs
To test the efficacy of siRNAs targeting Ebola genes in protecting guinea pigs from a lethal Ebola virus challenge via different administration routes and lipid formulations, the AD-11570 and control siRNA (AD-1955) were formulated in lipid particles containing E/F or P/Q formulation and administered intravenously (IV) or intraperitonealy (IP).
The E/F formulation contains (50%/10%/38.5%/1.5%) of: Cationic Lipid/DSPC/Cholesterol/PEG-C14. The P/Q formulation contains: (30%/30%/38.5%/1.5%) of: Catioinc Lipid/DSPC/Cholesterol/PEG-C14. XTC was the cationic lipid used in this example.
Venous access port (VAP) implanted Hartley guinea pigs (GP) were used in the study (app. 900 grams each). Guinea pigs were infected subcutaneously (s.c.) with 100 plaque forming units (PFU) of guinea pig-adapted Ebola-Zaire. The treatment route was via IV bolus where IV is indicated. The first treatment was given approximately 1 hour prior to infection. Table 12 summarizes the dosing regimen for each group of guinea pigs. Guinea pigs were used at n=3-8 per treatment group. Negative controls included PBS treated guinea pigs and guinea pigs treated with formulated luciferase siRNA (AD-1955). Guinea pigs were monitored for weight loss and survival. The original study design called for equal numbers of GP in all groups, however due to issues with VAP patency, animal numbers had to be adjusted. 4 of the 23 VAPs failed for injection and 1 animal died during surgery. Due to the VAP issues, 3 animals were treated with AD-1955 E/F IV on day 0 and then with AD-11570 P/Q IV on days 1, 2, and 4. Since we could not account for the effect of the day 0 treatment with the AD-1955 E/F formulation on the efficacy of the AD-11570 P/Q formulation, these animals were excluded from further analysis.
The results are shown in FIG. 15 and FIG. 16. 100% protection was observed with the E/F formulation dosed via IV administration. 60% protection was observed with the P/Q formulation dosed via IV administration (N=5). The less efficient protection in the AD-11570 IP treated group that was described above may be due to the use of larger animals in this study (900 grams v. 450 grams) and the larger amount of liposome per animal may exacerbate toxicity seen with the IP route resulting in more mortality. IV treatment shows no evidence for clinical signs of toxicity (e.g., swollen abdomens) observed with the IP treatment and is more efficacious and better tolerated.
Example 11
Dose Response Determination in Guinea Pigs with E/F Formulated AD-11570
To test the efficacy and optimal dose of siRNAs targeting Ebola genes in protecting guinea pigs from a lethal Ebola virus challenge, the AD-11570 and control siRNA (AD-1955) were formulated in lipid particles containing E/F formulation and administered intravenously (IV) at different concentrations.
The E/F formulation is described above. XTC was the cationic lipid used in this example.
VAP implanted Hartley guinea pigs (GP) were used in the study. Guinea pigs were infected subcutaneously (s.c.) with 100 plaque forming units (PFU) of guinea pig-adapted Ebola-Zaire. The treatment route was via IV bolus. The first treatment was given approximately 1 hour prior to infection. Table 13 summarizes the dosing regimen for each group of guinea pigs. Guinea pigs were used at n=6-8 per treatment group. Negative controls included guinea pigs treated with formulated luciferase siRNA (AD-1955). Guinea pigs were monitored for weight loss and survival for 14 days post infection.
The results are shown in FIG. 17 and FIG. 18. Protection with a 3 mg/kg dose was similar to that observed in the above Example. The death of one GP on day 5 was most likely due to heart failure related to the VAP placement and is unlikely to be test article or Ebola infection related. 100% protection was observed with 1 mg/kg dose with no significant weight loss. Partial efficacy was observed with the 0.3 mg/kg dose.
Example 12
Comparison of Different Lipid Formulations in Guinea Pigs with AD-11570
To test the efficacy of differently lipid formulated siRNAs targeting Ebola genes in protecting guinea pigs from a lethal Ebola virus challenge, the AD-11570 was formulated in lipid particles containing Lipid D, Lipid T, or Lipid M formulation and administered intravenously (IV).
All formulations tested in this Example contained 50 mol % of cationic lipid/10% DSPC/38.5% Cholesterol/1.5% PEG-DMG. The Lipid D formulation contained the XTC cationic lipid, described above. The Lipid T formulation contained TechG1, described above. The Lipid M formulation contained MC3, described above.
VAP implanted Hartley guinea pigs (GP) were used in the study. Guinea pigs were infected subcutaneously (s.c.) with 100 plaque forming units (PFU) of guinea pig-adapted Ebola-Zaire. The treatment route was via IV bolus. The first treatment was given approximately 1 hour prior to infection. Table 14 summarizes the dosing regimen for each group of guinea pigs. Guinea pigs were used at n=6-7 per treatment group. Guinea pigs were monitored for weight loss and survival for 15 days post infection.
The results are shown in FIG. 19 and FIG. 20. There was no apparent formulation-related toxicity. There was a lower level of protection with 1 mg/kg Lipid D treatment than observed in the previous Example. It is likely that the larger size of the GPs may be a contributing factor. No substantial protection was observed with Lipid D and Lipid M at 0.3 mg/kg. The 0.3 mg/kg dose of Lipid T provided similar protection as compared to the 1 mg/kg dose of Lipid D. This is consistent with the previously observed improved activity of Lipid T formulations in leukocyte and liver silencing in the mouse model.
Example 13
Post-Ebola Exposure Treatment of Guinea Pigs with AD-11570
To test the efficacy of siRNAs targeting Ebola genes in treating guinea pigs after a lethal Ebola virus challenge, the AD-11570 was formulated in lipid particles containing E/F formulation and administered intravenously (IV).
The E/F formulation is described above. XTC was the cationic lipid used in this example.
VAP implanted Hartley guinea pigs (GP) were used in the study. Guinea pigs were infected subcutaneously (s.c.) with 100 plaque forming units (PFU) of guinea pig-adapted Ebola-Zaire. The treatment route was via IV bolus. Table 15 summarizes the dosing regimen for each group of guinea pigs. Guinea pigs were used at n=4-6 per treatment group. Negative controls included guinea pigs treated with PBS via IP administration. Guinea pigs were monitored for weight loss, disease index, and survival for 15 days post infection.
The results are shown in FIG. 21, FIG. 22, and FIG. 23. A 48 hour delay of treatment post-infection provided a level of protection similar to that achieved with treatment at the time of infection. siRNA treated animals remained largely healthy throughout the study.
Example 14
Viral Load and Disease Pathology Determination in Guinea Pigs with E/F Formulated AD-11570
To test the effect of siRNAs targeting Ebola genes on viral load and disease pathology in guinea pigs, the AD-11570 and control siRNA (AD-1955) were formulated in lipid particles containing E/F formulation and administered intravenously (IV).
The E/F formulation is described above. XTC was the cationic lipid used in this example. The Lipid D formulation contained the XTC cationic lipid, described above.
Guinea pigs (GP) were used in the study. Guinea pigs were infected subcutaneously (s.c.) with 100 plaque forming units (PFU) of guinea pig-adapted Ebola-Zaire. The treatment route was via IV bolus. Table 16 summarizes the dosing regimen for each group of guinea pigs. Guinea pigs were used at n=4-12 per treatment group. Negative controls included guinea pigs treated with formulated luciferase siRNA (AD-1955), PBS, no treatment, and naïve. Guinea pigs were monitored for viral titer, serum chemistry, and pathology on days 5, 7, and 10. Table 17 summarizes the GP sacrifice schedule. Group 4 was used as a control to demonstrate the expected course of infection in naïve animals.
The results are shown in FIG. 24. Treatment with AD-11570 resulted in dramatic suppression of Ebola virus in the GP model. Only 1 of 10 animals treated with AD-11570 had measureable serum viremia at any of the timepoints examined (app. 4 log lower viremia than control animals). This demonstrates the anti-viral effect of treatment with Ebola specific siRNA. Evidence of liver enzyme elevations in the AD-11570 treated animals was observed. This may be related to formulation toxicity or a low level infection in the liver and is consistent with weight loss that typically accompanies XTC treatment in this model. Evidence of maintenance of platelet levels with AD-11570 treatment was observed.
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.









