Note: Descriptions are shown in the official language in which they were submitted.
W 0 ~2/03051 PCT/US91/057
DESCRIPTION ~S ~ il 1 b
INHIBITION OF HERPESVIRIDAE INFECTION BY ANTISENSE
OLIGONUCLEOTIDES.
Backaround Of The Invention
The present invention is directed to antisense
oligomers which are complementary to a vital region of a
viral genome which are active as antiviral agents.
The present invention is also directed to methods of
interfering with replication of a virus after infection of
host cells by the virus and to antisense oligomers which
are useful in interfering with viral replication.
In viral replication, viral genes are typically
activated and expressed in phases. In the replication of
a virus such as a virus o~ the Herpes family, genes are
expressed in three pha~es. Phase I involves the
expression of about five genes. 'rhese genes are mostly
regulatory in nature and are term~d "alpha genes." The
lS second set of genes expressed are called "beta genes;"
~heir function is to make protelns that a~act nucleic
acid metaboli~m and synthesis or replication of viral DNA.
The third set o~ genes, the gamma genes, comprise about 30
to 40 genes that control the synthesis sf structural
proteins of the virus. The gamma yenes are induced after
the onset of viral DNA synthesis. In the usual course of
viral infection and replication, the infected cell is
"killed", it may be killed outright, but alternatively may
be constructively "killed" by being unable to divide or
exprass its own genes. In order to prevent production of
viral progeny and/or minimize the number of viral progeny
produced, the viral replication process should be
interrupted at a s~age prior to where viral progeny are
produced. At a time soon after infe~tion, viral
components reprogram the infected cell's metabolism for
the production of viral progeny, and at this time, the
cell is slated for eventual death.
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W0~2/03051 PCT/US91/OS7~6
The use of antisense oligonucleotides that are
complementary to and bind to specific t-~rget nucleic
sequences, particularly specific messenger RNA's, has been
suggest~d as a means to deactivate specific genes. (See
Weintraub, "Antisense RNA and DNA, Scientific Americ~n,
pages 40 to 46 (January 1990)).
The use of antisense oligomers which are
complementary to certain splice junction mRNA's of herpes
simplex virus type 1 ("HSV-1") to specifically inhibit
virus replication has been reported (See Kulka et al.,
Proc. Nat. Acad. Sci. (USA) 86:6868-6872 (September,
1989))-
Summary Of The Invention
Accorcling to the present invention, antisense
oligomers are provided that are complementary to a vitalregion of a viral genome which act as antiviral agents.
Such vital regions comprise nucleic acid sequences
necessary for viral replicati~n and are included in one or
more essential genes. Thus, in one aspect, the present
invention is directQd to an oligo~ler complementary to such
a vital region or mRNA transcri.pt thereo~, which when
hybridized to said target sequence!, inhibits or interferes
with viral DNA synthesis or replication. In one preferred
aspect the target se~uence comprises a portion of a mRNA
transcript of a gene essential for viral DNA synthesis or
replication. Suitable target sequences include sequences
at or proximate to a 5'-terminal translational s~tart or a
3'-terminal polyadenylation signal of the gene.
The present invention is also directed to methods of
interfering with replication of a virus after it has
infected host cells. The present invention is also
directed to oligomers which are useful in interfering with
and/or inhibiting such viral replication. According to
methods of t~e present invention, said virus or viral DNA
3S or their environment is contacted with an oligomer which
is complementary to a target seguence which comprises a
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WO~2/03051 PCTt~S91/057~6
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vital region of the viral genome or mRNA transcript
thereof. Optionally, two or more oligomers may be used
wherein each oligomer is complementary to a different
target sequence. Target sequences may be portions of the
same gene or of different genes.
In one aspect, the present invention is directed to
a method of interfering with replication of a virus after
infection of host cells by the virus wherein the cells or
their growth environment i5 contacted with an amount of an
oligomar which is complementary to and which hybridizes
with a messenger RNA sPquence for a gene essential for
viral DNA synthesis and/or replication, that is effective
to interfere with expression or function of said gene.
In one preferred aspect of the present invention, the
method~ of the present invention are especially useful in
interferin~ with viral replicatior1 in infections resulting
from viruses of the family HerF\esviridae, particularly
human herpes viruses, especially Herpes Simplex viruses.
Thus, the present invention is als~o directed to methods of
in~ibiting or interfering with replication of a human
herpes virus, especially a Herpes Simplex vlrus, by
contacting the virus viral DNA or cells infected therewith
with an oligomer complemantary to a nucleic acid target
seguence essential for viral DNA synthesis or replication
and wherein the oligomer can selectively hybridize with
said target sequenced. For Herpes Simplex viruses,
preferably the target sequence comprises a mRNA transcript
of an essential ~-gene. Suitable ~ genes include UL5,
UL8, UL9, ULl5, UL29, UL30, UL42 and UL52. Suitable
regions of these genes for selection of a target sequence
include a sequence at or proximate to a 5'-translational
start or a 3'-polyadenyl~tion signal.
