U.S. patent application number 11/433213 was filed with the patent office on 2007-01-04 for antisense antiviral compound and method for treating influenza viral infection.
Invention is credited to Jianzhu Chen, Qing Ge, Patrick L. Iversen, David A. Stein, Dwight D. Weller.
Application Number | 20070004661 11/433213 |
Document ID | / |
Family ID | 36228476 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070004661 |
Kind Code |
A1 |
Stein; David A. ; et
al. |
January 4, 2007 |
Antisense antiviral compound and method for treating influenza
viral infection
Abstract
The invention provides antisense antiviral compounds and methods
of their use and production in inhibition of growth of viruses 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. The antisense antiviral
compounds are substantially uncharged, including partially
positively charged, morpholino oligonucleotides having 1) a
nuclease resistant backbone, 2) 12-40 nucleotide bases, and 3) a
targeting sequence of at least 12 bases in length that hybridizes
to a target region selected from the following: a) the 5' or 3'
terminal 25 bases of the negative sense viral RNA segment of
Influenzavirus A, Influenzavirus B and Influenzavirus C; b) the
terminal 25 bases of the 3' terminus of the positive sense cRNA
and; and c) the 50 bases surrounding the AUG start codon of an
influenza viral mRNA.
Inventors: |
Stein; David A.; (Corvallis,
OR) ; Ge; Qing; (Rolla, MO) ; Chen;
Jianzhu; (Brookline, MA) ; Iversen; Patrick L.;
(Corvallis, OR) ; Weller; Dwight D.; (Corvallis,
OR) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Family ID: |
36228476 |
Appl. No.: |
11/433213 |
Filed: |
May 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11259434 |
Oct 25, 2005 |
|
|
|
11433213 |
May 11, 2006 |
|
|
|
60622077 |
Oct 26, 2004 |
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Current U.S.
Class: |
514/44A ; 514/81;
536/23.1; 544/81 |
Current CPC
Class: |
C12N 2310/3233 20130101;
C12N 2310/3513 20130101; C12N 15/1131 20130101; C12N 2310/3145
20130101 |
Class at
Publication: |
514/044 ;
514/081; 536/023.1; 544/081 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/02 20060101 C07H021/02; C07F 9/6533 20060101
C07F009/6533 |
Claims
1. An antiviral compound comprising an oligonucleotide analog
having a) a nuclease-resistant backbone, b) 12-40 nucleotide bases,
and c) a targeting sequence of at least 12 bases in length that
hybridizes to a target region selected from the following: i) the
5' or 3' terminal 25 bases of a negative sense viral RNA segment of
Influenzavirus A, Influenzavirus B and Influenzavirus C, ii) the
terminal 25 bases of the 3' terminus of a positive sense cRNA of
Influenzavirus A, Influenzavirus B and Influenzavirus C, and iii)
the 50 bases surrounding the AUG start codon of an influenza viral
mRNA, wherein said oligonucleotide analog further has: a) the
capability of being actively taken up by mammalian host cells, and
b) the ability to form a heteroduplex structure with the viral
target region, wherein said heteroduplex structure is: i) composed
of the positive or negative sense strand of the virus and the
oligonucleotide compound, and ii) characterized by a Tm of
dissociation of at least 45.degree. C.
2. (canceled)
3. The compound of claim 1, wherein the oligonucleotide analog is
composed of morpholino subunits linked by phosphorous-containing
intersubunit linkages that join a morpholino nitrogen of one
subunit to a 5' exocyclic carbon of an adjacent subunit.
4. The compound of claim 3, wherein the morpholino subunits are
joined by phosphorodiamidate linkages in accordance with the
structure: ##STR4## where Y.sub.1.dbd.O, Z=O, Pj is a purine or
pyrimidine base-pairing moiety effective to bind, by base-specific
hydrogen bonding, to a base in a polynucleotide, and X is alkyl,
alkoxy, thioalkoxy, or alkyl amino.
5. The compound of claim 3, in which at least 2 and no more than
half of the total number of intersubunit linkages are positively
charged at physiological pH.
6. The composition of claim 5, wherein said morpholino subunits are
linked by phosphorodiamidate linkages, in accordance with the
structure: ##STR5## where Y.sub.1.dbd.O, Z=O, Pj is a purine or
pyrimidine base-pairing moiety effective to bind, by base-specific
hydrogen bonding, to a base in a polynucleotide, and X for the
uncharged linkages is alkyl, alkoxy, thioalkoxy, or an alkyl amino
of the form wherein NR.sub.2, where each R is independently
hydrogen or methyl, and for the positively charged linkages, X is
1-piperazine.
7. The compound of claim 1, wherein the oligonucleotide analog
hybridizes to a sequence selected from the group consisting of SEQ
ID NOs:1-9.
8. The compound of claim 1, wherein the viral target region
comprises SEQ ID NO:3 or SEQ ID NO:5.
9. The compound of claim 1, wherein the antisense compound has at
least 12 contiguous bases from one of the sequences selected from
the group consisting of SEQ ID NOs:10-24.
10. The compound of claim 1, wherein the targeting sequence
comprises SEQ ID NO:12 or SEQ ID NO:13.
11. The compound of claim 1, wherein the oligonucleotide analog is
conjugated to an arginine-rich polypeptide that enhances the uptake
of the compound into host cells.
12. The compound of claim 10, wherein the arginine-rich polypeptide
is selected from the group consisting of SEQ ID NOs:25-30.
Description
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 11/259,434, filed Oct. 25, 2005, which claims the benefit
of priority to U.S. Provisional Application No. 60/622,077, filed
Oct. 26, 2004. Both applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to antisense oligonucleotide compounds
for use in treating an influenza virus infection and antiviral
treatment methods employing the compounds.
REFERENCES
[0003] Agrawal, S., S. H. Mayrand, et al. (1990). "Site-specific
excision from RNA by RNase H and mixed-phosphate-backbone
oligodeoxynucleotides." Proc Natl Acad Sci USA 87(4): 1401-5.
[0004] Blommers, M. J., U. Pieles, et al. (1994). "An approach to
the structure determination of nucleic acid analogues hybridized to
RNA. NMR studies of a duplex between 2'-OMe RNA and an
oligonucleotide containing a single amide backbone modification."
Nucleic Acids Res 22(20): 4187-94. [0005] Bonham, M. A., S. Brown,
et al. (1995). "An assessment of the antisense properties of RNase
H-competent and steric-blocking oligomers." Nucleic Acids Res
23(7): 1197-203. [0006] Boudvillain, M., M. Guerin, et al. (1997).
"Transplatin-modified oligo(2'-O-methyl ribonucleotide)s: a new
tool for selective modulation of gene expression." Biochemistry
36(10): 2925-31. [0007] Cox, N. J. and K. Subbarao (1999).
"Influenza." Lancet 354(9186): 1277-82. [0008] Cox, N. J. and K.
Subbarao (2000). "Global epidemiology of influenza: past and
present." Annu Rev Med 51: 407-21. [0009] Cross, C. W., J. S. Rice,
et al. (1997). "Solution structure of an RNA.times.DNA hybrid
duplex containing a 3'-thioformacetal linker and an RNA A-tract."
Biochemistry 36(14): 4096-107. [0010] Dagle, J. M., J. L. Littig,
et al. (2000). "Targeted elimination of zygotic messages in Xenopus
laevis embryos by modified oligonucleotides possessing terminal
cationic linkages." Nucleic Acids Res 28(10): 2153-7. [0011] Ding,
D., S. M. Grayaznov, et al. (1996). "An oligodeoxyribonucleotide
N3'->P5' phosphoramidate duplex forms an A-type helix in
solution." Nucleic Acids Res 24(2): 354-60. [0012] Egholm, M., O.
Buchardt, et al. (1993). "PNA hybridizes to complementary
oligonucleotides obeying the Watson-Crick hydrogen-bonding rules."
Nature 365(6446): 566-8. [0013] Felgner, P. L., T. R. Gadek, et al.
(1987). "Lipofection: a highly efficient, lipid-mediated
DNA-transfection procedure." Proc Natl Acad Sci USA 84(21): 7413-7.
[0014] Gait, M. J., A. S. Jones, et al. (1974).
"Synthetic-analogues of polynucleotides XII. Synthesis of thymidine
derivatives containing an oxyacetamido- or an oxyformamido-linkage
instead of a phosphodiester group." J Chem Soc [Perkin 1] 0(14):
1684-6. [0015] Gee, J. E., I. Robbins, et al. (1998). "Assessment
of high-affinity hybridization, RNase H cleavage, and covalent
linkage in translation arrest by antisense oligonucleotides."
Antisense Nucleic Acid Drug Dev 8(2): 103-11. [0016] Lesnikowski,
Z. J., M. Jaworska, et al. (1990). "Octa(thymidine
methanephosphonates) of partially defined stereochemistry:
synthesis and effect of chirality at phosphorus on binding to
pentadecadeoxyriboadenylic acid." Nucleic Acids Res 18(8): 2109-15.
[0017] Mertes, M. P. and E. A. Coats (1969). "Synthesis of
carbonate analogs of dinucleosides. 3'-Thymidinyl 5'-thymidinyl
carbonate, 3'-thymidinyl 5'-(5-fluoro-2'-deoxyuridinyl) carbonate,
and 3'-(5-fluoro-2'-deoxyuridinyl) 5'-thymidinyl carbonate." J Med
Chem 12(1): 154-7. [0018] 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.
[0019] Nelson, M. H., D. A. Stein, et al. (2005). "Arginine-rich
peptide conjugation to morpholino oligomers: effects on antisense
activity and specificity." Bioconjug Chem 16(4): 959-66. [0020]
Strauss, J. H. and E. G. Strauss (2002). Viruses and Human Disease.
San Diego, Academic Press. [0021] Summerton, J. and D. Weller
(1997). "Morpholino antisense oligomers: design, preparation, and
properties." Antisense Nucleic Acid Drug Dev 7(3): 187-95. [0022]
Toulme, J. J., R. L. Tinevez, et al. (1996). "Targeting RNA
structures by antisense oligonucleotides." Biochimie 78(7): 663-73.
[0023] Williams, A. S., J. P. Camilleri, et al. (1996). "A single
intra-articular injection of liposomally conjugated methotrexate
suppresses joint inflammation in rat antigen-induced arthritis." Br
J Rheumatol 35(8): 719-24. [0024] Wu, G. Y. and C. H. Wu (1987).
"Receptor-mediated in vitro gene transformation by a soluble DNA
carrier system." J Biol Chem 262(10): 4429-32.
BACKGROUND OF THE INVENTION
[0025] Influenza viruses have been a major cause of human mortality
and morbidity throughout recorded history. Influenza A virus
infection causes millions of cases of severe illness and as many as
500,000 deaths each year worldwide. Epidemics vary widely in
severity but occur at regular intervals and always cause
significant mortality and morbidity, most frequently in the elderly
population. Although vaccines against matched influenza strains can
prevent illness in 60-80% of healthy adults, the rate of protection
is much lower in high-risk groups. Furthermore, vaccination does
not provide protection against unexpected strains, such as the H5
and H7 avian influenza outbreaks in Hong Kong in 1997 and Europe
and Southeast Asia in 2003 and 2004. Current anti-influenza drugs
are limited in their capacity to provide protection and therapeutic
effect (Cox and Subbarao 1999; Cox and Subbarao 2000).
[0026] Influenza A is a segmented RNA virus of negative-polarity.
Genome segments are replicated by a complex of 4 proteins: the 3
polymerase polypeptides (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).