TABLE 2








double overhang design













sense






strand

antisense strand
















position


Seq


Seq

duplex


in 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
GGAuGAuGAAGGuuuuAAUTsT
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
CAUGUGUCCUACUGAUUGCTsT
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
UUUCUUAAUCUUCAUCACUTsT
AD-










11610


 


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










11611


 


646-664
VP40
A18620
93
UGCCUGCUGCAACAUGGACTsT
A18621
94
GUCCAUGUUGCAGCAGGCATsT
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


 


2557-2575
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-










11648


 


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-










11656


 


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
AGuuAuAuccGccuuGGuuTsT
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
AAucccGAGGcGGcAAuucTsT
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
uGuAGcGcAAuuGAcuuuGTsT
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
AcccAAAcGGAcccAAuuuTsT
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
cAcAuccGcucucGAGGuGTsT
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-










12503


 


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
uccuGAcuAcGAGGAuucGTsT
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


 


1045-1063
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-










12007


 


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









parent
light


anti-
seq

seq



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























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





30768







 


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





30770







 


AD-11597
AD-3544
NP
A-
A-30773
999
uAGGAcAcAuGAuGGuGAudTsdT
1000
AUcACcAUcAUGUGUCCuAdTsdT





30772







 


AD-11598
AD-3545
NP
A-
A-30775
1001
GAuGGuGAuuuuccGuuuGdTsdT
1002
cAAACGGAAAAUcACcAUCdTsdT





30774







 


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





30776







 


AD-11603
AD-3547
NP
A-
A-30779
1005
cuGAGAAGcAAcuccAAcAdTsdT
1006
UGUUGGAGUUGCUUCUcAGdTsdT





30778







 


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





30780







 


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





30782







 


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





30784







 


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





30786







 


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





30788







 


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





30790







 


AD-11614
AD-3554
GP
A-
A-30793
1019
GcuGAAAAcuGcuAcAAucdTsdT
1020
GAUUGuAGcAGUUUUcAGCdTsdT





30792







 


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





30794







 


AD-11618
AD-3556
GP
A-
A-30797
1023
AAAAcuGcuAcAAucuuGAdTsdT
1024
UcAAGAUUGuAGcAGUUUUdTsdT





30796







 


AD-11621
AD-3557
GP
A-
A-30799
1025
AcuGcuAcAAucuuGAAAudTsdT
1026
AUUUcAAGAUUGuAGcAGUdTsdT





30798







 


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





30800







 


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





30802







 


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





30804







 


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





30806







 


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





30808







 


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





30810







 


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





30812



















TABLE 6







In vitro plaque
In vitro plaque




assay against
assay against




Ebola-Zaire
Ebola-Sudan



In vitro
(% inhibition
(% inhibition


modified
plasmid screen
relative to no
relative to no


duplex
IC50 (nM)
siRNA)
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







duplex
% of control
















IFN induction normalized


to positive control siRNA










AD-11546
131.6



AD-11558
98.8



AD-11570
0



AD-11588
147



AD-11590
0.0



AD-11594
17.8



AD-11597
22.0



AD-11598
25.7



AD-11599
136



AD-11600
13.5



AD-11603
69.0



AD-11606
33.2



AD-11609
126.9



AD-11610
138.2



AD-11611
43.2



AD-11613
167.0



AD-11614
162.1



AD-11615
171.2



AD-11618
137.3



AD-11621
0.0



AD-11622
37.2



AD-11623
58.0



AD-11624
63.2



AD-11627
6.4



AD-11628
0.0



AD-11630
0.0



AD-11631
0.0



AD-11644
0



AD-11650
0.0



AD-11659
0.0



AD-11673
9.2



AD-11678
0.0



AD-11683
0.0



AD-11684
0.0



AD-11691
0



AD-11695
0.0



AD-11698
5.3



AD-11706
0.0



AD-11707
0



AD-11710
0



AD-11721
0.0



AD-11725
0.0



AD-11732
0.0



AD-11743
16.1



AD-11756
0.0



AD-11757
0.0



AD-11758
0.0



AD-11759
0.0



AD-11773
0.0



AD-11780
0.0



AD-11789
0.0



AD-11804
0.0



AD-11811
0.0



AD-11814
0.0



AD-11816
0.0



AD-11822
9.8



AD-11823
0.0



AD-11832
0.0



AD-11836
0.0



AD-11939
0.0



AD-11976
0.0



AD-11982
6.9



AD-11990
12.2



AD-11992
7.3



AD-12007
0.0



AD-12013
0.0



AD-12019
24.6



AD-12024
0.0



AD-12035
0.0



AD-12475
0.0



AD-12484
21.0



AD-12491
0.0



AD-12500
12.6



AD-12502
101.6



AD-3542
0.0



AD-3543
0.0



AD-3544
10.1



AD-3545
11.8



AD-3546
0.0



AD-3547
0.0



AD-3548
0.0



AD-3549
0.0



AD-3550
7.4



AD-3551
0.0



AD-3552
0.0



AD-3553
0.0



AD-3554
0.0



AD-3555
0.0



AD-3556
0.0



AD-3557
0.0



AD-3558
0.0



AD-3559
0.0



AD-3560
0.0



AD-3561
0.0



AD-3562
0.0



AD-3563
0.0



AD-3564
0.0



AD-3621
0.0



AD-3622
0.0



AD-3623
5.7



AD-3624
10.0



AD-3625
0.0



AD-3626
0.0







TNF induction normalized


to positive control siRNA










AD-11546
30.1



AD-11558
57.2



AD-11570
0



AD-11588
0.0



AD-11590
130.5



AD-11594
124.8



AD-11597
242.9



AD-11598
180.3



AD-11599
183.9



AD-11600
141.0



AD-11603
81.4



AD-11606
79.8



AD-11609
46.3



AD-11610
48.5



AD-11611
41.0



AD-11613
52.6



AD-11614
48.1



AD-11615
60.0



AD-11618
54.6



AD-11621
325.8



AD-11622
28.5



AD-11623
26.0



AD-11624
29.2



AD-11627
125.0



AD-11628
101.1



AD-11630
170.3



AD-11631
156.0



AD-11644
0.0



AD-11650
0.0



AD-11659
69.0



AD-11673
0.0



AD-11678
0.0



AD-11683
19.6



AD-11684
24.3



AD-11691
0.0



AD-11695
5.2



AD-11698
14.0



AD-11706
0.0



AD-11707
0.0



AD-11710
0.0



AD-11721
0.0



AD-11725
0.0



AD-11732
0.0



AD-11743
0.0



AD-11756
0.0



AD-11757
0.0



AD-11758
0.0



AD-11759
0.0



AD-11773
0.0



AD-11780
7.9



AD-11789
5.7



AD-11804
0.0



AD-11811
0.0



AD-11814
0.0



AD-11816
0.0



AD-11822
0.0



AD-11823
0.0



AD-11832
0.0



AD-11836
0.0



AD-11939
0.0



AD-11976
0.0



AD-11982
15.1



AD-11990
0.0



AD-11992
0.0



AD-12007
0.0



AD-12013
0.0



AD-12019
5.0



AD-12024
19.8



AD-12035
8.5



AD-12475
0.0



AD-12484
9.5



AD-12491
13.1



AD-12500
53.3



AD-12502
55.5



AD-3542
0.0



AD-3543
0.0



AD-3544
0.0



AD-3545
0.0



AD-3546
0.0



AD-3547
31.0



AD-3548
5.9



AD-3549
10.8



AD-3550
0.0



AD-3551
0.0



AD-3552
0.0



AD-3553
0.0



AD-3554
11.5



AD-3555
6.2



AD-3556
9.1



AD-3557
0.0



AD-3558
5.8



AD-3559
5.3



AD-3560
0.0



AD-3561
0.0



AD-3562
0.0



AD-3563
0.0



AD-3564
0.0



AD-3621
0.0



AD-3622
0.0



AD-3623
0.0



AD-3624
0.0



AD-3625
0.0



AD-3626
0.0





















TABLE 8








In vitro
In vitro




plasmid screen
plasmid



duplex
single dose
screen



name
(% silencing)
IC50 (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

















TABLE 10







Summary of Guinea Pig Study Dosing Regimen



















Treat-

Dose/




Formula-
Dose

ment
Num-
group


Group
siRNA
tion
(mg/kg)
Route
on days
ber
(mg)

