Among other factors, in one preferred aspect, the
present invention is based on our finding tha~ oligomers
complementary to mRNA transcripts o~ genes that code for
the ~ group of polypeptides that are essential for viral
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WO~2/03~)5l PCT/US91/057~6
replication are especially effective in decreasing and/or
inhibiting viral replication in Herpes Simplex viruses.
According to an additional aspect of the present
Invention, methods of treating an organism infec~ed with
a Herpes-viradae virus are provided using these antisense
oligomers and methods.
Definitions
As used herein, the following terms have the
following meanings, unless expressly stated to the
contrary:
The term "nuclecside" includes a nucleosidyl unit and
is used interchangeably therewith.
The term "nucleotide" refers to a subunit of a
nucleic acid consisting of a phosphate group, a S carbon
sugar and a nitrogen containing base. In RNA the 5 carbon
sugar is ribose. In DNA, it is a 2-deoxyribose. The term
also includes analogs of such subunits.
The term "nucleotide multimer" refers to a chain of
nucleotides linked by phosphodiester bonds, or analogs
therQof.
An "oligonucleotide" is a nucleotide multimer
generally about 3 to about lO0 nucleotides in length, but
which may be greater than lOO nucleotides in length. They
ara usually considered to be synthesized from nucleotide
monomers.
A "deoxyribooligonucleotide" is an oligonuclaotide
consisting of deoxyribonucleotide monomers.
A "polynucleotide~' refers to a nucleotide multimer
generally about lOO nucleotides or more in length. These
are usually of biological origin or ara obtained by
enzymatic means.
A "nucleotide multimer probe" is a nucleotide
multimer having a nucleotide sequence complementary with
a target nucleotide sequence contained within a second
nucleotide multimer, usually a polynucleotide. Usually
the probe is selected to be perfectly complementary to the
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WC)92/03051 PCTIU~91/05~6
corresponding base in the target sequence. However, in
some cases it may be adequate or even desirable that one
or more nucleotides in the probe not be complementary to
the corresponding ba~e in the target sequence.
A "non-nucleotide monomeric unit" refers to a
monomeric unit which does not signi~icantly participate in
hybridization of an oligomer. Such monomeric units must
not, for example, participate in any significant hydrogen
bonding with a nucleotide, and optionally include
groupings capable of interacting after hybridization o~
oligomer to the target sequence, such as crosslinking
alkylation, intercalating and chelating agents.
A nucleotide/non-nucleotide polymer" refers to a
polymer comprised of nucleotide and non-nucleotide
monomeric uni_s.
An "oligonucleotide/non-nucllsotide multimer" is a
multimer generally of synthetic ori~in having less than
100 nucleotides, but which may contain in excess of 200
nucleotides and which contains one or more non-nucleotide
monomeric units.
A "monomeric unit" ro~ers to a unit of either a
nuclQotid~ r~agent or a non-nucleotide reagent o~ the
present invention, which the realgent contributes to a
polymer.
A "hybrid" is the complex formed ~etween two
nucleotide multimers by Watson-Crick base pairing s
between the complementary bases.
The term "oligomer" refers to oligonuc7eotides,
nonionic oligonucleoside alkyl- and aryl-phosphonate
analogs, phosphorothioate analo~s of oligonucleotides,
phosphoamidate analogs of oligonucleotides, neutral
phosphate ester oligonucleotide analogs, such as
phosphotriesters and other oligonucleotide analogs and
modified oligonucleotides, and also includes
nucleotide/non-nucleotide poly~ers. The term also
includes nucleotide/non-nucleotide polymers wherein one or
more of the phosphorous group linkages between monomeric
SUE3STITUTE SHET
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WC~2/~)3051 PCT/US91/05756
7~ 6
units has been replaced by a non-phosphorous linXage such
as a formacetal linkage or a carbamate linkage.
The term "alkyl- or aryl-phosphonate oligomer~' refers
to nucleotide oligomers (or nucleotide/non-nucleotide
polvmers) having internucleoside (or intermonomPr)
phosphorus group linkages wherein at least one alkyl- or
aryl- phosphonate linkage replaces a phosphodiester
linkage.
The term "methylphosphonate oligomer~ (or "MP-
oligomer") refers to nucleotide oligomers (ornucleotide/non-nucleotide polymer) having internucleoside
(or intermonomer) phosphorus group linkages wherein at
least one methylphosphonate internucleoside linkage
replaces a phosphodiester i~ternucleoside linkage.
In some of the various oligomer sequences listed
herein "p" in, e.g., as in ApA represents a phosphate
diester linkage, and "~" in, e.g." as in C~G represents a
methylphosphonate linkage. Certain other sequences are
depicted without the use of p or ~ to indicate the type of
20 phosphorus diester linkage. In such occurrences, A as in r
ATC indicates a phosphate diester linkage between the 3'-
carbon of A and the 5' carbon of T, whereas ~, as in ATC
or ATC indicates a methylphosphonate linkage between the
3'-carbon of A and the 5'-carbon of T or T.
