isolated antiviral antisense oligonucleotide and pharmaceutical composition comprising the same

专利摘要:
OLIVONUCLEOTIDE ANTISENSE ANTIVIRAL ISOLATED, COMPOSITION FOR REDUCTION OF INFLUENZA VIRUS REPLICATION AND PHARMACEUTICAL COMPOSITION The present invention relates to antisense antiviral compounds and methods of their use and production in inhibiting the growth of the virus of the Orthomyxoviridae family and in the treatment of a viral infection. The compounds are particularly useful in the treatment of influenza virus infection in a mammal. Exemplary antisense antiviral compounds are substantially uncharged, or partially positively charged, morpholine oligonucleotides having 1) a nuclease-resistant structure, 2) 12-40 nucleotide bases, and 3) a target sequence of at least 12 bases in length that hybridizes to a target region selected from the following: a) the 25 bases of the 5 'or 3' terminal of the negative sense viral RNA segment of Influenzavirus A, influenzavirus B and influenzavirus C; b) the 30 terminal bases of the 5 'or 3' terminal of a positive sense vcRNA; c) the 45 bases surrounding the AUG start codon of an influenza viral mRNA and; d) the 50 bases surrounding the donor site or splice acceptor of the influenza mRNAs subjected to alternative splicing.
公开号:BR112012011381B1
申请号:R112012011381-0
申请日:2010-11-12
公开日:2020-12-22
发明作者:Patrick L. Iversen
申请人:Sarepta Therapeutics, Inc.；
IPC主号:

专利说明:

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[001] This application claims the benefit under 35 U.S.C. § 119 (e) of U.S. Provisional Patent Application No. 61 / 261,278, filed on November 13, 2009; Provisional Patent Application No. 61 / 292,056, filed on January 4, 2010; and U.S. Provisional Patent Application No. 61 / 377,382, filed on August 26, 2010, each of which is incorporated by reference in its entirety. Statement regarding Sequence Listing
[002] The Sequence Listing with this order is provided in the next format instead of a paper copy, and is hereby incorporated by reference in the specification. The name of the text file containing the Sequence Listing is 120178_456PC_SEQUÊNCIA_LISTING.txt. The text file is 33 KB, created on November 11, 2010, and is being submitted electronically through EFS-Web. Field of the Invention
[003] The invention relates to antisense oligonucleotides for use in the treatment of an influenza virus infection and antiviral treatment methods using oligonucleotides. Background of the Invention
[004] Influenza viruses have been a major cause of human mortality and morbidity throughout recorded history. Infection of the Influenza A virus causes millions of cases of severe illness and as many as 500,000 deaths each year around the world. Epidemics vary widely in severity, but they occur at regular intervals and always cause significant mortality and morbidity, most often in the elderly population. Although vaccines against combined influenza strains can prevent disease in 60-80% of healthy adults, the rate of protection is much lower in high-risk groups. In addition, vaccination does not provide protection against unexpected strains, such as H5 and H7 avian influenza that broke out in Hong Kong in 1997 and Europe and Southeast Asia in 2003 and 2004. Current anti-influenza drugs are limited in their ability to provide protection and therapeutic effect (Cox and Subbarao 1999; Cox and Subbarao 2000).
[005] Influenza A is a negative polarity segmented RNA virus. Genome segments are replicated by a complex of 4 proteins: 3 polypeptide polymerase (PA, PB1 and PB2) and NP (Nucleoprotein). The 5 'and 3' terminal sequence regions of all 8 genome segments are highly conserved within a genotype (Strauss and Strauss 2002).
[006] Influenza A virus can be subdivided according to the antigenic and genetic nature of its surface glycoproteins; 15 hemagglutinin subtypes (HA) and 9 neuraminidases (NA) have been identified so far. Viruses having all known HA and NA subtypes have been isolated from avian hosts, but only viruses of the subtypes H1N1 (1918), H2N2 (1957/58), and H3N2 (1968) have been associated with disseminated epidemics in humans (Strauss and Strauss 2002).
[007] Since 1997, when H5N1 influenza virus was transmitted to humans and killed 6 of the 18 infected people, there have been multiple transmissions of avian influenza viruses to mammals. Either the total virus is transmitted directly or gene segments from the avian influenza virus are acquired by mammalian strains. Widespread infections of poultry with H5N1 viruses in Asia have caused increased concern that this subtype may achieve human-to-human spread and establish inter-species transmission. The species whose different types of influenza viruses are capable of infecting are determined by different forms of the virus glycoproteins (HA, NA). This provides a considerable species barrier between birds and humans that is not easily overcome. Pigs, however, provide a "mix" capable of being infected by both types of viruses and thus allowing the passage of avian viruses to humans. When an individual pig cell is co-infected with both avian and human influenza viruses, recombinant forms can emerge that carry an avian HA genotype, but easily infect humans. Avian HA can infect pigs, but not humans. In pigs, during the packing of the genome segment, it is possible to create a virus with several Avian and Human HA segments and / or NA segments (Cox and Subbarao 2000).
[008] The influenza virus infects humans and animals (for example, pigs, birds, horses) and can cause acute respiratory disease. There have been numerous attempts to produce effective vaccines against influenza viruses. None, however, has been completely successful, particularly on a long-term basis. This may be due, at least in part, to the segmented characteristics of the influenza virus genome, which makes it possible, by re-grouping the segments, into numerous ways to exist. For example, it has been suggested that there may be an exchange of RNA segments between animal and human influenza viruses, which may result in the introduction of new antigenic subtypes in both populations. So, a long-term vaccination approach failed due to the emergence of new subtypes (antigenic "change"). In addition, the virus's surface proteins, hemaglutinin and neuraminidase, constantly undergo minor antigenic changes (antigenic drift). This high degree of variation explains why specific immunity developed against a particular influenza virus has not established protection against new variants. So, alternative antiviral strategies are needed. Although influenza B and C viruses cause less clinical disease than types A, new antiviral drugs should also be useful in slowing down infections caused by these agents.
[009] Influenza viruses that occur naturally among birds are called avian influenza (avian influenza). Birds carry viruses in their intestines, but they usually don't get sick from the infection. However, migratory birds can carry bird flu to infect chickens, ducks and domestic turkeys causing illness and even death. Avian influenza does not easily infect humans, but when human exposure is more frequent, such as contact with domestic birds, human infections occur. A dangerous bird flu (H5N1) was first identified in sternidae in South Africa in 1961 and was identified as a potentially deadly form of influenza. H5N1 spread occurred in eight Asian countries in late 2003 and 2004. At that time more than 100 million birds in those countries either died or were killed in order to control the spread. Beginning in June 2004, new fatal spread of H5N1 has been reported in Asia that is currently progressive. Human H5N1 infections have been observed in Thailand, Vietnam and Cambodia with a mortality rate of around 50 percent. These infections have most often occurred from human contact with poultry, but a few cases of human-to-human spread of H5N1 have occurred.
[0010] A triple rearranged influenza A virus (H1) has been circulating since 1998 with segments from pigs (HA, NP, NA, M and NS), human (PB1), and birds (PB2 and PA). The newly described and newly described influenza A virus of swine origin (2009H1N1 ((S-OIV), which is responsible for the spread of the progressive international disease, is a genetically rearranged triple virus that includes genetic elements of this pre-existing virus that it rearranged with the neuraminidase (NA) and matrix (M) segments of a Eurasian pig virus (Research Team S-OIV, 2009). The influenza A (H1) triple rearranged virus has occasionally been transmitted to humans but has not spread efficiently from human to human, but the new S-OIV is very efficient in human-to-human transmission. Recently, 3440 laboratory-confirmed cases of S-OIV infection have been reported from 29 countries. Mexico, where a total of 1364 cases have been documented, resulting in 45 deaths (case fatality rate 3.3%). Outside Mexico, there have been only three reported deaths (case fatality rate 0.1 %) .The reason for this desq The geographical balance in the death rate is not clear at this time.
[0011] While S-OIV is currently sensitive to the neuraminidase inhibitors oseltamivir and zanamivir, seasonal influenza has been previously documented to involve mutations that confer resistance to the neuraminidase inhibitor. Will S-OIV replace human H1 as the seasonal influenza virus or will S-OIV rearrange with another strain of influenza to create another new variant Will it evolve to become more lethal These uncertainties are compounded by the time interval from the identification of a new virus to the manufacture and distribution of a new vaccine. Also, a sufficiently new viral hemagglutinin antigen may require the use of large doses of immunogen and a “prime boost” scheme, posing practical difficulties for mass vaccination campaigns that should promptly elicit protective immunity. In view of these considerations, there is an urgent need to create new forms of prophylaxis and therapy for S-OIV in particular, ideally with broad activity against various strains, subtypes and types of influenza viruses.
[0012] An urgent need exists for new forms of treatment for influenza A based on (a) the known propensity of this virus to pass both low-level and less frequent antigenic change, but unpredictable major antigenic change leading to pandemic disease, (b) the clear failure of vaccination, even when strains are reasonably combined, to prevent influenza-related illness in a significant proportion of vaccine recipients, and (c) the increased frequency of resistance to approved forms of influenza therapy (for example, adamantane derivatives and, more recently, the neuraminidase inhibitor, oseltamivir).
[0013] In view of the severity of diseases caused by the influenza virus, there is an immediate need for new therapies to treat influenza infection. Given the lack of prevention or effective therapies, it is thus an object of the present invention to provide therapeutic compounds and methods for treating a host infected with an influenza virus. summary
[0014] Modalities of the present invention include, in one aspect, an anti-viral compound effective in inhibiting replication within a host cell of a viral RNA having a single-stranded negative sense genome and selected from the Orthomyxoviridae family including the genera Influenzavirus A, Influenzavirus B and Influenzavirus C. The compound can target viral RNA sequences within a region selected from the following: 1) 25 bases of the 5 'or 3' terminal of the negative sense viral RNA segments; 2) the 25 terminal bases of the 5 'or 3' terminal of the positive sense nRNA; 3) 45 bases surrounding the AUG start codons of influenza and viral mRNAs; 4) 50 bases surrounding the donor site or splice acceptor of influenza mRNAs subjected to alternative splicing.
[0015] In certain embodiments, the antiviral compound may include an oligonucleotide characterized by: a) a nuclease-resistant structure, b) 12-40 nucleotide bases, and c) a targeting sequence of at least 10 bases with - length, which hybridizes to a target region selected from the following from the following: i) the 25 bases 5 'or 3' terminal of a negative sense viral RNA segment of Influenzavirus A, Influenzavirus B and Influenzavirus C, such as a segment comprising M1 or M2, ii) the 25 terminal bases of the 5 'or 3' terminal of a positive sense cRNA for Influenzavirus A, Influenzavirus B and Influenzavirus C, iii) the 45 bases surrounding the AUG initiation codon an influenza viral mRNA, such as M1 or M2 mRNA, and iv) 50 bases surrounding the donor or splice acceptor sites of Influenzavirus A, Influenzavirus B and Influenzavirus C mRNAs subject to alternative splicing, such as M1 or M2 mRNA.
[0016] An oligonucleotide can also be characterized by: a) the ability to be actively taken up by mammalian host cells, and / or b) the ability to form a heteroduplex structure with the viral target region, in which said heteroduplex structure is: i) composed of positive or negative sense streak of the virus and the oligonucleotide compound, and ii) characterized by a dissociation Tm of at least 45oC.
[0017] Modalities of the present invention include, in another aspect, an antiviral compound that inhibits, in a mammalian host cell, replication of an infectious influenza virus having a negative, segmented, single-stranded genome selected from the family Orthomyxoviridae. The compound can be administered to infected host cells as an oligonucleotide characterized by the elements described above. The compound can be administered to a mammalian patient infected with the influenza virus, or at risk of infection with the influenza virus.
[0018] The compound can be composed of morpholine subunits linked by inter-subunit bonds containing uncharged phosphorus, joining a morpholine nitrogen of a subunit to an exocyclic carbon 5 'of an adjacent subunit. In one embodiment, intersubunit bonds are phosphorodiamidated bonds, such as those having the structure:
where Y1 = O, Z = O, Pj is a purine or pyrimidine-based pairing fraction effective at linking, by base-specific hydrogen bonding, to a base on a poly-nucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkylamino, for example, where X = NR2, where each R is independently hydrogen or methyl.
[0019] The compound can be composed of morpholine subunits linked with the unloaded bonds described above scattered with bonds that are positively charged. The total number of positively charged connections is between 2 and no more than half the total number of connections. The positively charged bonds have the above structure, where X is 1-piperazine.
The compound can include a covalent conjugate of a fraction analogous to the oligonucleotide capable of forming such a heteroduplex structure with the positive and negative sense RNA strand of the virus, and an effective arginine-rich polypeptide to increase uptake of the compound host cells. Exemplary polypeptides comprise one of the sequences identified as SEQ ID NOs: 115-128.
[0021] In a related aspect, embodiments of the present invention include a heteroduplex complex formed between: (a) the 25 bases of the 5 'or 3' terminal of the negative sense and / or viral RNA; (b) the 25 terminal bases of the 5 'or 3' terminal of the positive sense and / or mRNA; (c) 45 bases surrounding the AUG initiation codon of the viral mRNA and / or; (d) 50 bases surrounding the donor and splice acceptor sites of influenza mRNAs subjected to alternative splicing and; (e) an oligonucleotide characterized by: (i) a nuclease-resistant structure; (ii) capable of being captured by mammalian host cells, (iii) containing between 12-40 nucleotide bases, where the said heteroduplex complex has a dissociation Tm of at least 45oC.
[0022] In certain embodiments, an exemplary oligonucleotide may be composed of morpholine subunits linked by unloaded phosphorus-containing subunit bonds, joining a morpholine nitrogen from a subunit to an exocyclic 5 'carbon of an adjacent subunit. The compound may have phosphorodynamic bonds, such as in the structure
where Y1 = O, Z = O, Pj is a purine or pyrimidine-based pairing fraction effective at linking, by base-specific hydrogen bonding, to a base on a poly-nucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkylamino. In a preferred compound, X = NR2, where each R is independently hydrogen or methyl. Compound can also be composed of morpholine subunits linked with the unloaded bonds described above interspersed with bonds that are positively charged. The total number of positively charged connections is between 2 and no more than half of the total number of connections. The positively charged bonds have the above structure, where X is 1-piperazine.
The compound can be the oligonucleotide alone or a conjugate of the oligonucleotide and an arginine-rich polypeptide capable of increasing the uptake of the compound in host cells. Exemplary polypeptides have one of the sequences identified as SEQ ID NOs: 115-128. In yet another aspect, modalities of the present invention include an antisense oligonucleotide and related methods inhibiting replication in mammalian host cells of an influenza virus, having a single-stranded, segmented, negative-sense RNA genome selected from the family. Orthomyxoviridae. The compound can be characterized by the viral RNA elements described here. In certain modes, the cell is in a patient, typically a patient having an influenza virus infection.
[0024] In some modalities, the patient has a secondary bacterial infection, and the method also involves administering a bacterial antibiotic, separately or concomitantly with the anti-viral antisense oligonucleotide. In specific embodiments, the secondary bacterial infection is an infection of streptococcal pneumonia (for example, Streptococcus pneumomoniae). In certain modalities, the antibiotic is a beta-lactam. In specific modalities, the antibiotic is selected from penicillin, amoxicillin, cephalosporin, chloramphenicol and clindamycin.
[0025] Also included are methods of reducing replication of an influenza virus, comprising administering an antisense oligonucleotide targeting an RNA molecule encoding CD200 or the CD200 receptor, separately or concurrently with one or more antiviral antisense oligonucleotides here described.
[0026] A pharmaceutical composition comprising an antiviral antisense oligonucleotide described herein, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a bacterial antibiotic, such as penicillin, amoxicillin, cephalosporins, chloramphenicol, or clindamycin. In preferred embodiments, the bacterial antibiotic is bacteriostatic. In some embodiments, the pharmaceutical composition further comprises an antisense oligonucleotide targeted against an RNA molecule encoding CD200 or the CD200 receptor.
[0027] For treatment of the Influenza virus, such as Influenza A virus, the targeting sequence can hybridize to a region associated with one of the group of sequences identified as SEQ ID NOs: 1-11. Preferred target sequences are those complementary to either the negative target strand of SEQ ID NO: 4 or the positive target strand of SEQ ID NO: 2. Morpholine antisense phosphorodiamidate oligomers (“PMOs” that target these two regions are listed as SEQ ID NOs: 23 and 12, respectively. Brief Description of the Figures
[0028] Figure 1A shows a structure of morpholine oligomer with a phosphorodiamidate bond.
[0029] Figure 1B shows a morpholine oligometer as in Figure 1A, but where the structural bonds contain a positively charged group in the form of a phosphorodiamidate (piperazine) bond.
[0030] Figure 1C shows a conjugate of an arginine-rich peptide and an antisense oligomer, according to an embodiment of the invention.
[0031] Figures 1D-G show the segment of the repeating subunit of exemplary oligonucleotides morpholine, designated D to G.
[0032] Figure 2 shows the structure of a preferred exemplary antisense compound of the invention in a PMOplus® form (M1 / M2-AUGplus; SEQ ID NO: 13). The three phosphorodiamidate (piperazine) (pip-PDA) bonds transmit a positive charge network, then the term PMOplus®.
[0033] Figure 3 shows the three different species of influenza virus RNA present in infected cells, vRNA, mRNA and vcRNA, and the target location of the intended PMO described here.
[0034] Figure 4A shows the conservation of the 5'-terminus sequence of 60 M1 / M2 nucleotides from important influenza serotypes: H1N1, H1N1 (s-OIV), H5N1, H3N2, H9N2 and H7N7.
[0035] Figure 4B shows the percentage of isolates having the base indicated as the number subscribed after each base for the target M1 / M2-AUG (SEQ ID NO: 12).
[0036] Figures 5A-5B show the location of the target sequences of the invention relative to the initiation codon AUG and the 5'terminal of the vcRNA, respectively.
[0037] Figure 6 shows a dose-dependent reduction in viral titer using the M1 / M2-AUG target compounds of the invention (SEQ ID NOs: 12 and 13) in a H3N2 murine model system.
[0038] Figures 7A-7D show ferrets treated with M1 / M2-AUG (SEQ ID NOs: 12 and 13) have reduced clinical signs of influenza life after infection with a pandemic swine flu isolate 2009H1N1 (S-OIV).
[0039] Figure 7E shows ferrets infected with S-IOV and treated with the M1 / M2-AUG compounds of the invention (SEQ ID NOs: 12 and 13) led to a 2.3 log inhibition of viral titer.
[0040] Figures 8A-C show the effect of target PPMO for the splice acceptor site on viral HA RNA, expression of M1 protein and M2 protein, respectively.
[0041] Figures 9A-B show the effects of the LNA antisense oligomers targeted for the AUG initiation codon on viral HA RNA and M2 protein expression.
[0042] Figures 10A-B show the effect of the 2'OMe antisense oligomers targets for the M1 / M2 AUG initiation codon and splice acceptor site on viral HA RNA and M2 protein expression.
[0043] Figure 11 shows the inhibition of M1 and M2 protein expression in MDCK cells infected with H1N1 PR8 treated with a target PPMO for the M1 / M2 AUG initiation codon. Detailed Description Definitions
[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those versed in the technique to which the invention belongs. Although any methods and materials similar or equivalent to them described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[0045] The article “one” is used here to refer to one or more than one (that is, at least one) of the grammatical object of the article. For example, "an element" means an element or more than an element.
[0046] “Fence” means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies as much as 30, 25, 20, 25, 10, 9, 8 , 7, 6, 5, 4, 3, 2 or 1% to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or reference length.
[0047] By "coding sequence" is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
[0048] Through this specification, unless the context requires otherwise, the words "understand", "understand", and "understanding" will be understood to imply the inclusion of a step or element or group of declared steps or elements , but not excluding any step or element or groups of steps or elements.
[0049] By "consisting of" is intended to include, and limited to, whatever follows the phrase "consisting of". Then, the phrase “consisting of” indicates that the elements listed are required or mandatory, and that no other elements may be present. By "consisting essentially" it is intended to include any element listed after the sentence, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the elements listed. Then, the phrase “essentially consisting of” indicates that the elements listed are required or mandatory, but that other elements are optional and may or may not be present depending on whether or not it materially affects the activity or action of the listed elements.
[0050] The terms "complementary" and "complementarity" refer to polynucleotides (ie, a sequence of nucleotides) related by basic matching rules. For example, the “A-G-T” sequence is complementary to the “T-C-A” sequence. Complementarity can be “partial”, in which only some of the nucleic acid bases are combined according to the basic pairing rules. Or it can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strips has significant effects on the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is always desired, some modalities may include one or more, but preferably 6, 5, 4, 3, 2 or 1 combinations with target RNA breather. Variations in any location between the oligomer are included. In certain modalities, variations in the sequence near the end of an oligomer are generally preferable to variations in the interior, and if present they are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of the 5 'and / or 3 'terminal.
[0051] The terms "cell-penetrating peptide" or "CPP" are used in a changeable manner and refer to cationic cell-penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains. The peptides, as shown here, have the ability to induce cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells in a given culture population. cell, including all integers between them, and allow macromolecular translocation within multiple tissues in vivo under systemic administration.
[0052] The terms "antisense oligomer" or "antisense compound" or "antisense oligonucleotide" or "oligonucleotide" are used in a way that can be changed and refer to a sequence of cyclic subunits, each giving a reading fraction base, linked by inter-subunit bonds that allow the base pairing fractions to hybridize to a targeting sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid heteroduplex: oligomer within the targeting sequence. Cyclic subunits can be based on a ribose or other pentose sugar or, in certain embodiments, a morpholine group (see morpholine oligomer description below). Also contemplated are nucleic acid peptides (PNAs), blocked nucleic acids (LNAs), 2'-O-methyl oligonucleotides and interfering RNA agents (siRNA agents), and other antisense agents known in the art.
[0053] Such an antisense oligomer can be designed to block or inhibit mRNA translation or to inhibit natural pre-mRNA splice processing, or induce degradation of target mRNAs, and can be referred to "directly at" or "targeted against ”A targeting sequence with which it hybridizes. In certain modalities, the targeting sequence includes a region including an AUG start codon from an mRNA, a 3 'or 5' splice site from a preprocessed mRNA, a branch point. The targeting sequence can be within an exon or within an intron. The targeting sequence for a splice site can include an mRNA sequence having its 5 'terminal 1 at about 25 base pairs downstream of a normal splice acceptor junction in a pre-processed mRNA. A preferred splice site targeting sequence is any region of a pre-processed mRNA that includes a splice site or is either entirely contained within an exon coding sequence or traverses a splice acceptor or donor site. An oligomer is more generally said to be "directed against" a biologically relevant target, such as a protein, virus, or bacterium, when it is directed against the target's nucleic acid in the manner described above.
[0054] Included are antisense oligonucleotides that comprise, consist essentially of, or consist of one or more of SEQ ID NOs: 12-114. Also included are variants of these antisense oligomers, including variant oligomers having 80%, 85%, 90%, 97%, 98% or 99% (including all integers between them) sequence identity or sequence homology for any of SEQ ID NOs: 12-114, and / or variants that differ from those sequences by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, preferably those variants that inhibit replication of influenza in a cell. Also included are oligonucleotides of any one or more SEQ ID NOs: 12-114, which comprise an appropriate number of charged bonds, as described herein, for example, up to about 1 for every 2-5 unloaded bonds, such as about 4-5 for every 10 unloaded bonds, and / or which comprise an Arg rich peptide attached thereto, as also described herein.
[0055] The terms "oligomer morpholine" or "PMO" (oligomer morpholine phosphoramidate or phosphotodiamidate) refer to an oligonucleotide analog composed of structures of the morpholine subunit, where (i) the structures are joined together by bonds containing bonds phosphorus, one to three atoms together, preferably two atoms together, and preferably uncharged or cationic, joining nitrogen morpholine from a subunit to an exocyclic carbon 5 'from an adjacent subunit, and (ii) each morphine ring has a purine or pyrimidine or an equivalent base pairing fraction effective to bind, by base-specific hydrogen bonding, to a base on a polynucleotide. See, for example, the structure in Figure 1A, which shows a preferred type of phosphorodiamidate bond. Variations can be made to this link as long as they do not interfere with the link or activity. For example, oxygen linked to phosphorus can be replaced with sulfur (thiophosphorodiamidate). The 5 'oxygen can be replaced with amino substituted with amino or lower alkyl. Pending phosphorus-bound nitrogen can be unsubstituted, mono-substituted or di-substituted with (optionally substituted) lower alkyl. See also the discussion of katonic bonds below. The purine or pyrimidine-based fraction is typically adenine, cytosine, guanine, uracil, thymine or inosine. The synthesis, structures, and binding characteristics of morpholine oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,127,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063 and 5,506,337, and Appn. PCT Nos. PCT / US07 / 11435 (cationic bonds) and PCT Order No. US2008 / 012804 (improved synthesis), all of which are incorporated by reference.
[0056] The term "oligonucleotide analog" refers to an oligonucleotide having (i) a modified backbone structure, for example, a structure other than the standard phosphodiester bond found in natural oligo- and polynucleotides, and (ii) optionally, modified sugar fractions, for example, morpholine fractions instead of ribose or deoxyribose fractions. Oligonucleotide analogues support bases capable of binding hydrogen by the Watson-Crick base paired with standard polynucleotide bases, where the analogous structure present in the base in a way to allow such hydrogen bonding in a sequence-specific form between the analogous molecule oligonucleotide and bases in a standard polynucleotide (for example, single-stranded RNA or single-stranded DNA). Preferred analogs are those having a substantially uncharged phosphor containing structure.
[0057] A structure containing substantially uncharged phosphor in an analogue oligonucleotide is one in which a majority of subunit bonds, for example, between 50-100%, typically at least 60% to 100% or 75% or 80% of their bonds are either uncharged or substantially uncharged, and contain a single phosphorus atom. Antisense oligonucleotides and oligonucleotide analogs can contain between about 8 and 40 subunits, typically about 8-25 subunits, and preferably about 12 to 25 subunits. In certain modalities, oligonucleotides may have exact sequence complementarity to the targeting sequence or next complementarity, as defined below.
[0058] An “subunit: of an oligonucleotide refers to a nucleotide unit (or nucleotide analog). The term can refer to the nucleotide unit with or without the linked intersubunit bond, although, when referring to a “charged subunit”, the charge typically resides within the intersubunit bond (for example, a phosphate or phosphorothioate bond or a bond cationic, as shown in Figure 1B).
[0059] The parantine fraction of the purine or pyrimidine base is typically adenine, cytosine, guanine, uracil, thymine or inosine. Also included are bases such as pyrin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trime115toxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (for example, 5 -methylcitidine), 5-alkyluridines (for example, ribotimidine), 5-halouridine (for example, 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (for example, 6-methyluridine), kickback, kenosine, 2- thiouridine, 4-thiouridine, wibutosine, wibutoxosine, 4-acetylitidine, 5- (carboxyhydroxymethyl) uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-methyluridine, β-D-galactosylqueosine, 1-methyllosine methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbo-nilmethyl 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine, tr-derivatives eonin and others (Burgin et al, 1996, Biochemistry, 35, 14090; Uhlman & Peyman, above). By "modified bases" in this respect are meant nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as illustrated above; such bases can be used at any position in the antisense molecule. Versed in the technique they will appreciate that depending on the uses of the oligomers. Ts and Us are possible to exchange. For example, with other antisense chemicals such as antisense 2'-O-methyl oligonucleotides that are more like RNA, the T bases may be known as U (see, for example, Sequence Listing).
[0060] An "amino acid subunit" or "amino acid residue" may refer to an α amino acid residue (-CO-CHR-NH-) or a β residue or other amino acid residue (for example, -CO- (CH2) nCHR -NH-), where R is a side chain (which may include hydrogen) and n is 1 to 7, preferably 1 to 4.
[0061] The term “naturally occurring amino acid” refers to an amino acid present in proteins found in nature, such as amino acids 20 (L) used during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylisin , desmosine, isodesmosine, homocysteine, citrulline and ornithine. The term “unnatural amino acids” refers to those amino acids not present in proteins found in nature, examples include beta-alanine (β-Ala), 6-amino-exanoic acid (Ahx) and 6-aminopentanoic acid. Additional examples of "unnatural amino acids" include, without limitation, (D) -amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to those skilled in the art.
[0062] “Isolated” means material that is substantially or essentially free of components that normally accompany it in its native state. For example, an "isolated polynucleotide" or "isolated oligonucleotide", as used herein, may refer to a polynucleotide that has been purified or removed from sequences that flank it in a naturally occurring state, for example, a fragment of DNA that has been removed from sequences that are normally adjacent to the fragment.
[0063] An "effective amount" or "therapeutically effective amount" refers to an amount of therapeutic compound, such as an antisense oligomer or RNA interference agent (eg siRNA), administered to a mammalian patient, either as a single dose or as part of a series of doses, which is effective in producing a desired therapeutic effect. For an antisense oligomer, this effect is typically caused by inhibiting translation or natural splice processing of a selected targeting sequence. An “effective amount”, directed against an infectious influenza virus, also relates to an effective amount to reduce the replication rate of the infecting virus, and / or viral load, and / or symptoms associated with viral infection.
[0064] By "increasing" or "increasing" or "elevating" or "raising", or "stimulating" or "stimulating" generally refers to the ability of one or more antisense compounds or compositions or RNAi to produce or cause an greater physiological response (ie, downstream effects) in a cell or an individual, as compared to the response caused by either no antisense compound or a control compound. An "increased" or "high" amount is typically an "statistically significant" amount, and may include an increase that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 , 40, 50 or more times (eg 500, 100 times) (including all integers and decimal points between them and above 1), (eg 1.5; 1.6; 1.7 ; 1.8 etc) the amount produced by the antisense compound (the absence of an agent) or a control compound.
[0065] The term "reduce" or "inhibit" may generally relate to the ability of one or more antisense compounds or RNAi of the invention to "decrease" a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as a measure according to routine techniques in the diagnostic technique. Physiological or cellular responses (in vivo or in vitro) will be apparent to those skilled in the art, and may include reductions in the symptoms or pathology of influenza infection, or reductions in viral replication or viral load. A “decrease” in an answer can be “statistically relevant” as compatible with the response produced by a non-antisense compound or a control composition, and can include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% decrease, including all integers between them.
[0066] The term "targeting sequence" refers to a portion of the target RNA against which the oligonucleotide or antisense agent is directed, that is, the sequence to which the oligonucleotide will hybridize by Watson-Crick based pairing. of a complementary sequence. In certain embodiments, the targeting sequence may be a contiguous region of the viral negative strand RNA or viral mRNA, or it may be composed of regions of the 5 'and 3' terminal sequences of the viral or complementary viral genomic RNA.
[0067] The term "targeting sequence" or "antisense targeting sequence" refers to the sequence in an oligonucleotide or other antisense agent that is complementary (meaning, in addition, substantially complementary) to the targeting sequence in RNA genome. The entire sequence, or just a portion, of the antisense compound can be complementary to the targeting sequence. For example, in an oligonucleotide having 20-30 bases about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, or 29 can be target sequences that are complementary to the target region. Typically, the targeting sequence is formed from contiguous bases, but can alternatively be formed from non-contiguous sequences which, when placed together, for example, from opposite ends of the oligonucleotide, constitute the sequence that transposes the targeting sequence.
[0068] The target and intended sequences can be selected as by binding the antisense compound to a region within; 1) the 25 bases of the 5 'or 3' terminal of the negative sense viral RNA; 2) the 30 terminal bases of the 5 'or 3' terminal of the positive sense mRNA; 3) 45 bases around the AUG start codons of the viral mRNA and / or; 4) 50 bases surrounding the donor splice or acceptor sites of viral mRNAs subjected to alternative splicing. In certain embodiments, the target region may include 1) the 25 bases of the 5 'or 3' terminal of the M1 or M2 region of the negative sense viral RNA; 2) the 30 terminal bases of 5 'or 3' terminal of the M1 or M2 positive sense mRNA; 3) 45 bases surrounding the AUG start codons of the M1 or M2 mRNA and / or; 4) 50 bases surrounding the donor splice or acceptor sites for the viral M1 or M2 mRNAs. In certain embodiments, the target region may comprise both the AUG codon and the surrounding bases or contributing to the viral RNA donor splice site (for example, M1 or M2 mRNA), such as a polypyrimidine tract or lariat-forming sequence. In certain embodiments, using a single antisense oligomer or RNAi agent for the target, both the AUG initiation codon and the proximal donor splice sequences (eg, polypyrimidine tract) of the M1 / M2 RNA can provide synergistic effects with respect to expression of the reducing target protein, reducing viral replication, or both.
[0069] Target or intended sequences are described as “complementary” to one another when hybridization occurs in an antiparallel configuration. A targeting sequence can have "close" or "substantial" complementarity to the targeting sequence and still works for the purpose of the present invention, that is, it can still be functionally "complementary". In certain embodiments, an oligonucleotide may have at most a mismatch with the targeting sequence of 10 nucleotides, and preferably at most a mismatch at 20.
Alternatively, an oligonucleotide may have at least 90% sequence homology, and preferably at least 95% sequence homology, with the exemplary antisense target sequences described here.
[0071] An oligonucleotide "specifically hybridizes" to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions, with a Tm substantially greater than 45 ° C, preferably at least 50 ° C, and typically 60 ° C- 80 ° C or greater. Such hybridization preferably corresponds to severe hybridization conditions. At a given ionic strength and pH, Tm is the temperature at which 50% of a targeting sequence hybridizes to a complementary polynucleotide. Again, such hybridization can occur with “close” or “substantial” complementarity of the antisense oligomer for the targeting sequence, as well as with exact complementarity.
[0072] "Homology" refers to the percentage and number of amino acids that are identical or constitute conservative substitutions. Homology can be determined using sequence comparison programs such as GAP (Deveraux et al, 1984, Nucleic Acids Research 12, 387-395). Thus, sequences of a length similar or substantially different to those mentioned here can be compared by inserting spaces in the alignment, such spaces being determined, for example, by comparing the algorithm used by the GAP.
[0073] The recitations "sequence identity" or, for example, comprising a "sequence 50% identical to", as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or a amino acid-by-amino acid basis on a comparison window. Then, a “percent sequence identity” can be calculated by comparing two sequences optimally aligned in a comparison window, determining the number of positions in which the identical nucleic acid base (for example, A , T, C, G, I) or the identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Asn, Gln, Cys and Met) occurs in both sequences to produce the number of combined positions, dividing the number of combined positions by the total number of positions in the comparison window (that is, the size of the window), and multiplying the result per 100 to produce the sequence identity percentage.
[0074] Terms used to describe the sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A “reference sequence” is at least 8 to 10 but often 15 to 18 and always at least 25 units of monomers, including nucleotide and amino acid residues, in length. Because two polynucleotides can each comprise (1) a sequence (that is, only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, a sequence of comparisons between two (or more) polynucleotides are typically done by comparing sequences of two polynucleotides in a “comparison window” to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 where a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can comprise additions or deletions (ie spaces) of about 20% or less as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of two sequences. Optimal sequence alignment for alignment of a comparison window can be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (that is, resulting in the highest percentage of homology with the comparison window) generated by any of the several selected methods. Reference can also be made to the BLAST family of programs as, for example, revealed by Altschul et al, 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 by Ausubel et al, “Curriculum Protocols in Molecular BIology” John Wiley & Sons Inc. 1994-1998. Chapter 15.
[0075] A "nuclease-resistant" oligomeric molecule (oligomer) refers to one whose structure is substantially resistant to nuclease cleavage, in unhybridized or hybridized form; by extracellular and intracellular nucleases common in the body; that is, the oligomer shows little or no nuclease cleavage under normal nuclease conditions in the body to which the oligomer is exposed.
[0076] A "heteroduplex" refers to a duplex between an antisense oligonucleotide and the complementary portion of a target RNA. A "nuclease-resistant heteroduplex" refers to a heteroduplex formed by the attachment of an antisense oligomer to its complementary target, such that the heteroduplex is substantially resistant to degradation in vivo by intracellular and extracellular nucleases, such as RNaseH, which are capable of cutting double-stranded RNA / RNA or RNA / DNA complexes.
[0077] An "intracellular base-specific binding event involving a target RNA" refers to the specific binding of an antisense oligonucleotide to a target RNA sequence within the cell. The Bse specificity of such a link is sequence dependent. For example, a single-stranded polynucleotide can specifically bind to a single-stranded polynucleotide that is complementary in sequence.
[0078] As used herein, the term "body fluid" comprises a variety of types of samples obtained from a patient including, urine, saliva, plasma, blood, spinal fluid, or other sample of biological origin, such as epithelial cells or dermal debris, and may refer to cells or cellular fragments suspended in it, or the liquid medium and its solutes.
[0079] The term “relative quantity” is used where a comparison is made between a test measure and a control measure. The relative amount of a reagent forming a complex in a reaction is the amount of reagent with a test specimen, compared to the amount of reagent with a control specimen. The specimen control can be run separately in the same assay, or it can be part of the same sample (for example, normal tissue surrounding a malignant area in a tissue section).
[0080] "Treatment" of an individual or a cell of any type of intervention provided as a means of altering the natural course of a disease or pathology in the individual or cell. Treatment includes, but is not limited to, the administration of, for example, a pharmaceutical composition, and can be done either prophylactically, or subsequent to the initiation of a pathological event or contact with an etiologic agent. Treatment includes any desirable effect on the symptoms or pathology of a disease or condition associated with influenza virus infection. The related term “improved therapeutic result” relating to a patient diagnosed as infected with a particular virus, can refer to a slow or slow growth in the virus, or viral load, or detectable symptoms associated with infection by this particular virus.
[0081] Also included are “prophylactic” treatments, which can be directed to reduce the rate of progression of the disease or condition being treated, delaying the onset of this disease or condition, or reducing the severity of its onset. "Treatment" or "prophylaxis" does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.
[0082] An agent is "actively taken by mammalian cells" when the agent can enter the cell by a mechanism other than passive diffusion across the cell membrane. The agent can be transported, for example, by “active transport”, referring to the transport of agents through a mammalian cell membrane by, for example, an ATP-dependent transport mechanism, or by “facilitated transport”, referring to the transport of antisense agents through the cell membrane by a transport mechanism that requires binding of the agent to a carrier protein, which then facilitates the passage of the bound agent through the membrane. For active and facilitated transport, oligonucleotide analogs preferably have a substantially unloaded structure, as defined below.
[0083] Alternatively, the antisense compound can be formulated in a complexed form, such as an agent having an anionic structure complexed with cationic lipids or liposomes, which can be captured by cells by an endocytic mechanism. The antisense oligonucleotide can also be conjugated, for example, at the 5 'or 3' terminal, to an arginine-rich peptide, such as a portion of the HIV TAT protein, polyarginine, or for combinations of arginine and other amino acids including amino acids 6-aminoexanoic acid (Ahx) and beta-alanine (βAla). Exemplary arginine-rich delivery peptides are listed as SEQ ID NOs: 115-128. These exemplary arginine-rich delivery peptides facilitate transport in the target host cell as described (Moulton, Nelson et al, 2004).
[0084] Therefore, included are methods of treating an influenza virus infection, by administering one or more antisense oligomers of the present invention (for example, SEQ ID NOs: 12-114, and variants thereof), optionally as part of a pharmaceutical formulation or dosage form, to a patient in need of it. A "patient", as used herein, can include any animal that exhibits a symptom, or is a risk by exhibiting a symptom, that can be treated with an antisense compound of the invention, such as a patient who has or is at risk for having an influenza virus infection. Suitable individuals (patients) include laboratory animals (such as mice, mice, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and, preferably, human patients, are included.
[0085] Also contemplated are alternating methods of interfering RNA (RNAi), such as those involving double-stranded RNA molecules, or dsRNA. The term "double strand" means two separate nucleic acid strands comprising a region in which at least a portion of the strands is sufficiently complementary to the hydrogen bond and form a duplex structure. The term "duplex" or "duplex structure" refers to the region of a double-stranded molecule in which the two separated strands are substantially complementary, and then hybridize to each other.
[0086] "dsRNA" refers to a ribonucleic acid molecule having a duplex structure comprising two complementary and antisense nucleic acid strands (that is, the sense and antisense strands). Not all nucleotides in a dsRNA must exhibit Watson-Crick base pairs; the two strands of RNA can be substantially complementary. RNA strands can have the same or different number of nucleotides. The term "sdRNA" also includes "siRNA" or short interference RNA.
[0087] It will be understood that the term "ribonucleotide" or "nucleotide" can, in the case of a modified RNA or substitute nucleotide, also refer to a modified nucleotide, or substitution fraction substituted in one or more positions. So, the dsRNA is or includes a region that is at least partially complementary to the target RNA. In certain embodiments, sdRNA is completely complementary to the target RNA. It is not necessary to have perfect complementarity between the dsRNA and the target, but the match should be sufficient to allow the dsRNA, or a cleavage product of the same, to target specific silencing sequence, such as by cleaving the RNAi from the target RNA. Complementarity, or degree of homology with the target tape, is more critical on the antisense tape. While perfect complementarity, particularly in antisense tape, some modalities are always desired which may include one or more, but preferably 6, 5, 4, 3, 2, or less mismatches with respect to the target RNA. Mismatches are more tolerated in the terminal regions, and if present they are preferably in a terminal region or regions, for example, within 6, 5, 4, or 3 nucleotides of the 5 'and / or 3' terminal. This sense tape only needs to be substantially complementary to the antiseptic tape to maintain the overall double-tape character of the molecule.
[0088] As used herein, "modified dsRNA" refers to a dsRNA molecule that comprises at least one alteration that gives more resistance to nucleases (for example, protein kinase) than an identical dsRNA molecule that recognizes the same RNA target. dsRNAs can include a single stranded nucleotide and / or at least one substituted nucleotide.
[0089] As used herein, a "protruding nucleotide" refers to an unpaired nucleotide or nucleotides that protrude from the duplex structure when a 3 'end of an RNA strand extends beyond the 5' end of the other strand complement, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at the termination of the dsRNA, that is, no protruding nucleotides. A blunt ended dsRNA is a dsRNA that is double-stranded over its entire length, that is, no nucleotides protruding from its terminations in the molecule.
[0090] The term "base base pair", as used herein, refers to the last nucleotide of the base pair at a terminal in the duplex region of a double stranded molecule. For example, if a dsRNA or other molecule is a "blunt ended" (that is, it has no protruding nucleotides), the last nucleotide base pair at both ends of the molecule are terminal base pairs. Where a dsRNA or other molecule has a nucleotide protruding at one or both ends of the duplex structure, the last nucleotide base pair (s) immediately adjacent to the nucleotide (s) ( s) protruding (s) is the terminal base pair at that end (s) of the molecule.
Also included are vector delivery systems that are capable of expressing the target sequences of the oligomeric influenza virus of the present invention, as well as vectors that express a polynucleotide sequence comprising any one or more of SEQ ID NOs: 12 -114, or variants thereof, or that which expresses a polynucleotide sequence that is complementary to any or more of the target sequences of SEQ ID NOs: 1-11. Included are vectors that express siRNA or other duplex-forming RNA interference molecules.
[0092] By "vector" or "nucleic acid construction" is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, in which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a parent cell or tissue thereof, or be integrable with the defined host genome such that the cloned sequence is reproducible. Consequently, the vector can be an autonomously replicating vector, that is, a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, for example, a closed linear or circular plasmid, a extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any meaning to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicated along with the chromosome (s) into which it has been integrated.
[0093] A vector or nucleic acid building system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the host cell genome, or a transposon. The choice of the vector will typically depend on the vector's compatibility with the host cell into which the vector is to be introduced. In the present case, the vector or nucleic acid construct is preferably one that is operationally functional in a mammalian cell, such as a muscle cell. The vector can also include a selection marker such as an antibiotic or drug resistance gene, or a reporter gene (i.e., green fluorescence protein, luciferase), which can be used for selection or identification of suitable transformants or transfectants. Exemplary delivery systems may include viral vector systems (ie, virus-mediated transduction) including, but not limited to, retroviral vectors (eg, lentivirus), adenovirus vectors, adeno-associated viral vectors, and herpes virus vectors, among others known in the art.
[0094] The term "operably linked" as used herein means placing an oligomer coding sequence under the regulatory control of a promoter, which then controls the transcription of the oligomer.
[0095] A wild gene or gene product is one that is most frequently observed in a population and is then arbitrarily referred to as the “normal” or “wild” form of the gene.
[0096] "Alkyl" refers to a monovalent complementary saturated radical containing carbon and hydrogen, which can be branched, linear, or cyclic (cycloalkyl). Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl, n-heptyla, isopropyl, cyclopropyl, cyclopentyl, ethylcyclopentyl, and cyclohexyl. Generally preferred are alkyl groups having one to six carbon atoms, referred to as "lower alkyl", and exemplified by methyl, methyl, n-butyl, i-butylam-butyl, isoamyl, n-pentyl, and isopentyl. In one embodiment, lower alkyl refers to C1 to C4 alkyl.
[0097] "Alkenyl" refers to an unsaturated monovalent radical containing carbon and hydrogen, which can be branched, linear, or cyclic. The alkenyl group can be monounsaturated or polyunsaturated. Generally preferred are alkynyl groups having one to six carbon atoms, referred to as "lower alkenyl".
[0098] "Alquinyl" refers to a single or branched unsaturated branched hydrocarbon radical containing from 2 to 18 carbons comprising at least one carbon-carbon triple bond. Examples include without limitation ethynyl, propynyl, thal-propynyl, butynyl, thal-butynyl, tert-butynyl, pentynyl and hexynyl. The term "lower alkynyl" refers to an alkynyl group, as defined herein, containing between 2 to 8 carbohydrates.
[0099] "Cycloalkyl" refers to a mono- or poly-cyclic alkyl radical. Examples include, without limitation, cyclobutyl, cyclopentyl, cyclohexyl, cycloeptyl and cyclooctyl.
[00100] "Aryl" refers to a substituted or unsubstituted monovalent aromatic radical, usually having a single ring (for example, phenyl) or condensed rings (for example, naphthyl). This term includes heteroaryl groups, which are aromatic ring groups having one or more nitrogen, oxygen, or sulfur atoms, such as furyl, pyrrolyl, and indolyl. By "substituted" is meant that one or more hydrogen rings in the aryl group are replaced with a halide such as fluorine, chlorine, or bromine; with a lower alkyl group containing one or two carbon atoms; nitro, amino, methylamino, dimethylamino, methoxy, halomethoxy, halomethyl, or haloethyl. Preferred substitutes include halogen, methyl, ethyl, and methoxy. Generally preferred are aryl groups having a single ring.
[00101] "Aralkyl" refers to an alkyl, preferably lower alkyl (C1- C4, more preferably C1-C2), substituent which is further substituted with an aryl group; examples are benzyl (-CH2C6H5) and phenethyl (-CH2CH2C6H5).
[00102] "Thioalkoxy" refers to a radical of the formula -SRc where Rc is an alkyl radical as defined herein. The term "lower thioalkoxy" refers to an alkoxy group, as defined herein, containing between 1 and 8 carbons.
[00103] "Aloxy" refers to a radical of the formula -Orda where Rd is an alkyl radical as defined herein. The term "lower alkoxy" refers to an alkoxy group, as defined herein, containing between 1 and 8 carbons. Examples of alkoxy groups include, without limitation, methoxy and ethoxy. '
[00104] "Alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group.
[00105] "Carbonyl" refers to the radical -C (= O) -.
[00106] "Guanidinyl" refers to the radical H2N (C = NH2) -NH-.
[00107] "Amidinyl" refers to the radical H2N (C = NH2) CH-.
[00108] "Amino" refers to the radical -NH2.
[00109] "Alkylamino" refers to a radical of the formula -NHRd or -NRdRd where each Rd is, independently, an alkyl radical as defined herein. The term "lower alkylamino" refers to an alkylamino group, as defined herein, containing between 1 and 8 carbons.
[00110] "Heterocycle" means a 5- to 7-membered monocyclic ring, or 7- to 10-membered bicyclic ring, which is either saturated, unsaturated, or aromatic, and which contains 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur, and in which the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocyclics are fused to a benzene ring. The heterocycle can be attached through any heteroatom or carbon atom. Preferably, the atoms in the ring include 3 to 6 carbon atoms. Such heterocycles include, for example, pyrrolidine, piperidine, piperzine, and morpholine.
[00111] Heterocycles include heteroaryls as defined below. So, in addition to the heteroaryls listed below, heterocycles also include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizinyl, hydantoinyl, valerolactamil, oxiranyl, oxetanil, tetrahydrofuranil, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl, tetrahydropyridyl.
[00112] "Heteroaryl" means an aromatic heterocycle ring of 5 to 10 members and having at least one heteroatom selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono ring systems - and bicyclic. Representative heteroaryls are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazole, thiazolyl, benzoatiazolyl, isoxazolyl, pyrazolyl, pyridine, pyrololyl, pyridine, pyridine, cinolinyl, phthalazinyl and quinazolinyl.
[00113] The term "substituted", with respect to an alkyl, alkenyl, alkynyl, aryl, aralkyl, or alkaryl group, refers to the replacement of a hydrogen atom with a substituent containing heteroatom, such as, for example , halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, oxo (keto), nitro, cyano, or various acids or esters such as carboxylic, sulfonic or phosphonic.
[00114] The term "substituted", particularly with respect to an alkyl, alkoxy, thioalkoxy, or alkylamino group, refers to the replacement of a hydrogen atom on carbon with a substituent containing heteroatom, such as, for example, example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, oxo (keto), nitro, cyano, or various acids or esters such as carboxylic, sulfonic or phosphonic. It can also refer to the replacement of a hydrogen atom in a heteroatom (such as an amino hydrogen) with a group containing alkyl, carbonyl or another carbon.
[00115] In certain embodiments, the terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkoxy", "optionally substituted thioalkoxy", "substituted lower alkenyl", "optionally substituted lower alkoxy" ”,“ Optionally substituted lower thioalkoxy ”,“ optionally substituted lower amino alkyl ”and“ optionally substituted heterocyclyl ”means that, when substituted, at least one hydrogen atom is substituted with a substituent. In the case of an oxo substituent (= O), two hydrogen atoms are replaced. In this respect, substituents include: deuterium, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted cycloalkyl, oxo, halogen, -CN, -ORx, NRxRy, NRxC (= O ) Ry, NRxSO2Ry, -NRxC (= O) NRxRy, C (= O) Rx, C (= O) ORx, C (= O) NRxRy, -SOmRx and - SomNRxRy, where m is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted cycloalkyl and each of said optionally substituted alkyl substituents, alkenyl optionally substituted, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocycle and optionally substituted cycloalkyl can still be substituted c om one or more of oxo, halogen, -CN, -ORx, NRxRy, NRxC (= O) Ry, NRxSO2Ry, -NRxC (= O) NRxRy, C (= O) Rx, C (= O) ORx, C ( = O) NRxRy, - SOmRx and -SomNRxRy.
[00116] The selection of target sequences capable of inhibiting the replication of the influenza virus genome is discussed below. Target Virus
[00117] Modalities of the present invention are based, in part, on the discovery that effective inhibition of single-stranded, segmented, negative-sense RNA viruses can be achieved by exposing animals infected with influenza viruses to antisense oligonucleotide compounds ( i) that targets 25 bases of the 5 'or 3' terminal of the negative sense viral RNA; 2) the 30 terminal bases of the 5 'or 3' terminal of the positive sense mRNA; 3) 45 bases surrounding the AUG start codons of the viral and / or mRNA; 4) 50 bases surrounding the donor or splice acceptor sites of influenza mRNAs for alternative splicing and (ii) having physical and pharmacokinetic characteristics that allow effective interaction between the antisense compound and the virus within the host cells. In certain embodiments, oligomers can be used to treat a mammalian patient infected with influenza viruses.
[00118] Certain modalities of target RNA viruses having genomes which are: (i) single strand, (ii) segmented and (iii) negative polarity. Target viruses also synthesize two different versions of a positive polarity negative RNA (vRNA) genomic complement: 1) cRNA that is used as a standard for replicating negative sense RNA, and 2) a sense RNA complementary positive (mRNA) that is used for translating viral proteins. Figure 3 is a schematic example showing these different RNA species and the target location of the antisense PMO described in the present invention.
[00119] Target virus families include members of the Orthomyxoviridae family including the genera Influenzavirus B, Influenzavirus B and Influenzavirus C. Various physical, morphological and biological characteristics of members of the Orthomyxoviridae family can be found, for example, in the Textbook of Human Virology, R. Belshe, Ed. 2nd Edition, Mosby, 1991, in the Universal Virus Database of the International Committee on Virus Taxonomy (www.ncbi.nlm.nih.gov/ICTVdb/index.htm) and in human virology textbooks (see, for example (Strauss and Strauss 2002)). Some of these key biological characteristics of the Orthomyxoviridae family of viruses are described below.
[00120] Influenza A, influenza B and influenza C viruses are the only members of the genus Influenzavirus A, Influenzavirus B and Influenzavirus B, respectively. These viruses are membrane-enclosed viruses whose genomes are segmented sensory-negative (ie, less) strands of RNA ((-) RNA). The ten influenza virus genes are present in eight segments of the single-stranded RNA of strains A and B, and in seven segments of strain C. The segments vary in size (from 890 to 2341 nucleotides in length) and each it is a standard for synthesis of different mRNAs. The influenza virus virion contains virus-specific RNA polymerases necessary for mRNA synthesis from these standards, and in the absence of such specific polymerases, the strand less than the influenza virus RNA is not infectious. Beginning of mRNA transcription occurs when the influenza virus mRNA polymerase takes 12 to 15 nucleotides from the 5 'end of a cell precursor mRNA or mRNA and use of the copied oligonucleotide as a primer. This process has been termed "cap-snatching" because it places a 5 'cap structure on the viral mRNA. Generally, mRNAs made through this process encode only one protein. The viral RNA segments of gene M and NS gene also code for spliced mRNAs, which resulted in the production of two different proteins for each of these two segments.
[00121] RNA replication of the influenza virus occurs in the nucleus and involves the synthesis of three different RNA species. A schematic of this process is shown in Figure 3. After infection of a virgin cell, the strand-less virus RNA (vRNA) is transported to the nucleus where RNA for translation (mRNA) is synthesized using 10-13 nucleotide primers from the 5 'terminal cleaved by virus-encoded enzymes from cellular pre-RNA molecules (ie, cap-snatching). Synthesis of each mRNA continues until near the end of the genome segment where an enlarged oligo (U) is found and a poly (A) tail is added. The dedicated viral mRNAs are transported to the cytoplasm for translation and after sufficient viral proteins are transported back to the nucleus, synthesis of vRNA for nascent virions is initiated. An exact antigenomic copy of vRNA is synthesized (called cRNA) which is a perfect complement to genomic vRNA and serves as a standard for producing new vRNA. The different RNAs synthesized during replication of the influenza virus are shown schematically in Figure 3.
[00122] GenBank reference for exemplary viral nucleic acid target sequences representing influenza A genomic segments are listed in Table 1 below. The nucleotide sequence numbers in Table 1 are derived from the Genbank reference for the positive RNA strand. It will be appreciated that these sequences are only illustrative of other sequences in the Orthomyxoviridae family, as may be available from literature databases or available gene sequence patents. The strings below, identified as SEQ ID NOs: 1-11, are also listed in the Sequence Listing at the end of the specification.
[00123] Table 1 lists the targets for the influenza A, M1 and M2 viral genes codified by genomic segment 7. The target sequences in Table 1 represent; 1) the 25 bases of the 3 'terminal of the negative sense viral RNA (SEQ ID NO: 4); 2) the 25 terminal bases of the 5 'terminal of the positive sense mRNA (SEQ ID NO: 3); 3) 45 bases surrounding the AUG initiation codon of the indicated influenza virus genes (SEQ ID NO: 2). The sequences shown are the positive strand sequence (i.e., antigenic or mRNA) of the 5 'or 3' orientation except for SEQ ID NO: 4 which is the minus strand sequence (that is, genomic or virion RNA). It will be apparent that when the target is the vRNA strand minus the targeting sequence, it is the complement of the sequence listed in Table 1 unless otherwise noted, for example, SEQ ID NO: 4.
[00124] The M1 and M2 proteins are components of the viral matrix protein and ion channel activity, respectively. The two proteins are produced from alternative splice forms of segment 7 vcRNA using the same AUG starting site. The M2 protein is the target of two current anti-influenza therapies, amantadine and rimantadine. An exemplary targeting sequence for the AUG initiation codon region (-20 to +25 relative to the AUG initiation codon) of the M1 / M2 genes is represented as SEQ ID NO: 2 which is a subsequence of the terminal 60 nucleus region listed as SEQ ID NO: 1. The 3 '(25 nucleotide) terminal targeting sequence of the M1 / M2 segment is represented by SEQ ID NO: 3 which is also a subsequence of the terminal nucleotide region 60 and can be targeted on both positive strips (vcRNA) and negative strand (vRNA) of the segment. The 5'terminal sequence (SEQ ID NO: 3) can be a successful target on the strip less shown below as SEQ ID NO: 4. SEQ ID NOs: 1-4 are from the 2009H1N1 (S-OIV) virus and derived of an exemplary virus isolate found in the GenBank database under accession number GQ332646. 60 nucleotide regions of the 5 'terminal of other influenza A subtypes are listed in Table 1 as SEQ ID NOs: 5, 6, 7, 8 for H1N1, H5N1, H3N1, H3N2 and H2N2, respectively. Corresponding terminal AUG and target regions can be derived from these viral sequences using the guide described above.
[00125] It is also possible to target the donor and acceptor splice regions of the M1 / M2 segment. The splice donor and splice acceptor sites are nucleotides 51 and 740, respectively. Target of each of the splice junction using antisense compounds of the invention is contemplated. In addition, it is possible to block both the AUG starting sites and the splice donor sites using appropriately designed antisense compounds (for example, SEQ ID NOs: 12-16 and 19-22). The splice acceptor target region is shown below for the 2009H1N1 (S-OIV) subtype as SEQ ID NO: 10. The corresponding region for the H5N1 subtype is listed in Table 1 as SEQ ID NO: 9.
[00126] Furthermore, it is contemplated that any sensitive region of translation, sensitive to splice or sensitive to replication of the M1 / M2 segment can be targeted using compounds of the invention. The M1 / M2 sequence reference (segment 7) for the prototype H1N1 subtype (Puerto Rico / 8/34) is shown in Table 1 as SEQ ID NO: 11 and can be found in the GenBank Reference Sequence database on NC_00216. Corresponding M1 / M2 segment sequences can be obtained from publicly available sequence databases. It is contemplated that antisense compounds of the invention can be targets for other regions of this segment with the expectation that target regions sensitive to additional translation, splice and / or replication can be identified. Table 1: Exemplary Influenza Virus Nucleic Acid Target Sequences