[0027] Influenza A viruses can be subtyped according to the
antigenic and genetic nature of their surface glycoproteins; 15
hemagglutinin (HA) and 9 neuraminidase (NA) subtypes have been
identified to date. Viruses bearing all known HA and NA subtypes
have been isolated from avian hosts, but only viruses of the H1N1
(1918), H2N2 (1957/58), and H3N2 (1968) subtypes have been
associated with widespread epidemics in humans (Strauss and Strauss
2002).
[0028] Since 1997, when H5N1 influenza virus was transmitted to
humans and killed 6 of 18 infected persons, there have been
multiple transmissions of avian influenza viruses to mammals.
Either the whole 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 increasing concern that this subtype may achieve
human-to-human spread and establish interspecies transmission. The
species which different types of influenza viruses are able to
infect are determined by different forms of the virus glycoproteins
(HA, NA). This provides a considerable species barrier between
birds and humans which is not easily overcome. Pigs, however,
provide a "mixing pot"-able to be infected by both types of virus
and thereby 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 readily infect humans. Avian HA can infect pigs,
but not humans. In pigs, during genome segment packaging, it is
possible to create a virus with several Avian segments and Human HA
and/or NA segments (Cox and Subbarao 2000).
[0029] Influenza viruses infect humans and animals (e.g., pigs,
birds, horses) and may cause acute respiratory disease. There have
been numerous attempts to produce vaccines effective against
influenza virus. None, however, have been completely successful,
particularly on a long-term basis. This may be due, at least in
part, to the segmented characteristic of the influenza virus
genome, which makes it possible, through re-assortment of the
segments, for numerous forms to exist. For example, it has been
suggested that there could be an interchange of RNA segments
between animal and human influenza viruses, which would result in
the introduction of new antigenic subtypes into both populations.
Thus, a long-term vaccination approach has failed, due to the
emergence of new subtypes (antigenic "shift"). In addition, the
surface proteins of the virus, hemagglutinin and neuraminidase,
constantly undergo minor antigenic changes (antigenic "drift").
This high degree of variation explains why specific immunity
developed against a particular influenza virus does not establish
protection against new variants. Hence, alternative antiviral
strategies are needed. Although influenza B and C viruses cause
less clinical disease than the A types, new antiviral drugs should
also be helpful in curbing infections caused by these agents.
[0030] Influenza viruses that occur naturally among birds are
called avian influenza (bird flu). The birds carry the viruses in
their intestines but do not generally get sick from the infection.
However, migratory birds can carry the bird flu to infect domestic
chickens, ducks and turkeys causing illness and even death. Avian
flu 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 terns in
South Africa in 1961 and was identified as a potentially deadly
form of flu. Outbreaks of H5N1 occurred in eight Asian countries in
late 2003 and 2004. At that time more than 100 million birds in
these countries either died or were killed in order to control the
outbreak. Beginning in June of 2004 new deadly outbreaks of H5N1
were reported in Asia which is currently ongoing. Human infections
of H5N1 have been observed in Thailand, Vietnam and Cambodia with a
death rate of about 50 percent. These infections have mostly
occurred from human contact with infected poultry but a few cases
of human-to-human spread of H5N1 have occurred.
[0031] Currently, there is no vaccine to protect humans against
H5N1 but research efforts are underway. There are four currently
approved influenza medications, amantadine, rimantadine,
oseltamivir and zanamivir. Unfortunately, the H5N1 virus is
resistant to both amantadine and rimantidine. The remaining
oseltamivir and zanamivir may show some efficacy to H5N1 but need
to be evaluated more extensively.
[0032] In view of the severity of the diseases caused by influenza
viruses there is an immediate need for new therapies to treat
influenza infection. Given the lack of effective prevention or
therapies, it is therefore an object of the present invention to
provide therapeutic compounds and methods for treating a host
infected with an influenza virus.
SUMMARY OF THE INVENTION
[0033] The invention includes, in one aspect, an anti-viral
compound effective in inhibiting replication within a host cell of
an RNA virus having a single-stranded, negative sense genome and
selected from the Orthomyxoviridae family including the
Influenzavirus A, Influenzavirus B and Influenzavirus C genera. The
compound targets viral RNA sequences within a region selected from
the following: 1) the 5' or 3' terminal 25 bases of the negative
sense viral RNA segments; 2) the terminal 25 bases of the 3'
terminus of the positive sense cRNA and; 3) 50 bases surrounding
the AUG start codons of influenza viral mRNAs.
[0034] The antiviral compound consists of an oligonucleotide analog
characterized by: a) a nuclease-resistant backbone, b) 12-40
nucleotide bases, and c) a targeting sequence of at least 12 bases
in length, that hybridizes to a target region selected from the
following: i) the 5' or 3' terminal 25 bases of a negative sense
viral RNA segment of Influenzavirus A, Influenzavirus B and
Influenzavirus C, ii) the terminal 30 bases of the 3' terminus of a
positive sense cRNA of Influenzavirus A, Influenzavirus B and
Influenzavirus C, and iii) the 50 bases surrounding the AUG start
codon of an influenza viral mRNA.
[0035] The oligonucleotide analog also has: a) the capability of
being actively taken up by mammalian host cells, and b) the ability
to form a heteroduplex structure with the viral target region,
wherein said heteroduplex structure is: i) composed of the positive
or negative sense strand of the virus and the oligonucleotide
compound, and ii) characterized by a Tm of dissociation of at least
45.degree. C.
[0036] The invention includes, in another aspect, an antiviral
compound that inhibits, in a mammalian host cell, replication of an
infecting influenza virus having a single-stranded, segmented,
negative-sense genome and selected from the Orthomyxoviridae
family. The compound is administered to the infected host cells as
an oligonucleotide analog characterized by the elements described
above on pp. 5-6. The compound may be administered to a mammalian
subject infected with the influenza virus, or at risk of infection
with the influenza virus.
[0037] The compound may be composed of morpholino subunits linked
by uncharged, phosphorus-containing intersubunit linkages, joining
a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an
adjacent subunit. In one embodiment, the intersubunit linkages are
phosphorodiamidate linkages, such as those having the structure:
##STR1## where Y.sub.1.dbd.O, Z=O, Pj is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and X is alkyl, alkoxy,
thioalkoxy, or alkyl amino, e.g., wherein X.dbd.NR.sub.2, where
each R is independently hydrogen or methyl.
[0038] The compound may be composed of morpholino subunits linked
with the uncharged linkages described above interspersed with
linkages that are positively charged at physiological pH. The total
number of positively charged linkages is between 2 and no more than
half of the total number of linkages. The positively charged
linkages have the structure above, where X is 1-piperazine.
[0039] The compound may be a covalent conjugate of an
oligonucleotide analog moiety capable of forming such a
heteroduplex structure with the positive or negative sense strand
of the virus, and an arginine-rich polypeptide effective to enhance
the uptake of the compound into host cells. Exemplary polypeptides
have one of the sequences identified as SEQ ID NOs:25-30.
[0040] In a related aspect, the invention includes a heteroduplex
complex formed between: [0041] (a) the 5' or 3' terminal 25 bases
of the negative sense viral RNA and/or; [0042] (b) the terminal 25
bases of the 3' terminus of the positive sense mRNA and/or; [0043]
(c) 50 bases surrounding the AUG start codons of viral mRNA of an
influenza virus selected from the Orthomyxoviridae family and,
[0044] (d) an oligonucleotide analog compound characterized by:
[0045] (i) a nuclease-resistant backbone, [0046] (ii) capable of
uptake by mammalian host cells, [0047] (iii) containing between
12-40 nucleotide bases,
[0048] where said heteroduplex complex has a Tm of dissociation of
at least 45.degree. C. and disruption of a stem-loop secondary
structure.
[0049] An exemplary oligonucleotide analog is composed of
morpholino subunits linked by uncharged, phosphorus-containing
intersubunit linkages, joining a morpholino nitrogen of one subunit
to a 5' exocyclic carbon of an adjacent subunit. The compound may
have phosphorodiamidate linkages, such as in the structure ##STR2##
where Y.sub.1.dbd.O, Z=O, Pj is a purine or pyrimidine base-pairing
moiety effective to bind, by base-specific hydrogen bonding, to a
base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or
alkyl amino. In a preferred compound, X.dbd.NR.sub.2, where each R
is independently hydrogen or methyl. The compound may also be
composed of morpholino subunits linked with the uncharged linkages
described above interspersed with linkages that are positively
charged at physiological pH. The total number of positively charged
linkages is between 2 and no more than half of the total number of
linkages. The positively charged linkages have the structure above,
where X is 1-piperazine. The compound may be the oligonucleotide
analog alone or a conjugate of the analog and an arginine-rich
polypeptide capable of enhancing the uptake of the compound into
host cells. Exemplary polypeptides have one of the sequences
identified as SEQ ID NOs:25-30.
[0050] In still another aspect, the invention includes an
oligonucleotide analog compound for use in inhibiting replication
in mammalian host cells of an influenza virus having a
single-stranded, segmented, negative-sense RNA genome and selected
from the Orthomyxoviridae family. The compound is characterized by
the elements described above on pp. 5-6.
[0051] An exemplary oligonucleotide analog is composed of
morpholino subunits linked by uncharged, phosphorus-containing
intersubunit linkages, joining a morpholino nitrogen of one subunit
to a 5' exocyclic carbon of an adjacent subunit. The compound may
have phosphorodiamidate linkages, such as in the structure ##STR3##
where Y.sub.1.dbd.O, Z=O, Pj is a purine or pyrimidine base-pairing
moiety effective to bind, by base-specific hydrogen bonding, to a
base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or
alkyl amino. In a preferred compound, X.dbd.NR.sub.2, where each R
is independently hydrogen or methyl. The compound may be composed
of morpholino subunits linked with the uncharged linkages described
above interspersed with linkages that are positively charged at
physiological pH. The total number of positively charged linkages
is between 2 and no more than half of the total number of linkages.
The positively charged linkages have the structure above, where X
is 1-piperazine.
[0052] The compound may be the oligonucleotide analog alone or a
conjugate of the analog and an arginine-rich polypeptide capable of
enhancing the uptake of the compound into host cells. Exemplary
polypeptides have one of the sequences identified as SEQ ID
NOs:25-30.
[0053] For treatment of Influenza A virus as given below, the
targeting sequence hybridizes to a region associated with one of
the group of sequences identified as SEQ ID NOs:1-9. Preferred
targeting sequences are those complementary to either the minus
strand target of SEQ ID NO:4 or the positive-strand target of SEQ
ID NO:3. Exemplary antisense phosphorodiamidate morpholino
oligomers ("PMOs") that target these two regions are listed as SEQ
ID NOs:12 and 13, respectively.
BRIEF DESCRIPTION OF THE FIGURES
[0054] FIGS. 1A-1D show the repeating subunit segment of several
preferred morpholino oligonucleotides, designated A through D,
constructed using subunits having 5-atom (A), six-atom (B) and
seven-atom (C-D) linking groups suitable for forming polymers.
[0055] FIGS. 2A-2G show examples of uncharged linkage types in
oligonucleotide analogs. FIG. 2H shows an example of a preferred
cationic linkage group.
[0056] FIG. 3 shows the three different species of influenza virus
RNA present in infected cells, vRNA, mRNA and cRNA, and the target
location of targeting PMO described herein.
[0057] FIG. 4 shows the conservation of target sequences in two
important stereotypes of influenza, H1N1 and H5N1, for each base of
two preferred PMOs (PB1-AUG and NP-3' terminus; SEQ ID NOs:13 and
12). The percentage of isolates having the indicated base is the
subscript number after each base.
[0058] FIGS. 5A-5B show the effect of 20 mM AUG-targeted and
termini-targeted PMO on influenza virus replication in infected
Vero cells. FIG. 5C describes the experimental protocol.