The term "antisense oligomer~ refers to an oligomer
which is complementary ~o the "sense" strand of a DNA
duplex and to the mRNA transcript synthesized ~rom that
sequence. A DNA duplex is comprised of two co~plementary
DNA strands, one termed the "sense" strand and one termed
the "antisense" strand. Messenger RNA transcripts ar~
synthesized using the antisense DNA strand as a template
and hence are homologous ~with the replacement of T with
A) to the sense strand.
ThQ term "vital region" of a viral geno~e or viral
DNA refers to a nucleic acid sequence which is necessary
for viral replication such that if the sequence is deleted
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or rendered nonfunctional, the virus is incapable of
replication.
The term "deblocking conditions" describes the
conditions used to remove the blocking (or protectin~)
S group from the 5'-OH group on ~ ribose or deoxyribose
group.
The term "deprotecting conditions" describes the
conditions used to remove the protecting groups from the
nucleoside bases.
The term "tandem oligonucleotide~' or "tandem
oligomer" refers to an oligonucleotide or oligomer which
is complemlentary to a sequence 5' or 3' to a target
nucleic acid sequence and which is co hybridized with the
oligomer complementary to the target sequence. Tandem
lS oligamers may improve hybridization of these oligomers to
the target by helping to make the target sequence more
accessible to such oligomers, such as by decreasing the
secondary structure of the target nucleic acid sequence.
The melting temperature or "Tm" of a duplex ~such as
a dou~le strand~d nucleic acid DNA:DNA or RNA:DNA) is
de~ined a the temperature at which half ~he helical
structure is lost.
Detailed De,scri~tion Of The Invention
Th~ present invention is directed to antisense
oligomers useful as antiviral agents and to methods of
interfering with viral replication in a host cell after
its infe~tion using such antisense oligomers, wherein said
oligomers are complementary to (and which hybridi~e with)
a target nucleic acid sequence of a gene essential for
viral replication or a viral messenger RNA transcript of
said gene.
~re~errad ~arget Sequences
In general, pre~erred are target nucleic acid
seguences which comprise a ~ital region of the viral
genome. Thesa target sequences may comprise portions of
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WO~)2/0305l PCT/US91/05756
an essential gene for viral DNA replication or a mRNA
transcript thereof which are "available", i.e. are in a
state where the complementary oligomer is able to
hybridize with the target sequence. Thus, these target
sequences are preferably single stranded and relatively
free of secondary structure and bound protein.
Preferred target sequences include mRNA transcripts
o~ genes which are "essential" for DNA replication.
Moreover, mRNA transcripts which are present in low
numbers comprise particularly advantageous target
sequences for this antisense therapy. With fewer mRNA
transcripts, a lower con~entration of oligomer can
hybridize with and interfere with the function of a larger
percentage of the mRNA from a particular gene.
Certain g~nes which code for ~RNAIs which are present
in large amounts comprise less pre~erred target sequences,
since if the function of these ~NA's is only partially
blocked, the unhybridized mRNA's may proceed with the
normal replicative cycle of th~! virus. Moreover, a
proportionally larger amount of oligomer would ~e required
~o block an equivalent frac~lon oi8 the mRNA.
Preferred are essential genes which are expressed
during the earlier stages of DNA replication, but after
the cell is "committed" to death due to infection by the
2S virus. By blocking DNA replication at such an earlier
stage, few if any functional virus particles are made.
Since the cell has already been "committed" to death it
will die whether or not functional virus particles are
made and after death, will be dealt with by the host
organism's immune system. These cells include cells
which, due to the viral infection, are unable to divide
and/or express their own genes. If viral DNA replication
is blocked before the cell has been committed to death,
the viral DNA in the cell will not be destroyed and viral
DNA replication may recommence later on.
SUBSTITUTE SHEET
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W0~2/n3051 PCT~US91/05756
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Suitable target sequences include sequences which are
at or proximate to a 5~-terminal translational start or a
3'-terminal polyadenylation signal.
Preferred target sequencas include those which have
a relatively high loc'al G-C base content. Sequences
having a relatively high local G-C content are preferred
in part because they tend to hybridize more tightly to the
complementary oligomer and exhibit a correspondingly
higher Tm. Especially preferred target sequences hav~ a
high G-C base content on both ends of the target sequence
that is complementary to and hybridizes with the
complementary oligomer, which enables the ends of the
oligomer to hybridize more tightly to the target sequence.
Pr~ferred Oliqome~s
These oligomers may comprise either ribonucleoside or
deoxyribonucleoside monomeric: units; however,
deoxyribonucleoside monomeric unil:s are preferred.
Preferred are oliqomers which comprise from about 6
to about 40 nucleotides, more prei.erably ~rom about 12 to
about 20 nucleo~ides. Although oligomers which comprise
more than a~out 20 nucleotidec3 may be used, where
complementarity to a longer sequence is desired, it may be
advantageous to employ shorter tandem oligomers to
maximize solubility and transpor~ across cell membranes
while competing for the development of a secondary
structure of the target nucleic acid, such as a mRNA.