[00127] Figure 4 shows the conservation of the 60 nucleotides of the 5 'terminal of the M1 / M2 segment from important influenza A subtypes: H1N1, H1N1 (S-OIV), H5N1, H3N2, H9N2 and H7N7. Figure 4B shows conservation of the target sequences in an important influenza serotype, H1N1 (2009), also known as swine influenza (S-OIV), for each base of a preferred PMO (M1 / M2-AUG; SEQ ID NO: 12) based on the NCBI influenza database of genomic sequences (Bao Y., P. Bolotov, D. Dernovoy, B. Kiryutin, L. Zaslavsky, T. Tatusova, J. Ostell, and D. Lipman. The Influenza Virus Resource at the National Center for Biotechnology Information. J. Virol. 2008 Han; 82 (2): 596-601). The capital letter indicates the target base and the number subscribed near the base indicates the conservation percentage for this base for H1N1 (2009) isolated in the database as indicated above the sequence. These data indicate that no base position shows any significant variation for the target M1 / M2-AUG for H1N1 (2009).
[00128] In certain embodiments, antisense target sequences are designed to hybridize to a region of one or more of the target sequences listed in Table 1. Selected antisense target sequences can be made shorter, for example, about 12 bases, or longer, for example, about 40 bases, and include a small number of mismatches, as soon as the sequence is sufficiently complementary to the effect of translation, splice and / or inhibition of replication on hybridization with the target, and forms with the viral RNA, a heteroduplex having a Tm of 45 ° C or more.
[00129] In certain modalities, the degree of complementarity between the target and the antisense targeting sequence is sufficient to form a stable duplex. The region of complementarity of the antisense oligomers with the target RNA sequence can be as short as 8-11 bases, but is preferably 12-15 bases or more, for example, 12-20 bases, or 12-25 bases, including all numbers integers between these variations. An antisense oligomer of about 14-15 bases is generally long enough to have a unique complementary sequence in the viral genome. In certain embodiments, a minimum length of complementary bases may be required to achieve the requirement of connection Tm, as discussed below.
[00130] In certain modalities, oligomers as long as 40 bases may be suitable, where at least a minimum number of bases, for example, 10-12 bases are complementary to the targeting sequence. In general, however, facilitated or active uptake in cells is optimized at oligomer lengths of less than about 30. For PMO oligomers, described in additional below, an optimal balance of binding and uptake stability generally occurs at lengths of 18 -25 bases. Included are antisense oligomers (for example, PNAs, LNAs, 2'-OMe) and PMO oligomers consisting of about 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bases, where at least about 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 contiguous or non-contiguous bases are complementary to the target sequences of SEQ ID NOs: 1-11, or variants thereof.
[00131] In certain embodiments, antisense oligomers may be 100% complementary to the viral nucleic acid targeting sequence, or they may include mismatches, for example, favored variants, as soon as a heteroduplex formed between the oligomer and viral nucleic acid targeting sequence is sufficiently stable to resist the action of cellular nucleases and other modes of degradation that can occur in vivo. Structures of oligomers that are less susceptible to nuclease breakdown are discussed below. Mismatches, if present, are less destabilizing to the terminal regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligomer, the percentage of base pairs G: C in the duplex, and the position of mismatch (s) in the duplex, according to the well-understood principles of stability duplex. Although such an antisense oligomer is not necessarily 100% complementary to the viral nucleic acid targeting sequence, it is effective to stably and specifically bind to the targeting sequence, such that a biological activity of the target nucleic acid, for example, protein expression viral (s), is modulated.
[00132] The stability of the duplex formed between an oligomer and a targeting sequence is a function of the binding Tm and the susceptibility of the duplex to cellular enzymatic cleavage. The Tm of an antisense compound with respect to complementary sequence RNA can be measured by conventional methods, such as those described by Hames et al, Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C.G. and Wallace R.B., 1987, Oligonucleotide Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. In certain modalities, antisense oligomer may have a Tm ligand, with respect to a complementary sequence RNA, with a better body temperature and preferably greater than about 45 ° C or 50 ° C. Tm in the range of 60-80 ° C or larger is preferred. According to well-known principles, the Tm of an oligomer compound, with respect to a complementary hybrid base RNA, can be increased by increasing the proportion of the paired C: G bases in the duplex, and / or by increasing the length ( base pairs) of the heteroduplex. At the same time, for purposes of optimizing cell uptake, it may be advantageous to limit the size of the oligomer. For this reason, compounds that show high Tm (45-50 ° Cou greater) over a length of 25 bases or less are generally preferred over those requiring more than 25 bases for high Tm values.
[00133] In certain embodiments, such as PMO oligomers, the antisense activity of an oligomer can be increased by using a mixture of uncharged and cationic phosphorodiamidate bonds, as exemplified in Figure 1B. The total number of cationic bonds in the oligomer can range from 1 to 10 (including all integers between them), and be spread across the oligomer. Preferably the number of charged connections is at least 2 and not more than half of the total structural connections, for example, between 2, 3, 4, 5, 6, 7 or 8 positively charged connections, and preferably each charged connection it is separated with the structure by at least 1, 2, 3, 4 or 5 unloaded connections. The antisense activity of various oligomers can be measured in vitro by fusing the target region of the oligomer to the 5 'terminal of a reporter gene (eg, firefly luciferase) and then measuring the inhibition of the mRNA translation of the fusion gene transcribed in the assays cell-free translation. The inhibitory properties of oligomers containing a mixture of uncharged and cationic bonds can be increased by approximately five to 100 times in cell free translation assays. A preferred antisense oligomer of the invention (M1 / M2-AUG) in a form that contains three cationic bonds, as illustrated in Figure 1B and Figure 2, spread across the oligomer is shown as SEQ ID NO: 13 in Table 2 below. A series of exemplary antisense oligomers that target M1 / M2 AUG and contain three scattered cationic bonds are shown in SEQ ID NOs: 34-47.
[00134] Table 2 below shows exemplary target sequences, in a 5'-to-3 'orientation, which are complementary to the influenza A virus. The listed sequences provide a collection of the target sequences from which target sequences can selected according to the general class rules discussed above. While the listed target sequences can be used for any antisense analog oligonucleotide chemistry (for example, PNA, LNA or 2'-OMe) the sequences in Table 2 are preferred for use as PMO antisense oligomers. SEQ ID NOs: 12- 22, 25-29 and 34-47 are antisense to the positive strand (mRNA or vcRNA) of the virus although SEQ ID NOs: 23 and 24 are antisense to the minus strand (vRNA). So, for example, in selecting a target against the 3 'end of the ribbon minus the segment encoding M1 / M2 (influenza A segment 7) SEQ ID NO: 4, or a portion of the effective sequence to block the function of the 3 'less ribbon terminal can be selected. SEQ ID NOs: 12-29 and 34-47 targeting the M1 / M2 segment of Influenza A H1N1 subtype (S-OIV) although SEQ ID NOs: 30-33 targeting the PB1 or NP segments as indicated. Table 2. Exemplary Antisense Target Sequences