[0059] FIGS. 6A-6C show the dose response of AUG-targeted PMO on
influenza virus replication in Vero cells using the hemagglutinin
assay (6A) and the plaque assay techniques (6B). FIG. 6C describes
the experimental protocol.
[0060] FIGS. 7A-7B show the dose response of termini-targeted PMO
on influenza virus replication in Vero cells using the same assays
as in FIG. 6. FIG. 7C describes the experimental protocol.
[0061] FIGS. 8A-8C show the suppression of transcription of vRNA to
mRNA and cRNA by PMOs that target the 3' terminus of NP vRNA. FIG.
8D describes the experimental protocol.
[0062] FIG. 9A shows the synergistic effect of PMO that target the
termini of the NP segment and the NP gene AUG start codon on
influenza A virus replication in Vero cells. FIG. 9B describes the
experimental protocol.
[0063] FIG. 10 shows the synthetic steps to produce subunits used
to produce +PMO containing the (1-piperazino) phosphinylideneoxy
cationic linkage as shown in FIG. 2H.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0064] The terms below, as used herein, have the following
meanings, unless indicated otherwise:
[0065] "Alkyl" refers to a fully saturated monovalent radical
containing carbon and hydrogen, which may be branched, linear, or
cyclic (cycloalkyl). Examples of alkyl groups are methyl, ethyl,
n-butyl, t-butyl, n-heptyl, 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, ethyl, n-butyl, i-butyl,
t-butyl, isoamyl, n-pentyl, and isopentyl. In one embodiment, lower
alkyl refers to C.sub.1 to C.sub.4 alkyl.
[0066] "Alkenyl" refers to an unsaturated monovalent radical
containing carbon and hydrogen, which may be branched, linear, or
cyclic. The alkenyl group may be monounsaturated or
polyunsaturated. Generally preferred are alkenyl groups having one
to six carbon atoms, referred to as "lower alkenyl".
[0067] "Aryl" refers to a substituted or unsubstituted monovalent
aromatic radical, generally having a single ring (e.g., benzene) or
two condensed rings (e.g., naphthyl). This term includes heteroaryl
groups, which are aromatic ring groups having one or more nitrogen,
oxygen, or sulfur atoms in the ring, such as furyl, pyrrole,
pyridyl, and indole. By "substituted" is meant that one or more
ring hydrogens in the aryl group is 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
substituents include halogen, methyl, ethyl, and methoxy. Generally
preferred are aryl groups having a single ring.
[0068] "Aralkyl" refers to an alkyl, preferably lower
(C.sub.1-C.sub.4, more preferably C.sub.1-C.sub.2) alkyl,
substituent which is further substituted with an aryl group;
examples are benzyl (--CH.sub.2C.sub.6H.sub.5) and phenethyl
(--CH.sub.2CH.sub.2C.sub.6H.sub.5).
[0069] "Heterocycle" refers to a non-aromatic ring, preferably a 5-
to 7-membered ring, whose ring atoms are selected from the group
consisting of carbon, nitrogen, oxygen and sulfur. Preferably, the
ring atoms include 3 to 6 carbon atoms. Such heterocycles include,
for example, pyrrolidine, piperidine, piperazine, and
morpholine.
[0070] The term "substituted", with respect to an alkyl, alkenyl,
alkynyl, aryl, aralkyl, or alkaryl group, refers to replacement of
a hydrogen atom with a heteroatom-containing substituent, 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.
[0071] The terms "oligonucleotide analog" refers to an
oligonucleotide having (i) a modified backbone structure, e.g., a
backbone other than the standard phosphodiester linkage found in
natural oligo- and polynucleotides, and (ii) optionally, modified
sugar moieties, e.g., morpholino moieties rather than ribose or
deoxyribose moieties. The analog supports bases capable of hydrogen
bonding by Watson-Crick base pairing to standard polynucleotide
bases, where the analog backbone presents the bases in a manner to
permit such hydrogen bonding in a sequence-specific fashion between
the oligonucleotide analog molecule and bases in a standard
polynucleotide (e.g., single-stranded RNA or single-stranded DNA).
Preferred analogs are those having a substantially uncharged,
phosphorus containing backbone.
[0072] A substantially uncharged, phosphorus containing backbone in
an oligonucleotide analog is one in which a majority of the subunit
linkages, e.g., between 50-100%, are uncharged at physiological pH,
and contain a single phosphorous atom. The analog contains between
8 and 40 subunits, typically about 8-25 subunits, and preferably
about 12 to 25 subunits. The analog may have exact sequence
complementarity to the target sequence or near complementarity, as
defined below.
[0073] A "subunit" of an oligonucleotide analog refers to one
nucleotide (or nucleotide analog) unit of the analog. The term may
refer to the nucleotide unit with or without the attached
intersubunit linkage, although, when referring to a "charged
subunit", the charge typically resides within the intersubunit
linkage (e.g. a phosphate or phosphorothioate linkage).
[0074] A "morpholino oligonucleotide analog" is an oligonucleotide
analog composed of morpholino subunit structures of the form shown
in FIGS. 1A-1D, where (i) the structures are linked together by
phosphorus-containing linkages, one to three atoms long, joining
the morpholino nitrogen of one subunit to the 5' exocyclic carbon
of an adjacent subunit, and (ii) P.sub.i and P.sub.j are purine or
pyrimidine base-pairing moieties effective to bind, by
base-specific hydrogen bonding, to a base in a polynucleotide. The
purine or pyrimidine base-pairing moiety is typically adenine,
cytosine, guanine, uracil or thymine. The synthesis, structures,
and binding characteristics of morpholino oligomers are detailed in
U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,
5,166,315, 5,521,063, and 5,506,337, all of which are incorporated
herein by reference.
[0075] The subunit and linkage shown in FIG. 1B are used for
six-atom repeating-unit backbones, as shown in FIG. 1B (where the
six atoms include: a morpholino nitrogen, the connected phosphorus
atom, the atom (usually oxygen) linking the phosphorus atom to the
5' exocyclic carbon, the 5' exocyclic carbon, and two carbon atoms
of the next morpholino ring). In these structures, the atom Y.sub.1
linking the 5' exocyclic morpholino carbon to the phosphorus group
may be sulfur, nitrogen, carbon or, preferably, oxygen. The X
moiety pendant from the phosphorus is any stable group which does
not interfere with base-specific hydrogen bonding. Preferred X
groups include fluoro, alkyl, alkoxy, thioalkoxy, and alkyl amino,
including cyclic amines, all of which can be variously substituted,
as long as base-specific bonding is not disrupted. Alkyl, alkoxy
and thioalkoxy preferably include 1-6 carbon atoms. Alkyl amino
preferably refers to lower alkyl (C.sub.1 to C.sub.6) substitution,
and cyclic amines are preferably 5- to 7-membered nitrogen
heterocycles optionally containing 1-2 additional heteroatoms
selected from oxygen, nitrogen, and sulfur. Z is sulfur or oxygen,
and is preferably oxygen.
[0076] A preferred morpholino oligomer is a
phosphorodiamidate-linked morpholino oligomer, referred to herein
as a PMO. Such oligomers are composed of morpholino subunit
structures such as shown in FIG. 2B, where X.dbd.NH2, NHR, or NR2
(where R is lower alkyl, preferably methyl), Y.dbd.O, and Z=O, and
Pi and Pj are purine or pyrimidine base-pairing moieties effective
to bind, by base-specific hydrogen bonding, to a base in a
polynucleotide, as seen in FIG. 2G. Also preferred are morpholino
oligomers where the phosphordiamidate linkages are uncharged
linkages as shown in FIG. 2G interspersed with cationic linkages as
shown in FIG. 2H where, in FIG. 2B, X=1-piperazino. In another FIG.
2B embodiment, X=lower alkoxy, such as methoxy or ethoxy, Y.dbd.NH
or NR, where R is lower alkyl, and Z=O The term "substituted",
particularly with respect to an alkyl, alkoxy, thioalkoxy, or
alkylamino group, refers to replacement of a hydrogen atom on
carbon with a heteroatom-containing substituent, 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. It may also
refer to replacement of a hydrogen atom on a heteroatom (such as an
amine hydrogen) with an alkyl, carbonyl or other carbon containing
group.
[0077] As used herein, the term "target", relative to the viral
genomic RNA, refers to a viral genomic RNA, and specifically, to a
region associated with stem-loop secondary structure within the
5'-terminal end 40 bases of the positive-sense RNA strand of a
single-stranded RNA (ssRNA) virus described herein.
[0078] The term "target sequence" refers to a portion of the target
RNA against which the oligonucleotide analog is directed, that is,
the sequence to which the oligonucleotide analog will hybridize by
Watson-Crick base pairing of a complementary sequence. As will be
seen, the target sequence may be a contiguous region of the viral
positive-strand RNA, or may be composed of complementary fragments
of both the 5' and 3' sequences involved in secondary
structure.
[0079] The term "targeting sequence" is the sequence in the
oligonucleotide analog that is complementary (meaning, in addition,
substantially complementary) to the target sequence in the RNA
genome. The entire sequence, or only a portion, of the analog
compound may be complementary to the target sequence. For example,
in an analog having 20 bases, only 12-14 may be targeting
sequences. Typically, the targeting sequence is formed of
contiguous bases in the analog, but may alternatively be formed of
non-contiguous sequences that when placed together, e.g., from
opposite ends of the analog, constitute sequence that spans the
target sequence. As will be seen, the target and targeting
sequences are selected such that binding of the analog is to a
region within; 1) the 5' or 3' terminal 25 bases of the negative
sense viral RNA; 2) the terminal 25 bases of the 3' terminus of the
positive sense mRNA and/or; 3) 50 bases surrounding the AUG start
codons of viral mRNA.
[0080] Target and targeting sequences are described as
"complementary" to one another when hybridization occurs in an
antiparallel configuration. A targeting sequence may have "near" or
"substantial" complementarity to the target sequence and still
function for the purpose of the present invention, that is, it may
still be "complementary." Preferably, the oligonucleotide analog
compounds employed in the present invention have at most one
mismatch with the target sequence out of 10 nucleotides, and
preferably at most one mismatch out of 20. Alternatively, the
antisense oligomers employed have at least 90% sequence homology,
and preferably at least 95% sequence homology, with the exemplary
targeting sequences as designated herein.
[0081] An oligonucleotide analog "specifically hybridizes" to a
target polynucleotide if the oligomer hybridizes to the target
under physiological conditions, with a Tm substantially greater
than 45.degree. C., preferably at least 50.degree. C., and
typically 60.degree. C.-80.degree. C. or higher. Such hybridization
preferably corresponds to stringent hybridization conditions. At a
given ionic strength and pH, the Tm is the temperature at which 50%
of a target sequence hybridizes to a complementary polynucleotide.
Again, such hybridization may occur with "near" or "substantial"
complementary of the antisense oligomer to the target sequence, as
well as with exact complementarity.
[0082] A "nuclease-resistant" oligomeric molecule (oligomer) refers
to one whose backbone is substantially resistant to nuclease
cleavage, in non-hybridized or hybridized form; by common
extracellular and intracellular nucleases 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.
[0083] A "heteroduplex" refers to a duplex between an
oligonculeotide analog and the complementary portion of a target
RNA. A "nuclease-resistant heteroduplex" refers to a heteroduplex
formed by the binding of an antisense oligomer to its complementary
target, such that the heteroduplex is substantially resistant to in
vivo degradation by intracellular and extracellular nucleases, such
as RNAseH, which are capable of cutting double-stranded RNA/RNA or
RNA/DNA complexes.