1
AD-11570
LNP09
2-3
IP
0, 3, 6
8
36


2
AD-11570
DODMA
2-3
IP
0, 3, 6
8
36


3
AD-1955
LNP09
2-3
IP
0, 3, 6
8
36


4
AD-1955
DODMA
2-3
IP
0, 3, 6
8
36


5
saline
—
—
IP
0, 3, 6
8
36
















TABLE 11







Summary of Guinea Pig Study with Different Dosing Regimens
















Dose

Treatment on



Group
siRNA
Formulation
(mg/kg)
route
days
N





1
AD-11570
LNP09
3
IP
0, 2, 4, 6, 8
8


2*
AD-11570
LNP09
1
IP
Daily (0-8)
8


3#
AD-11570
LNP09
1
IP
Daily (0-8)
8


4
AD-1955
LNP09
3
IP
0, 2, 4, 6, 8
8


5
AD-1955
LNP09
1
IP
Daily (0-8)
8


6
Saline
—
—
IP
Daily (0-8)
8





*Group 2 animals are dosed with siRNA at 2 hours prior to virus infection for the Day 0 dose.


#Group 3 animals are dosed with siRNA 30 minutes after virus infection for the Day 0 dose.


















TABLE 12







formu-


# of


Group
siRNA
lation
Route
Dose and timing
guinea pigs




















1
11570
P/Q
IV
3 mg/kg, 0, 2, 4, dpc
5


2
11570
E/F
IV
3 mg/kg, 0, 2, 4, dpc
8


3
1955
E/F
IV
3 mg/kg, 0, 2, 4, dpc
3


4
11570
E/F
IP
3 mg/kg, 0, 2, 4, dpc
8


5
PBS

IP
0, 2, 4, dpc
8






















TABLE 13











# of


Group
siRNA
Formulation
Route
Dose
Regimen
GP







1
11570
E/F
IV
3 mg/kg
D 0, 2, 4
6


2
11570
E/F
IV
1 mg/kg
D 0, 2, 4
6


3
11570
E/F
IV
0.3 mg/kg  
D 0, 2, 4
6


4
 1955
E/F
IV
3 mg/kg
D 0, 2, 4
5


5
N/A
N/A


D 0, 2, 4
8






















TABLE 14





Group
siRNA
Formulation
Route
Dose
Regimen
# of GP







1
11570
Lipid D
IV
0.3 mg/kg
D 0, 2, 4
6


2
11570
Lipid T
IV
0.3 mg/kg
D 0, 2, 4
6


3
11570
Lipid M
IV
0.3 mg/kg
D 0, 2, 4
6


4
11570
Lipid D
IV
  1 mg/kg
D 0, 2, 4
5


5
N/A
N/A


D 0, 2, 4
7























TABLE 15











2nd
#




Formu-



and 3rd
of


Group
siRNA
lation
Route
Dose
1st dose
dose
GP







1
11570
E/F
IV
3 mg/kg
Day 0, 1 hr
2, 4,
6







before
dpc







virus


2
11570
E/F
IV
3 mg/kg
Day 0, 1 hr
2, 4,
6







after virus
dpc


3
11570
E/F
IV
3 mg/kg
Day 1, 24 hr
2, 4,
6







after
dpc







virus


4
11570
E/F
IV
3 mg/kg
Day 2 48 hr
4, 6,
6







after virus
dpc


5
PBS

IP
0


4






















TABLE 16










# of




Treat-
Formula-

Dose and
guinea


Group
ment
tion
Route
Regimen*
pigs
Challenge





















1
11570
Lipid
IV
3 mg/kg,
12
100 pfu




D E/F

0, 2, 4, dpc

EBOV


2
 1955
Lipid
IV
3 mg/kg,
8
100 pfu




D E/F

0, 2, 4, dpc

EBOV


3
PBS
None
IP
0, 2, 4, dpc
8
100 pfu








EBOV


4
None
None
None
None
8
100 pfu








EBOV


5
Naïve
None
None
None
4
None




















TABLE 17





Group #
Treatment
day 5
day 7
day 10



















1
11570
n = 4
n = 4
n = 4


2
1955
n = 4
n = 4


3
PBS
n = 4
n = 4


5
Naïve
n = 4

















NP Chimeric sequence (SEQ ID NO: 1043):



GGTACCCTCGAGGAGGAAGATTAATAATTTTCCTCTCATTGAAATTTATATCGGAATTTAAATTG


 


AAATTGTTACTGTAATCACACCTGGTTTGTTTCAGAGCCACATCACAAAGATAGAGAACAACCTA


 


GGTCTCCGAAGGGAGCAAGGGCATCAGTGTGCTCAGTTGAAAATCCCTTGTCAACACCTAGGTCT


 


TATCACATCACAAGTTCCACCTCAGACTCTGCAGGGTGATCCAACAACCTTAATAGAAACATTAT


 


TGTTAAAGGACAGCATTAGTTCACAGTCAAACAAGCAAGATTGAGAATTAACCTTGGTTTTGAAC


 


TTGAACACTTAGGGGATTGAAGATTCAACAACCCTAAAGCTTGGGGTAAAACATTGGAAATAGTT


 


AAAAGACAAATTGCTCGGGTTTACCTGAGAGCCTACAACATGGATAAACGGGTGAGAGGTTCATT


 


GGCGCCGAGTCTCACTGAATCTGACATGGATTACCACAAGATCTTGACAGCAGGTCTGTCCGTTC


 


AACAGGGGATTGTTCGGCAAAGAGTCATCCCAGTGTATCAAGTAAACAATCTTGAAGAAATTTGC


 


CAACTTATCATACAGGCCTTTGAAGCAGGTGTTGATTTTCAAGAGAGTGCGGACAGTTTCCTTCT


 


CATGCTTTGTCTTCATCATGCGTACCAGGGAGATTACAAACTTTTCTTGGAAAGTGGCGCAGTCA


 


AGTATTTGGAAGGGCACGGGTTCCGTTTTGAAGTCAAGAAGCGTGATGGAGTGAAGCGCCTTGAG


 


GAATTGCTGCCAGCAGTATCTAGTGGAAAAAACATTAAGAGAACACTTGCTGCCATGCCGGAAGA


 


GGAGACAACTGAAGCTAATGCCGGTCAGTTTCTCTCCTTTGCAAGTCTATTTCTACCCAAACTTG


 


TCGTTGGAGAAAAGGCTTGCCTTGAGAAGGTTCAAAGGCAAATTCAAGTACATGCAGAGCAAGGA


 


CTGATACAATATCCAACAGCTTGGCAATCAGTAGGACACATGATGGTGATTTTCCGTTTGATGCG


 


AACAAATTTTCTGATCAAATTTCTCCTAATACACCAAGGGATGCACATGGTTGCCGGGCATGATG


 


CCAACGATGCTGTGATTTCAAATTCAGTGGCTCAAGCTCGTTTTTCAGGCTTATTGATTGTCAAA


 


ACAGTACTTGATCATATCCTACAAAAGACAGAACGAGGAGTTCGTCTCCATCCTCTTGCAAGGAC


 


CGCCAAGGTAAAAAATGAGGTGAACTCCTTTAAGGCTGCACTCAGCTCCCTGGCCAAGCATGGAG


 


AGTATGCTCCTTTCGCCCGACTTTTGAACCTTTCTGGAGTAAATAATCTTGAGCATGGTCTTTTC


 