Antisense Oligonucleotide Compounds
[00135] As detailed above, the antisense oligonucleotide (the term "antisense" indicates that the compound is directed against either virus positive sense or negative sense or minus strand RNA) typically comprises a base sequence targeted by a region that includes one or more of the following: 1) the 25 bases of the 5 'or 3' terminal of the negative sense viral RNA; 2) the 30 terminal bases of the 5 'or 3' terminal of the positive sense vcRNA; 3) 45 bases surrounding the AUG start codons of the viral mRNA and / or '4) 50 bases surrounding the donor or splice acceptor sites of the influenza mRNAs for alternative splicing. In addition, the oligomer is capable of effectively targeting infecting viruses when administered to a host cell, for example, in an infected mammalian patient, such as by reducing the expression of the target protein (for example, M1 or M2 or both ), by reducing viral replication or both. This requirement is typically met when the oligomer compound (a) has the ability to be actively captured by mammalian cells, and (b) once captured, forms a duplex with the target RNA with a Tm greater than about 45o C.
[00136] In certain embodiments, the structure of the oligomer can be substantially unloaded, and, preferably, it can be recognized as a substrate for active or facilitated transport across the cell membrane. The ability of the oligomer to form a stable duplex with the target RNA can also relate to other characteristics of the oligomer structure, including the length and degree of complementarity of the antisense oligomer with respect to the target, the ratio of combinations of bases G: C to A: T, and the positions of any mismatched bases. The ability of the antisense oligomer to resist cellular nucleases can promote survival and final distribution of the agent to the cell cytoplasm. Sequences of exemplary antisense oligomers of the invention using the PMO structure chemistry are listed in Table 2. Target sequences using alternative chemistries are listed below in Table 3 and 4 for PNA and LNA chemistries, respectively. In general, PNA and LNA chemists use shorter target oligomers due to their relatively high target binding strength compared to PMO and 2’O-Me oligomers.
[00137] Peptide nucleic acids (PNAs) are DNA analogues in which the structure is structurally homomorphic with a deoxyribose structure, consisting of N- (2-aminoethyl) glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying the Watson-Crick base matching rules, and mimic DNA in terms of base matching recognition (Egholm, Buchardt et al, 1993). The structure of PNAs is formed by peptide bonds instead of phosphodiester bonds, making them well suited for antisense applications (see structure below). The structure is unloaded, resulting in the PNA / DNA or PNA / RNA duplexes that exhibit better than normal thermal stability. PNAs are not recognized by nucleases or proteases.
[00138] PNAs can be produced synthetically using any technique known in the art. A PNA is a DNA analogue in which a polyamide structure replaces the traditional DNA ribose phosphate ring, as illustrated below.

[00139] Despite a radical structural change in the natural structure, PNAs are able to bind the specific sequence in a helical form to DNA or RNA. Characteristics of PNAs include a high affinity of binding to complementary DNA or RNA, a destabilizing effect caused by a mismatch of single base, resistance to nucleases and proteases, hybridization with DNA or RNA regardless of salt concentration and triplex formation with homopurine DNA . PANAGENE® has developed its proprietary BTS PNA monomers (Bts; benzotriazol-2-sulfonyl group) and oligomerization process property. PNA oligomerization using Bts PNA monomers is made up of repeated cycles of deprotection, coupling and coverage. Exemplary patents to this technology include US Patent Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006 and 7,179,896. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, Pat. USA Nos. 5,539,082; 5,714,332; and 5,719,262, each of which is incorporated by reference. Additional teaching of PNA compounds can be found in Nielsen et al, Science, 254: 1497-1500, 1991.
[00140] Exemplary PNA compounds for practicing the invention are listed below in Table 3. These oligonucleotides can be essentially according to the procedures disclosed in the references cited here. Table 3. Exemplary Antisense PNA Target Sequences


[00141] Oligonucleotide compounds of the present invention can also contain "blocked nucleic acid" (LNAs) subunits. LNA structures can be found, for example, in Wengel, et al, Chemical Communication (1998) 455; Tetrahedron (1998) 54: 3607, and Accounts of Chem. Research (1999) 32: 301); Obika, et al, Tetrahedron Letters (1997) 38: 8735; (1998) 39: 5401, and Bioorganic Medicinal Chemistry (2008) 16: 9230. Exemplary non-limiting LNA structures are illustrated below:

[00142] Compounds of the invention can incorporate one or more LNAs; in some cases, the compounds may be entirely composed of LNAs. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are known in the art: US Patent Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461. Typical intersubunit linkers include phosphodiester and phosphorothioate fractions; alternatively, linkers containing non-phosphorus can be employed. A preferred embodiment is a compound containing LNA where each LNA subunit is separated by a DNA subunit. Still preferred compounds are composed of alternating LDA and DNA subunits where the linker intersubunit is phosphorothioate.
[00143] The following compounds are prepared essentially according to the procedures disclosed in the references cited above. Exemplary compounds containing LNA subunits (LNAs are capitalized, DNAs are in a smaller case, and the sequences are read from 5'to 3 ') are shown below in Table 4. Table 4. Exemplary LNA Antisense Target Sequences



[00144] A preferred oligomer structure employs subunits based on morpholine having fractions paired with the bases, joined by unloaded bonds, as described above. Especially preferred is a substantially uncharged phosphorodiamidate-linked morpholine oligomer. Morpholine oligonucleotides, including antisense oligomers, are detailed, for example, in U.S. Patent with Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063 and 5,506,337, and PCT application No. US2008 / 012804, all of which are expressly incorporated by reference.
[00145] Certain properties of the morpholino-based subunits include: the ability to be linked in an oligomeric form by unloaded and stable structural bonds; the ability to support a nucleotide base (eg, adenine, guanine or uracil cytosine) such that the polymer formed can hybridize to a complementary base target nucleic acid, including target RNA, with a high Tm, even with such short oligomers with 10-14 bases; the ability of the oligomer to be actively transported in mammalian cells; and the ability of the hetero-duplex oligomer: RNA to resist RNase degradation.
[00146] Examples of morpholine oligonucleotides having structural bonds containing phosphorus are illustrated in Figs. 1A-1C. Especially preferred is a morpholine oligonucleotide bound to phosphorodiamidate, as shown in Figure 1B, which is modified, in accordance with an aspect of the present invention, to contain positively charged groups in preferably 10% -50% of its structural bonds. Morpholine oligonucleotides with uncharged structural bonds, including antisense oligonucleotides, are detailed, for example, in (Summerton and Weller, 1997) and in co-owned U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063 and 5,506,337, and in PCT application No. US2008 / 012804, all of which are incorporated herein by reference . Exemplary morpholine oligonucleotides with charged structural bonds and / or modified end groups, including antisense oligonucleotides, are detailed in PCT application No. US2007 / 011435; Co-pending US Provisional Order No. 61 / 349,783; and US Provisional Order co-pending No. 61 / 361,878, each of which is incorporated by reference in its entirety.
[00147] Properties of the morpholine-based subunits include: 1) the ability to be linked in an oligomeric form by stable, uncharged or positively charged structural bonds; 2) the ability to support a nucleotide base (for example, adenine, cytosine, guanine, thymidine, uracil and hypoxanthine) such that the polymer formed can hybridize with a target nucleic acid with complementary base, Tm values above about 45 ° C in relatively short oligonucleotides (for example, 10-15 bases); 3) the ability of the oligonucleotide to be actively or passively transported in memory cells; and 4) the ability of the antisense oligonucleotide heteroduplex: RNA to resist the degradation of RNase and RNaseH, respectively.
[00148] Exemplary structural structures for antisense oligonucleotides of the claimed dirt matter include the types of morpholine subunits shown in Figs. 1D-1G, each linked by subunit bonds containing uncharged or positively charged phosphorus. Fig. 1D shows a phosphorus-containing bond that forms the unit structure of five repeated atoms where the morpholine rings are linked by a 1-atom phosphoamide bond. Fig. 1E shows a bond that produces a structure of repeated units of 6 atoms. In this structure, the Y atom linking carbon 5 'morpholine to the phosphorus group can be sulfur, nitrogen, carbon or, preferably, oxygen. The fraction X pending from the phosphorus can be fluorine, a substituted alkyl or alkyl, a substituted alkoxy or alkoxy, a substituted thio-alkoxy or thioalkoxy, or unsubstituted, mono-substituted or disubstituted nitrogen, including cyclic structures, such as like morpholines or piperidines. Alkyl, alkoxy and thioalkoxy preferably include 1-6 carbon atoms. The Z fractions are sulfur or oxygen, and are preferably oxygen.
[00149] The connections shown in Figs. 1F and 1G are designed for 7-atom unit length structures. In structure 1F, fraction X is Structure 1E, and fraction Y can be methylene, sulfur, or preferably oxygen. In Structure 1G, fractions X and Y are as in Structure 1E. Particularly preferred morpholine oligonucleotides include those composed of morpholine subunit structures as shown in Fig. 1E, where X = NH2, N (CH3) 2, or 1-piperazine or another charged group, Y = O, and Z = O.
[00150] As noted above, the substantially unloaded oligonucleotide can be modified, according to one aspect of the invention, to include unloaded links, for example, up to about 1 per 2-5 links does not load - das, such as about 4-5 for every 10 unloaded connections. In certain modalities, an excellent improvement in antisense activity can be seen when about 25% of structural bonds are cationic. In certain embodiments, an increase can be seen with a small number, for example, 10-20% of cationic bonds, or where the number of cationic bonds is in the range of 50-80%, such as about 60%.
[00151] Additional experiments conducted in support of the present invention indicate that the increase seen with charges in the cationic structure can, in some cases, be further increased by distribution of the load volume of the structural bonds of the “central region” of the oligonucleotide antisense, for example, in a 20-mer oligonucleotide with 8 cationic structural bonds, with at least 70% of these charged bonds located in the 10 most central bonds.
[00152] In certain embodiments, antisense compounds can be prepared by solid-phase synthesis step by step, employing methods detailed in the references cited above, and below with respect to the synthesis of oligonucleotides having cationic and uncharged structural bonds or mixture . In some cases, it may be desirable to add additional chemical fractions to the antisense compound, for example, to increase pharmacokinetics or to facilitate the capture or detection of the compound. Such a fraction can be covalently attached, typically to an end of the oligomer, according to standard synthetic methods. For example, adding a fraction of polyethylene glycol or another hydrophilic polymer, for example, one having 10-100 monomeric subunits, can be useful in increasing solubility. One or more charged groups, for example, charged anionic groups such as an organic acid, can increase cell uptake.
[00153] A reporter fraction, such as a fluorescein group or a radio-cadmium can be attached for detection purposes. Alternatively, the reporter's tag attached to the oligomer can be a linker, such as an antigen or biotin, capable of binding a labeled antibody or streptavidin. In selecting a fraction for binding or modifying an antisense compound, it is generally clearly desirable to select chemical compounds from groups that are biocompatible and are likely to be tolerated by a patient without unwanted side effects.
[00154] As noted above, certain antisense compounds can be constructed to contain a selected number of scattered cationic bonds with uncharged bonds of the type described above. The inter-subunit bonds, both uncharged and cationic, are preferably phosphorus-containing bonds, having the structure:
where: W is S or O, and is preferably O, X = NR1R2 or OR6, Y = O or NR7, and each said link in the oligomer is selected from: (a) uncharged link (a), where each of R1, R2, R6 and R7 are independently selected from hydrogen and lower alkyl; (b1) cationic bond (b1), where X = NR1R2 and Y = O, and NR1R2 represents an optionally substituted piperazine group, such that R1R2 = - CHRCHRN (R3) (R4) CHRCHR-, where each R is independently H or CH3 , R4 is H, CH3, or an electron pair, and R3 is selected from H, lower alkyl, for example, CH3, C (= NH) NH2, ZL-NHC (= NH) NH2, and [C ( 0) CHR'NH] mH, where Z is C (O) or a direct bond, L is an optional linker up to 18 atoms in length, preferably up to 12 atoms, and more preferably up to 8 atoms in length, having selected bonds at from alkyl, alkoxy, and alkylamino, R 'is a side chain of a naturally occurring amino acid or a homologue of one or two carbons of the same, in is 1 to 6, preferably 1 to 4; (b2) cationic bond (b2), where X = NR1R2 and Y = 0, R1 = H or CH3, and R2 = LNR3R4R5, where L, R3, and R4 are as defined above, and R5 is H, lower alkyl, or lower (alkoxy) alkyl; and (b3) cationic bond (b3), where Y = NR7 and X = OR6, and R7 = LNR3R4R5, where L, R3, R4 and R5 are as defined above, and R6 is H or lower alkyl; and at least one such bond is selected from the cationic bonds (b1), (b2), and (b3).
[00155] In certain embodiments, an oligomer can include at least two consecutive bonds of type (a) (that is, uncharged bonds). In additional embodiments, at least 5% of the bonds in the oligomer are cationic bonds (i.e., type (b1), (b2), or (b3); for example, 10% to 60%, and preferably 20-50% of the alloys - tions can be cationic bonds.
[00156] In one embodiment, at least one bond is of type (b1), where, preferably, each R is H, R4 is H, CH3, or an electron pair, and R3 is selected from H, lower alkyl , for example, CH3, C (= NH) NH2, and C (0) -L-NHC (= NH) NH2. The last two modalities of R3 provide a guanidine fraction, either directly linked to the piperazine ring, or pendant to a linker group L, respectively. To facilitate synthesis, the variable Z in R3 is preferably C (O) (carbonyl), as shown.
[00157] The linker group L, as noted above, contains bonds in their structure selected from alkyl (for example, -CH2-CH2-), alkoxy (-CO-), and alkylamino (for example, -CH2-NH-), with the proviso that the terminal atoms in L (for example, those adjacent to cabonyl or nitrogen) are carbon atoms. Although branched linkages (for example, -CH2-CHCH3-) are possible, the linker is preferably unbranched. In one embodiment, the linker is a hydrocarbon linker. Such a linker may have the structure - (CH2) n-, where n is 1-12, preferably 2-8, and more preferably 2-6.
[00158] The morpholine subunits have the structure:
where Pi is a base pairing fraction, and the bonds in spite of the above connect to the nitrogen atom of (i) to the carbon 5 'of an adjacent subunit. The Pi-based pairing fractions can be the same or different, and are generally designed to provide a sequence that binds to a target nucleic acid.
[00159] The use of the types of bond types (b1), (b2) and (b3) above to link morpholine subunits can be illustrated graphically as follows:

[00160] Preferably, all cationic bonds in the oligomer are of the same type; that is, all of type (b1), all of type (b2), or all of type (b3).
[00161] In additional modalities, the cationic bonds are selected from the bonds (b1 ') and (b1 ") as shown below, where (b1") is referred to here as a "Pip" bond and (b1 ") is referred to here as a “GuX” link:

[00162] In the above structures, W is S or O, and is preferably O; each of R1 and R2 is independently selected from hydrogen and lower alkyl, and is preferably methyl; and A represents hydrogen or a non-interfering substituent on one or more carbon atoms in (b1 ') and (b1' ''). Preferably, the carbon rings on the piperazine ring are unsubstituted; however, they can include non-interfering substitutes, such as methyl or fluorine. Preferably, at most one or two carbon atoms are then replaced. In additional embodiments, at least 10% of the connections are of the (b1 ') or (b1' ') type; for example, 10% -60% and preferably 20% to 50% of the connections can be of the (b1 ') or (b1' ') type.
[00163] In certain embodiments, the oligomer does not contain bonds of the type (b1 ') above. Alternatively, the oligomer does not contain bonds of type (b1) where each R is H, R3 is H or CH3, and R4 is H, CH3, or an electron pair.
[00164] The morpholine subunits can also be linked by non-phosphorus-based interunit bonds, as described above, where at least one bond is modified with a pending cationic group as described above.
[00165] Other analogous oligonucleotide bonds that are uncharged in their unmodified state but which may also have a pendent amino substituent can be used. For example, a 5 'nitrogen atom in a morpholino ring can be used in a sulfamide or urea bond (where phosphorus is replaced with carbon or sulfur, respectively) and modified in a way analogous to the 5' nitrogen atom in structure (b3) above.
[00166] Oligomers having any number of cationic bonds are provided, including oligomers completely bonded to cationic. Preferably, however, the oligomers are partially charged, having, for example, 10% -80%. In preferred embodiments, about 10% to 60%, and preferably 20% to 50% of the bonds are cationic.
[00167] In one embodiment, the cationic bonds are spread across the structure. The partially charged oligomers preferably contain at least two uncharged bonds; that is, the oligomer preferably does not have a strictly alternating pattern along its entire length.
[00168] Also considered are oligomers having cationic bonding blocks and uncharged bonding blocks; for example, a central block of unloaded bonds can be flanked by cationic bonds, or vice versa. In one embodiment, the oligomer has approximately equal length 5 ', 3' and central regions, and the percentage of cationic bonds in the central region is greater than about 50%, preferably greater than about 70%.
[00169] Oligomers for use in antisense applications generally range in length from about 10 to about 40 subunits, more preferably about 10 to 30 subunits, and typically 15-25 bases. For example, an oligomer of the invention having 19-20 subunits, a useful length for an antisense compound, can ideally have two to ten, for example, four to eight, cationic bonds, and the remaining uncharged bonds. An oligomer having 14-15 subunits can ideally have two to seven, for example, 3, 4 or 5 cationic bonds and the remaining uncharged bonds.
[00170] Each morpholine ring structure supports a base pairing fraction, to form a sequence of base pairing fractions that is typically designed to hybridize to a selected antisense target in a cell or a patient being treated. The base pairing fraction may be a purine or pyrimidine found in native DNA or RNA (for example, A, G, C, T or U) or an analog, such as hypoxanthine (the component base of the nucleoside inosine) or 5-methyl cytosine. Transporter Peptides
[00171] In certain embodiments, the antisense compounds of the invention can include an oligonucleotide fraction conjugated to a fraction of arginine-rich transporter peptide effective to increase the transport of the compound in cells. The transport fraction can be connected to an oligomer terminal, as shown, for example, in Fig. 1C. The peptide carrier fraction preferably comprises 6 to 16 subunits selected from subunits X ', subunits Y', and subunits Z ', where (a) each subunit X' independently represents lysine, arginine or an arginine analogue, the said analogous sense a cationic α amino acid comprising a side chain of structure R1N = C (NH2) R2, where R1 is H or R; R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may also include oxygen or nitrogen; R1 and R2 can together form a ring; and the side chain is linked to said amino acid via R1 or R2. (b) each Y 'subunit independently represents a neutral amino acid -C (0) - (CHR) n-NH-, where n is 2 to 7 and each R is independently H or methyl; and (c) each Z 'subunit independently represents an amino acid α having a neutral aralkyl side chain; wherein the peptide comprises a sequence represented by one of (X'Y'X ') P, (X'Y') m, (X ') m, and (X'Z'Z') p, where p is 2 a 5 in is 2 to 9. Certain modalities include various combinations selected independently from (X'Y'X ') P, (X'Y') m, (X ') m, and / or (X'Z' Z ') P, including, for example, peptides having the sequence (X'Y'X') (X'Z'Z ') (X'Y'X') (X'Z'Z ') (SEQ ID NO : 129).
[00172] In selected modalities, for each X ', the side chain fraction is guanidyl, as in the amino acid subunit arginine (Arg). In additional modalities, each Y 'is -CO- (CH2) n-CHR-NH-, where n is 2 to 7 and R is H. For example, when n is 5 and R is H, Y' is a 6-aminoexanoic acid subunit, abbreviated here as Ahx; when n is 2 and R is H, Y 'is a β-alanine subunit, abbreviated here as B. Certain modalities relate to carrier peptides having a combination of different amino acids, including, for example, peptides comprising the sequence - RAhxRRBRRAhxRRBRAhxB- (SEQ ID NO: 124), which contains both β-alanine and 6-aminoexanoic acid.
Preferred peptides of this type include those comprising arginine dimers alternating with unique Y 'subunits, where Y' is preferably Ahx. Examples include peptides having the formula (RY'R) p or the formula (RRY ') p, where Y' is preferably Ahx. In one embodiment, Y 'is a 6-amino-exanoic acid subunit, R is arginine and p is 4.
[00174] Certain modalities include several linear combinations of at least two of (RY'R) P and (RRY ') P, including, for example, illustrative peptides having the sequence (RY'R) (RRY') (RY 'R) (RRY') (SEQ ID NO: 130), or (RRY ') (RY'R) (RRY') (SEQ ID NO: 131). Other combinations are contemplated. In an additional illustrative embodiment, each Z 'is phenylalanine, and m is 3 or 4.
[00175] The conjugated peptide is preferably attached to an end of the oligomer via an Ahx-B linker, where Ahx is a subunit of 6-aminooxanoic acid and B is a β-alanine subunit, as shown, for example , in Fig. 1C. Alternative linkers between the peptide and oligomer include glycine and cysteine. These and related linkers can be conjugated via an amide or bisulfide bond.
[00176] In selected modalities, for each X ', the side chain fraction is independently selected from the group consisting of guanidyl (HN = C (NH2) NH-), amidinyl (HN = C (NH2) C <), 2-aminodihydropyrimidyl, 2-aminotetrahydropyramidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl, and is preferably selected from guanidyl and amidinyl. In one embodiment, the side chain fraction is guanidyl, as in the amino acid subunit arginine (Arg).
[00177] In certain embodiments, the Y 'subunits can be either contiguous, in which no X' subunit intervenes between Y 'subunits, or spreads only between X' subunits. In certain embodiments, the linker subunit can be between Y 'subunits. In one embodiment, the Y 'subunits are at one end of the conveyor; in other embodiments, they are flanked by X 'subunits. In still preferred embodiments, each Y 'is -CO- (CH2) n-CHR-NH-, where n is 2 to 7 and R is H. For example, when n is 5 and R is H, Y' is a subunit 6-amino-xanoic acid, abbreviated here as Ahx.
[00178] In modalities selected from this group, each X 'comprises a fraction of the guanidyl side chain, as in an arginine subunit. Preferred peptides of this type include those comprising arginine dimers alternating with Y 'subunits, where Y' is preferably Ahx. Examples include peptides having the formula (RY'R) 4 (SEQ ID NO: 132) or the formula (RRY ') 4 (SEQ ID NO: 133), where Y' is preferably Ahx. In the latter case, the nucleic acid analogue is preferably linked to a Y 'terminal subunit, preferably at the C-terminal, as shown, for example, in Fig. 1C. An exemplary linker is of the AhxB structure, where Ahx is a subunit of 6-aminoexanoic acid and B is a β-alanine subunit. Alternative linkers include cysteine and glycine.
[00179] The transport fractions as described here have been shown to greatly increase the cell entrenchment of linked oligomers, relative to uptake of the oligomer in the absence of the linked transport fraction, and relative to uptake by a missing linked transport fraction the hydrophobic Y 'subunits. Such increased uptake is preferably evidenced by at least a two-fold increase, and preferably a four-fold increase, in the uptake of the compound in mammalian cells relative to uptake of the agent by a linked transport fraction lacking the hydrophobic Y 'subunits. Uptake is preferably increased at least twenty times, and more preferably forty times, relative to the unconjugated compound.
[00180] An additional benefit of the transport fraction is to expect the ability to stabilize a duplex between an antisense compound and its target nucleic acid sequence, presumably because of electrostatic interaction between the positively charged transport fraction and the negatively nucleic acid loaded. The number of subunits loaded on the carrier is less than 14, as noted above, and preferably between 8 and 11.
[00181] The use of arginine-rich peptide transporters (i.e., cell-penetrating peptides) is particularly useful in the practice of certain embodiments of the present invention. Certain transporter peptides have been shown to be highly effective in the distribution of antisense compounds in primary cells including hematopoietic and muscle cells (Marshall, Oda et al. 2007; Jearawiriyapaisarn, Moulton et al. 2008; Wu, Moulton et al. 2008). In addition, compared to other transporter peptides such as Penetratin and the Tat peptide, the transporter peptides described here, when conjugated to an antisense PMO, demonstrate an increased ability to alter the splicing of various gene transcripts (Marshall, Oda et al, 2007). Exemplary peptides in these studies include P007 (SEQ ID NO: 118), CP04057 (SEQ ID NO: 123), and CP06062 (SEQ ID NO: 124).
[00182] Exemplary carrier peptides, including linkers (B, AhxB, C, or G) are given below in Table 5. In certain embodiments, the exemplary carrier peptides listed in Table 5 can be conjugated to the PMO via disulfide or amide. Table 5. Exemplary Carrier Peptides
RNA Interference Agents
[00183] The influenza target regions described herein (for example, M1, M2; SEQ ID NOs: 1-11) can also be targeted by a variety of RNA-based methods of interference. Interference RNA (RNAi) is a mechanism for silencing an evolutionary gene, usually conserved, originally discovered in studies of the nematode Caenorhabditis elegans (Lee et al, Cell 75: 843,1993; Reinhart et al, Nature 403: 901, 2000). It can work by introducing dsRNA into cells expressing the appropriate molecular machinery, which then degrades the corresponding endogenous mRNA. The mechanism involves conversion of dsRNA into short RNAs that directional ribonucleases to homologous mRNA targets (summarized, for example, by Ruvkun, Science 2294: 797, 2001).
[00184] In certain embodiments, the methods provided here may use double-stranded ribonucleic acid (dsRNA) molecules and modulating agents to reduce the replication of the influenza virus, such as by interfering with M1 protein expression or M2. dsRNAs generally comprise two single strands. A dsRNA strand comprises a nucleotide sequence that is substantially identical to a portion of the target gene or target region (the "sense" strand), and the other strand (the "complementary" or "antisense" strand) comprises a sequence that it is substantially complementary to a portion of the target region. The tapes are sufficiently complementary to hybridize to form a duplex structure. In certain embodiments, the complementary RNA strand can be less than 30 nucleotides, less than 25 nucleotides in length, or 19 to 24 nucleotides in length. In certain aspects, the complementary nucleotide sequence can be 20-23 nucleotides in length, or 22 nucleotides in length.
[00185] In certain embodiments, at least one RNA strand comprises a highlighted nucleotide 1 to 4 nucleotides in length. In other embodiments, the dsRNA may further comprise at least one chemically modified nucleotide. In certain aspects, a dsRNA comprising a single strand protruding from 1 to 4 nucleotides can comprise a molecule in which the unpaired nucleotide of the protruding single strand which is directly adjacent to the pair of terminal nucleotides contains a purine base. In other respects, the last complementary nucleotide pairs at both ends of a dsRNA are a G-C pair, or at least two of at least four pairs of terminal nucleotides are G-C pairs.
[00186] Certain embodiments of the present invention may comprise microRNAs. MicroRNAs represent a large group of small RNAs naturally produced in organisms, some of which regulate the expression of target genes. Micro-RNAs are formed from approximately 70 single-stranded nucleotides by stapling the precursor transcript by Dicer. (See Ambros et al. Current Biology 13: 807, 2003). Micro-RNAs are not translated into proteins, but instead bind to specific messenger RNAs, thus blocking translation. Micro-RNAs are thought to loosely match the base with their targets to inhibit translation. Certain micro-RNAs can be transcribed as stapled RNA precursors, which are then processed into two mature forms by Dicer enzyme.
[00187] In certain embodiments, the modulating agent, or RNAi oligonucleotide, is single-stranded. In other embodiments, the modulating agent, or RNAi oligonucleotide, is double-stranded. Certain modalities may also employ short-acting RNAs (siRNA). In certain embodiments, the first strand of the double-stranded oligonucleotide contains two or more nucleoside residues than the second strand. In other modes, the first strand and the second strand have the same number of nucleosides; however, the first and second strands are offset, such that the two terminal nucleosides on the first and second strands are not paired with a residue on the complementary strand. In certain examples, the two nucleosides that are not paired are thymidine residues.
[00188] In examples when the modulating agent comprises siRNA, the agent must include a region of sufficient homology to the target region, and be of sufficient length in terms of nucleotides, such that the siRNA agent, or a fragment thereof , can mediate negative regulation of the target RNA. It will be understood that the term "ribonucleotide" or "nucleotide" can, in the case of a modified RNA or substituted nucleotide, also refer to a modified nucleotide, or alternate substitution fraction at one or more positions. So, a siRNA agent is or includes a region that is at least partially complementary to the target RNA. There is no need for perfect complementarity between the siRNA agent and the target, but the match must be sufficient to allow the siRNA agent, or a cleavage product thereof, to direct the silencing of the specific sequence, such as by cleavage of RNAi from the target RNA. Complementarity, or degree of homology with the target tape, is more critical on the antisense tape. As perfect complementarity, particularly in the antisense tape, it is always desired that some modalities include one or more but preferably 10, 8, 6, 5, 4, 3, 2 or less bad combinations with respect to the target RNA. Mismatches are more tolerated in the terminal regions, and if present, they are preferably in a region or terminal regions, for example, within 6, 5, 4 or 3 nucleotides of the 5 'and / or 3' terminal. The sense tape only needs to be sufficiently complementary with the antiseptic tape to maintain the overall double-tape character of the molecule.
[00189] In addition, a siRNA modulating agent can be modified or include substitution nucleosides. Single-stranded regions of a siRNA agent can be modified or include replacement nucleosides, for example, the unpaired region or regions of a stapled structure, for example, a region linking two complementary regions, may have modifications or nucleosides substitutes. Modification to stabilize one or more 3'or 5'terminal of a siRNA agent, for example, against exonucleases, or in favor of the antisense siRNA agent to enter RISC are also useful. Modifications may include C3 amino linkers (or C6, C7, C12), thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagent if become phosphoramidites and have another hydroxyl group protected with DMT, allowing multiple couplings during RNA synthesis.
[00190] siRNA agents can include, for example, molecules that are long enough to have the interferon response (which can be cleaved by Dicer (Bernstein et al., Nature, 409: 363-366, 2001) and enter a RISC (silencing complex induced by RNAi), in addition to molecules that are short enough that they do not carry the interferon response (whose molecules will also be cleaved by Dicer and / or enter a RISC), for example, molecules that they are of a size that allows you to enter a RISC, for example, molecules that look like Di cleavage products. Molecules that are short enough that they do not lead to an interferon response are called siRNA agents or shorter RNAi agents here "SiRNA agent or shorter RNAi agent" as used herein refers to a siRNA agent that is short enough that it does not induce a deleterious interferon response in a human cell, for example, it has a duplex region of less than 60 but prefer - substantially less than 50 , 40 or 30 nucleotide pairs. A siRNA modulating agent, or a cleavage product thereof, can down-regulate a target gene, for example, by inducing RNAi with respect to a target RNA, preferably an influenza target RNA such as M1 or M2.
[00191] Each strand of a siRNA modulating agent can be equal to or less than 35, 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strip is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred siRNA agents have a double region of 17, 18, 19, 21, 22, 23, 24 or 25 pairs of nucleotides, and one or more spares, preferably one or two spare 3 ', of 2-3 nucleotides.
[00192] In addition to the homology of the target RNA and the ability to downregulate a target gene, a siRNA modulating agent can have one or more of the following properties: it can, despite modifications, even a very large number , or all nucleosides, has an antisense strip that can have bases (or modified bases) in the appropriate three-dimensional structure in order to be able to form the correct base pairing and form a duplex structure with a homologous target RNA that is sufficient to allow negative regulation of the target, for example, by cleavage of the target RNA; it may, despite modifications, even a very large number, or all of the nucleosides, still have “RNA-like” properties, that is, it may have the general structure, chemical and physical properties of an RNA molecule, even if not exclusively, or even partially, of the ribonucleotide-based content. For example, a siRNA agent can contain, for example, a sense and / or antisense tape in which all the nucleotide sugars contain, for example, 2'fluor in place of 2'hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound in theory, electronegative fluoride prefers an axial orientation when attached to the ribose's C2 'position. This spatial preference of fluorine can, in turn, force sugars to adopt a C23-endo fold. This is the same folding mode as seen in the RNA molecules and gives rise to the family-type helix The RNA characteristic. Also, since fluorine is a good hydrogen bridge acceptor, it can participate in the same hydrogen bonding interaction with water molecules that are known to stabilize RNA structures. Generally, it is preferred that a fraction modified at the 2 'position be able to enter the H bond that is more characteristic of the OH fraction of a ribonucleotide than the H fraction of a deoxyribonucleotide.
[00193] A "single stranded RNAi agent" as used herein, is an RNAi agent that is made up of a simple molecule. It can include a duplex region, formed by intra-tape pairing, for example, it can be, or include, a stapled or extended structure. Such simple RNAi modulating agents are preferably antisense with respect to the target molecule. A single-stranded RNAi agent must be long enough that it can enter the RISC and participate in the RISC-mediated cleavage of a target mRNA. A single-stranded RNAi agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40 or 50 nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.
[00194] Stapled RNAi modulating agents may have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24 or 25 pairs of nucleotides. The duplex region can preferably be equal to or less than 200, 100 or 50 in length. Certain variations for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 pairs of nucleotides in length. The clamp may have a single spare tape or terminal unpaired region, preferably 3 ', and preferably on the antisense side of the clamp. In certain embodiments, spares are 2-3 nucleotides in length.
[00195] Certain modulating agents used according to the methods provided herein can comprise RNAi oligonucleotides such as chimeric oligonucleotides, or "chimeras", which contain two or more chemically distinct regions, each made of at least one monomer unit, i.e. , a nucleotide in the case of an oligonucleotide compound. Such oligonucleotides typically contain at least one region in which the oligonucleotide is modified to confer on the increased resistance of the oligonucleotide to the target nucleic acid. Consequently, comparable results can always be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to oligodeoxynucleotide phosphorothioate. Chimeric oligonucleotides can be formed as structures composed of two or more oligonucleotides, modified oligonucleotides, oligonucleotides and / or oligonucleotide mimicry as described above. Such oligonucleotides have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, Pat. USA Nos. 5,013,830, 5,149,797, 5,220,007, 5,256,775, 5,366,878, 5,403,711, 5,491,133, 5,565,350, 5,623,065, 5,652,355, 5,652,356, 5,700,922, and 5,955 .589, each of which is incorporated by reference. In certain embodiments, the chimeric oligonucleotide is RNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, or RNA-DNA-RNA-DNA, where the oligonucleotide is between 5 and 60 nucleotides in length.
[00196] In one aspect of the invention, modulating agents, such as RNAi agents, relate to an oligonucleotide comprising at least one linker attached to an altered or unnatural nucleobase. A large number of compounds can function as the altered base. The structure of the altered base is important to the extent that the altered base must not substantially prevent binding of the oligonucleotide to its target, for example, mRNA. In certain embodiments, the altered base is difluortolil, nitropyrrolyl, nitroimidazolyl, nitroindolyl, naphthalenyl, anthracenyl, pyridinyl, quinolinyl, pyrenyl or the divalent radical of any of the unnatural nucleobases described herein. In certain embodiments, the unnatural nucleobase is difluortolil, nitropyrrolyl, or nitroimidazolyl. In certain embodiments, the unnatural nucleobase is difluortolil. A wide variety of binders are known in the art and accessible to the present invention. For example, the linker can be a steroid, bile acid, lipid, folic acid, pyridoxal, B12, riboflavin, biotin, aromatic compound, polycyclic compound, anchor ether, intercalator, cleavage molecules, protein binding agent, or carbohydrate. In certain embodiments, the binder is a steroid or aromatic compound. In certain examples, the linker is cholesteryl.
[00197] In other embodiments, the RNAi agent is an oligonucleotide attached to a ligand for the purposes of improving cell direction and uptake. For example, an RNAi person can be attached to an antibody, or antigen-binding fragment of it. As an additional example, an RNAi agent can be attached to a specific ligand binding molecule, such as a polypeptide or fragment of the polypeptide that specifically binds to a particular cell surface receptor.
[00198] In other embodiments, the modulating agent comprises an unnatural nucleobase. In certain embodiments, the unnatural nucleobase is difluortolyl, nitroimidazolyl, nitroindolyl, or nitropyrrolyl. In certain embodiments, the modulating agents provided herein relate to a double-stranded oligonucleotide sequence, where only one of the two strands contains an unnatural nucleobase. In certain embodiments, modulating agents as used herein relate to a double-stranded oligonucleotide sequence, wherein both strands independently comprise at least one unnatural nucleobase.
[00199] In certain embodiments, the fraction of ribose sugar that naturally occurs in nucleosides is replaced with a hexose sugar. In some respects, hexose sugar is an alose, altrose, glucose, mannose, sugar, idose, galactose, thalose, or a derivative thereof. In a preferred embodiment, hexose is D-hexose. In certain examples, the fraction of ribose sugar that naturally occurs in nucleosides is replaced with a polycyclic heteroalkyl ring or cyclohexenyl group. In certain examples, the polycyclic heteroalkyl group is a bicyclic ring containing an oxygen atom in the ring. In certain examples, the polycyclic heteroalkyl group is an eptane bicycle [2.2.1], an octane bicycle [3.2.1], or a nonane bicycle [3.3.1]. In certain embodiments, the structure of the oligonucleotide has been modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. In certain embodiments, at least one of the bases or at least one of the sugars in the oligonucleotide has been modified to improve the therapeutic or diagnostic properties of the oligonucleotide compound. In examples when the oligonucleotide is double stranded, the two strands are complementary, partially complementary, or chimeric oligonucleotides.
[00200] Examples of modified RNAi agents intended for use in the methods of the present invention include oligonucleotides containing modified structures or unnatural internucleoside bonds. As defined herein, oligonucleotides having modified structures or internucleoside bonds include those that have a phosphorus atom in the structure and those that do not have a phosphorus atom in the structure. Modified oligonucleotides that do not have a phosphorus atom in their inter-sugar structure can also be considered to be oligonucleotides. Specific oligonucleotide chemical changes are described below. It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the following modifications can be incorporated into a single oligonucleotide compound or even a single nucleotide of it.
[00201] Examples of modified internucleoside bonds or structures include, for example, phosphorothioates, chiral phosphotothioates, phosphorodithioates, phosphotriets, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and 3 phosphonates, chiral phosphonates, 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thioalalkylphosphotriesters, and boranophosphates having normal 3'-5' bonds, 2'-5 'analogs attached to them, and those having inverted polarity in the adjacent nucleic units of adjacent nucleic pairs they are linked to 3'-5 'to 5'-3' or 2'-5 'to 5'-2'. Various salts, mixed salts and free acid forms are also included.
[00202] Representative United States patents that teach the preparation of the above phosphorus-containing bonds include, but are not limited to, Pat. USA 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 incorporated herein by reference.
[00203] Examples of modified internucleoside bonds or structures that do not include a phosphorus atom in it (i.e., oligonucleotides) have structures that are formed by short-chain alkyl or cycloalkyl inter-sugar bonds, mixed heteroatom and inter-bonds alkyl or cycloalkyl sugars, or one or more short chain heteroatomic or heterocyclic inter-sugar bonds. These include those having morpholine bonds (formed in part from the sugar portion of a nucleoside); siloxane structures; sulfide, sulfoxide and sulfone structures; structural formacetyl and thiophormacetyl; methylene formacetyl and thiophormacetyl structures; structures containing alkenes; sulfanate structures; methyleneimino and methylene-drazin structures; sulfonate and sulfonamide structures; amide structures; and others having parts of components N, O, S and CH2 mixed.
[00204] Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, Pat. USA 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 incorporated herein by reference.
[00205] In other examples of mimetic oligonucleotide, both the sugar bond and the internucleoside, that is, the structure of the nucleoside units can be replaced with new groups. Nucleobase units are maintained by hybridization with an appropriate nucleic acid target compound. Such an oligonucleotide, a mimetic oligonucleotide, which has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar structure of an oligonucleotide is replaced with a structure containing amide, in particular an aminoethylglycine structure. The nucleobases are retained and are attached directly or indirectly to the atoms of the amide portion of the structure. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, Pat. No. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Additional teachings of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497.
[00206] The present invention further comprises oligonucleotides employing ribozymes. Synthetic RNA molecules and derivatives thereof that catalyze highly specific endoribonuclease activities are known as ribozymes (See, generally, U.S. Pat. No. 5,543,508 to Haseloff et al, and U.S. Pat. No. 5,545,729 to Goodchild et al). Cleavage reactions are catalyzed by the RNA molecules themselves. In naturally occurring RNA molecules, self-catalyzed cleavage sites are located within highly conserved regions of secondary structure RNA (Buzayan et al., Proc. Natl. Acad. Sci. USA 83: 8859- 62.1986; Forster et al., Cell. 50: 9-16,1987). Naturally occurring auto-catalytic RNA molecules have been modified to generate ribozymes that can be targets for a particular cellular or pathogenic RNA molecule with a high degree of specificity. Therefore, ribozymes serve the same general purpose as antisense oligonucleotides (ie, modulation of the expression of a specific gene) and, like oligonucleotides, are nucleic acids having significant portions of the single strand.
[00207] In certain examples, RNAi agents for use with the methods provided here can be modified by the non-binding group. A number of non-binding molecules have been conjugated to oligonucleotides in order to increase the activity, cell distribution, cell target, or cell uptake of the oligonucleotide, and procedures for making such conjugations are available in the scientific literature. Such non-binding fractions have included lipid fractions, such as cholesterol (Letsinger et al, Proc. Natl. Acad. Sci. USA, 86: 6553-56,1989), cholic acid (Manoharan et al, Bioorg. Med. Chem Lett. 4: 1053,1994), a thioester, for example, hexyl-5-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 660: 306, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3: 2765, 1993), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20: 533, 1992), an aliphatic chain, for example, dodecandiol or undecyl residues (Saison-Behmoaras et al , EMBOJ. 10: 111,1991; Kabanov et al, FEBS Lett. 259: 327,1990; Svi- narchuk etal., Biochimie. 75: 49,1993), a phospholipid, for example, dihexadecyl-rac-glycerol or 1,2-di-O-hexadenyl-rac-glycero-3-H-phosphonate triethylammonium (Manoharan et al., Tetrahedron Lett., 36: 3651, 1995; Shea et al, Nucl. Acids Res. 18: 3777, 1990), a pliamine chain or a polyethylene glycol (Manoharan et al., Nucleosides & Nucleotides. 14: 969, 1995), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett. 36: 3651, 1995), a palmitile fraction (Mishra et al, Biochim. Biophys. Acta. 1264: 229, 1995), or an octadecylamine or hexylamino-carbonoxy-oxycholesterol fraction (Crooke et al., J. Pharmacol. Exp. Ther. 277: 923, 1996). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides having an aminoligator at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be carried out either with the oligonucleotide still attached to the solid support or following cleavage of the oligonucleotide in the solution phase. Purification of the oligonucleotide conjugate by HPLC typically produces the pure conjugate.
[00208] Additional examples of modulating agents, such as RNAi oligonucleotides, can be found in US Application Publication Nos. 2007/0275465, 2007/0054279, 2006/0287260, 2006/0035254, 2006/0008822, which are incorporated by reference. Inhibition of Influenza Virus Replication
[00209] The antisense compounds detailed above are useful in inhibiting the replication of segmented, negative sense, single-stranded RNA viruses of the Orthomyxoviridae family. In one embodiment, such inhibition is effective in treating the infection of a host animal by these viruses. Consequently, the method comprises, in one modality, contacting an infected cell as a virus with an effective antisense agent to inhibit the replication of the specific virus. In this fashion, the antisense agent is administered to a mammalian patient, for example, human or domestic animal, infected with a given virus, in a suitable pharmaceutical carrier. It is contemplated that the antisense oligonucleotide stops the growth of the RNA virus in the host. The RNA virus can be reduced in number or eliminated with little or no detrimental effect on the normal growth or development of the host.
[00210] In the present invention as described in the Examples, Morpholine Phosphorodiamidate Oligomers (PMOs), designed to hybridize to the M1 / M2 gene segment of influenza A virus (ie, segment 7), were evaluated for their ability to inhibit the production of influenza virus in two animal models. PMOs were either conjugated to an arginine-rich peptide to facilitate entry into cells or made as PMOplus® compounds containing cationic bonds. The compounds target the AUG translation starting site of the M1 matrix protein (M1) and the ion channel protein (M2), both of which are expressed from the same AUG initiation codon using splice forms of the M1 / M2 mRNA.
[00211] The M1 / M2-AUG target antisense compounds of the invention lead to the inhibition of viral titer in an H3N2 mouse model as described in Example 1. The M1 / M2-AUG target compounds of the invention also demonstrate signs reduced flu infection clinics and reduced viral titers in nasal lavages in the 2009H1N1 pandemic swine flu ferret model (S-OIV) as described in Example 2. Consequently, the antisense oligonucleotides and RNAi agents exemplified here can be used in the treatment viral infections, mainly those attributable to segmented RNA virus, negative sense and simple strand of the family Orthomyxoviridae.
[00212] Modalities of the present invention also include combination of therapies and related compositions. For example, the antiviral antisense oligonucleotides (i.e., viral target) and RNAi agents provided herein can be used in combination with antisense oligonucleotides with the host's molecular target or RNAi agents. In this concept, antisense or RNAi targeting of a host immune response gene and / or its receptor can be used to improve the immune response and thus prevent or reduce subsequent infections, whether viral or bacterial (for example, secondary bacterial infections). As an example, it has been shown that CD200 / R - / - mice do not develop sepsis following influenza infection. CD200 is a negative regulator of innate immune responses resulting in negative regulation of the innate immune response in general. Therefore, certain treatment methods may include administration of antisense agents and / or RNAi targeting a host RNA molecule encoding CD200 and / or the CD200 receptor (see, for example, Hatherly et al., EurJ Immunol. 34 : 1688-94, 2004) in combination with the administration (concurrently or separately) of any one or more of the influenza targeting agents described herein. Also included are compositions that comprise an antisense agent or RNAi targeted against CD200 and / or CD200 receptor (for example, targeted in its AUG initiation codon or a splice site) in combination with an antisense agent or targeted RNAi against the influenza virus, as described herein. These methods and compositions can be used to treat influenza virus infections alone, and / or secondary bacterial infections (eg, Streptococcal pneumonia) associated with influenza virus infections.
[00213] Modalities of the present invention also include combination therapies for the treatment of viral infections (e.g., influenza infections) accompanied by secondary bacterial infections. Most deaths in the 1918-1919 influenza pandemic probably resulted from secondary bacterial pneumonia caused by common upper respiratory tract bacteria, such as Streptococcus pneumoniae, and recent evidence from the 2009 H1N1 pandemic indicates that secondary bacterial infections remain an important cause of death (see, for example, Louie etal., Clin Infect Disease. 50: e59-62, 2010; Jain etal., N Engl] Med. 361: 1935-44, 2009; Jamieson etal., Cell Host Microbe. 7: 103-14, 2010). The standard of care for streptococcal pneumonia includes antibiotics. Primary antibiotics include bactericidal beta-lactam agents such as penicillin and amoxicillin, second-line agents include cephalosporins, and third-line agents include chloramphenicol or clindamycin. Accordingly, embodiments of the present invention include methods and compositions related to the administration (concurrently or separately) of one or more bacteriostatic or bactericidal antibiotics (e.g., penicillin, amoxicillin, cephalosporin, chloramphenicol, clindamycin) in combination with one or more antisense agents. or influenza targeting RNAi provided herein, primarily to treat or control secondary bacterial infections associated with influenza virus infection.
[00214] As another example, the antisense oligonucleotides and RNAi agents of the present invention can be administered (concurrently or separately) in combination with other therapies targeting the influenza virus, such as oseltamivir phosphate (TAMIFLU®). In certain aspects, the combination of one or more antisense oligonucleotides (for example, AVI-7100) and oseltamivir can achieve synergistic effects in reducing the influenza virus titer and / or other symptoms of influenza virus infection (for example, alveolitis , infiltration of immune cells), related to the use of oseltamivir alone or antiviral antisense oligonucleotides alone. Also included are compositions comprising oseltamivir in combination with an antisense oligonucleotide antiviral agent or RNAi targeting the influenza virus, as described here. In specific modalities, these compositions and methods can be used in the treatment of different infections of the influenza virus resistant to oseltamivir. Identification of an Infective Agent
[00215] The specific virus causing the infection can be determined by methods known in the art, for example, serological or culture methods.
[00216] Serological identification employs a viral sample or a culture isolated from a biological specimen, for example, saliva, feces, urine, cerebrospinal fluid, blood, etc., from the patient. Immunoassay for the detection of the virus is generally performed by methods routinely employed by those skilled in the art, for example, ELISA or Western blot. In addition, monoclonal antibodies specific to particular strains or viral species are always commercially available.
[00217] Culture methods can be used to isolate and identify particular types of viruses, by employing techniques including, but not limited to, comparison of characteristics such as growth rates and morphology under various culture conditions.
[00218] Another method for identifying the viral infectious agent in an infected patient employs RNA isolation from a biological specimen followed by nucleic acid amplification using specific PCR primers that target suspicious viral agents, for example, influenza Seasonal H1N1, pandemic H1N1 S-OIV, H5N1 avian influenza or H3N2 swine influenza. Formulations and Administration
[00219] In certain embodiments, the present invention provides formulations or compositions suitable for the therapeutic delivery of antisense oligomers, as described herein. Therefore, in certain embodiments, the present invention provides pharmaceutically acceptable compositions that comprise a therapeutically effective amount of one or more of the oligomers described herein, formulated together with one or more pharmaceutically acceptable vehicles (additives) and / or diluents. While it is possible for an oligomer of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
[00220] Methods for the distribution of nucleic acid molecules are described, for example, in Akhtar et al., 1992, Trends Cell Bio., 2: 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar; Sullivan et al., PCT WO 94/02595. These and other protocols can be used to deliver virtually any nucleic acid molecule, including the isolated oligomers of the present invention.
[00221] As detailed herein, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those suitable for the following: (1) oral administration, for example, liquid medication (aqueous solutions or suspensions) or non-aqueous), tablets, for example, those targeted for oral, sublingual, and systemic absorption, pills, powders, granules, pastes for application on the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection such as, for example, a sterile solution or suspension, or sustained release formulation; (3) topical application, for example, as a cream, ointment, or a controlled release adhesive or spray applied to the skin; (4) intravaginally or intra-rectally, for example, as a vaginal suppository, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
[00222] The phrase "pharmaceutically acceptable" is used here to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk / benefit ratio.
[00223] The phrase "pharmaceutically acceptable vehicle" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (eg, lubricant, such magnesium, stearate calcium or zinc, or steric acid), or material encapsulating solvent, involved in the distribution or transport of the subject compound from one organ, or body part, to another organ, or body part. Each vehicle must be "acceptable" in the sense of being compatible with other ingredients of the formulation and not harmful to the patient.
[00224] Some examples of materials that can serve as pharmaceutically acceptable carriers include, without limitation: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oils, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH of buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other compatible non-toxic substances used in pharmaceutical formulations.
[00225] Additional non-limiting examples of agents suitable for formulation with the antisense oligomers of the present invention include: PEG-conjugated nucleic acids, phospholipid-conjugated nucleic acids, nucleic acids containing lipophilic fractions, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can increase the entry of drugs into various tissues; biodegradable polymers, such as poly microspheres (DL-lactide-coglycolide) for controlled release distribution after implantation (Emerich, DF et al., Cell Transplant. 8: 47- 58,1999) Alkermes, Inc. Cambridge, Mass .; and charged nanoparticles such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood-brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
[00226] The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (modified, branched and unbranched PEG- or combinations thereof, or long-circulating liposomes or stealth liposomes). Oligomers of the invention can also comprise covalently linked PEG molecules of varying molecular weights. These formulations offer a method to increase drug accumulation in target tissues. This drug vehicle treatment resists opsonization and elimination by mononuclear phagocytic system (MPS or RES), thus allowing a greater number of times of blood circulation and increased tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995 , 95, 2601-2627; Ishiwata etal., Chem. Pharm. Bull. 1995, 43.1005-1011). Such liposomes have been shown to selectively accumulate in tumors, presumably by extravasation and capture in neo-vascularized target tissues (Lasic et al., Science. 267: 1275-1276,1995; Oku etal., Biochim. Biophys. Acta. 1238 : 86-90.1995). The long circulation of liposomes increases the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes that are known to accumulate in MPS tissues (Liu etal.J. Biol. Chem. 42: 24864-24870, 1995; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., PCT International Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to prevent accumulation in metabolically aggressive MPS tissues such as liver and spleen.
[00227] In an additional embodiment, the present invention includes oligomer compositions prepared for distribution as described in U.S. Patent Nos. 6,692,911, 7,163,695 and 7,070,807. In this concept, in one embodiment, the present invention provides an oligomer of the present invention in a composition comprising copolymers of lysine and histidine (HK) as described in U.S. Patent Nos. 7,163,695, 7,070,807, and 6,692,911 either alone or in combination with PEG (for example, branched or unbranched PEG or a mixture of both), in combination with PEG and a target fraction or any of the foregoing combinations with a cross-linking agent. In certain embodiments, the present invention provides antisense oligomers in compositions comprising gluconic acid-modified polyistidine or gluconylated polystidine / transferrin-polylysine. One skilled in the art will also recognize that amino acids with properties similar to His and Lys can be substituted within the composition.
[00228] Certain modalities of the oligomers described here may contain a basic functional group, such as amino or alkylamino, and are then capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this regard refers to non-toxic inorganic or organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ in the delivery vehicle or the manufacturing process of the dosage form, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolation of the salt then formed during the subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate salts. mesylate, glucoeptonate, lactobionate, and lauryl sulfonate and the like (See, for example, Berge et al.J. Pharm. Sci. 66: 1-19, 1977).
[00229] Pharmaceutically acceptable salts of the disclosed oligomers include conventional non-toxic salts or quaternary ammonium salts of the compounds, for example, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymalean, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2 -acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, disulfonic, oxalic, isothionic ethane, and the like.
[00230] In certain embodiments, the oligomers of the present invention may contain one or more acidic functional groups, and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically accessible bases. The term "pharmaceutically acceptable salts" in these examples refers to a non-toxic inorganic or organic base addition salt of compounds of the present invention. These salts can also be prepared in situ in the administration vehicle or the manufacturing process of the dosage form, or by separate reaction of the purified compound in its free acid form with a suitable base, such as hydroxide, carbohydrate or bicarbonate of a pharmaceutically acceptable cation metal, with ammonia, or with a pharmaceutically acceptable primary, secondary or tertiary amine. Suitable alkaline or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of basic addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethylanolamine, piperazine and the like. (See, for example, Berge et al, above).
[00231] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions .
[00232] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfide, sodium sulfite and the like; (2) oil-soluble anti-oxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[00233] Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations can conveniently be presented in the form of a single dose and can be prepared by any methods well known in the pharmaceutical art. The amount of the active ingredient that can be combined with a material carrier to produce a single dose form will vary depending on the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a material carrier to produce a single dose form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will vary from 0.1 percent to about ninety-nine percent of the active ingredient, preferably from about 5 percent to about 70 percent, more preferably about 10 percent to about 30 percent.
[00234] In certain embodiments, a formulation of the present invention comprises an excipient selected from cyclodextrins, celluloses, liposomes, micelle-forming agents, for example, bile acids, and polymeric vehicles, for example, polyesters and polyanidides; and an oligomer of the present invention. In certain embodiments, a formulation mentioned above gives oral bioavailability of an oligomer of the present invention.
[00235] Methods of preparing such formulations or compositions include the step of bringing an oligomer of the present invention in association with the vehicle and, optionally, one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately combining a compound of the present invention with liquid vehicles, or solidly divided solid vehicles, or both, and then, if necessary, shaping the product.
[00236] Formulations of the invention suitable for oral administration can be in the form of capsules, seals, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as lozenges (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. An oligomer of the present invention can also be administered as a mass, electuary or paste.
[00237] In solid dose forms of the invention for oral administration (capsules, tablets, pills, pills, powders, granules, lozenges and the like), the active ingredient can be mixed with one or more pharmaceutically acceptable vehicles , such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as amides, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidine, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate (5) solution retarding agents, such as paradine; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as polyxamer and sodium lauryl sulfate; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents, and (11) controlled release agents such as crospovidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard-coated gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
[00238] A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium cell carboxymethyl), surface active agent or surface dispersal. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
[00239] Tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as pills, capsules, pills and granules, can optionally be scored or prepared with coatings and coatings, such as enteric coatings and other coatings well known in the art. pharmaceutical formulation technique. They can also be formulated to provide slow or controlled release of the active ingredient using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymeric matrices, liposomes and / or microspheres. They can be formulated for quick release, for example, freeze-drying. They can be sterilized by, for example, filtration through a bacteria retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injection medium immediately before of use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient (s) only, or preferably, in a certain portion of the gastrointestinal tract, optionally, in a delayed form. Examples of soaked compositions that can be used include polymeric substances and waxes. The active ingredient can also be a micro-encapsulated form, if appropriate, with one or more of the excipients described above.
[00240] Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, prolylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed oil, peanut oil, corn, germ, olive, castor and gerbera), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof.
[00241] In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, colorants, perfumers and preservatives.
[00242] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydride, bentonite, agar and tragacanth, and mixtures thereof. .
[00243] Formulations for rectal or vaginal administration can be present as a suppository, which can be prepared by mixing one or more compounds of the invention with one or more more suitable excipients or non-irritating vehicles comprising, for example , cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which is solid at room temperature but liquid at body temperature and thus will melt in the rectum or vaginal activity and release the active compound.
[00244] Formulations or dosage forms for topical or transdermal administration of an oligomer as provided herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, adhesives and inhalants. The active oligomers can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. Ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, ben- tonites, silicic acid, talc and zinc oxide, or mixtures thereof.
[00245] Powders and sprays may contain, in addition to an oligomer of the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain normal propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
[00246] Transdermal patches have the added advantage of providing controlled distribution of an oligomer of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the oligomer in the medium itself. Absorption enhancers can also be used to increase the flow of the agent through the skin. The rate of such flow can be controlled by or providing a rate of membrane control or dispersion of the agent in a polymeric matrix or gel, among other methods known in the art.
[00247] Pharmaceutical compositions suitable for parenteral administration may comprise one or more oligomer of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous and non-aqueous isotonic solutions, dispersions or emulsions, or sterile powders which can be re-sterile - made up of sterile injectable solutions or dispersions well before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that give the isotonic formulation with the blood of the intended container or suspending or thickening agent. Examples of suitable aqueous and non-aqueous vehicles that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as oil olive oil, and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials, such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.
[00248] Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms on the revealed oligomers can be guaranteed by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, sorbic acid phenol and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form as it is brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
[00249] In some cases, in order to prolong the effect of a drug, it is desired to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material having poor water solubility, among other methods known in the art. The rate of absorption of the drug then depends on its rate of dissolution which, in turn, may depend on the size of the crystal and crystalline shape. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oily vehicle.
[00250] Injectable deposit forms can be made by forming microcapsule matrices of the revealed oligomers in biodegradable polymers such as polylactic-polyglycolide. Depending on the rate of oligomer to polymer, and the nature of the particular polymer employed, the rate of release of the oligomer can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations can also be prepared by capturing the drug in liposomes or microemulsions that are compatible with body tissues.
[00251] When the oligomers of the present invention are administered as pharmaceuticals, for humans and animals, they can be given by themselves or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) active ingredient in combination with a pharmaceutically acceptable carrier.
[00252] As noted above, the formulations or preparations of the present invention can be given orally, parenterally, topically or rectally. They are typically given in the appropriate forms for each route of administration. For example, they are administered in a tablet or capsule formula, by injection, inhalation, eye lotion, ointment, suppository etc., administration by injection, infusion or inhalation; topic by lotion or ointment; and rectal by suppository.
[00253] The phrases "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular injection and infusion , intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachoid, intraspinal and intrasternal.
[00254] The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and is then subjected to metabolism and other types of processes, for example, subcutaneous administration.
[00255] Regardless of the selected route of administration, the oligomers of the present invention, which can be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, can be formulated in dosage forms pharmaceutically acceptable by methods conventional methods known to those skilled in the art. Current dosage levels of the attic ingredients in the pharmaceutical compositions of this invention may vary in order to obtain a quantity of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, without being unacceptable. potentially toxic to the patient.
[00256] The dosage level selected will depend on a variety of factors including the activity of the particular oligomer of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular oligomer being employed, the rate and extent of absorption, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular oligomer used, age, sex, weight, condition, general health and previous medical history of the patient being treated, and types of factors well known in medical techniques.
[00257] A doctor or veterinarian being skilled in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the doctor or veterinarian may initiate doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than those required in order to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, a suitable daily dose of a compound of the invention will be the amount of the compound that is the lowest effective dose to produce the therapeutic effect. Such an effective dose will generally depend on the factors described above. Generally, oral, intravenous, intracerebrocentric and subcutaneous doses of the compounds of this invention to a patient, when used for the indicated purposes, will vary from about 0.0001 to about 100 mg per kilogram of body weight per day.
[00258] If desired, the effective daily dose of the active compound can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in single dose forms. In certain situations, dosage is one administration per day. In certain modalities, dosage is one or more administrations every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to reduce replication of the influenza virus.
[00259] Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar with the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels , cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art. In certain embodiments, microemulsification technology can be used to improve the bioavailability of lipophilic pharmaceutical agents (insoluble in water). Examples include Trimethrin (Dordunoo, SK, et al, Drug Development and Industrial Pharmacy, 17: 1685-1713,1991) and REV 5901 (Sheen, P. C „et al.J Pharm Sci. 80: 712- 714, 1991). Among other benefits, microemulsification provides increased bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thus bypasses the liver, and prevents destruction of compounds in the hepatobiliary circulation.
[00260] In one aspect of the invention, the formulations contain micelles formed from an oligomer as provided herein and at least one amphiphilic vehicle, wherein the micelles have an average diameter of less than about 100 nm. Most preferred modalities provide micelles having an average diameter of less than about 50 nm, and even more preferred modalities provide micelles having an average diameter of less than about 30 nm, or even less than about 20 nm.
[00261] While all suitable amphiphilic vehicles are contemplated, presently preferred vehicles are generally those with Generally Recognized Safe (GRAS) status, and which can both solubilize the compound of the present invention and microemulsify it in one stage late when the solution comes into contact with a complex aqueous phase (such as one found in human gastro-intestinal treatment). Usually, amphiphilic ingredients that satisfy these requirements have HLB (hydrophilic to lipophilic balance) values of 2-20, and their structures contain single-chain aliphatic radicals in the range of C-6 to C-20. Examples are polyethylene glycolized fatty glycerides and polyethylene glycols.
[00262] Examples of amphiphilic vehicles include monounsaturated polyethylene glycolized fatty acid glycerides, such as those obtained from various partially hydrogenated vegetable oils. Such oils may advantageously consist of tri-, di- and mono-glycerides of fatty acid and di- and polyethylene glycol monoesters of the corresponding fatty acids, with a particularly preferred fatty acid composition including capric acid 4-10, capric acid 3-9, lauric acid 40-50, moristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%. Another useful class of amphiphilic vehicles includes sorbitan and / or partially esterified sorbitol, with saturated or monounsaturated fatty acids (SPAN series) or corresponding ethoxylated analogues (TWEEN series).
[00263] Commercially available amphiphilic vehicles may be particularly useful, including Gelucire, Labrafil, Labrasol, or Lauroglycol series (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-monooleate, PEG-di -oleate, PEG-mono-laurate and di-laurate, lecithin, polysorbate 80 etc (produced and distributed by a number of companies in the USA and around the world).
[00264] In certain embodiments, the distribution may occur by the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of this invention in suitable host cells. In particular, the compositions of the present invention can be formulated for delivery to or encapsulated in a lipid particle, lymphoma, vesicle, nanosphere, nanoparticle or the like. The formulation and use of such distribution vehicles can be carried out using known and conventional techniques.
[00265] Hydrophilic polymers suitable for use in the present invention are those that are easily soluble in water, can be covalently attached to a vesicle-forming lipid, and are tolerated in vivo without toxic effects (i.e., they are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also called polylactide), polyglycolic acid (also called poliglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol. In certain embodiments, polymers have a molecular weight of about 100 or 120 daltons to about 5000 or 1000 daltons, or about 300 daltons to about 5000 daltons. In other embodiments, the polymer is polyethylene glycol having a molecular weight of about 100 to about 5000 daltons, or having a molecular weight of about 300 to about 5000 daltons. In certain embodiments, the polymer is 750 dalton polyethylene glycol (PEG (750)). Polymers can also be defined by the number of monomers thereof; a preferred embodiment of the present invention uses a polymer of at least three monomers, such PEG polymers of the three monomers (approximately 150 daltons).
[00266] Other hydrophilic polymers that may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and cellulose derivatives such as hydroxymethylcellulose or hydroxyethylcellulose.
[00267] In certain embodiments, a formulation of the present invention comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, acrylic polymers and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses, polypropylene, polypropylene, polyethylene, polystyrene, lactic acid and glycolic polymers, polyanhydrides, poly (ortho) esters, poly (boric acid), poly (valeric acid), poly (lactide-co-caprolactone ), polysaccharides, proteins, polyaluronic acids, polycyanoacrylates, and combinations, mixtures or copolymers of the same.
[00268] Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8 glucose units, designated by the Greek letter α, β or Y, respectively. The glucose units are linked by α-1,4-glycosidic bonds. As a consequence of the chair forming of the sugar units, all the secondary hydroxyl groups (in C-2, C-3) are located on one side of the ring, while all the primary hydroxyl groups in C-6 are located in the other side. As a result, the outer faces are hydrophilic, making the cyclodextrins soluble in water. In contrast, the cyclodextrin cavities are hydrophobic, since they are marked by hydrogen atoms of C-3 and C-5 atoms, and by ether-type oxygen. These matrices allow complexation with a variety of relatively hydrophobic compounds, including, for example, steroid compounds, such as 17α-estradiol. Complexation occurs through Van der Waals interactions and hydrogen bonding. For a general review of cyclodextrin chemistry, see, Wenz, Agnew, Chem. Int. Ed. Engl., 33: 803-822, 1994.
[00269] The physicochemical properties of cyclodextrin derivatives depend strongly on the type and degree of substitution. For example, its solubility in water ranges from insoluble (for example, triacetyl-beta-cyclodextrin) to 147% soluble (w / v) (G-2-beta-cyclodextrin). In addition, they are soluble in many organic solvents. The properties of cyclodextrins allow control over the solubility of various components of the formulations by increasing or decreasing their solubility.
[00270] Numerous cyclodextrins and methods for their preparation have been described. For example Parmeter (I) et al (U.S. Pat. No. 3,453,259) and Gramera et al (U.S. Pat. No. 3,459,731) described electroneutral cyclodextrins. Other derivatives include cyclodextrins with cationic properties [Parmeter (II), Pat. No. 3,453,257], insoluble cross-linked cyclodextrin (Solms, U.S. Pat. No. 3,420,788), and cyclodextrins with anionic properties (Parmeter (III), U.S. Pat. No. 3,426,011]. Among the cyclodextrin derivatives with anionic properties, carboxylic acids, phosphorous acids, phosphoric acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulfinic acids, and sulfonic acids have been attached to parent cyclodextrin [see, Parmeter (III) , above] In addition, sulfoalkyl ether cyclodextrin derivatives have been described by Stella et al (U.S. Pat. No. 5,134,127).
[00271] Liposomes consist of at least one lipid bilayer membrane closing an aqueous internal compartment. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and typically vary between 0.02 and 0.05 μm in diameter; large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Large olidolamellar vesicles and multilamellar vesicles have multiple layers, usually concentric of membranes and are typically larger than 0.1 μm. Liposomes with several non-concentric membranes, that is, several smaller vesicles contained within a larger vesicle, are called multivesicular vesicles.
[00272] One aspect of the present invention relates to formulations comprising liposomes containing an oligomer of the present invention, where the liposome membrane is formulated to provide a liposome with increased carrying capacity. Alternatively or in addition, the compound of the present invention can be contained within, or adsorbed to, the liposomal bilayer of the liposome. An oligomer of the present invention can aggregate with a lipid surfactant and carried within the internal space of the liposome; in these cases, the liposome membrane is formulated to resist the rupture effects of the active-surfactant aggregate.
[00273] According to one embodiment of the present invention, the lipid layer of a liposome contains lipids derived with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the encapsulated inner space by the liposome, and extends from the outside of the lipid bilayer into the surrounding environment.
[00274] Active agents contained within liposomes of the present invention are in solubilized form. Aggregates of surfactants and active agent (such as emulsions or micelles containing the active agent of interest) can be captured within the interior space of the liposomes according to the present invention. A surfactant acts to disperse and solubilize the active agent, and can be selected from any aliphatic, cycloaliphatic or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholines (LPCs) of various chain lengths (for example, from about C14 to about C20). Polymer-derived lipids such as PEG-lipids can also be used for micelle formation as they will act to inhibit micelle / membrane fusion, and how adding a polymer to tensile molecules decreases the CMC of the surfactant and helps micelle formation. Preferred are surfactants with CMCs in the micromolar variation; Higher CMC surfactants can be used to prepare micelles captured within the liposomes of the present invention.
[00275] Liposomes according to the present invention can be prepared by any of a variety of methods that are known in the art. See, for example, Pat. US No. 4,235,871; Published PCT application WO96 / 14057; New RRC, Liposomes: A Practical Approach, IRL Press, Oxford (1990), pages 33-104; Lasic DD, Liposomes from physics to applications, Elsevier Science 25 Publishers BV, Amsterdam, 1993. For example, liposomes of the present invention can be prepared by diffusing a lipid derived with a hydrophilic polymer into the preformed liposomes, such as by exposure of preformed liposomes to micelles composed of polymers grafted with lipid, in lipid concentrations corresponding to the percentage of final mol of the derived lipid that is desired in the liposome. Liposomes containing a hydrophilic polymer can also be formed by homogenization, lipid field hydration, or extrusion techniques, as are known in the art.
[00276] In another exemplary formulation procedure, the active agent is first dispersed by sonication in a lysophosphatidylcholine or other surfactant with low CMC (including polymer-grafted lipids) that easily solubilize hydrophobic molecules. The resulting micellar suspension of the active agent is then used to rehydrate a dry lipid sample that contains an appropriate mol% of the lipid grafted with polymer, or cholesterol. The lipid and active agent suspension is then formed into liposomes using extrusion techniques as shown in the art, and the resulting liposomes separated from the unencapsulated solution by standard column separation.
[00277] In one aspect of the present invention, liposomes are prepared to have substantially homogeneous sizes in a selected size range. An effective size adjustment method involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a uniformly selected pore size; the pore size of the membrane will roughly correspond to the larger sizes of the liposomes produced by the reagents such as DharmaFECT® and Lipofectamine® can be used to introduce polynucleotides or proteins into cells.
[00278] The release characteristics of a formulation of the present invention depend on the encapsulating material, the concentration of the encapsulated drug, and the presence of release modifiers. For example, release can be manipulated to be pH dependent, for example, using a pH sensitive coating that releases only at a low pH, such as in the stomach, or a higher pH, such as in the intestine. An enteric coating can be used to prevent release from occurring until after passing through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stoma, followed by late release in the intestine. Release can also be manipulated by including salts or pore-forming agents, which can increase water uptake or drug release by diffusion from the capsule. Excipients that modify the drug's solubility can also be used to control the rate of release. Agents that increase the degradation of the matrix or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (ie, as particulates), or they can be co-dissolved in the polymeric phase depending on the compound. In most cases the amount should be between 0.1 to thirty percent (w / w polymer). Types of degradation enhancers include inorganic salts such as ammonium and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine, spermine, choline, ethanolamine, diethanolamine, and triethanolamine and surfactants such as Tween® and Pluronic®. Pore-forming agents that add microstructure to the matrices (ie, water-soluble compounds such as inorganic salts and sugars) are added as particulates. The variation is typically between one and thirty percent (w / w polymer).
[00279] The uptake can also be manipulated by alternating the resistance time of the particles in the intestine. This can be achieved, for example, by coating the particle with, or selecting as the encapsulating material, a mucosal adhesive polymer. Examples include most polymers with free carboxyl groups, such as chitosin, celluloses, and especially polyacrylates (as used herein, polyacrylates refer to polymers including modified acrylate groups and groups such as cyanoacrylates and methacrylates).
[00280] An oligomer can be formulated to be contained within, or, adapted for release by a surgical or medical device or implant. For example, hydrogels, or other polymers, such as biocompatible and / or biodegradable polymers, can be used to coat an implant with the compositions of the present invention (i.e., the composition can be adapted for use with the medical device by using hydrogel or other polymer). Polymers and copolymers for coating devices measured with an agent well known in the art. Examples of implants include, but are not limited to, stents, stents eluting drugs, sutures, prostheses, vascular catheters, dialysis catheters, vascular grafts, prosthetic heart valves, cardiac pacemakers, implanted cardioverter defibrillators IV needles, devices for adjustment and bone formation, such as pins, screws, plates and other devices, and matrices of artificial tissues for wound healing.
[00281] In addition to the methods provided herein, oligomers for use according to the invention can be formulated for administration in any form convenient for use in human or veterinary medicine, by analogy with other pharmacists. The antisense oligomers and their corresponding formulations can be administered with other therapeutic strategies in the treatment of influenza virus infection (for example, Oseltamivir, which is sold under the trademark TAMIFLU®).
[00282] In accordance with the invention, antisense oligomer delivery routes include, but are not limited to, various systemic routes, including oral and parenteral routes, for example, intravenous, subcutaneous, intraperitoneal, and intramuscular, as well as distribution by inhalation, transdermal, pulmonary and topical. The appropriate route can be determined by one skilled in the art, as appropriate to the patient's condition under treatment. For example, an appropriate route for delivering an antisense oligomer for the treatment of a viral skin infection is topical delivery, while delivery of an antisense oligomer for the treatment of a viral respiratory infection (eg influenza A) is by delivery via inhalation. , intranasal or pulmonary. The oligomer can also be delivered directly to the viral infection site, or into the bloodstream.
[00283] The antisense oligomer can be administered in any convenient vehicle that is physiologically acceptable. Such a composition can include any of a variety of standard pharmaceutically acceptable vehicles employed by those skilled in the art. Examples include, but are not limited to, saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions, such as oil / water emulsions or triglyceride emulsions, tablets and capsules. The choice of physiologically acceptable vehicle will vary depending on the choice of mode of administration.
[00284] In some examples, as noted above, liposomes can be used to facilitate the uptake of antisense oligonucleotides in cells (See, for example, Williams, SA, Leukemia. 10 (12): 1980-1989, 1996; Lappalainen et al, Antiviral Res. 23: 119,1994; Uhlmann et al., Antisense Oligonucleotides: A New Therapeutic Principle, Chemical Reviews, Volume 90, No. 4, pages 544-584,1990; Gre- goriadis, G. , Chapter 14, Liposomes, Drug Carriers in Biology and Medicine, pp. 287- 341, Academic Press, 1979). Hydrogels can also be used as vehicles for administering antisense oligomers, for example, as described in WO 93/01286 or PCT Application No. US1992 / 005305. Alternatively, oligonucleotides can be administered in microspheres or microparticles. (See, for example, Wu, G.Y. and Wu, C.H Biol. Chem. 262: 4429-4432, 1987). Alternatively, the use of gas-filled microtubules complexed with antisense oligomers may increase distribution to target tissues, as described in U.S. Patent No. 6,245,747.
[00285] Controlled release compositions can also be used. These can include semi-permeable polymeric matrices in the form of molded articles such as films or microcapsules.
[00286] In one aspect of the method, the patient is a human patient, for example, a patient diagnosed as having a localized or systemic viral infection. The condition of a patient can also dictate the prophylactic administration of an antisense oligomer of the invention, for example, in the case of a patient who (1) is immunocompromised; (2) is a burn victim; (3) has a permanent catheter; or (4) you are about to have or have recently had surgery. In a preferred embodiment, the oligomer is a phosphorodiamidate morpholino oligomer, contained in a pharmaceutically acceptable carrier, and is distributed orally. In another preferred embodiment, the oligomer is a morpholine phosphorodiamidate oligomer, contained in a pharmaceutically acceptable carrier, and is delivered intravenously (i.v.).
[00287] The antisense compounds can be administered in an amount and effective way to result in a peak blood concentration of at least 200-400 nM antisense oligomers. Typically, one or more doses of the antisense oligomer are administered, usually at regular intervals, over a period of about one to two weeks. Preferred doses for oral administration are about 1-100 mg of oligomer per 70 kg. In some cases, doses greater than 100 mg of oligomer / patient may be necessary. For i.v. administration, preferred doses are about 1 mg to 500 mg of oligomer per 70 kg. The antisense oligomer can be administered at regular intervals for a short period of time, for example, daily for two weeks or less. However, in some cases the oligomer is administered intermittently over a long period of time. Administration can be followed by, or concurrent with, administration of an antibiotic or other therapeutic treatment. The treatment regimen can be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and the patient's physiological examination on treatment. Treatment Monitoring
[00288] An effective in vivo treatment regimen using the antisense oligonucleotides of the invention may vary according to the duration, dose, frequency and route of administration, as well as the condition of the patient under treatment (ie prophylactic administration versus administration in response to localized or systemic infection). Consequently, such in vivo therapy will always require monitoring by appropriate tests of the particular type of viral infection under treatment, and corresponding adjustments in the dose or treatment regimen, in order to achieve an optimal therapeutic result. Treatment can be monitored, for example, by general infection indicators, such as complete blood count (CBC), nucleic acid detection methods, immunodiagnostic tests, viral culture, or heteroduplex detection.
[00289] The effectiveness of an antisense oligomer administered in vivo of the invention in inhibiting or eliminating the growth of one or more types of RNA viruses can be determined from biological samples (tissue, blood, urine, etc.) taken from a patient before of, during and subsequent the administration of the antisense oligomer. Tests of such samples include (1) monitoring the presence or absence of heteroduplex formation with target and non-target sequences, using procedures known to those skilled in the art, for example, electrophoretic gel mobility assay; (2) monitoring the amount of viral protein production, as determined by standard techniques such as ELISA or Western blotting, or (3) measuring the effect of the viral titer, for example, by the Spearman-Karber method. (See, for example, Pari, GS et al., Antimicrob. Agents and Chemotherapy. 39 (5): 1157-1161, 1995; Anderson, KP et al., Antimicrob. Agents and Chemotherapy. 40: 2004-2011, 1996 ; Cottral, GE (ed) in: Manual of Standard Methods for Veterinary Microbiology, pp. 60-93, 1978). References Abes, R., H. M. Moulton, et al. (2008). "Delivery of steric block morpholino oligomers by (R-X-R) 4 peptides: structure-activity studies." Nucleic Acids Res. Cox, N. J. and K. Subbarao (1999). "Influenza." Lancet 354 (9186): 1277-82. Cox, N. J. and K. Subbarao (2000). "Global epidemiology of influenza: past and present." Annu Rev Med 51: 407-21. Egholm, M., O. Buchardt, et al. (1993). "PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules." Nature 365 (6446): 566-8. Jearawiriyapaisarn, N., H. M. Moulton, et al. (2008). "Sustained Dystrophin Ex-pression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice." Mol Ther. Marshall, N. B., S. K. Oda, et al. (2007). "Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing." Journal of Immunological Methods 325 (1-2): 114-126. Moulton, H. M., M. H. Nelson, et al. (2004). "Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides." Bioconjug Chem 15 (2): 290-9. Munster, V. J., E. de Wit, et al. (2009). "Pathogenesis and Transmission of Swine-Origin 2009 A (H1N1) Influenza Virus in Ferrets." Science. Stein, C. A., J. B. Hansen, et al. (2010). "Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents." Nucleic Acids Res 38 (1): e3. Strauss, J. H. and E. G. Strauss (2002). Viruses and Human Disease. San Diego, Academic Press. Summerton, J. and D. Weller (1997). "Morpholino antisense oligomers: design, preparation, and properties." Antisense Nucleic Acid Drug Dev 7 (3): 187-95. Wu, B., H. M. Moulton, et al. (2008). "Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer." Proc Natl Acad Sci IJSA 105 (39): 14814-9.
[00290] All publications and patent applications cited in this specification are hereby incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
[00291] Although the foregoing invention has been described in some details by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications can be made in it, without departing from the spirit or purpose of the attached claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will easily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results. Examples A. Materials and Methods
[00292] All peptides were usually synthesized by Global Peptide Sevices (Ft. Collins, CO) or in AVI BioPharma (Corvallis, OR) and purified to> 90% purity (see Example 2 below). PMOs were synthesized in AVI Bi-oPharma according to known methods, as described, for example, in ((Summerton and Weller 1997) and US Patent No. 5,185,444 and further described in PCT application No. US08 / 012804 Exemplary PMO structures are as shown in Figures 1A-C. 2'-OMe oligomers were synthesized by Integrated DNA Technologies Inc., Skokie, IL. LNA oligomers were produced by Biosyntheisis, Inc. Lewisville, TX.
[00293] Some of the PMO oligomers were conjugated at the 3 'terminal with an arginine-rich peptide ((RAhxRRBR) 2AhxB or (RAhxR) 4AhxB; SEQ ID NOs: 124 and 118, respectively) to form peptide-conjugated PMOs (PPMOs) to increase cell uptake as described (U.S. Patent No. 7,468,418, PCT Application No. US08 / 008168 e (Marshall, Oda et al. 2007; Abes, Moulton et al. 2008)).
[00294] A synthetic pathway that can be used to make morpholine subunits containing a phosphinylideneoxy (1-piperazine) bond is described in PCT application No. US07 / 011435 and further experimental detail for a representative synthesis is provided below. Reaction of piperazine and trityl chloride gave trityl piperazine, which was isolated as the succinate salt. Reaction with ethyl trifluoracetate in the presence of a weak base (such as diisopropylethylamine or DIEA) provided 1-trifluoracetyl-4-trityl piperazine, which was immediately reacted with HCl to provide the salt in good production. The dichlorophosphoryl fraction was introduced with phosphorus oxychloride in toluene.
[00295] The acid chloride is reacted with morpholine (moN) subunits, which can be prepared as described in U.S. Patent No. 5,185,444 or in Summerton and Weller 1997 (cited above) and further described in the PCT application No. US08 / 012804, to provide the activated subunits. Suitable protecting groups are used for nucleoside bases, where necessary; for example, benzoyl for adenine and cytosine, phenylacetyl for guanine, and pivaloylmethyl for inosine. Subunits containing the phosphinylideneoxy (1-piperazine) bond can be incorporated into the existing PMO synthesis protocol, as described, for example, in Summerton and Weller (1997), without modification. EXAMPLE 1 INFLUENCE A VIRUS INHIBITION IN A MODEL MURINE SYSTEM
[00296] A murine model of influenza A virus infection was used to determine the in vivo efficacy of antisense oligomers representative of the infection. Influenza A subtype H2N3 (Port Chalmers / 1/73) was used to infect female Balb / c mice through intranasal administration of approximately 4x104 plaque-forming units in a volume of 50 microliters. The studies used 12 mice per group with their removed on day two to determine the viral titer and six removed on day six to determine the viral titer. Secondary objectives included prevention of weight loss and survival.
[00297] Three test oligomer compounds, PB1-AUG + 15, M1 / M2-AUG and NP-v3 '(SEQ ID NOs: 12.13 and 30-33) as listed in Table 1 and below in Table 6 were evaluated as both, peptide conjugated (PPMO) and chemical bonding to positive charge (PMOplus®). PPMOs were synthesized using the CP06062 peptide (SEQ ID NO: 124) conjugated to the PMO 3'terminal. Each test agent was evaluated at three dose levels (10, 30 and 100 micrograms) to establish dose-dependent relationships. Dosing was through the intranasal route starting 4 hours before infection on Day 0 and then daily through Day 4 for a total of 5 doses. The primary goal of the study was to reduce the viral titer in the lung measured as plaque-forming units per gram of lung tissue. Table 6. Antisense oligomers used in the Murino H3N2 model