[0084] A "base-specific intracellular binding event involving a
target RNA" refers to the specific binding of an oligonucleotide
analog to a target RNA sequence inside a cell. The base specificity
of such binding is sequence specific. For example, a
single-stranded polynucleotide can specifically bind to a
single-stranded polynucleotide that is complementary in
sequence.
[0085] An "effective amount" of an antisense oligomer, targeted
against an infecting influenza virus, is an amount effective to
reduce the rate of replication of the infecting virus, and/or viral
load, and/or symptoms associated with the viral infection.
[0086] As used herein, the term "body fluid" encompasses a variety
of sample types obtained from a subject including, urine, saliva,
plasma, blood, spinal fluid, or other sample of biological origin,
such as skin cells or dermal debris, and may refer to cells or cell
fragments suspended therein, or the liquid medium and its
solutes.
[0087] The term "relative amount" is used where a comparison is
made between a test measurement and a control measurement. The
relative amount of a reagent forming a complex in a reaction is the
amount reacting with a test specimen, compared with the amount
reacting with a control specimen. The control specimen may be run
separately in the same assay, or it may be part of the same sample
(for example, normal tissue surrounding a malignant area in a
tissue section).
[0088] "Treatment" of an individual or a cell is any type of
intervention provided as a means to alter the natural course of the
individual or cell. Treatment includes, but is not limited to,
administration of e.g., a pharmaceutical composition, and may be
performed either prophylactically, or subsequent to the initiation
of a pathologic event or contact with an etiologic agent. The
related term "improved therapeutic outcome" relative to a patient
diagnosed as infected with a particular virus, refers to a slowing
or diminution in the growth of virus, or viral load, or detectable
symptoms associated with infection by that particular virus.
[0089] An agent is "actively taken up by mammalian cells" when the
agent can enter the cell by a mechanism other than passive
diffusion across the cell membrane. The agent may be transported,
for example, by "active transport", referring to transport of
agents across a mammalian cell membrane by e.g. an ATP-dependent
transport mechanism, or by "facilitated transport", referring to
transport of antisense agents across the cell membrane by a
transport mechanism that requires binding of the agent to a
transport protein, which then facilitates passage of the bound
agent across the membrane. For both active and facilitated
transport, the oligonucleotide analog preferably has a
substantially uncharged backbone, as defined below. Alternatively,
the antisense compound may be formulated in a complexed form, such
as an agent having an anionic backbone complexed with cationic
lipids or liposomes, which can be taken into cells by an
endocytotic mechanism. The analog may also be conjugated, e.g., at
its 5' or 3' end, to an arginine-rich peptide, such as a portion of
the HIV TAT protein, polyarginine, or to combinations of arginine
and other amino acids including the non-natural amino acids
6-aminohexanoic acid (Ahx) and beta-alanine (.beta.Ala). Exemplary
arginine-rich delivery peptides are listed as SEQ ID NOs:25-30.
These exemplary arginine-rich delivery peptides facilitate
transport into the target host cell as described (Moulton, Nelson
et al. 2004; Nelson, Stein et al. 2005).
[0090] Rules for the selection of targeting sequences capable of
inhibiting replication of the influenza viral genome are discussed
below.
II. TARGETED VIRUSES
[0091] The present invention is based on the discovery that
effective inhibition of single-stranded, segmented, negative-sense
RNA viruses can be achieved by exposing cells infected with
influenza virus to antisense oligonucleotide analog compounds (i)
that target 1) the 5' or 3' terminal 25 bases of the negative sense
viral RNA; 2) the terminal 25 bases of the 3' terminus of the
positive sense mRNA and/or; 3) 50 bases surrounding the AUG start
codons of viral mRNA; and (ii) having physical and pharmacokinetic
features which allow effective interaction between the antisense
compound and the virus within host cells. In one aspect, the
oligomers can be used in treating a mammalian subject infected with
influenza virus.
[0092] The invention targets RNA viruses having genomes that are:
(i) single stranded, (ii) segmented and (iii) negative polarity.
The targeted viruses also synthesize two different versions of a
genomic complement of the negative sense virion RNA (vRNA) with
positive polarity: 1) cRNA that is used as a template for
replication of negative sense virion RNA, and 2) a complementary
positive sense RNA (mRNA) that is used for translation of viral
proteins. FIG. 3 is a schematic that shows these different RNA
species and the target location of antisense PMO described in the
present invention. In particular, targeted viral families include
members of the Orthomyxoviridae family including the Influenzavirus
A, Influenzavirus B and Influenzavirus C genera. Various physical,
morphological, and biological characteristics of members of the
Orthomyxoviridae family can be found, for example, in Textbook of
Human Virology, R. Belshe, ed., 2.sup.nd Edition, Mosby, 1991, at
the Universal Virus Database of the International Committee on
Taxonomy of Viruses (www.ncbi.nlm.nih.gov/ICTVdb/index.htm) and in
human virology textbooks (see, for example (Strauss and Strauss
2002). Some of the key biological characteristics of the
Orthomxyoviridae family of viruses are described below.
[0093] Influenza Viruses
[0094] Influenza A, influenza B and influenza C viruses are the
only members of the Influenzavirus A, Influenzavirus B and
Influenzavirus C genera, respectively. These viruses are
membrane-enclosed viruses whose genomes are segmented
negative-sense (i.e. minus) strands of RNA ((-)RNA). The ten
influenza virus genes are present on eight segments of the
single-stranded RNA of strains A and B, and on seven segments of
strain C. The segments vary in size (from 890 to 2341 nucleotides
in length) and each is a template for synthesis of different mRNAs.
The influenza virus virion contains virus-specific RNA polymerases
necessary for mRNA synthesis from these templates and, in the
absence of such specific polymerases, the minus strand of influenza
virus RNA is not infectious. Initiation of transcription of the
mRNAs occurs when the influenza virus mRNA polymerase takes 12 to
15 nucleotides from the 5' end of a cellular mRNA or mRNA precursor
and uses the borrowed oligonucleotide as a primer. This process has
been termed "cap-snatching" because it places a 5' cap structure on
the viral mRNA. Generally, the mRNAs made through this process
encode only one protein. The M gene and NS gene viral RNA segments
also code for spliced mRNAs, which results in production of two
different proteins for each of these two segments.
[0095] Replication of influenza viral RNA occurs in the nucleus and
involves the synthesis of three different species of RNA. A
schematic of this process is shown in FIG. 3. After infection of a
naive cell, the minus strand virion RNA (vRNA) is transported to
the nucleus where RNA destined for translation (mRNA) is
synthesized using 5'-terminal 10-13 nucleotide primers cleaved by
viral-encoded enzymes from capped cellular pre-mRNA molecules (i.e.
cap-snatching). Synthesis of each mRNA continues to near the end of
the genome segment where an oligo(U) stretch is encountered 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 into the nucleus, synthesis of vRNA destined
for nascent virions is initiated. An exact antigenomic copy of vRNA
is synthesized (termed cRNA) which is a perfect complement of the
genomic vRNA and serves as a template for production of new vRNA.
The different RNAs synthesized during influenza virus replication
are shown schematically in FIG. 3.
[0096] GenBank references 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-strand RNA. It
will be appreciated that these sequences are only illustrative of
other sequences in the Orthomyxoviridae family, as may be available
from available gene-sequence databases of literature or patent
resources. The sequences below, identified as SEQ ID NOs:1-9, are
also listed in the Sequence Listing at the end of the
specification.
[0097] The target sequences in Table 1 represent; 1) the 5' or 3'
terminal 25 bases of the negative sense viral RNA (SEQ ID NOs:4-9);
2) the terminal 25 bases of the 3' terminus of the positive sense
mRNA (SEQ ID NOs:4-9) and; 3) 50 bases surrounding the AUG start
codons of the indicated influenza virus genes (SEQ ID NOs:1-3). The
sequences shown are the positive-strand (i.e., antigenomic or mRNA)
sequence in the 5' to 3' orientation. It will be obvious that when
the target is the minus-strand vRNA the targeted sequence is the
complement of the sequence listed in Table 1.
[0098] Table 1 lists the targets for three different influenza A
viral genes, PB2, PB1 and nucleoprotein (NP), encoded by genomic
segments 1, 2 and 5, respectively. The PB1, PB2 and NP proteins are
components of the viral RNA polymerase and PB2 also functions as
the "cap-snatching" enzyme. The target sequences for the AUG start
codons of the three genes are represented as SEQ ID NOs:1-3. The 3'
terminal sequences of the three genomic segments are represented by
SEQ ID NOs:4, 6 and 8 and can be targeted on both the positive
strand and the negative strand of those segments. The 5' terminal
sequences (SEQ ID NOs:5, 7 and 9) can be successfully targeted on
the minus strand. TABLE-US-00001 TABLE 1 Exemplary Influenza Viral
Nucleic Acid Target Sequences Nucle- SEQ GenBank otide ID Name No.
Region Sequence (5' to 3') NO NP-31 J02147 21-
UCACUCACUGAGUGACAUCAAAAUCA 1 70 UGGCGUCCCAAGGCACCAAACGGU PB2-11
V00603 1- AGCGAAAGCAGGUCAAUUAUAUUCAA 2 50 UAUGGAAAGAAUAAAAGAACUAAG
PB1- J02151 1- AGCGAAAGCAGGCAAACCAUUUGAAU 3 AUG 50
GGAUGUCAAUCCGACCUUACUUUU NP- J02147 1541- AAAGAAAAAUACCCUUGUUUCUACU
4 3'term 1565 NP- J02147 1- AGCAAAAGCAGGGUAGAUAAUCACU 5 5'term 25
PB1- J02151 2317- CAUGAAAAAAUGCCUUGUUCCUACU 6 3'term 2341 PB1-
J02151 1- AGCGAAAGCAGGCAAACCAUUUGAA 7 5'term 25 PB2- V00603 2317-
GUUUAAAAACGACCUUGUUUCUACU 8 3'term 2341 PB2- V00603 1-
AGCGAAAGCAGGUCAAUUAUAUUCA 9 5'term 25
[0099] FIG. 4 shows conservation of target sequences in two
important serotypes of influenza, H1N1 and H5N1, for each base of
two preferred PMOs (PB1-AUG and NP-3'term; SEQ ID NOs:13 and 12)
based on Los Alamos National Laboratory (LANL) influenza database
of genome sequences (Macken, C., Lu, H., Goodman, J., & Boykin,
L., "The value of a database in surveillance and vaccine
selection," in Options for the Control of Influenza IV. A. D. M. E.
Osterhaus, N. Cox & A. W. Hampson (Eds.) Amsterdam: Elsevier
Science, 2001, 103-106). The same search was conducted with the
National Library of Medicine GenBank database which is composed of
different sequences for influenza and virtually identical results
were obtained. The capital letter indicates the PMO base and the
subscript number next to the base indicates the percent
conservation for that base for all the isolates in the database.
These data indicate only base positions 15 and 16 show any
variation for the 3'(-)NP terminus and even better conservation of
sequence in the PB1-AUG target.
[0100] Targeting sequences are designed to hybridize to a region of
the target sequence as listed in Table 1. Selected targeting
sequences can be made shorter, e.g., 12 bases, or longer, e.g., 40
bases, and include a small number of mismatches, as long as the
sequence is sufficiently complementary to disrupt the stem
structure(s) upon hybridization with the target, and forms with the
virus positive-strand, a heteroduplex having a Tm of 45.degree. C.
or greater.
[0101] More generally, the degree of complementarity between the
target and targeting sequence is sufficient to form a stable
duplex. The region of complementarity of the antisense oligomers
with the target RNA sequence may be as short as 8-11 bases, but is
preferably 12-15 bases or more, e.g. 12-20 bases, or 12-25 bases.
An antisense oligomer of about 14-15 bases is generally long enough
to have a unique complementary sequence in the viral genome. In
addition, a minimum length of complementary bases may be required
to achieve the requisite binding Tm, as discussed below.