CCTCAACTATCGGCAATTGCACTCGGAGTCGCCACAGCACACGGGAGTACCCTCGCAGGAGTAAA


 


TGTTGGAGAACAGTATCAACAACTCAGAGAGGCTGCCACTGAGGCTGAGAAGCAACTCCAACAAT


 


ACGCAGAGTCTCGCGAACTTGACCATCTTGGACTTGATGATCAGGAAAAGAAAATTCTTATGAAC


 


TTCCATCAGAAAAAGAACGAAATCAGCTTCCAGCAAACAAACGCTATGGTAACTCTAAGAAAAGA


 


GCGCCTGGCCAAGCTGACAGAAGCTATCACTGCTGCGTCACTGCCCAAAACAAGTGGACATTACG


 


ATGATGATGACGACATTCCATTTCCCGGGCCGATCTATGATGACGACAATCCTGGCCATCAAGAT


 


GATGATCCGACTGACTCACAGGATACGACCATTCCCGATGGTGTTGTTGACCCGTATGATGGAAG


 


CTACGGCGAATATCCTGACTACGAGGATTCGGCTGAAGGTGCACCAGATGACTTGGTCCTATTCG


 


ATCTAGACGAGGACGACGAGGACACTAAGCCAGTGCCTAATAGATCGACCAAGGGTGGACAACAG


 


AAGAACAGTCAAAAGGGCCAGCATATAGAGGGCAGACAGATCCGACCTTGGACGGAGCGAAAAAG


 


GTGCCGGAGTTGCAGAACAATCCACCACGCCAGTGCGCCACTCACGGACAATGACAGAAGAAATG


 


AACCCTCCGGCTCAACCAGCCCTCGCATGCTGACACCAATTAACGAAGAGGCAGACCCACTGGAC


 


GATGCCGACGACGAGAGTCTCACATCCCTGCCCTTGGAGTCAGATGATGAAGAGCAGGACAGGGA


 


CGGAACTTCCAACCGCACACCCACTGTCGCCCCACCGGCTCCCGTATACAGAGATCACTCTGAAA


 


AGAAAGAACTCCCGCAAGACGAGCAACAAGATCAGGACCACACTCAAGAGGCCAGGAACCAGGAC


 


AGTGACAACACCCAGTCAGAACACTCTTTTGAGGAGATGTATCGCCACATTCTAAGATCACAGGG


 


GCCATTTGATGCTGTTTTGTATTATCACCTAATGAGTGATGAGCCTGTAGTTTTCAGTACCAGTG


 


ATGGCAAAGAGTACACGTATCCAGACTCCCTTGAAGAGGAATATCCACCATGGCTCACTGAAAAA


 


GAGGCTATGAATGAAGAGAATAGATTTGTTACATTGGATGGTCAACAATTTTATTGGCCGGTGAT


 


GAATCACAAGAATAAATTCATGGCAATCCTGCAACATCATCAGTGAATGAGCATGGAACAATGGG


 


ATGATTCAACCGACAAATAGCTAACATTAAGTAGTCAAGGAACGAAAACAGGAAGAATTTTTGAT


 


GTCTAAGGTGTGAATTATTATCACAATAAAAGTGATTCTTATTTTTGAATTTAAAGCTAGCTTAT


 


TATTACTAGCCGTTTTTCAAAGTTCAATTTGAGTCTTAATGCAAATAGGCGTTAAGCCACAGTTA


 


TAGCCATAATTGTAACTCAATATTCTAACTAGCGATTTATCTAAATTAAATTACATTATGCTTTT


 


ATAACTTACCTACTAGCCTGCCCAACATTTACACGATCGTTTTATAATTAAGAAAAAAGCGGCCG


 


CAGAGCTC


 


GP Chimeric sequence (SEQ ID NO: 1044):


GGTACCCTCGAGGATGAAGATTAAGCCGACAGTGAGCGTAATCTTCATCTCTCTTAGATTATTTG


 


TTTTCCAGAGTAGGGGTCGTCAGGTCCTTTTCAATCGTGTAACCAAAATAAACTCCACTAGAAGG


 


ATATTGTGGGGCAACAACACAATGGGCGTTCTTAGCCTACTCCAATTGCCTCGTGATCGATTCAA


 


GAGGACATCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCACTTGGAG


 


TCATCCACAATAGCACATTACAGGTTAGTGAGATTGACCAGCTAGTCTGCAAGGATCATACTGAT


 


ATGCCATCTGCAACTAAAAGATGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGA


 


AGCTGGTGAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCGGACGGGAGCGAATGCT


 


TACCCGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGGTATGTGCACAAAGTATCAGGA


 


ACGGGACCGTGTGCCGGAGACTTTGCCTTCCATAAAGAGGGTGCTTTCTTCCTGTATGATCGACT


 


TGCTTCCACAGTTATCTACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGC


 


CCCAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAATGCAACGGAGGAC


 


CCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAGGCTACCGGTTTTGGAACCAATGAGAC


 


AGAGTACTTGTTCGAGGTTGACAATTTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGT


 


TTCTGCTCCAGCTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAAAACTA


 


ATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGGAGTGGGCCTTCTGGGAAACTAAAAA


 


AACCTCACTAGAAAAATTCGCAGTGAAGAGTTGTCTTTCACAGTTTTATCGCTCAACGAGACAGA


 


CATCAGTGGTCAGAGTCCGGCGCGAACTTCTTCCGGAAGAATCTCCGACCGGGCCACTGAAGACC


 


ACAAAATCATGGCTTCAGAAAATTCCTCTGCAATGGTTCAAGTGCACAGTCAAGGAAGGGAAACA


 


ACATTGCCGTCTCAGAATTCGACAGAAGGTCGAAGAGCGAGTCCCCAATCCCTCACAACCAAACC


 


AGGTCCGGACAACAGCACCCATAATACACCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAG


 


TTGAACAACATCACCGCAGAACAGACAACGACAGCACAGCCTCCGACACTCCCTCTGCCACGACC


 


GCAGCCGGACCCCCAAAAGCAGAGAACACCAACACGAGCAAGAGCACTGACTTCCTGGACCCCGC


 


CACCACAACAAGTCCCCAAAACCACAGCGAGACCGCTGGCAACAACAACACTCATCACCAAGATA


 


CCGGAGAAGAGAGTGCCAGCAGCGGGAAGCTAGGCTTAATTACCAATACTATTGCTGGAGTCGCA


 


GGACTGATCACAGGCGGGAGAAGAACTCGAAGAGAAGCAATTGTCAATGCTCAACCCAAATGCAA


 


CCCTAATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATCGGACTGGCCTGGATACCAT


 


ATTTCGGGCCAGCAGCCGAGGGAATTTACATAGAGGGGCTAATGCACAATCAAGATGGTTTAATC


 


TGTGGGTTGAGACAGCTGGCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAAC


 


GGAGCTGCGGACATATACCATACTCAACCGTAAGGCAATTGATTTCTTGCTGCAGCGATGGGGCG


 


GCACATGCCACATTCTGGGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACA


 


GACAAAATTGATCAGATTATTCATGATTTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGA


 


CAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTATAATTG


 


CAGTTATCGCTTTATTCTGTATATGCAAATTTGTCTTTTAGTTTTTCTTCAGATTGCTTCATGGA


 


AAAGCTCAGCCTCAAATCAATGAAACCAGGATTTAATTATATGGATTACTTGAATCTAAGATTAC


 


TTGACAAATGATAATATAATACACTGGAGCTTTAAACATAGCCAATGTGATTCTAACTCCTTTAA


 


ACTCACAGTTAATCATAAACAAGGTTTGACATCAATCTAGTTATCTCTTTGAGAATGATAAACTT


 


GATGAAGATTAAGAAAAAGCGGCCGCAGAGCTC


 


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):


GGCGCGCCTCGAGGAGGAAGATTAAGAAAAACTGCTTATTGGGTCTTTCCGTGTTTTAGATGAAG


 


CAGTTGAAATTCTTCCTCTTGATATTAAATGGCTACCCAACATACACAATACCCAGACGCTAGTT


 