[00298] Figure 6 shows the effect on viral titer on Day 6 post-infection. Each viral titer is the average of six animals treated with PPMO and six with PMOplus®. Target compounds M1 / M2-AUG (SEQ ID NOs: 12 and 13) showed substantially greater activity compared to other tested compounds. The viral title from the treatment of Dengue - negative control shown in Figure 6 was obtained using an irrelevant PPMO and PMOplus® sequence that targets the Dengue virus. EXAMPLE 2 INFLUENZA A VIRUS INHIBITION IN A FERN MODEL SYSTEM
[00299] An observation in support of the present invention was the demonstration of the antiviral efficacy of the compounds of the invention in the model system of domestic ferret animal (Mustela putorius furo) using the new virus H1N12009 (S-OIV). Advantages of the ferret model include the ability to use natural human influenza virus isolates, as opposed to strains adapted to mice, and the development of most clinical signs seen in humans such as fever and nasal discharge (Munster, de Wit et al. 2009).
[00300] Six ferrets were infected with a 2009 H1N1 strain resistant to Tamiflu obtained from the Centers for Disease Control (pandemic swine flu). The viral infection route was intranasal (4x104 plaque-forming units) on Day 1 and dosing was either by intraperitoneal (ip) injection for PMOplus® compounds or intranasally (in) for PPMO compounds. The PMOplus® compounds (30 mg / kg ip dose) and PPMO (1.5 mg / kg dose) negative control directed to Dengue were administered as described above in Example 1. The dose for PMOplus® compounds was administered as described above in Example 1. The dose for PMO-plus® compounds was 10 and 30 mg / kg for PPMO M1 / M2-AUGplus (SEQ ID NO: 13; AVI-7100) and 0.5 and 1.5 mg / kg for M1 / M2-AUG (SEQ ID NO: 12) conjugated at the 3 'end of SEQ ID NO: 124). Dosing was done four hours before infection and on Days 1, 2 and 5. Tamiflu (Oseltamivir) was administered (10 mg / kg dose) as a positive antiviral control in parallel with the antisense compounds. Saline was also included as a negative control.
[00301] Observations during life included weight gain (Figure 7A), sneezing (Figure 7B), nasal discharge (Figure 7C) and respiratory distress (Figure 7D). The target M1 / M2-AUG compounds prevented weight loss and reduced sneezing, nausea discharge and respiratory distress stress. Viral nasal lavage titers for Day 1 through Day 5 post-infection are shown in Figure 7E as the area on the curve (AUC) of the infectious tissue culture dose (TCID). The M1 / M2-AUG agent showed a reduction of 2.3 log relative to saline (99.6% reduction) and 1.1 log reduction greater than Tamiflu (94.4% greater).
[00302] To further assess the effectiveness of the AVI-7100 (SEQ ID NO: 13), a PMO-plus targeting the M1 / M2 segment of the influenza translation site was tested on ferrets infected with a pandemic influenza virus (SOIV) H1N1 resistant to non-adapted oseltamivir. A total of 36 male ferrets were used in this study. Male ferrets with a combined body weight of about 700 g at the start of the study were randomly assigned to one of 5 treatment groups (shown in Table 7 below), and placed in Hepa filter cages (four per cage) to monitor the transmission cage to virus cage. The cages were kept inside the BSL-2 laboratory at the University of Tulano Medical Center. Table 7. Study design on ferrets