Alternatively, it may be advantageous to use moro than one
oligomer, each oligomer comple~entary to a distinct target
sequence which may be part of the same gene or a different
gen~.
Although nucleotide oligomers (i.e., having the
phosphodiester internucleoside linkages present in natural
nucleotide oligomers, as well as other oligonucleotide
analogs) may be used according to the present invention,
preferred oligomers comprise oligonucleoside alkyl and
aryl~phosphonate analogs, phosphorothioate oligonucleoside
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analogs, phosphoro-amidate analogs and phosphotriester
oligonucleotide analogs. However, especially preferred
are oligonucleoside alkyl- and aryl-analogs which contain
phosphonate linkages replacing the phosphodiester linkages
which connect two nucleosides. The preparation of such
oligonucleoside alkyl and aryl-phosphonate analogs and
their use to inhibit expression of preselected nucleic
acid sequences is disclosed in U.S. Patent Nos. 4,469,863;
4,511,713; 4,757,055; 4,507,433; and 4,591,614, the
disclosures of which are incorporated herein by reference.
A particularly preferred class of those phosphonate
analogs are methylphosphonate oligomers.
Such alkyl- and aryl-phosphonate oligomers
advantageously have a nonionic phosphorus backbone -~hich
l!j allows better uptake of oligomers by cells. Also, the
alkyl- and aryl-phosphonate int~srmonomQric linkages of
such alkyl- and aryl-phosphonate oligomers are
advantageously resistant to nuclQases.
WherQ the oligomers comprise alkyl- or aryl-
phosphona~c oligomers, it may~ be advanta~eous toincorporate nuclQosidQ monomeric units having modi~ied
ribosyl moieties. The use of nucleotide units having 2'-
O-alkyl- and in particular 2'-O-methyl-, ribosyl moieties,
in these alkyl or aryl phosphonate oligomers may
advantageously improve hybridization of the oligomer to
its complementary target nucleic acid sequence.
Synthetic methods for preparing methylphosphate
oligomers ("MP-oligomers") are described in Lee, BL. et
al., ~iochemistxy 27:3197-3203 (1988) and Miller, P.W., et
al., Biochemistry 25:5092-5097 (1986), the disclosure of
which are incorporated herein by reference.
Preferred are oligonucl~oside alkyl- and aryl-
phosphonate analogs wherein at least one of the
phosphodiester internucleoside linkages is replaced by a
3' - 5' linked internucleoside methylphosphonyl (MP) group
(or "methyl-phosphonate"). The methylphosphonate linkage
is isosteric with respect to the phosphate groups of
$~B~r~
WO92/(~3051 PCT/US91/~57~6
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oligonucleotides. Thus, these methylphosphonate oligomers
("MP-oligomers") should present minimal steric
restrictions to interaction with complementary
polynucleotides or single-stranded regions of nucleic acid
molecules. These MP-oliyomers should be more resistant to
hydrolysis by various nuclease and esterase activities,
since the methylphosphonyl group is not found in naturally
occurring nucleic acid molecules. It has been found that
certain MP-oligomers are more resistant to nuclease
hydrolysis, are taken up in intact form by mammalian cells
in culture and can exert specific inhibitory effects on
cellular DNA and protein synthesis (See, e.g., U.S. Patent
No. 4,469,863).
If desired, labeling groups such as psoralen,
chemiluminescent groups, cross-linking agents,
intercala~ing agents such as acricline, alkylating agents
or groups capable of cleaving the targeted portio~ of the
viral nucleic acid such as molecular scis90rs like o-
phenanthroline-copper or EDTA-iron may be incorporated in
the MP-oligomers.
Preferred are MP-oligomers h~lving at least about 6
nucleosid~s which i~ usually~ su~e~icient to allow for
specific binding to the desired nucleic acid sequence.
More preferred are MP-oligomers having from about 6 to
about 40 nucleosides, especially preferred are those
having from about 10 to about 25 nucleosides. Due to a
combination of ease of preparation, with specificity for
a selected sequence and minimization o~ intra-oligomer-
internucleoside interactions such as folding and coiling,
particularly preferred are MP;oligomers of from about 12
to 20 nucleosides.
One group of preferred MP-oligomars includes MP-
oligomers where the 5'-internucleoside linkage is a
phosphodiester linkage and the remainder of the
internucleoside linkages are methylphosphonyl (or
methylphosphonate~ linkages. Having a phosphodiester
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W092/030sl PCT/~S91/OS7~6
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linkage on the 5'-end of the MP-oligomer pe~mits kinase
labelling and electrophoresis of the oligomer.
Preferred Embodiment of the Invention .