ATCATCACCAATTGTATTGGACCAATGTGACCTAGTCACTAGAGCTTGCGGGTTATATTCATCAT


 


ACTCCCTTAATCCGCAACTACGCAACTGTAAACTCCCGAAACATATCTACCGTTTGAAATACGTG


 


TAACTGTTACCAAGTTCTTGAGTGATGTACCAGTGGCGACATTGCCCATAGATTTCATAGTCCCA


 


GTTCTTCTCAAGGCACTGTCAGGCAATGGATTCTGTCCTGTTGAGCCGCGGTGCCAACAGTTCTA


 


GATGAAATCATTAAGTACACAATGCAAGATGCTCTCTTCTTGAAATATTATCTCAAAAATGTGGG


 


TGCTCAAGAAGACTGTGTTGATGAACACTTTCAAGAGAAAATCTTATCTTCAATTCAGGGCAATG


 


ATTTTTACATCAAATGTTTTTCTGGTATGATCTGGCTATTTTAACTCGAAGGGGTAGATTAAATC


 


GAGGAAACTCTAGATCAACATGGTTTGTTCATGATGATTTAATAGACATCTTAGGCTATGGGGAC


 


TTGTTTTTTGGAAGATCCCAATTTCAATGTTACCACTGAACACACAAGGAATCCCCCATGCTGCT


 


ATGGACTGGTATCAGGCATCAGTATTCAAAGAAGCGGTTCAAGGGCATACACACATTGTTTCTGT


 


TTTACTGCCGACGTCTTGATAATGTGCAAAGATTTAATTACATGTCGATTCAACACAACTCTAAT


 


CTCAAAAATAGCAGAGATTGAGGATCCAGTTTGTTCTGATTATCCCAATTTTAAGATTGTGTCTA


 


TGCTTACCAGAGCGGAGATTACTTACTCTCCATATTAGGGTCTGATGGGTATAAAATTATTAAGT


 


TCCTCGAACCATTGTGCTTGGCCAAAATTCAATTATGCTCAAAGTACACTGAACGAAAAGGGCGG


 


TTTTAACACAAATGCATTTAGCTGTAAATCACACCCTAGAAGAAATTACAGAAATGCGTGCACTA


 


AAGCCTTCACAGGCTCAAAAGATCCGTGAATTCCATAGAACATTGATAAGGCTGGAGATGACGCC


 


ACAACACTTTGTGAGCTATTTTCCATTCAAAAACACTGGGGGCATCCTGTGCTACATAGTGAAAC


 


AGCAATCCAAAAAGTTAAAAAACATGCTACGGTGCTAAAAGCATTACGCCCTATAGTGATTTTCG


 


AGACATCTGTGTTTTTAAATATAGTATTGCCAAACATTATTTTGATAGTCAAGGATCTTGGTACA


 


GTGTTACTTCAGACCGATGTTTAACGCCGGGATTGAATTCTTATATCAAAAGAAATCAATTCCCT


 


CCGTTGCAATGATTAAAGAACTACTATGGGAATTTTACCACCTTGACCACCCTCCACTTTTCTCA


 


ACCAAAATTATTAGTGACTTAAGTATTTTTATAAAAGACAGAGCTACCGCAGTAGAAAGGACATG


 


CTGGGATGAGTATTCGAGCCTAATGTTCTAGGATATAATCCACCTCACAAATTTAGTACTAAACG


 


TGTACCGGAACAATTTTTAGAGCAAGAAAACTTTTCTATTGAGAATGTTCTTTCATACGCCCAAG


 


AACTTAGGTTCTACTACCACAATATCGGAACTTTTCTTTCTCATTGAAAGAGAAAGAGTTGAATG


 


TAGGTAGAACCTTCGGAAAATTGCCTTATCCGACTCGCAATGTTCAAACACTTTGTGAAGCTCTG


 


TTAGCTGATGTCTTGCTAAAGCATTTCCTAGCAATATGATGGTAGTTACGGAACGTGAGCAAAAA


 


GAAAGCTTATTGCATCAAGCATCATGGCACCACACAAGTGATGATTTTGGTGAACATGCCACAGT


 


TAGAGGGAGTACTTTGTAACTGATTTAGAGAAATACAATCTTGCATTTAGATATGAGTTTACAGC


 


ACCTTTTATAGAATATTGCAACCGTTGCTATGGTGTTAAGAATGTTTTTAATTGGATGCATTATA


 


CAATCCCACAGTTTATATGCATGTCAGTGATTATTATAATCCACCACATAACCTCACACTGGAGA


 


ATCGAGACAACCCCCCCGAAGGGCCTAGTTCATACAGGGGTCATATGGGAGGGATTGAAGGACTG


 


CAACAAAAACTCTGACAAGTATTTCATGTGCTCAAATTTCTTTAGTTGAAATTAAGACTGGTTTT


 


AAGTTACGCTCAGCTGTGATGGGTGACAATCAGTGCATTACTGTTTTATCAGTCTTCCCCTTAGA


 


GACTGACGCAGACGGCAGGAACAGAGCGCCGAAGACAATGCAGCGAGGGTGGCCGCCAGCCTAGC


 


AAAAGTTACAAGTGCCTGTGGAATCTTTTTAAAACCTGATGAGACTTTCGTACACTCAGGTTTTA


 


TCTATTTTGGAAAAAACAATATTTGAATGGGGTCCAATTGCCTCAGTCCCTTAAAACGGCTACAA


 


GAATGGCACCATTGTCTGATGCAATTTTTGATGATCTTCAAGGGACCCTGGCTAGTATAGGCACT


 


GCTTTTGAGCGATCAACTCCGAAACTAGACATATCTTTCCTTGCAGGATAACCGCAGCTTTCCAT


 


ACGTTTTTTTCGGTGAGAATCTTGCAATATCATCATCTCGGGTTCAATAAAGGTTTTGACCTTGG


 


ACAGTTAACACTCGGCAACCTCTGGATTTCGGAACAATATCATTGGCACTAGCGGTACCGCAGGT


 


GCTTGGAGGGTTATCCTTCTTGAATCCTGAGAAATGTTTCTACCGGAATCTAGGAGATCCAGTTA


 


CCTCAGGCTTATTCCAGTAAAAACTTATCTCCGAATAGAGACCTATTGAGCTCCACCGCGGTGGC


 


GGCCGCTCTAGCCCGGGCGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGT


 


CGACCTCGAGGGGGGGCCCGTACCTTACATCGCGTTAATTAACTAGTGGATCGATCCCCAATTCG


 


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


CGCGACGTAATACGACTCACTATAGGGCGAATTGGGCGCGCCCCGTCTCAGAATGATTGAGATGG


 


ATGATTTATTCTTACCTTTAATTGCGAAGAACCCTGGGAATTGTAGCGCAATTGACTTTGTGTAA


 


ATCCTAGCGGATTAAATGTCCCTGGGTCGCAAGACTTAACTTCATTTCTGCGCCAGATTGTACGC


 


AGGACCATCACCCTAAGTGCGAAAAACAAACTTATTAATACCTTATTTCATGCGTCAGCTGACTC


 


GAAGACGAAATGGTTTGTAAATGGCTATTATCATCAACTCCTGTTATGAGTCGTTTTGCGGCCGA


 


TATCTTTTCACGCACGCCGAGCGGGAAGCGATTGCAAATTCTAGGATACCTGGAAGGAACACGCC


 


ATTATTAGCCTCTAAGATCATCAACAATAATACAGAGACACCGGTTTTGGACAGACTGAGGAAAA


 


TAACATTGCAAAGGTGGAGCCTATGGTTTAGTTATCTTGATCATTGTGATAATATCCTGGCGGAG


 


CTTTAACCCAAATAACTTGCACAGTTGATTTAGCACAGATTCTGAGGGAATATTCATGGGCTCAT


 


ATTTTAGAGGGAAGACCTCTTATTGGAGCCACACTCCCATGTATGATTGAGCAATTCAAAGTGTT


 