[00303] Ferrets were treated with AVI-7100 1 to 4 hours before viral deficiency. The route of administration was intraperitoneal for groups 2, 3, and 5; and oral for group 1. The dose interval was 4 hours, 24, 48, 72, 96 and 120 hours after the viral discount. Groups 5 were treated as follows: Group 1 received oseltamivir at 5 mg / kg every 12 hours orally, Group 2 received AVI-7100 (a PMOplus compound; 5'- CGG T + TA GAA GAC + TCA TC + TTT -3 ') at 10 mg / kg dose via ip, Group 3 received AVI-7100 (a PMOplus compound) at 30 mg / kg dose via ip, Group 4 received sterile saline control via ip, Group 5 received AVI -7100 to 10 mg / kg once a day via ip and oseltamivir at 5 mg / kg twice daily. The reason for differences in group sizes between groups 1-2 (8 ferrets each) and groups 4-5 (6 ferrets each) was due to the limited availability of seronegative influenza A ferrets at the start of the study.
[00304] All ferrets involved in this study survived at the end of the study, day eight post-infection, suggesting either that these animals were very healthy, or that this particular virus was not very pathogenic in this model. Nevertheless, as shown below, these results not only show that treatment with AVI-7100 significantly reduces symptoms of influenza virus infection relative to controls untreated or treated with oseltamivir, but also illustrates the synergistic effects that can be achieved with the combination of AVI-7100 and osel- tamivir. A summary of the clinical observations is shown in Table 8 below. Taba e 8: Clinical Observations