Infections due to viruses of the family Herpesvirdae
comprise particularly suitable targets for ~herapy using
the antisense oligo~ers and methods of the present
invention. Herpes viruses vary greatly in their
biological properties. Some have a wide host cell range,
multiply efficiently and rapidly destroy the cells which
they infect (HSV-l, HSV-2). Others have a narrow host
cell range. A ubiquitous property of these herpes viruses
~ ~ is their capacity to remain latent in the host in which
they multiply. The mechanism by which the virus
perpetuates itself appears to re~lect a function of
dedicated viral genes as well as association with
appropriate cells. In general, infections caused by
herpes viruses have been found to be persistant. Herpes
viruses for which therapy using these anti~ense oligomers
appears promising include human herpes viruses 1 to 7
which include Herpes Simplex Virus Type 1, Herpes Simplex
Virus Type 2, Varicella-Zoster virus, Epstein-Barr virus,
Cytomegalovirus and human herpes viruses 6 and 7.
In one preferred embodiment, ~le present invention is
directed to antisense oligomers which are useful as
antiviral agents against herpes simplex virus ("HSV"),
particularly type 1 ~"HSV-l"), and to methods of
controlling HSV-l infections by inhibiting~ and/or
interfering with replication of HSV-l.
According to the present invention, antisense
oligomers are provided that are complementary to
"essential" genes.
In Herpes Yiruses, as ~uch as about one half of viral
genes are non essential; ~hat is, they may be deleted or
at least reduced in expres~ion or treated with antisense
oligomers and not effect viral replication.
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Preferred target sequences for these antisense
oligomers comprise essential genes, that is genes which
when deleted or their function is compromised,
significantly affect viral replication, particularly the
synthesis and/or replication of DNA. Also, as noted,
preferred target sequences inclu~e mR~A transcripts of
such essential genes, wherein copies are present only in
low n~mbers. For this reason, we have found that
essential ~ genes of HSV-l to comprise particularly
suitable target sequences for these antisense oligomers.
HSV-l has about 15 ~ genes, of which at least about
8 have ~een reported to be essential. These essential
genes include the genes termed UL5, UL8, UL9, UL15, UL29,
UL30, UL42 and UL52. These genes have been reported to
l'; code ~or protelns which are necessary for viral DNA
synthesis and/or replication. Seven of these genes have
been reported to be required for viral-origin-dependent
DNA synthesis and to map in the I, component of the viral
DNA. These seven genes have been reported to speci~y the
~ollowing: a DNA polymera~e ~UL30) with an apparent
molecular wQi~h~ of 140,000; a singl~-strand speci~ic-
DNA-binding protein designated ~s ICP8 (UL29) with an
apparent molecular weight of 124,000; a protein binding to
the origin of viral DNA synthesis (uLs) with a translated
molecular weight of 94,000; a protein that binds to
double-stranded DNA (UL42) with a molecular weight of
62,000; and three additional proteins (UL5, predicted
molecular weight of 99,000; UL8, predicted ~olecular
weight of 80,000; and UL52, predicted molecular weight of
114,000). ~hese three proteins form a complex in which
each protein is present in equimolar ratios and which
functions as a primase and helicase. The protein
specified by UL5 has independently been shown to act as a
DNA dependent ATPase.
The above noted seven proteins app~ar to be all that
is necessary for ori5-dependent amplification of DNA
transfected into cells. For this reason, oligomers which
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W0')2/0305l PCT/US91/057~6
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are complementary to the mRNA of one of these seven genes
are particularly preferred and comprise especially
suitable antiviral agents against HSV-1.
Of the above noted seven essential genes, preferred
are the genes denoted UL5, UL8 and UL52. It is believed
that the mRNA transcripts of these genes comprise target
se~lences which are particularly susceptible to inhibition
using these antisense oligomers.
Portions of these essential genes which ~ay be
relatively more available to these antisense oligomers
comprise esp~ecially suitable target sequences. It is
believed that sequences that are proximate to the 5'-
terminal translational start of these mRNA transcripts or
to the 3'-terminal polyadenylation signal comprise
especially suitable target sequences in view of their
demonstrated susceptibility to inhibition of viral
function due to hybridization of an antisense oligomer.
Preferred target sequences include ~portions of these mRNA
transcripts in which it appears that secondary structure
of the mRNA does not inter~ere with its ability to
hybridize to a complementary oligomer.
Antisense oligomers complement2lry to selected xegions
of mRNA transcripts of these seven genes have been assayed
for antiviral activity using a Virus Titer Reduction Assay
(see Example A) and a Direct Plaque Assay (Example B) and
have been found to demonstrate antiviral activity (see
Tables II, III and IV).
To assist in understanding tha present invention, the
following examples are included which described the
results of a series of experiments. The following
examples relating to this invention should not, of course,
be co~strued in specifically limiting the invention and
such variations of the invention, now known or later
developed, which would be within the purview of one
skilled in ~he art are considered to fall within the scope
of the present invention as hereinafter claimed.