TGGCTGAAACCCTACGAACAATGTCCGCAGTGTTCAAATGCAAAGCAACCAGGTGGGAAACCATT


 


CGTGTCAGTGGCAGTCAAGAAACATATTGTTAGTGCATGGCCGAACGCATCCCGAATAAGCTGGA


 


CTTCGGGGATGGAATCCCATACATTGGATCAAGGACAGAAGATAAGATAGGTCAGCCCGCTATTA


 


AGCCGAGGTGTCCTTCCGCAGCCTTAAGAGAGGCCATTGAATTGGCGTCCCGTTTAACATGGGTA


 


ACTAAGGCAGTTCGAACAGTGACTTGCTAATAAAACCATTTTTGGAAGCACGAGTAAATTTAAGT


 


GTTCAAGAAATACTTCAAATGACCCCTTCACATTACTCAGGAAATATTGTGCATCGGTATAACGA


 


TCAAACAGTCCTCATTCTTTCATGGCCAATCGTATGAGTAATTCAGCAACGCGCTTGATGGTATC


 


TACAAACACTTTAGGTGAGTTTTCAGGAGGTGGCCAGTCTGCACGCGACAGCAATATTATTTTCC


 


AGAATTTATAAATTATGCAGTTGCACTGTTCGATATTAAATTTAGAAACACTGAGGCTACAGATA


 


TCCAATATAATCGTGCTCACCTTCATCTAACTAAGTGTTGCACCCGGGAAGTACCAGCTCAGTAT


 


TTAACAACACAACCACGCTAAATCTAGATTTAACAAGATACCGAGAAAACGAATTGATTTATGAC


 


AGTAATCCTCTAAAAGGAGGACTCAATTGCAACTTATCGATTGACAGTCCTTTTTTCCAAGGTAA


 


ACGGCTGACATTATAGAAGATGATCTTATTCGACTGCCTCACTTATCTGGATGGGAGCTAGCCAA


 


GACCATCATGCAATCAATTATTTCAGATAGCAACAATTCATCTACAGACCCAATTAGCAGTGGAG


 


AAACAAGACATTCACTACCCATTTCTTAACTTATCCCAAGATAGGACTTCTGTACAGTTTTGGGG


 


CCTTTGTAAGTTATTATCTTGGCAATACAATTCTTTGCACGAAAAAGATCGGACTTGACAATTTT


 


TTATATTACTAACTACTCAAATTCATAATCTACCACATCGCTCATTGCGAATACTTAAGCCAACA


 


TTCAAACATGCAAGCGTTATGTCACGGTTAATGAGTATTGATCCTCATTTTTCTATTTACATAGG


 


CGGTGCTGCAGTGACAGAGGACTCTCAGATGCGGCCAGGTTATTTTTGAGAACGTCCATTTCATC


 


TTTTCTTACATTTGTAAAAGAATGGATAATTAATCGCGGAACAATTGTCCCTTTATGGATAGTAT


 


ATCCGCTAGAGGTCAAAACCCAACACCTGTGAATAATTTTCTCTATCAGATCGTAGAACTGCTGG


 


TGCATGATTCATCAAGACAACAGGCTTTTAAAACTACCATAAGTGATCATGTACATCCTCACGAC


 


AATCTTGTTTACCATGTAAGAGTACAGCCAGCAATTTCTTCCATGCATCATTGGCGTACTGGAGG


 


AGCAGACACAGAAACAGCAACCGAAAATACTTGGCAAGAGACTCTTCAACTGGATCAAGCACAAA


 


CAACAGTGATGGTATATTGAGAGAAGTCAAGAACAAACCACCAGAGATCCACATGATGGCACTGA


 


ACGGAATCTAGTCCTACAAATGAGCCATGAAATAAAAAGAACGACAATTCCACAAGAAAACACGC


 


ACCAGGGTCCGTCGTCCAGTCCTTTCTAAGTGACTCTGCTTGTGGTACAGCAAATCCAAAACTAA


 


ATTTCGATCGATCGAGACACAATGTGAAATTTCAGGATCATAACTCGGCATCCAAGAGGGAAGGT


 


CATCAAATAATCTCAACCGTCTAGTCCTACCTTTCTTTACATTATCTCAAGGGACACGCCAATTA


 


ACGTCATCCAATGAGTCACAAACCCAAGACGAGATATCAAAGTACTTACGGCAATTGAGATCCGT


 


CATTGATACTACCATAATTGTCGCTTCACCGGTATAGTCTCGTCCATGCATTACAAACTTGATGA


 


GGTCCTTTGGGAAATAGAGAGTTTCAAGTCGGCTGTGACGCTAGCAGAGGGAGAAGGTGCTGGTG


 


CCTTACTATTGATTCAAAATACGGCGTTAAGAAGTTATTTTTCAACACGCTAGCTACTGAGTCCA


 


GTATAGAGTCAGAAATAGTATCAGGAATGACTACTCCTAGGATGCTTCTACCTGTTATGTCAAAA


 


TTCCATAATGACCAAATTAGATTATTCTTAACAACTCAGCAAGCCAAATAACAGACATAACAAAT


 


CCTACTTGGTTTAAAGACCAAAGAGCAAGGCTACCTAAGCAAGTCGAGGTTATAACCATGGATGC


 


AGAGACAACAGAGAATATAACAGATCGAAATTGTACGAAGCTGTATATAAATTGATCTTACACCA


 


TATTGATCCTAGCGTATTGAAAGCAGTGGTCCTTAAAGTCTTTCTAAGTGATACTGAGGGTATGT


 


TATGGCTAAATGATAATTTACCCCGTTTTTTGCCACTGGTTATTTAATTAAGCCAATAACGTCAA


 


GTGCTAGATCTAGTGAGTGGTATCTTTGTCTGACGAACTTCTTATCAACTACACGTAAGATGCCA


 


CACCAAAACCATCTCAGTTGTAACAGGTAATACTTACGGCATTGCAACTGCAAATTCAACGAAGC


 


CCATACTGGCTAAGTCATTTAACTCAGTATGCTGACTGTGAGTTACATTTAAGTTATATCCGCCT


 


TGGTTTTCCATCATTAGAGAAATACTATACCACAGGTATAACCTCGTCGATTCAAAAAGAGGTCC


 


ACTAGTCTCTATCACTCAGCACTTAGCACATCTTAGAGCAGAGATTCGAGAATTAACTAATGATT


 


ATAATCAACAGCGACAAAGTCGACCCAGACTTATCATTTTATTCGTACTGCAAAAGGACGGATAA


 


CTAAACTAGTCAATGATTATTTAAAATTCTTTCTTATTGTGCAAGCATTAAAACATAATGGGACA


 


TGGCAAGCTGAGTTTAAGAAATTACAGAGTTGATTAGTGTGTGCAATAGGTTCTACCATATTAGA


 


GATTGCAATTGTGAAGAACGTTTCTTAGTTCAAACCTTATATTTACATAGAATGCAGGATTCTGA


 


AGTTAAGCTTATCGAAAGGCTGACAGGCTTCTGAGTTTATTTCCGGATGGTCTCTACAGGTTTGA


 


TTGAATTACCGTGCATAGTATCCTGATACTTGCAAAGGTTGGTTATTAACATACAGATTATAAAA


 


AAGCGGCCGCAGAGCTCCAGCGGTGGGGCCGCCGGCGTCTAGCCCGGGCGGATCCCTGCAGGAAT


 


TCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGCCCATGCAGGCCGGCCAGGTACCTTAGT


 


TAATTAACAGCTTTTGTTCCCTTTAGTAGGGTTAATTGACGCGCTC


 


VP24 (SEQ ID NO: 1045):


GGCGCGCCTCGAGGATGAAGATTAATGCGGAGGTCTGATAAGAATAAACCTTATTATTCAGATTA


 


GGCCCCAAGAGGCATTCTTCATCTCCTTTTAGCAAAGTACTATTTCAGGGTAGTCCAATTAGTGG


 