[00305] As an additional indicator, observations of cells that infiltrate the upper respiratory tract are measures of the severity of the infection. The summary of macrophage cellularity in the nasal lavage is included in Table 9 below. In addition, untreated ferrets treated only with oseltamivir showed significant lung congestion with marked alveolitis (inflammation of the lung), abundant infiltrating cells including lymphocytes and neutrophils, and moderate alveolar wall thickness of the lung. In contrast, ferrets treated with AVI-7100 (with or without oseltamivir) showed no congestion in the lung, only mild alveolitis, and few infiltrating cells. Table 9. Macrophage Cellularity in the Upper Respiratory Tract

[00306] As shown in Table 10 below, peak viremia in the nasal lavage was observed on day 1. No nasal lavage was collected on day 2, 4, 6 and 7 in order to minimize the unpleasant influence of the nasal lavage collection on the progression of viral infection. Significant benefit was observed in the group treated with AVI-7100 relative to either saline or oseltamivir. Synergistic effects were also observed with the combination of AVI-7100 (10 mg / kg) and oseltamivir, relating to treatment with AVI-7100 only (10 mg / kg) and oseltamivir only. Here, the AUC for viral titer in the nasal lavage for the combination (AVI-7100 and oseltamivir) shows a reduction greater than 4 log relative to the tamiflu-only group and a reduction greater than 3 log relative to the saline group. The combination also shows a much greater reduction in viral titer relative to the equivalent amount of AVI-7100 alone (from AUC of 5.515 to AUC of 2.999), suggesting that AVI-7100 may increase the anti-viral effects of oseltamivir. This result is surprising because the virus used in this study is otherwise resistant to oseltamivir. Table 10: Viral Title
EXAMPLE 3 INFLUENZA A VIRUS INHIBITION IN TISSUE CULTURE USING ANTI-SENSITIVE OLIGOMERS WITH TARGET ON THE splice SITE
[00307] One aspect of the present invention is the inhibition of replication of influenza A virus by multiple site antisense target within the M1 / M2 segment. In addition to inhibiting translation by targeting the common M1 / M2 AUG starting site, splice donor sites and splice acceptors can also be targets using compounds of the invention. Two PMO targeting the splice acceptor site at position 740 were synthesized as peptide conjugated PPMO, SA740 and SA746 (SEQ ID NOs: 26 and 29, respectively) and replaced in an in vitro tissue culture replication system for strain H1N1 PR8. The P007 cell penetrating peptide (SEQ ID NO: 118) was conjugated to the 3 'terminal of the PMO.
[00308] Alveolar murine macrophage cell line (ATCC; AMJ2-C11) was infected at 0.1 MOI with H1N1 (strain PR8) and 1 hour post-infection PPMOs were added. Cells were incubated at 35 degrees C for one night. Viral supernatant was then taken and incubated with VNAR protease to release the viral RNA. HA RNA was quantified by quantitative real-time PCR (qRT-PCR). Cells were washed, fixed and permeabilized. M1 and M2 proteins were then tested with monoclonal antibodies for 30 min at 37 degrees C. Cells were washed and anti-mouse IgG conjugated to Alexa 646 was added for 15 min at room temperature. M1 and M2 were then tested by flow cytometry. To determine levels of M1 and M2 protein, the percentage of positive M1 or M2 cells was multiplied by the mean fluorescence intensity of M1 or M2. Each sample was then divided by the untreated control to generate the percentage of M1 or M2 compared to untreated mixed controls.
[00309] Figure 8A shows the reduction in viral levels of HA RNA (measured using qRT-PCR). Both SA740 and Sa746 inhibited the production of HA RNA indicating an inhibition of viral replication compared to the mixed control. The most profound effect was observed in 10 micromolar with an approximate reduction of two logs using SA746 and a reduction of one log with SA740. Figures 8B and 8C show the effect of SA740 and SA746 on protein levels M1 and M2, respectively. The flow cytometry method described above was used to determine the relative protein levels. Both oligomers inhibited the production of M2 protein, although levels of M1 protein were reduced by SA740. EXAMPLE 4 INFLUENZA A VIRUS INHIBITION IN TISSUE CULTURE USING BLOCKED NUCLEIC ACID OLIGOMERS
[00310] The compounds of the present invention include oligonucleotide analogs comprised of different chemical entities than PMO. A series of blocked nucleic acids that target the region of the AUG starting site of the M1 / M2 segment have been synthesized (LNA-AUG1, LNA-AUG12, LNA-AUG13 and LNA-AUG10; SEQ ID NOs: 63, 74 , 75 and 72, respectively) and tested in the same assay for protein expression of viral RNA and M2 as described above in Example 3. Intracellular distribution of LNA oligomers was through “gymnotic” distribution (Stein, Hansen et al, 2010) . AMJ2-C11 cells were infected with PR8 for 1 h and then washed. The cells were then plated in a 96-well plate with LNA or 2'OMe compounds and allowed to incubate overnight at 35 degrees C. Levels of viral RNA and M2 protein expression were evaluated at this time (approximately 18 hours total). incubation time). Figure 9A shows the effect of four different LNAs on viral RNA levels (the HA segment). At 7.5 micromolar there is a reduction of approximately 3 log in the levels of viral HA RNA for the oligomer LNA-AUG1 compared to a reduction of approximately 1.5 log for the compound LNA-AUG12 (SEQ ID NOs: 63 and 74, respectively ). LNA-AUG is a 20mer although LNA-AUG12 is a 16mer. There is an order of effectiveness according to the length for all four LNA oligomers indicating that the most London LNAs are preferred embodiments of the invention. This relationship is also observed in the measure of the M2 protein expression shown in Figure 9B with the LNA-AUG1 oligo being more effective when compared to the LNA-AUG10 compound in 7.5 micromolar (SEQ ID NOs: 63 and 72, respectively). The relatively short LNA-AUG10 compound consisting of a base 10 targeting sequence was at least effective in both HA RNA and M2 protein expression assays. EXAMPLE 5 INFLUENZA A VIRUS INHIBITION IN TISSUE CULTURE USING 2'OMe OLIGOMERS
[00311] The compounds of the present invention also include analog antisense oligomers consisting of 2'OMe residues linked by phosphorothioate bonds. Three 2'OMe oligos were produced by IDT, 2'OMe-AUGl, 2'0Me-AUG2 and 2'OMe-SAl; SEQ ID NOs: 12, 20 and 26, respectively. These oligomers were designed to target the AUG initiation codon of the M1 / M2 segment of the splice acceptor site located at nucleotide 740. The 2'Me-SA1 (SEQ ID NO: 26) sequence matches that of the compound PPMO described in Example 3 above as SA740. The 2'Ome compounds were tested in the same assay for their ability to inhibit viral HA RNA levels and M2 protein expression as described above in Examples 3 and 4. Intracellular distribution was achieved through “gymnosis” as described above for LNAs in Example 4.
[00312] All three 2'Ome compounds were effective in reducing levels of viral HA RNA from between 2.5 and 4.5 logs to 7.5 micromolar as shown in Figure 10A. The relative effectiveness of the three compounds was also observed in the M2 protein measurement assays as shown in Figure 10B. The most effective compound was 2'OMe-AUG2 24mer that targets the region of the AUG starting site (SEQ ID NO: 20). Similarly effective was the 2'OMe-SA1 oligomer (SEQ ID NO: 26) which has the M1 / M2 splice acceptor site downstream. EXAMPLE 6 INHIBITION OF M1 AND M2 IN VITRO PROTEIN EXPRESSION
[00313] The effect of an exemplary compound of the invention on the expression of the M1 and M2 protein was evaluated using a western blot analysis of the treated and infected AMJ2-C11 cells. An exemplary PPMO compound of the M1 / M2 PPMO invention; P007-M1 / M2-AUG; SEQ ID NO: 12 conjugated at the 3 'terminal of SEQ ID NO: 118) was used to treat MDCK cells for one night at 3 micromolar. The cells were then subsequently infected with H1N1-PR8 at 0.01 MOI for 1 hour and washed. 18 hours post-infection the cells were lysed and protein extracted. Equal amounts were loaded onto gels for subsequent analysis by a standard (western) immunoblot assay using monoclonal antibodies that react with M1, M2 and actin proteins. As shown in Figure 11, the expression of both M1 and M2 proteins was reduced compared to an untreated control and an irrelevant PPMO control (Dengue). Signal strength analysis indicated that M2 protein expression was inhibited by M1 / M2 PPMO to a greater extent than M1 protein expression as shown in Figure 11 (i.e., 9% for M2 versus 27% for M1). The comparison signal for M1 and M2 was normalized for actin control. List of Strings

权利要求:
Claims (9)
[0001]
1. Isolated antiviral antisense oligonucleotide CHARACTERIZED by the fact that it comprises: a) a nuclease-resistant structure; b) 20-40 nucleotide bases, and c) a targeting sequence that hybridizes, or is complementary to, the 45 bases that surround the AUG initiation codon of an influenza M1 or M2 viral mRNA, in which the oligonucleotide is a morpholine oligonucleotide, in that the targeting sequence comprises SEQ ID NO: 12 or 13; or where the oligonucleotide comprises one or more blocked nucleic acid (LNA) subunits, where the targeting sequence comprises SEQ ID NO: 63; or where the oligonucleotide is a peptide nucleic acid (PNA), where the targeting sequence comprises SEQ ID NO: 48.
[0002]
2. Oligonucleotide, according to claim 1, CHARACTERIZED by the fact that the oligonucleotide has the ability to form a heteroduplex structure with the viral target region, in which said heteroduplex structure is: a) composed of the positive sense ribbon of the virus and oligonucleotide, and b) has a dissociation Tm of at least 45 ° C.
[0003]
3. Oligonucleotide, according to claim 1, CHARACTERIZED by the fact that the subunits of the morpholino oligonucleotide are joined by bonds containing phosphorus according to the structure:
[0004]
4. Oligonucleotide, according to claim 3, CHARACTERIZED by the fact that X is 1-piperazine for at least 2 and not more than half of the total number of phosphorus-containing bonds, and in which the remaining bonds are X = NR2 , where each R is independently hydrogen or methyl.
[0005]
5. Oligonucleotide, according to claim 4, CHARACTERIZED by the fact that the morpholino oligonucleotide comprises the targeting sequence of SEQ ID NO: 13, and has 3 phosphorus-containing bonds, where X is 1-piperazine as indicated by (+) in SEQ ID NO: 13.
[0006]
6. Antisense antiviral oligonucleotide, according to claim 1, CHARACTERIZED by the fact that it consists of the following structure:
[0007]
7. Oligonucleotide, according to claim 1, CHARACTERIZED by the fact that the oligonucleotide is conjugated to an arginine-rich polypeptide that increases oligonucleotide uptake in host cells.
[0008]
8. Oligonucleotide, according to claim 7, CHARACTERIZED by the fact that the arginine-rich polypeptide is selected from the group consisting of SEQ ID NOs: 115 to 128.
[0009]
9. Pharmaceutical composition CHARACTERIZED by the fact that it comprises an antisense oligonucleotide as defined in any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-06-12| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-06-26| B07B| Technical examination (opinion): publication cancelled [chapter 7.2 patent gazette]|

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2019-02-12| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-03-31| B25D| Requested change of name of applicant approved|Owner name: SAREPTA THERAPEUTICS, INC. (US) |

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2020-06-16| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-10-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-22| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 22/12/2020, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 12/11/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US26127809P| true| 2009-11-13|2009-11-13|

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US61/261,278|2009-11-13|

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US29205610P| true| 2010-01-04|2010-01-04|

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US61/292,056|2010-01-04|

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US37738210P| true| 2010-08-26|2010-08-26|

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US61/377,382|2010-08-26|

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PCT/US2010/056613|WO2011060320A1|2009-11-13|2010-11-12|Antisense antiviral compound and method for treating influenza viral infection|

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