[0102] Oligomers as long as 40 bases may be suitable, where at
least a minimum number of bases, e.g., 12 bases, are complementary
to the target sequence. In general, however, facilitated or active
uptake in cells is optimized at oligomer lengths less than about
30, preferably less than 25. For PMO oligomers, described further
below, an optimum balance of binding stability and uptake generally
occurs at lengths of 15-22 bases.
[0103] The oligomer may be 100% complementary to the viral nucleic
acid target sequence, or it may include mismatches, e.g., to
accommodate variants, as long as a heteroduplex formed between the
oligomer and viral nucleic acid target sequence is sufficiently
stable to withstand the action of cellular nucleases and other
modes of degradation which may occur in vivo. Oligomer backbones
which are less susceptible to cleavage by nucleases are discussed
below. Mismatches, if present, are less destabilizing toward the
end 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 G:C base pairs in the duplex, and the position of the
mismatch(es) in the duplex, according to well understood principles
of duplex stability. Although such an antisense oligomer is not
necessarily 100% complementary to the viral nucleic acid target
sequence, it is effective to stably and specifically bind to the
target sequence, such that a biological activity of the nucleic
acid target, e.g., expression of viral protein(s), is
modulated.
[0104] The stability of the duplex formed between the oligomer and
the target 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
may 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. Each antisense oligomer should have a binding Tm, with
respect to a complementary-sequence RNA, of greater than body
temperature and preferably greater than 50.degree. C. Tm's in the
range 60-80.degree. C. or greater are preferred. According to well
known principles, the Tm of an oligomer compound, with respect to a
complementary-based RNA hybrid, can be increased by increasing the
ratio of C:G paired bases in the duplex, and/or by increasing the
length (in base pairs) of the heteroduplex. At the same time, for
purposes of optimizing cellular uptake, it may be advantageous to
limit the size of the oligomer. For this reason, compounds that
show high Tm (50.degree. C. or greater) at a length of 20 bases or
less are generally preferred over those requiring greater than 20
bases for high Tm values.
[0105] The antisense activity of the oligomer may be enhanced by
using a mixture of uncharged and cationic phosphorodiamidate
linkages as shown in FIGS. 2G and 2H. The total number of cationic
linkages in the oligomer can vary from 1 to 10, and be interspersed
throughout the oligomer. Preferably the number of charged linkages
is at least 2 and no more than half the total backbone linkages,
e.g., between 2-8 positively charged linkages, and preferably each
charged linkages is separated along the backbone by at least one,
preferably at least two uncharged linkages. The antisense activity
of various oligomers can be measured in vitro by fusing the
oligomer target region to the 5' end a reporter gene (e.g. firefly
luciferase) and then measuring the inhibition of translation of the
fusion gene mRNA transcripts in cell free translation assays. The
inhibitory properties of oligomers containing a mixture of
uncharged and cationic linkages can be enhanced between,
approximately, five to 100 fold in cell free translation
assays.
[0106] Table 2 below shows exemplary targeting sequences, in a
5'-to-3' orientation, that are complementary to influenza A virus.
The sequences listed provide a collection of targeting sequences
from which targeting sequences may be selected, according to the
general class rules discussed above. SEQ ID NOs:10-12, 15, 17, 20,
23 and 24 are antisense to the positive strand (mRNA or cRNA) of
the virus whereas SEQ ID NOs:13, 14, 16, 18, 19, 21 and 22 are
antisense to the minus strand (vRNA). Thus, for example, in
selecting a target against the 3' terminus of the minus strand of
the NP encoding segment (segment 5 of influenza A) SEQ ID NOs:13 or
16, or a portion of either sequence effective to block the function
of the 3' terminus of the minus strand can be selected.
TABLE-US-00002 TABLE 2 Exemplary Antisense Oligomer Sequences
Target Targeting SEQ. Nucle- GenBank Antisense Oligomer ID PMO
otides Acc. No. (5' to 3') NO. NP-AUG 39- J02147
CTTGGGACGCCATGATTTTG 10 58 PB2-AUG 24- V00603 CTTTTATTCTTTCCATATTG
11 43 PB1-AUG 13- J02151 GACATCCATTCAAATGGTTTG 12 33 (-)NP- 1-
J02147 AGCAAAAGCAGGGTAGATAATC 13 3'trm 22 (-)NP- 1544- J02147
GAAAAATACCCTTGTTTCTACT 14 5'trm 1565 (+)NP- 1544- J02147
AGTAGAAACAAGGGTATTTTTC 15 3'trm 1565 Flu(-) 1- J02147 AGCAAAAGCAGG
16 3'trm 12 Flu(+) 1553- J02147 AGTAGAAACAAGG 17 3'trm 1565 (-)PB1-
1- J02151 AGCGAAAGCAGGCAAACCAT 18 3'trm 20 (-)PB1- 2320- J02151
GAAAAAATGCCTTGTTCCTACT 19 5'trm 2341 (+)PB1- 2320- J02151
AGTAGGAACAAGGCATTTTTTC 20 3'trm 2341 (-)PB2- 1- V00603
AGCGAAAGCAGGTCAATTAT 21 3'trm 20 (-)PB2- 2320- V00603
TAAAAACGACCTTGTTTCTACT 22 5'trm 2341 (+)PB2- 2320- V00603
AGTAGAAACAAGGTCGTTTTTA 23 3'trm 2341 (+)NP- 1- J02147
AGTCTCGACTTGCTACCTCA 24 5'trm 20
III. ANTISENSE OLIGONUCLEOTIDE ANALOG COMPOUNDS
[0107] A. Properties
[0108] As detailed above, the antisense oligonucleotide analog
compound (the term "antisense" indicates that the compound is
targeted against either the virus' positive-sense strand RNA or
negative-sense or minus-strand) has a base sequence targeting a
region that includes one or more of the following; 1) the 5' or 3'
terminal 30 bases of the negative sense viral RNA; 2) the terminal
30 bases of the 3' terminus of the positive sense mRNA and/or; 3)
50 bases surrounding the AUG start codons of viral mRNA. In
addition, the oligomer is able to effectively target infecting
viruses, when administered to a host cell, e.g. in an infected
mammalian subject. This requirement is met when the oligomer
compound (a) has the ability to be actively taken up by mammalian
cells, and (b) once taken up, form a duplex with the target RNA
with a Tm greater than about 45.degree. C.
[0109] As will be described below, the ability to be taken up by
cells requires that the oligomer backbone be substantially
uncharged, and, preferably, that the oligomer structure is
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 will also depend on the oligomer
backbone, as well as factors noted above, the length and degree of
complementarity of the antisense oligomer with respect to the
target, the ratio of G:C to A:T base matches, and the positions of
any mismatched bases. The ability of the antisense oligomer to
resist cellular nucleases promotes survival and ultimate delivery
of the agent to the cell cytoplasm.
[0110] Below are disclosed methods for testing any given,
substantially uncharged backbone for its ability to meet these
requirements.
[0111] B. Active or Facilitated Uptake by Cells
[0112] The antisense compound may be taken up by host cells by
facilitated or active transport across the host cell membrane if
administered in free (non-complexed) form, or by an endocytotic
mechanism if administered in complexed form.
[0113] In the case where the agent is administered in free form,
the antisense compound should be substantially uncharged, meaning
that a majority of its intersubunit linkages are uncharged at
physiological pH. Experiments carried out in support of the
invention indicate that a small number of net charges, e.g., 1-2
for a 15- to 20-mer oligomer, can in fact enhance cellular uptake
of certain oligomers with substantially uncharged backbones. The
charges may be carried on the oligomer itself, e.g., in the
backbone linkages, or may be terminal charged-group appendages.
Preferably, the number of charged linkages is no more than one
charged linkage per four uncharged linkages. More preferably, the
number is no more than one charged linkage per ten, or no more than
one per twenty, uncharged linkages. In one embodiment, the oligomer
is fully uncharged.
[0114] An oligomer may also contain both negatively and positively
charged backbone linkages, as long as opposing charges are present
in approximately equal number. Preferably, the oligomer does not
include runs of more than 3-5 consecutive subunits of either
charge. For example, the oligomer may have a given number of
anionic linkages, e.g. phosphorothioate or N3'.fwdarw.P5'
phosphoramidate linkages, and a comparable number of cationic
linkages, such as N,N-diethylenediamine phosphoramidates (Dagle,
Littig et al. 2000). The net charge is preferably neutral or at
most 1-2 net charges per oligomer.
[0115] In addition to being substantially or fully uncharged, the
antisense agent is preferably a substrate for a membrane
transporter system (i.e. a membrane protein or proteins) capable of
facilitating transport or actively transporting the oligomer across
the cell membrane. This feature may be determined by one of a
number of tests for oligomer interaction or cell uptake, as
follows.
[0116] A first test assesses binding at cell surface receptors, by
examining the ability of an oligomer compound to displace or be
displaced by a selected charged oligomer, e.g., a phosphorothioate
oligomer, on a cell surface. The cells are incubated with a given
quantity of test oligomer, which is typically fluorescently
labeled, at a final oligomer concentration of between about 10-300
nM. Shortly thereafter, e.g., 10-30 minutes (before significant
internalization of the test oligomer can occur), the displacing
compound is added, in incrementally increasing concentrations. If
the test compound is able to bind to a cell surface receptor, the
displacing compound will be observed to displace the test compound.
If the displacing compound is shown to produce 50% displacement at
a concentration of 10.times. the test compound concentration or
less, the test compound is considered to bind at the same
recognition site for the cell transport system as the displacing
compound.
[0117] A second test measures cell transport, by examining the
ability of the test compound to transport a labeled reporter, e.g.,
a fluorescence reporter, into cells. The cells are incubated in the
presence of labeled test compound, added at a final concentration
between about 10-300 nM. After incubation for 30-120 minutes, the
cells are examined, e.g., by microscopy, for intracellular label.
The presence of significant intracellular label is evidence that
the test compound is transported by facilitated or active
transport.
[0118] The antisense compound may also be administered in complexed
form, where the complexing agent is typically a polymer, e.g., a
cationic lipid, polypeptide, or non-biological cationic polymer,
having an opposite charge to any net charge on the antisense
compound. Methods of forming complexes, including bilayer
complexes, between anionic oligonucleotides and cationic lipid or
other polymer components, are well known. For example, the
liposomal composition Lipofectin.RTM. (Felgner, Gadek et al. 1987),
containing the cationic lipid DOTMA
(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and
the neutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine),
is widely used. After administration, the complex is taken up by
cells through an endocytotic mechanism, typically involving
particle encapsulation in endosomal bodies.
[0119] The antisense compound may also be administered in
conjugated form with an arginine-rich peptide linked covalently to
the 5' or 3' end of the antisense oligomer. The peptide is
typically 8-16 amino acids and consists of a mixture of arginine,
and other amino acids including phenyalanine and cysteine. The use
of arginine-rich peptide-PMO conjugates can be used to enhance
cellular uptake of the antisense oligomer (See, e.g. (Moulton,
Nelson et al. 2004; Nelson, Stein et al. 2005). Exemplary
arginine-rich peptides for use in practicing the invention are
listed as SEQ ID NOs:25-30. Non-natural amino acids can be used in
combination with naturally occuring amino acids as shown in the
Sequence listing table for SEQ ID NOs:26-30. In these examples
6-aminohexanoic acid (Ahx) and/or beta-alanine (.beta.-Ala) are
used.