CACGTCTTTTAGCTGTATATCAGTCGCCCCTAGGCTAGGGTTTATAGTTGTCTCTAAGCTAAATT


 


GGTCTTGAACTAGTCTACTCGCAGAATCCTACCGGGAATAGACTAATTGAACTTAGCCGTTTAAA


 


ATTTAGTGCATAAATCTGGGCTAACACCACCAGGTCAACTCCATTGGCTGAAAAGAAGCTTACCT


 


ACAACGAACATCACTTTGAGCGCCCTCACAATTAAAAAATAGGAACGTCGTTCCAACAATCGAGC


 


GCAAGGTTTCAATATTATACGGGTCCATTAATTTCAACAAAATATTGATACTCCAGACACCAAGC


 


AAGACCTGAGAAAAAACCATGGCTAAAGCTACGGGACGATACAATCTAATATCGCCCAAAAAGGA


 


CCTGGAGAAAGGGGTTGTCTTAAGCGACCTCTGTAACTTCTTAGTTAGCCAAACTATTCAGGGGT


 


GGAAGGTTTATTGGGCTGGTATTGAGTTTGATGTGAACCAAAAGGGTATTACCCTATTGCATAGA


 


CTGAAAACTAATGACTTTGCCCCTGCATGGTCAATGACAAGGAATCTCTTTCCTCATTTATTTCA


 


AAATCCGAATTCCACAATTGAATCACCGCTGTGGGCATTGAGAGTCATCCTTGCAGCAGGGATAC


 


AGGACCAGCTGATTGACCAGTCTTTGATTGAACCCTTAGCAGGAGCCCTTGGTCTGATCTCTGAT


 


TGGCTGCTAACAACCAACACTAACCATTTCAACATGCGAACACAACGTGTCAAGGAACAATTGAG


 


CCTAAAAATGCTGTCGTTGATTCGATCCAATATTCTCAAGTTTATTAACAAATTGGATGCTCTAC


 


ATGTCGTGAACTACAACGGATTGTTGAGCAGTATTGAAATTGGAACTCAAAATCATACAATCATC


 


ATAACTCGGACTAATATGGGTTATCTTGTCGAGCTCCAAGAACCCGACAAATCTGCGATGGATAT


 


ACGACACCCTGGGCCGGCGAAATTTTCCTTACTACATGAATCGACACTTAAAGCATTTACACAAG


 


GATCCTCGACACGAATGCAAAGTTTGATTCTTGAATTTAATAGCTCTCTTGCTATCTAACTAAGG


 


TAGAAAAAATTGTACGATAGGGCTAACATTATGCTGACTCAATAGTTATCTTGACATCTCTGCTT


 


TCATAATCAGATATATAAGCATAATAAATAAATACTCATATTTCTTGATAATTTGTTTAACCACA


 


GATAAATCCTCACTGTAAGCCAGCTTCCAAGTTGACACCCTTACAAAAACCAGGACTCAGAATCC


 


CTCAAACAAGAGATTCCAAGACAACATCATAGAATTGCTTTATTATATGAATAAGCATTTTATCA


 


CCAGAAATCCTATATACTAAATGGTTAATTGTAACTGAACCCGCAGGTCACATGTGTTAGGTTTC


 


ACAGATTCTATATATTACTAACTCTAGAGCCCAAATTAACACGGTATAAGTAGATTAAGAAAAAA


 


GCCTGAGGAAGATTAAGAAAAAGCGGCCGCATTAATTAA


 


VP30 (SEQ ID NO: 1046):


GGCGCGCCTCGAGGATGAAGATTAAGAAAAAGGTAATCTTTCGATTATCTTTAATCTTCATCCTT


 


GATTCTACAATCATGACAGTTGTCTTTAGTGACAAGGGAAAGAAGCCTTTTTATTAAGTTGTAAT


 


AATCAGATCTGCGAACCGGTAGAGTTTAGTTGCAACCTAACACACATAAAGCATTGGTCAAAAAG


 


TCAATAGAAATTTAAACAGTGAGTGGAGACAACTTTTAAATGGAAGCTTCGTGAGCGCGGGAGAT


 


CAAGGAATTCACGTGCCGACCAGCAAGGGATGGACACGACCACCATGTTCGAGCACGATCATCAT


 


CCAGAGAGAATTATCGAGGTGAGTACCGTCAATCAAGGAGCGCCTCACAAGTGCGCGTTCCTACT


 


GTATTTCATAAGAAGAGAGTTGAACCATTAACAGTTCCTCCAGCACCTAAAGACATATGTCCGAC


 


CTTGAAAAAAGGATTTTTGTGTGACAGTAGTTTTTGCAAAAAAGATCACCAGCTTGAAAGCCTAA


 


CCGACCGGGAATTACTCCTACTAATCGCCCGTAAGACTTGTGGATCAGTTGATTCATCGCTTAAT


 


ATAACTGCACCCAAGGACTCGCGCTTAGCAAATCCAACGGCTGATGATTTCCAGCAAGAGGAAGG


 


TCCAAAAAATTACTAGTCGAGACTGCTCAAGACGGCAGAACACTGGGCGAGACAAGACATCAGAA


 


CCATAGAGGATTCAAAATTAAGAGCATTGTTGACTCTATGTGCTGTGATGACGAGGAAATTCTCA


 


AAATCCCAGCTGAGTCTTTTATGTGAGACACACCTAAGGCGCGAGGGGCTTGGGCAAGATCAGGC


 


AGAACCCGTTCTCGAAGTATATCAACGATTACACAGTGATAAAGGAGGCAGTTTTGAAGCTGCAC


 


TATGGCAACAATGGGACCGACAATCCCTAATTATGTTTATCACTGCATTCTTGAATATTGCTCTC


 


CAGTTACCGTGTGAAAGTTCTGCTGTCGTTGTTTCAGGCCTACGCTTACTTGCCCCCCCAAGCGT


 


TAATGAAGAAGCTTCAACCAACCCGGGGACATGCTCATGGTCTGATGAGGGTACCCCTTAATAAG


 


GCTGACTAAAACACTATATAACCTTCTACTTGATCACAATACTCCGTATACCTATCATCATATAT


 


TTAATCAAGACGATATCCTTTAAAACTTATTCAGTACTATAATCACTCTCGTTTCAAATTAATAA


 


GATGTGCATGATTGCCCTAATATATGAAGAGGTATGATACAACCCTAACAGTGATCAAAGAAAAT


 


CATAATCTCGTATCGCTCGTAATATAACCTGCCAAGCATACTCCCTAGAAGCGTTGAATCTTGTA


 


CACAAATAATGTTTTACTCTACAGGAGGTAGCAACGATCCATCCCATCAAAAAATAAGTATTTCA


 


TGACTTACTAATGATCTCTTAAAATATTAAGAAAAAGCGGCCGCATTAATTAA


 


VP35 (SEQ ID NO: 1047):


GGTACCGCGATCGCGATGAAGATTAAAACCTTCATCATCCTTACGTCAATTGAATTCTCTAGCAC


 


TCGAAGCTTATTGTCTTCAATGTAAAAGAAAAGCTGGTCTAACAAGATGACAACTAGAACAAAGG


 


GCAGGGGCCATACTGCGGCCACGACTCAAAACGACAGAATGCCAGGCCCTGAGCTTTCGGGCTGG


 


ATCTCTGAGCAGCTAATGACCGGCAAAATACCGCTAACCGACATCTTCTGTGATATTGAGAACAA


 


TCCAGGATTATGCTACGCATCCCAAATGCAACAAACGAAGCCAAACCCGAAGACGCGCAACAGTC


 


AAACCCAAACGGACCCAATTTGCAATCATAGTTTTGAGGAGGTAGTACAAACATTGGCTTCATTG


 


GCTACAGCTGTGCGTCGGCAAACCATCGCATCAGAATCATTAGAACAACGCATTACGAGTCTTGA


 


GAATGGTCTAAAGCCAGTTTATGATATGGCAAAAACAATATCATCCCTGAATCGCAGCTGTGCTG


 