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WO(~2/03~SI PCT/US91tO575
ExamPle 1
PreParation Of Phos~hate Diester Oli~omers
Phosphate diester oligomers are prepared using a
Biosearch model 8750 DNA synthesizer using standard
phosphoro-amidite chemistry (M.H. Caruthers, et al.,
Methods of Enzymol. 154:287-313 (1985)) according to the
manufacturers recommendations. The 5'-dimethoxytrityl
protecting group is left on at the end of the synthesis to
permit purification on a Sep-Pak~ C18 cartridge
(Millipore/Waters, Bedford, MA) as described by X.M. Lo et
al. (Proc. Natl. Acad. Sci. (USA) 81:2~85-22~9 (1984)).
During this procedure, the dimethoxytrityl protecting
group was removed.
Exam~le 2
PreParation Of Meth~lphos~honate Oli omers
Methylphosphonate oligomers are synthesized using
me~hylphosph~namidite monomers~ according to the chemical
m~thods described by P.S. ~iller et al. (Nucleic Acids
Re~ 6225-6~242 (1983)), A. Jager and J. Engels
(Tetrahedron Letters 25:1437-1440 tl984)) and M.A. Dorman
et al. (Tetrahedron Letters 40:95-102 ~1984)). Solid
phase synthesis is per~ormed on a Biosearch Model 8750 DNA
synthesizer according to the manufacturer's recommenda-
tions with the following modifications: 'IG'' and 'IC''
monomers are dissolved in 1:1 acetonitrile/dichloromethane
at a concentration of 100 mM. "A" and "T" monomers are
dissolved in acetonitrile at a concentration of 100 mM.
DEBLOCK reagent = 2~5% dichloroacetic acid in
dichloromethane. OXIDIZER reagent = 25 g/L iodine in 2~5%
water, 25% 2,6-lutidine, 72.5% tetrohydrofuran. CAP A=
10% acetic anhydride in acetonitrile. ~AP B = 0.625% N,N-
dimethylaminopyridine in pyridine. The 5'-dimethoxytrityl
protecting group is left on at the end of the synthesis to
facilitate purification of the oligomers, as described
below.
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WO92/03051 PCTtUS91/0~756
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The crude, protected methylphosphonate oligomers are
removed from the solid support by mixing with concentrated
ammonium hydroxide for two hours at room temperature. The
solution is drained from the support using an Econo-
ColumnT~ (Bio-Rad, Richmond, CA) and the support is washed
five times with 1:1 acetonitxile/water. The eluted
oligomer is evaporated to dryness under vacuum at room
temperature. Next, the protecting groups are removed from
the bases with a solution of ethylenediamine/ethanol/
acetonitrile/water (50:23.5:23.5:2.5) for 6 hours at room
temperature. The resulting solutions are then evaporated
to dryness under a vacuum.
The 5'-dimethoxytrityl ("trityl") containing
oligomers are purified from non-tritylated failure
sequences using a Sep-PakT~ C18 cartridge (Millipore/Waters
Bed~ord, MA) as follows: The cartridge is washed with
acetonitrile, 50% acetonitrile in 100 mM, triethylammonium
bicarbonate (TEAB, pH 7.5) and 25 mM TEAB. Next, the
crude methylphosphonate oligomer is dissolved in a small
volume of 1:1 acetonitrile/water and then diluted with 25
mM TEAB to a final concentration of 5% acetonitrile. This
solution is then passed through th~ cartridgs. N~xt, the
cartridge is washed with 15-20% acetonitrile in 25 mM TEA~3
to elute failure sequences from the cartridge. The
trityl-on oligomer remaining bound to the cartridge is
then detritylatPd by washing with 25 mM TEAB, 2
trifluoroacetic acid, and 2S mM TEAB, in that order.
Finally, the trityl-selected oligomer is eluted~from the
cartridge with 50~ acetonitrile/water and evaporated to
dryness under vacuum a~ room temperature.
The methylphosphonate oligomers are further purified
by reverse-phase HPLC chromatography as follows: A
Beckman System Gold HPLC is used with a Hamilton PRP-l
column (Reno, NV, 10 ~, 7 mm i.d. x 30S mm long). Buffer
A = 50 mM triethylammonium acetate (pH 7); Buffer B - 50%
acetonitrile in 50 mM triethylammonium acetate (pH 7).
The sample, dissolved in a small volume of 10-50
.~
:~ '
W092tO3~1 PCT/US9t/~ 6
17
acetonitrile/water, is loaded onto the column while
flowing at 2.5-3 ml/minute with 100% Buffer A. Next, a
linear gradient of 0-70% Buffer B is run over about
30-50 minutes at a flow rate of about 2.5-30 ml/minute.
Fractions containing ~ull length methylphosphonate
oligomer are collected, evaporated under vacuum and
resuspended in 50~ acetonitrile/water.
Example 3
Inhibition Of Herpes Sim~lex Virus-l Replication
Oligomers complementary to designated sequences o~
c~rtain~ genes of herpes simplex virus-l ("HSV-l") were
prepared. Sequences are reported in Tables I and IV.