[0120] In some instances, liposomes may be employed to facilitate
uptake of the antisense oligonucleotide into cells. (See, e.g.,
Williams, S. A., 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; Gregoriadis, G., Chapter 14,
Liposomes, Drug Carriers in Biology and Medicine, pp. 287-341,
Academic Press, 1979). Hydrogels may also be used as vehicles for
antisense oligomer administration, for example, as described in WO
93/01286. Alternatively, the oligonucleotides may be administered
in microspheres or microparticles. (See, e.g. Wu and Wu 1987).
Alternatively, the use of gas-filled microbubbles complexed with
the antisense oligomers can enhance delivery to target tissues, as
described in U.S. Pat. No. 6,245,747.
[0121] Alternatively, and according to another aspect of the
invention, the requisite properties of oligomers with any given
backbone can be confirmed by a simple in vivo test, in which a
labeled compound is administered to an animal, and a body fluid
sample, taken from the animal several hours after the oligomer is
administered, assayed for the presence of heteroduplex with target
RNA. This method is detailed in subsection D below.
[0122] C. Substantial Resistance to RNaseH
[0123] Two general mechanisms have been proposed to account for
inhibition of expression by antisense oligonucleotides. (See e.g.,
Agrawal, Mayrand et al. 1990; Bonham, Brown et al. 1995;
Boudvillain, Guerin et al. 1997). In the first, a heteroduplex
formed between the oligonucleotide and the viral RNA acts as a
substrate for RNaseH, leading to cleavage of the viral RNA.
Oligonucleotides belonging, or proposed to belong, to this class
include phosphorothioates, phosphotriesters, and phosphodiesters
(unmodified "natural" oligonucleotides). Such compounds expose the
viral RNA in an oligomer:RNA duplex structure to hydrolysis by
RNaseH, and therefore loss of function.
[0124] A second class of oligonucleotide analogs, termed "steric
blockers" or, alternatively, "RNaseH inactive" or "RNaseH
resistant", have not been observed to act as a substrate for
RNaseH, and are believed to act by sterically blocking target RNA
nucleocytoplasmic transport, splicing or translation. This class
includes methylphosphonates (Toulme, Tinevez et al. 1996),
morpholino oligonucleotides, peptide nucleic acids (PNA's), certain
2'-O-allyl or 2'-O-alkyl modified oligonucleotides (Bonham, Brown
et al. 1995), and N3'.fwdarw.P5' phosphoramidates (Ding, Grayaznov
et al. 1996; Gee, Robbins et al. 1998).
[0125] A test oligomer can be assayed for its RNaseH resistance by
forming an RNA:oligomer duplex with the test compound, then
incubating the duplex with RNaseH under a standard assay
conditions, as described in Stein et al. After exposure to RNaseH,
the presence or absence of intact duplex can be monitored by gel
electrophoresis or mass spectrometry.
[0126] D. In Vivo Uptake
[0127] In accordance with another aspect of the invention, there is
provided a simple, rapid test for confirming that a given antisense
oligomer type provides the required characteristics noted above,
namely, high Tm, ability to be actively taken up by the host cells,
and substantial resistance to RNaseH. This method is based on the
discovery that a properly designed antisense compound will form a
stable heteroduplex with the complementary portion of the viral RNA
target when administered to a mammalian subject, and the
heteroduplex subsequently appears in the urine (or other body
fluid). Details of this method are also given in co-owned U.S.
patent application Ser. No. 09/736,920, entitled "Non-Invasive
Method for Detecting Target RNA" (Non-Invasive Method), the
disclosure of which is incorporated herein by reference.
[0128] Briefly, a test oligomer containing a backbone to be
evaluated, having a base sequence targeted against a known RNA, is
injected into a mammalian subject. The antisense oligomer may be
directed against any intracellular RNA, including a host RNA or the
RNA of an infecting virus. Several hours (typically 8-72) after
administration, the urine is assayed for the presence of the
antisense-RNA heteroduplex. If heteroduplex is detected, the
backbone is suitable for use in the antisense oligomers of the
present invention.
[0129] The test oligomer may be labeled, e.g. by a fluorescent or a
radioactive tag, to facilitate subsequent analyses, if it is
appropriate for the mammalian subject. The assay can be in any
suitable solid-phase or fluid format. Generally, a solid-phase
assay involves first binding the heteroduplex analyte to a
solid-phase support, e.g., particles or a polymer or test-strip
substrate, and detecting the presence/amount of heteroduplex bound.
In a fluid-phase assay, the analyte sample is typically pretreated
to remove interfering sample components. If the oligomer is
labeled, the presence of the heteroduplex is confirmed by detecting
the label tags. For non-labeled compounds, the heteroduplex may be
detected by immunoassay if in solid phase format or by mass
spectroscopy or other known methods if in solution or suspension
format.
[0130] When the antisense oligomer is complementary to a
virus-specific region of the viral genome (such as those regions of
influenza RNA, as described above) the method can be used to detect
the presence of a given influenza virus, or reduction in the amount
of virus during a treatment method.
[0131] E. Exemplary Oligomer Backbones
[0132] Examples of nonionic linkages that may be used in
oligonucleotide analogs are shown in FIGS. 2A-2G. In these figures,
B represents a purine or pyrimidine base-pairing moiety effective
to bind, by base-specific hydrogen bonding, to a base in a
polynucleotide, preferably selected from adenine, cytosine, guanine
and uracil. Suitable backbone structures include carbonate (3A,
R.dbd.O) and carbamate (2A, R.dbd.NH.sub.2) linkages (Mertes and
Coats 1969; Gait, Jones et al. 1974); alkyl phosphonate and
phosphotriester linkages (2B, R=alkyl or --O-alkyl) (Lesnikowski,
Jaworska et al. 1990); amide linkages (2C) (Blommers, Pieles et al.
1994); sulfone and sulfonamide linkages (2D, R.sub.1,
R.sub.2.dbd.CH.sub.2); and a thioformacetyl linkage (2E) (Cross,
Rice et al. 1997). The latter is reported to have enhanced duplex
and triplex stability with respect to phosphorothioate antisense
compounds (Cross, Rice et al. 1997). Also reported are the
3'-methylene-N-methylhydroxyamino compounds of structure 2F. Also
shown is a cationic linkage in FIG. 2H wherein the nitrogen pendant
to the phosphate atom in the linkage of FIG. 2G is replaced with a
1-piperazino structure. The method for synthesizing the
1-piperazino group linkages is described below with respect to FIG.
10.
[0133] As noted above, the substantially uncharged oligomer may
advantageously include a limited number of charged backbone
linkages. One example of a cationic charged phophordiamidate
linkage is shown in FIG. 2H. This linkage, in which the
dimethylamino group shown in FIG. 2G is replaced by a 1-piperazino
group as shown in FIG. 2G, can be substituted for any linkage(s) in
the oligomer. By including between two to eight such cationic
linkages, and more generally, at least two and no more than about
half the total number of linkages, interspersed along the backbone
of the otherwise uncharged oligomer, antisense activity can be
enhanced without a significant loss of specificity. The charged
linkages are preferably separated in the backbone by at least 1 and
preferably 2 or more uncharged linkages.
[0134] Peptide nucleic acids (PNAs) are analogs of DNA in which the
backbone is structurally homomorphous with a deoxyribose backbone,
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
Watson-Crick base-pairing rules, and mimic DNA in terms of base
pair recognition (Egholm, Buchardt et al. 1993). The backbone of
PNAs is formed by peptide bonds rather than phosphodiester bonds,
making them well-suited for antisense applications. The backbone is
uncharged, resulting in PNA/DNA or PNA/RNA duplexes which exhibit
greater than normal thermal stability. PNAs are not recognized by
nucleases or proteases.
[0135] A preferred oligomer structure employs morpholino-based
subunits bearing base-pairing moieties, joined by uncharged
linkages, as described above. Especially preferred is a
substantially uncharged phosphorodiamidate-linked morpholino
oligomer, such as illustrated in FIGS. 1A-1D, and FIG. 2G.
Morpholino oligonucleotides, including antisense oligomers, are
detailed, for example, in co-owned U.S. Pat. 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, all of which are expressly incorporated by reference
herein.
[0136] Important properties of the morpholino-based subunits
include: the ability to be linked in a oligomeric form by stable,
uncharged backbone linkages; the ability to support a nucleotide
base (e.g. adenine, cytosine, guanine or uracil) such that the
polymer formed can hybridize with a complementary-base target
nucleic acid, including target RNA, with high Tm, even with
oligomers as short as 10-14 bases; the ability of the oligomer to
be actively transported into mammalian cells; and the ability of
the oligomer:RNA heteroduplex to resist RNAse degradation.
[0137] Exemplary backbone structures for antisense oligonucleotides
of the invention include the .beta.-morpholino subunit types shown
in FIGS. 1A-1D, each linked by an uncharged, phosphorus-containing
subunit linkage. FIG. 1A shows a phosphorus-containing linkage
which forms the five atom repeating-unit backbone, where the
morpholino rings are linked by a 1-atom phosphoamide linkage. FIG.
1B shows a linkage which produces a 6-atom repeating-unit backbone.
In this structure, the atom Y linking the 5' morpholino carbon to
the phosphorus group may be sulfur, nitrogen, carbon or,
preferably, oxygen. The X moiety pendant from the phosphorus may be
fluorine, an alkyl or substituted alkyl, an alkoxy or substituted
alkoxy, a thioalkoxy or substituted thioalkoxy, or unsubstituted,
monosubstituted, or disubstituted nitrogen, including cyclic
structures, such as morpholines or piperidines. Alkyl, alkoxy and
thioalkoxy preferably include 1-6 carbon atoms. The Z moieties are
sulfur or oxygen, and are preferably oxygen.
[0138] The linkages shown in FIGS. 1C and 1D are designed for
7-atom unit-length backbones. In Structure 1C, the X moiety is as
in Structure 1B, and the Y moiety may be methylene, sulfur, or,
preferably, oxygen. In Structure 1D, the X and Y moieties are as in
Structure 1B. Particularly preferred morpholino oligonucleotides
include those composed of morpholino subunit structures of the form
shown in FIG. 1B, where X.dbd.NH.sub.2 or N(CH.sub.3).sub.2,
Y.dbd.O, and Z=O.
[0139] As noted above, the substantially uncharged oligomer may
advantageously include a limited number of charged linkages, e.g.
up to about 1 per every 5 uncharged linkages, more preferably up to
about 1 per every 10 uncharged linkages. Therefore a small number
of charged linkages, e.g. charged phosphoramidate or
phosphorothioate, may also be incorporated into the oligomers.
[0140] The antisense compounds can be prepared by stepwise
solid-phase synthesis, employing methods detailed in the references
cited above. In some cases, it may be desirable to add additional
chemical moieties to the antisense compound, e.g. to enhance
pharmacokinetics or to facilitate capture or detection of the
compound. Such a moiety may be covalently attached, typically to a
terminus of the oligomer, according to standard synthetic methods.
For example, addition of a polyethyleneglycol moiety or other
hydrophilic polymer, e.g., one having 10-100 monomeric subunits,
may be useful in enhancing solubility. One or more charged groups,
e.g., anionic charged groups such as an organic acid, may enhance
cell uptake. A reporter moiety, such as fluorescein or a
radiolabeled group, may be attached for purposes of detection.
Alternatively, the reporter label attached to the oligomer may be a
ligand, such as an antigen or biotin, capable of binding a labeled
antibody or streptavidin. In selecting a moiety for attachment or
modification of an antisense oligomer, it is generally of course
desirable to select chemical compounds of groups that are
biocompatible and likely to be tolerated by a subject without
undesirable side effects.