AGATGGTTGCAAAATATGATCTTCTGGTGATGACAACCGGTCGGGCAACAGCAACCGCTGCGGCA


 


ACTGAGGCTTATTGGGCCGAACATGGTCAACCACCACCAGGCCCATCATTGTACGAGGATGGTGC


 


GATTCGGGGTAAATTGAAAGATCCGAACGGGACCGTCCCTCAAAGTGTTAGGGAGGCATTCAACA


 


ATCTAAACAGTACCACTTCACTAACTGAGGAAAATTTCGGGCGACCTTACATTTCGGCAAAGGAT


 


TTGAGAAACATTATGTATGATCACTTGCCTGGTTTTGGAACTGCTTTCCACCAATTAGTACAAGT


 


GATTTGTAAATTGGGAAAAGATAGCAACTCATTGGACATCATTCATGCTGAGTTCCAGGCCAGCC


 


TGGCTGAAGGAGACTCTCCTCAATGTGCCCTAATTCAAATTACAAAAAGAGTTCCAATCTTCCAA


 


GATGCTGCTCCACCTGTCATCCACATCCGCTCTCGAGGTGACATTCCCCGAGCTTGCCAGAAAAG


 


CTTGCGTCCAGTCCCACCATCGCCCAAGATTGATCGAGGTTGGGTATGTGTTTTTCAGCTTCAAG


 


ATGGTAAAACACTTGGACTCAAAATTTGAGCCAATGTAAGCTCATTTTGCGATGGGCGAATAATA


 


GCAGAGGCTTCAACTGCTGAACTATAGGGTACGTTACATTAATGATACACTTGTGAGTATCAGCC


 


CTGGATAATATAAGTCAATCCTAATCAATTGATAATATTGTTCATATCTCGCTAGCAGCTTAAAA


 


TATAAATGTAATAGGAGCTATATCTCTGACAGTATTATAATCAATTGTTATTAAGTAACCCAAAC


 


CAAAAGTGATGAAGATTAAGAAAAAGCGGCCGCAGAGCTC


 


VP40 (SEQ ID NO: 1048):


GGTACCTCGAGGATGAAGATTAAGAAAAACCTACCTCGGCTGAGAGAGTGTTTTTTCATTAACCT


 


TCATCTTGTAAACGTTGAGCAAAATTGTTAAAAATATGAGGCGGGTTATATTGCCTACTGCTCCT


 


CCTGAATATATGGAGGCCATATACCCTGTCAGGTCAAATTCAACAATTGCTAGAGGTGGCAACAG


 


CAATACAGGCTTCCTGACACCGGAGTCAGTCAATGGGGACACTCCATCGAATCCACTCAGGCCAA


 


TTGCCGATGACACCATCGACCATGCCAGCCACACACCAGGCAGTGTGTCATCAGCATTCATCCTT


 


GAAGCTATGGTGAATGTCATATCGGGCCCCAAAGTGCTAATGAAGCAAATCCCTATTTGGTTGCC


 


TCTAGGTGTCGCTGATCAAAAGACCTACAGCTTTGACTCAACTACGGCCGCAATTATGCTCGCAT


 


CTTATACGATCACCCATTTCGGCAAGGCAACCAACCCCCTCGTTAGAGTGAATCGACTGGGTCCT


 


GGAATCCCGGATCATCCCCTCAGGCTCCTGCGAATTGGAAACCAGGCTTTCCTCCAGGAGTTCGT


 


TCTTCCGCCAGTCCAACTACCCCAGTATTTCACCTTTGATTTGACAGCACTCAAACTGATCACCC


 


AACCACTGCCTGCTGCAACATGGACCGATGACACTCCAACAGGATCAAATGGAGCGTTGCGTCCA


 


GGAATTTCATTTCATCCAAAACTTCGCCCCATTCTTTTACCCAACAAAAGTGGGAAGAAGGGGAA


 


CAGTGCCGATCTAACATCTCCGGAGAAAATCCAAGCAATAATGACTTCACTCCAGGACTTTAAGA


 


TCGTGCCAATTGATCCAGCCAAGAGTATCATTGGGATCGAGGTGCCAGAAACTCTGGTCCACAAG


 


CTGACCGGTAAGAAGGTGACTTCTAAAAATGGACAACCAATCATCCCTGTTCTTTTGCCAAAGTA


 


CATTGGGTTGGACCCGGTGGCTCCAGGAGACCTCACCATGGTAATCACACAGGATTGTGACACGT


 


GTCATTCTCCTGCAAGTCTTCCAGCTGTGATTGAGAAGTAATTGCAATAATTGACTCAGATCCAG


 


TTTTATAGAATCTTCTCAGGGATAGCAACTCAATCGACTTTTAGGACCGTCCATTAGAGGAGACA


 


CTTTTAATTGAAAAATGTACTAATCGGGTCAAGGACCATTGTCTTTTTTCTCTCCTAAATGTAGA


 


ACTTAACAAAAGACTCATAATATACTTGTTTTTAAAGGATTGATTGATGAAAGAACATGCATAAG


 


CGATCCATACTTCGCCCTACTATAATCAATACGGTGATTCAAATGTTAATCTTTCTCATTGCACA


 


TACTTTTTGCCCTTATCCTCAAATTGCCTGCATGCTTACATCTGAGGATAGCCAGTGTGACTTGG


 


ATTGGAAATGTGGAGAAAAAATCGGGACCCATTTCTAGGTTGTTCACAATCCAAGTACAGACATT


 


GCCCTTCTAATTAAGAAAAAAGCGGCCGCAGAGCTC






Other embodiments are in the claims.

## Claims

1. A pharmaceutical composition for inhibiting the expression of a gene from an Ebola virus in an organism, comprising a double-stranded ribonucleic acid (dsRNA) and a lipid formulation comprising XTC/DSPC/Cholesterol/PEG-C14 in a ratio of 50%/10%/38.5%/1.5%; and
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 complementarity 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 pharmaceutical composition of claim 1, wherein said first sequence of said dsRNA consists of the sequence of SEQ ID NO:1027, and said second sequence consists of the sequence of SEQ ID NO:1028.

3. The pharmaceutical composition 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.

4. The pharmaceutical composition of claim 1, wherein said dsRNA comprises at least one modified nucleotide.

5. The pharmaceutical composition of claim 3, wherein said dsRNA comprises at least one modified nucleotide.

6. The pharmaceutical composition of claim 4, wherein said modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorthioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

7. The pharmaceutical composition of claim 4, 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.

8. A method for inhibiting the expression of a gene from an Ebola virus in a cell, the method comprising:
(a) introducing into the cell the pharmaceutical composition 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.

9. A method of treating, preventing or managing pathological processes mediated by Ebola expression comprising administering to a subject in need of such treatment, prevention or management a therapeutically of prophylactically effective amount of the pharmaceutical composition of claim 1.

10. The pharmaceutical composition of claim 1, wherein the dsRNA targets the VP35 of Ebola.

11. The pharmaceutical composition 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% compared to a control.

12. The pharmaceutical composition of claim 1, wherein said region of complementarity is 15-30 nucleotides in length.

13. The pharmaceutical composition of claim 1, wherein said region of complementarity is 19-24 nucleotides in length.

14. A method of increasing life-span of, decreasing viral titre in, or sustaining platelet count in a subject infected with an Ebola virus, comprising administering to the subject the pharmaceutical composition of claim 1 in an amount sufficient to increase the life-span of, decreasing viral titre in, or sustaining platelet count in the subject.

15. A pharmaceutical composition for inhibiting the expression of a gene from an Ebola virus in an organism, comprising a double-stranded ribonucleic acid (dsRNA) and a lipid formulation comprising a compound of formula X, selected from the group consisting of:



and 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 virus, and wherein said region of complementarity if less than 30 nucleotides in length.

16. The pharmaceutical composition of claim 15, wherein said first sequence of said dsRNA consists of the sequence of SEQ ID NO:1027, and said second sequence consists of the sequence of SEQ ID NO:1028.