These oligomers were assayed for their ability to
inhibit HSV-l replication according to the Virus Titer
Rf~duction Assay (Example A) and/or ~hel Direct Plaque Assay
(Example B). Results are reported in Tables II, III, IV
and V.
Ex~mple ~
Vixus Titer Reduction
~0 The Virus Titer Reduction assay measures the ability
of an oligomer to inhibit the total yield of virus
produced by a group of cells. This test consists of two
parts. First, a plate of cells is infected with virus;
second, oligomer in media or media alone is added; and
third, the cells are incubated for one to several
replications of virus. The cells are then harvested ~rom
the well and various dilutions of this harvest are used to
infect frèsh monolayers of cells. The amount of virus
~ound, measured by plaque foxmation, is the total amount
of virus produced by the initial cells. A comparison of
the total virus produced by treated and untreated cells is
a measure of the i~hibition of virus by the test drug.
P~esult~ are reported in Tables II, III, IV (under "VTR"),
and V.
SIJ~STITUTE SHEET
` .
WO ~2/0~05l P~r/US9l/057~6
lR
~,~
oliyomers complementary to designated sequence~ of
certain genes of herpes simplex virus-1 ("HSV-1") were prepared.
Sequences are reported in Tables I and IV.
These oligomer~ werc assayed ~or their ability to
inhibit HSv-l replication according to the Virus Titar Reduction
A say (Example A) and/or ~he Direct Plaque A~ay (Example B).
Results ara reported in Table~ II, III, IV and V.
VIRUS TITER REDUCTION
The Virus Titer ~eduction as~ay mQaaure~ tho ability of
an oligomer to inhlblt the total yield o~ virus produced by a
group of cQlls. This te3t con~ists of two parts. Firqt, a plate
0~ cnllg i9 in~ected w$th vi~uss secon~, ollgomer in m~dia or
medla alono is addeds and thlrd, the cl~lls are incubated ~or one
to s~veral replications o~ virus. Th~ colls aro thon harvested
~rom th~ woll and various dilutions o~ thls h~rv~t ar~ used to
infect freqh monolayer~ Or cells. The acount of virus ~ound,
mea3urad by plaqus rOrmation, is the total ~ount of virus
produced by the initial cell~. A co~parlson o~ thQ total viru~
produced ~y tr~ated and untreatad c~lls i9 a m~a~uro of th~
inhibltion o~ viru~ by th~ te~t drug. Results ar~ report~d in
Ta~le~ II, III, rv (undar "VT~, and V.
~ h~ Dlrec~t Plaqu~ ~s~ay maa ur~- tha ability of an
oli~o~er to inhiblt in~ection and virus r~plic~tlon. C~lls are
in~ected with enough virus to in~ect only a f~actlon o~ the
cells. Th~n an ov~rlay with or wl~hout ollgom~, i3 add~d. This
SUEISTITUTE SHEET
-
.
': ' ' ' ' ' ,
. "' .. . .
' . ' ' ' ,:
WO g2/030~1 PCT/US91~05756
' ~ ~ Y ~
18/1
overlay limits all spread of virus from cell to cell ~hrough the
extracellular fluid, but not by direct contact or ~xtension. The
resultant plaques can th~n be counted and obsarvQd ~or ~iZB- A
co~parison of plaqu~ number and 8iZQ in tr~at~d ~nd untrQated
cells provides tha infor~ation deslrad. R~ults ara reported in
Table IV under "PLAQUE."
SUE3STiTUTE SHEET
.'; '' '~'`'~. ' ' ' .
', ' ` "' - ,,
'.'. "' "' '' ' .
W() '~2/03(151 PCI/US~l/OS7~6
19
) U ~
TA~LE I
A. COM~LEMENTARY TO AREA AROUND TR~NSL~LoNAL S~ART
OLIG0~ER_~O~ LOCATION SE0UENCE
0015 UL5 -15 +3 5'-CAT-~C-C~C-~Ç_- CG-CTC-3'
0013 UL8-5 ~ +13 5l-CTa-ÇQQ-~9~-ÇC~-TC~-ÇAC-3'
0021 UL8~1 - +18 5'-GA~-~5_-$QQ-9~-GTC-ÇAT-3'
0028 UL8ll9 - +35 s'-C~Ç-~Ç- ÇC-~ÇQ-C~C-AC-3~
0046 UL8 5~-cc~-~5~-~ç~-aça-GC~ 3'
0047 --~ UL8 5'-GGC-ççC-~a-9~ ça-cGa-3'
0007 UL9_5 - +13 5'-C~-C~A-~a~-Ç~-~ga-Ç-3~
0056 UL15-6 - +12 5'-C -ACC~ a~-CCC-Q~-3'
0054 UL29-5 ~ +13 sl-Gç~-~3Q-~Q~-Qça-~9~--ç~-3
0039 UL30_3 _ +15 sl-GÇQ-~ÇQ-Cga-aaA-Ç~-Ç9--3'
0016 UL42-7 - +11 5'-G~ ÇQ-aTC-a3Q-ÇQ~-Ag~-3'
0052 ULS2-21 - -4 5~-GIQ-ÇgQ-9ÇQ-ÇCÇ-~aQ-aQC-3'
0019 ULS2-3 - +lS 5l-G~ç~5~Q-ç$Q-~QQ-sa~-~sç-3
0053 UL52+16 - +33 5l-c~-Q3~-~cç-~ca-s~ ç~-3
A, C, G or T ~ Pho~phate diester linkage
~ S~ ~ or ~ ~ Methylphosphon~te linkage
TITUTE SH~ET
.. . . . . .. .