IV. INHIBITION OF INFLUENZA VIRAL REPLICATION
[0141] The antisense compounds detailed above are useful in
inhibiting replication of single-stranded, negative-sense,
segmented RNA viruses of the Orthomyxoviridae family. In one
embodiment, such inhibition is effective in treating infection of a
host animal by these viruses. Accordingly, the method comprises, in
one embodiment, contacting a cell infected with the virus with a
antisense agent effective to inhibit the replication of the
specific virus. In this embodiment, the antisense agent is
administered to a mammalian subject, e.g., human or domestic
animal, infected with a given virus, in a suitable pharmaceutical
carrier. It is contemplated that the antisense oligonucleotide
arrests the growth of the RNA virus in the host. The RNA virus may
be decreased in number or eliminated with little or no detrimental
effect on the normal growth or development of the host.
[0142] In the present invention as described in the Examples,
Phosphorodiamidate Morpholino Oligomers (PMOs), designed to
hybridize to various gene segments of influenza A virus, were
evaluated for their ability to inhibit influenza virus production
in Vero cell culture. The PMOs were conjugated to a short
arginine-rich peptide to facilitate entry into cells in culture.
Vero cells were incubated with PMO compounds, inoculated with
influenza virus, and viral titer determined by hemagglutinin assay
and/or plaque-assay. The compounds targeting the AUG translation
start-sites of polymerase component PB1 and nuclear capsid protein
(NP), the 5' and 3' ends of vRNA NP segment and the 3'end of cRNA
NP segment were very effective, reducing the titer of influenza
virus by 1 to 3 orders of magnitude compared to controls, in a
dose-dependent and sequence-specific manner over a period of 2
days. Combinations of some of the PMOs exhibited a synergistic
antiviral effect as described in Example 3. These data indicate
that several of the PMOs tested in this study are potential
influenza A therapeutics.
[0143] The effective anti-influenza A PMO compounds were observed
not to alter the titer of influenza B virus grown in Vero cells due
to the lack of homology between the influenza A virus-specific PMOs
and the corresponding influenza B virus targets. However, the PMO
described herein (SEQ ID NOs:10-24) will target most, if not all,
influenza A virus strains because of the high degree of homology
between strains at the respective targets (SEQ ID NOs:1-9). An
example of the sequence conservation at two preferred targets is
shown in FIG. 4. In this case the sequence conservation between
multiple isolates of H5N1 (e.g. bird flu) and H1N1 was determined
for the targets of the PB1-AUG and (-) NP-3'trm PMOs (SEQ ID NOs:12
and 13, respectively). FIG. 4 shows a very high level of
conservation at these target sites.
[0144] A. Identification of the Infective Agent
[0145] The specific virus causing the infection can be determined
by methods known in the art, e.g. serological or cultural methods,
or by methods employing the antisense oligomers of the present
invention.
[0146] Serological identification employs a viral sample or culture
isolated from a biological specimen, e.g., stool, urine,
cerebrospinal fluid, blood, etc., of the subject. Immunoassay for
the detection of virus is generally carried out by methods
routinely employed by those of skill in the art, e.g., ELISA or
Western blot. In addition, monoclonal antibodies specific to
particular viral strains or species are often commercially
available.
[0147] Culture methods may be used to isolate and identify
particular types of virus, by employing techniques including, but
not limited to, comparing characteristics such as rates of growth
and morphology under various culture conditions.
[0148] Another method for identifying the viral infective agent in
an infected subject employs one or more antisense oligomers
targeting broad families and/or genera of viruses. Sequences
targeting any characteristic viral RNA can be used. The desired
target sequences are preferably (i) common to broad virus
families/genera, and (ii) not found in humans. Characteristic
nucleic acid sequences for a large number of infectious viruses are
available in public databases, and may serve as the basis for the
design of specific oligomers.
[0149] For each plurality of oligomers, the following steps are
carried out: (a) the oligomer(s) are administered to the subject;
(b) at a selected time after said administering, a body fluid
sample is obtained from the subject; and (c) the sample is assayed
for the presence of a nuclease-resistant heteroduplex comprising
the antisense oligomer and a complementary portion of the viral
genome. Steps (a)-(c) are carried for at least one such oligomer,
or as many as is necessary to identify the virus or family of
viruses. Oligomers can be administered and assayed sequentially or,
more conveniently, concurrently. The virus is identified based on
the presence (or absence) of a heteroduplex comprising the
antisense oligomer and a complementary portion of the viral genome
of the given known virus or family of viruses.
[0150] Preferably, a first group of oligomers, targeting broad
families, is utilized first, followed by selected oligomers
complementary to specific genera and/or species and/or strains
within the broad family/genus thereby identified. This second group
of oligomers includes targeting sequences directed to specific
genera and/or species and/or strains within a broad family/genus.
Several different second oligomer collections, i.e. one for each
broad virus family/genus tested in the first stage, are generally
provided. Sequences are selected which are (i) specific for the
individual genus/species/strains being tested and (ii) not found in
humans.
[0151] B. Administration of the Antisense Oligomer
[0152] Effective delivery of the antisense oligomer to the target
nucleic acid is an important aspect of treatment. In accordance
with the invention, routes of antisense oligomer delivery include,
but are not limited to, various systemic routes, including oral and
parenteral routes, e.g., intravenous, subcutaneous,
intraperitoneal, and intramuscular, as well as inhalation,
transdermal and topical delivery. The appropriate route may be
determined by one of skill in the art, as appropriate to the
condition of the subject under treatment. For example, an
appropriate route for delivery of an antisense oligomer in the
treatment of a viral infection of the skin is topical delivery,
while delivery of a antisense oligomer for the treatment of a viral
respiratory infection is by inhalation. The oligomer may also be
delivered directly to the site of viral infection, or to the
bloodstream.
[0153] The antisense oligomer may be administered in any convenient
vehicle which is physiologically acceptable. Such a composition may
include any of a variety of standard pharmaceutically acceptable
carriers employed by those of ordinary skill 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 suitable physiologically acceptable carrier will vary
dependent upon the chosen mode of administration.
[0154] In some instances, liposomes may be employed to facilitate
uptake of the antisense oligonucleotide into cells. (See, e.g.,
Williams, S. A., 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; Gregoriadis, G., Chapter 14,
Liposomes, Drug Carriers in Biology and Medicine, pp. 287-341,
Academic Press, 1979). Hydrogels may also be used as vehicles for
antisense oligomer administration, for example, as described in WO
93/01286. Alternatively, the oligonucleotides may be administered
in microspheres or microparticles. (See, e.g., Wu, G. Y. and Wu, C.
H., J. Biol. Chem. 262:4429-4432, 1987). Alternatively, the use of
gas-filled microbubbles complexed with the antisense oligomers can
enhance delivery to target tissues, as described in U.S. Pat. No.
6,245,747.
[0155] Sustained release compositions may also be used. These may
include semipermeable polymeric matrices in the form of shaped
articles such as films or microcapsules.
[0156] In one aspect of the method, the subject is a human subject,
e.g., a patient diagnosed as having a localized or systemic viral
infection. The condition of a patient may also dictate prophylactic
administration of an antisense oligomer of the invention, e.g. in
the case of a patient who (1) is immunocompromised; (2) is a burn
victim; (3) has an indwelling catheter; or (4) is about to undergo
or has recently undergone surgery. In one preferred embodiment, the
oligomer is a phosphorodiamidate morpholino oligomer, contained in
a pharmaceutically acceptable carrier, and is delivered orally. In
another preferred embodiment, the oligomer is a phosphorodiamidate
morpholino oligomer, contained in a pharmaceutically acceptable
carrier, and is delivered intravenously (i.v.).
[0157] In another application of the method, the subject is a
livestock animal, e.g., a chicken, turkey, pig, cow or goat, etc,
and the treatment is either prophylactic or therapeutic. The
invention also includes a livestock and poultry food composition
containing a food grain supplemented with a subtherapeutic amount
of an antiviral antisense compound of the type described above.
Also contemplated is, in a method of feeding livestock and poultry
with a food grain supplemented with subtherapeutic levels of an
antiviral, an improvement in which the food grain is supplemented
with a subtherapeutic amount of an antiviral oligonucleotide
composition as described above.
[0158] The antisense compound is generally administered in an
amount and manner effective to result in a peak blood concentration
of at least 200-400 nM antisense oligomer. Typically, one or more
doses of antisense oligomer are administered, generally at regular
intervals, for a period of about one to two weeks. Preferred doses
for oral administration are from about 1-100 mg oligomer per 70 kg.
In some cases, doses of greater than 100 mg oligomer/patient may be
necessary. For i.v. administration, preferred doses are from about
0.5 mg to 100 mg oligomer per 70 kg. The antisense oligomer may be
administered at regular intervals for a short time period, e.g.,
daily for two weeks or less. However, in some cases the oligomer is
administered intermittently over a longer period of time.
Administration may be followed by, or concurrent with,
administration of an antibiotic or other therapeutic treatment. The
treatment regimen may be adjusted (dose, frequency, route, etc.) as
indicated, based on the results of immunoassays, other biochemical
tests and physiological examination of the subject under
treatment.
[0159] C. Monitoring of Treatment
[0160] 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 subject under treatment (i.e., prophylactic
administration versus administration in response to localized or
systemic infection). Accordingly, such in vivo therapy will often
require monitoring by tests appropriate to the particular type of
viral infection under treatment, and corresponding adjustments in
the dose or treatment regimen, in order to achieve an optimal
therapeutic outcome. Treatment may be monitored, e.g., by general
indicators of infection, such as complete blood count (CBC),
nucleic acid detection methods, immunodiagnostic tests, viral
culture, or detection of heteroduplex.
[0161] The efficacy of an in vivo administered antisense oligomer
of the invention in inhibiting or eliminating the growth of one or
more types of RNA virus may be determined from biological samples
(tissue, blood, urine etc.) taken from a subject prior to, during
and subsequent to administration of the antisense oligomer. Assays
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, e.g., an
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 on viral
titer, e.g. by the method of Spearman-Karber. (See, for example,
Pari, G. S. et al., Antimicrob. Agents and Chemotherapy
39(5):1157-1161, 1995; Anderson, K. P. et al., Antimicrob. Agents
and Chemotherapy 40(9):2004-2011, 1996, Cottral, G. E. (ed) in:
Manual of Standard Methods for Veterinary Microbiology, pp. 60-93,
1978).
[0162] A preferred method of monitoring the efficacy of the
antisense oligomer treatment is by detection of the antisense-RNA
heteroduplex. At selected time(s) after antisense oligomer
administration, a body fluid is collected for detecting the
presence and/or measuring the level of heteroduplex species in the
sample. Typically, the body fluid sample is collected 3-24 hours
after administration, preferably about 6-24 hours after
administering. As indicated above, the body fluid sample may be
urine, saliva, plasma, blood, spinal fluid, or other liquid sample
of biological origin, and may include cells or cell fragments
suspended therein, or the liquid medium and its solutes. The amount
of sample collected is typically in the 0.1 to 10 ml range,
preferably about 1 ml or less.
[0163] The sample may be treated to remove unwanted components
and/or to treat the heteroduplex species in the sample to remove
unwanted ssRNA overhang regions, e.g., by treatment with RNase. It
is, of course, particularly important to remove overhang where
heteroduplex detection relies on size separation, e.g.,
electrophoresis of mass spectroscopy.
[0164] A variety of methods are available for removing unwanted
components from the sample. For example, since the heteroduplex has
a net negative charge, electrophoretic or ion exchange techniques
can be used to separate the heteroduplex from neutral or positively
charged material. The sample may also be contacted with a solid
support having a surface-bound antibody or other agent specifically
able to bind the heteroduplex. After washing the support to remove
unbound material, the heteroduplex can be released in substantially
purified form for further analysis, e.g., by electrophoresis, mass
spectroscopy or immunoassay.
V. EXAMPLES
[0165] The following examples illustrate but are not intended in
any way to limit the invention.
Materials and Methods
[0166] Standard recombinant DNA techniques were employed in all
constructions, as described in Ausubel, F M et al., in CURRENT
PROTOCOLS IN MOLBCULAR BIOLOGY, John Wiley and Sons, Inc., Media,
Pa., 1992 and Sambrook, J. et al., in MOLECULAR CLONING: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., Vol. 2, 1989).
[0167] All peptides were custom synthesized by Global Peptide
Services (Ft. Collins, Colo.) or at AVI BioPharma (Corvallis,
Oreg.) and purified to >90% purity (see Example 2 below). PMOs
were synthesized at AVI BioPharma in accordance with known methods,
as described, for example, in ((Summerton and Weller 1997) and U.S.
Pat. No. 5,185,444. The structure of the PMO is as shown in FIG.
2G.
[0168] PMO oligomers were conjugated at the 5' end with an
arginine-rich peptide (R.sub.5F.sub.2R.sub.4C-5'-PMO, SEQ ID NO:25)
to enhance cellular uptake as described (U.S. Patent Application
60/466,703 and (Moulton, Nelson et al. 2004; Nelson, Stein et al.
2005).
[0169] A schematic of a synthetic pathway that can be used to make
morpholino subunits containing a (1-piperazino) phosphinylideneoxy
linkage is shown in FIG. 10; further experimental detail for a
representative synthesis is provided in Materials and Methods,
below. As shown in the Figure, reaction of piperazine and trityl
chloride gave trityl piperazine (1a), which was isolated as the
succinate salt. Reaction with ethyl trifluoroacetate (1b) in the
presence of a weak base (such as diisopropylethylamine or DIEA)
provided 1-trifluoroacetyl-4-trityl piperazine (2), which was
immediately reacted with HCl to provide the salt (3) in good yield.
Introduction of the dichlorophosphoryl moiety was performed with
phosphorus oxychloride in toluene.
[0170] The acid chloride (4) is reacted with morpholino subunits
(moN), which may be prepared as described in U.S. Pat. No.
5,185,444 or in Summerton and Weller, 1997 (cited above), to
provide the activated subunits (5,6,7). Suitable protecting groups
are used for the nucleoside bases, where necessary; for example,
benzoyl for adenine and cytosine, isobutyryl for guanine, and
pivaloylmethyl for inosine. The subunits containing the
(1-piperazino) phosphinylideneoxy linkage can be incorporated into
the existing PMO synthesis protocol, as described, for example in
Summerton and Weller (1997), without modification.
Example 1
Inhibition of Influenza A virus in Cell Culture with
Phosphorodiamidate Morpholino Oligomers
[0171] Phosphorodiamidate Morpholino Oligomers (PMOs), designed to
hybridize to various gene segments of influenza A virus, were
evaluated for their ability to inhibit influenza virus production
in Vero cell culture. The PMOs were conjugated to a short
arginine-rich peptide (R.sub.5F.sub.2R.sub.4C) to facilitate entry
into cells in culture. Vero cells were incubated with PMO
compounds, inoculated with influenza A virus (strain PR8, H1N1),
and viral titer determined by hemagglutinin assay (HA) and/or
plaque-assay (CFU). The PMO compounds targeting the AUG translation
start-sites of polymerase component PB1 and nuclear capsid protein
(NP) (SEQ ID NOs: 10 and 11), the 5' and 3' ends of the NP gene
(SEQ ID NOs:13 and 14) encoded by the viral RNA (i.e. vRNA) and the
3' end of the NP gene (SEQ ID NO:15) encoded by the complementary
RNA (cRNA) were very effective in reducing the titer of influenza
virus by 1 to 3 orders of magnitude compared to controls, in a
dose-dependent and sequence-specific manner over a period of 2
days.
[0172] FIG. 5 shows the effect of 20 .mu.M AUG-targeted and 5' and
3' termini-targeted PMOs on influenza A virus production in Vero
cells compared to untreated (NT) and a scramble control PMO (Scr).
Vero cells were preincubated with PMO for 6 hours followed by virus
infection at a multiplicity of infection (M.O.I.) of 0.05. At
various times post-infection, supernatant was collected for
determination of virus titer as measured by a Hemaglutinin Assay
(HA). Three PMO targeting AUG start codons of the NP, PB1 and PB2
genes were effective at reducing influenza virus replication (SEQ
ID NOs:10-12) as shown in FIG. 5. Two PMOs that target the 5' and
3'-termini of vRNA, NP(-)5' and NP(-)3' (SEQ ID NOs:13 and 14) and
one PMO that targets the 3' termini of the cRNA, NP(+)3' (SEQ ID
NO:15) were effective at reducing influenza virus replication in
this assay as shown in FIG. 5. The PMO that targets the 5' termini
of the cRNA, NP(+)5' (SEQ ID NO:24), was less effective but still
demonstrated anti-viral activity.
[0173] PMOs that target the AUG start codons of three influenza
virus genes, NP-AUG, PB2-AUG and PB1-AUG (SEQ ID NOs: 0-12,
respectively) were assayed for their ability to inhibit influenza A
virus replication in a dose response assay using the hemagglutinin
assay and the plaque-forming assay. The results are shown in FIG.
6. For all three PMOs the concentration that effectively resulted
in a 50% reduction in viral replication (EC50) was found to be less
than 1 .mu.M. A three-log reduction in viral replication using the
plaque assay was observed for two of the PMOs, NP-AUG and PB1-AUG
at 10 .mu.M. Identical assays were performed using the
termini-targeted PMO: NP(-)3', NP(-)5', NP(+)3' and NP(+)5' (SEQ ID
NOs:13, 14, 15 and 24, respectively). All four PMOs demonstrated
significant reduction in viral titer as shown in FIG. 7.
Example 2
Effect of PMO Targeting the 3'-Terminus of NP vRNA on NP mRNA and
cRNA Transcription
[0174] Quantitative RTPCR was used to determine the effect of one
of the termini-targeted PMO, NP(-)3' (SEQ ID NO:13) on the
transcription of the NP vRNA segment into mRNA and cRNA species
(i.e., see FIG. 4). The mRNA transcription product is
positive-sense RNA whereas the cRNA is a negative-sense RNA. The
NP(-)3' PMO was incubated with Vero cells for 6 hours followed by
influenza A virus infection at an MOI of 0.05. Three hours
post-infection, RNA was isolated and RNA species specific reverse
transcription (RT) was performed followed by quantitative PCR on
the reaction product. FIG. 8 shows that the NP(-)3' PMO (SEQ ID
NO:13) that targets the 3' end of the vRNA strongly suppressed the
transcription of NP mRNA and cRNA.
Example 3
Synergistic Inhibition of Influenza a Virus Replication in Cell
Culture Using Combinations of Anti-influenza PMO
[0175] Combinations of some of the PMOs exhibited a synergistic
antiviral effect. FIG. 9 shows the synergistic effect of various
combinations of PMO that target the NP vRNA termini and the NP-AUG
region. PMO treatment and influenza A virus infection were as
described in Example 1. The plaque assay was used to measure virus
replication. Three termini-targeted PMO, NP(-)32', NP(-)5', NP(+)3'
(SEQ ID NOs:13-15) and the NP-AUG PMO (SEQ ID NO:10) were mixed in
various combinations as shown in FIG. 8. One combination, NP(+)3'
with NP(-)5' did not produce antiviral activity as this pair of PMO
are predicted to hybridize to each other. All the other PMO
combinations demonstrated significant inhibition of influenza A
viral replication.
Sequence CWU 1
1
30 1 50 RNA Influenza A virus 1 ucacucacug agugacauca aaaucauggc
gucccaaggc accaaacggu 50 2 50 RNA Influenza A virus 2 agcgaaagca
ggucaauuau auucaauaug gaaagaauaa aagaacuaag 50 3 50 RNA Influenza A
virus 3 agcgaaagca ggcaaaccau uugaauggau gucaauccga ccuuacuuuu 50 4
25 RNA Influenza A virus 4 aaagaaaaau acccuuguuu cuacu 25 5 25 RNA
Influenza A virus 5 agcaaaagca ggguagauaa ucacu 25 6 25 RNA
Influenza A virus 6 caugaaaaaa ugccuuguuc cuacu 25 7 25 RNA
Influenza A virus 7 agcgaaagca ggcaaaccau uugaa 25 8 25 RNA
Influenza A virus 8 guuuaaaaac gaccuuguuu cuacu 25 9 25 RNA
Influenza A virus 9 agcgaaagca ggucaauuau auuca 25 10 20 DNA
Artificial Sequence Synthetic Oligomer 10 cttgggacgc catgattttg 20
11 20 DNA Artificial Sequence Synthetic Oligomer 11 cttttattct
ttccatattg 20 12 21 DNA Artificial Sequence Synthetic Oligomer 12
gacatccatt caaatggttt g 21 13 22 DNA Artificial Sequence Synthetic
Oligomer 13 agcaaaagca gggtagataa tc 22 14 22 DNA Artificial
Sequence Synthetic Oligomer 14 gaaaaatacc cttgtttcta ct 22 15 22
DNA Artificial Sequence Synthetic Oligomer 15 agtagaaaca agggtatttt
tc 22 16 12 DNA Artificial Sequence Synthetic Oligomer 16
agcaaaagca gg 12 17 13 DNA Artificial Sequence Synthetic Oligomer
17 agtagaaaca agg 13 18 20 DNA Artificial Sequence Synthetic
Oligomer 18 agcgaaagca ggcaaaccat 20 19 22 DNA Artificial Sequence
Synthetic Oligomer 19 gaaaaaatgc cttgttccta ct 22 20 22 DNA
Artificial Sequence Synthetic Oligomer 20 agtaggaaca aggcattttt tc
22 21 20 DNA Artificial Sequence Synthetic Oligomer 21 agcgaaagca
ggtcaattat 20 22 22 DNA Artificial Sequence Synthetic Oligomer 22
taaaaacgac cttgtttcta ct 22 23 22 DNA Artificial Sequence Synthetic
Oligomer 23 agtagaaaca aggtcgtttt ta 22 24 20 DNA Artificial
Sequence Synthetic Oligomer 24 agtctcgact tgctacctca 20 25 12 PRT
Artificial Sequence Synthetic Peptide 25 Arg Arg Arg Arg Arg Phe
Phe Arg Arg Arg Arg Cys 1 5 10 26 14 PRT Artificial Sequence
Synthetic Arginine-rich peptide VARIANT (2)..(13) Xaa =
6-aminohexanoic acid VARIANT (14)..(14) Xaa = beta-Alanine 26 Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa 1 5 10 27 17
PRT Artificial Sequence Synthetic Arginine-rich peptide VARIANT
(2)..(16) Xaa = 6-aminohexanoic acid VARIANT (17)..(17) Xaa =
beta-Alanine 27 Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Arg Xaa 1 5 10 15 Xaa 28 9 PRT Artificial Sequence Synthetic
arginine-rich peptide VARIANT (2)..(8) Xaa = 6-aminohexanoic acid
VARIANT (9)..(9) Xaa = beta-Alanine 28 Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Xaa 1 5 29 8 PRT Artificial Sequence Synthetic arginine-rich
peptide VARIANT (2)..(7) Xaa = 6-aminohexanoic acid VARIANT
(8)..(8) Xaa = beta-Alanine 29 Arg Xaa Arg Arg Xaa Arg Xaa Xaa 1 5
30 11 PRT Artificial Sequence Synthetic Arginine-rich peptide
VARIANT (2)..(10) Xaa = 6-aminohexanoic acid VARIANT (11)..(11) Xaa
= beta-Alanine 30 Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa 1 5
10
* * * * *