WO ~2/~3051 PCl`~US9t/0~756
2û ~ u ~
8. CoMPTFMENT~RY TO PO~Y ~ SIGNAL
OLIGOMER N0. ~IÇ5~UQ~ ~S~IEY~
0005 UL5-3 - +15 5'-T~-g~-a~C-5~-Aa~-GGA-3'
0006 UL5+5 - +22 5~-C~-9~-95~-~~-TGT-C~-3'
0020 UL5+12 - +29 5'-Aa~-~5Q-GCT-GG~-TG~-~$~-3'
0008 UL5+23 - +40 5'-Aa~aQ-~~ a-a3~-~Q9-3'
0023 UL5+41 - +58 5-_GçQ_~g~_5~ _~a_AT~_3~
0059 UL5+59 - +76 5'-AC~-Ç~ -GCC-ÇGT-CGC-3'
0051 UL8-15 - +3 5'-A~ -aa~-G~C-~3~-5~-3'
0024 UL8+4 - +21 5'-A$~-GG5-_~a-~ a_-GCA-3'
0014 UL8+22 - +39 5'-TÇ~-C~G-G~C-Aa~-95~-r3~-3'
0022 UL8+40 - +57 5'-C~Q-9Qg-q9~-~ÇQ-CTC~ 3'
OOS7 UL15-13 - +5 5'-T~A-55g-ggQ-9Q~-CAC-9~Q-3'
0055 U~29+4 - +21 5'-A5Q-5CQ-5aQ-~ a-55~-3'
0040 UL30+3 - ~20 s'-G~-9Q~-Ç9Q-~A-Ç~ 3~-3'
0025 UL42-6 - ~12 5'-AÇ~-~aQ-33~-25~-TAC-~5~-3'
0017 UL42+4 - +21 S'-T~Q-9g~-A~ S~-~a9-~-3'
0018 UL42+22 ~ +39 5~-GS9-~a-CGC-gQg-~a-g~Q-3l
0026 ULA2+40 - +58 5'-Ca~ a-9~-9~a-CCA-C~C-3'
0151 UL42~59 - +76 5l-GA9-gg~-~G~cqc-Gcc-A~o-3l
0027 UL52+3 - +20 5'-G~-CG~ C-AT~-~a~ -3'
0038 UL52~21 - ~38 5'~~ ~ag~AQa~Ça~$a~~3'
0035 U~52+39 - ~56 s~-c~-çc~-gç~ Q-Aça-~aa-3
A, C, G or T ~ Pho~p~ate diester linXage
~, Ç, Q os ~ ~ Methylp~osp~onata linkaga
glJ~3$TlTUTE St;EET
` . `
.... .. . ...
., ~ . . . .
.
.
.. ....... . ... .. -.. . .
W O ~2/030~1 PCTtUS91/0S756
TAB~E II
ANTI-HERPES SI~PLEX VIRUS TYPE 1 ACTIVITY
(TRANSLATIONAL START)
OLIGoME~ NO ! GE~ OCATION % IN~I~ITIoN
(Gene Product) IATG STA~ +1) 1~8I~9~1
+l
0015 DNA DNP. ATP~e
-15--~ 3 63%
u~a
~ELICASE~PRIMASE
0021 +1-------~18 _.45%
002a . ~ +19---------~35 62
~2
ORIGIN 3INDING PROTEIN
0007 -5--------+1:1 49
SPLICE JUNCTION
0056 -6---------112 7
U~2
DNA 3INDING PROTEIN
0054 -S--------~l'l 68
~Q
DNA PO~YMERA5E
0039 -3---------~15 50%
U~42
DNA SYXTHE525 pRorEIN
00~6 -7------~ 11 26
U~2
HELICASE/PRIMASE
0052 -21------- -4 63S
0019 -3--~ --+15 79~
0053 ~16--------+33 74%
- . . , .,, ,. ,.-. , : ,
.:: . . . : -
:. ~ , ,. - ,-- ::
WO !)2t03051 PCI`/US91/05756
22
~L~
ANTI-HERPES SIMPT~X VIRUS TYPE 1 ACTIVITY
(POLY A SIGNAL)
OLIGOMER ~0.G~E LOC~TION S INHIBITION
(Gene Pr~duct) +l ~$AxI~uM)
A(A)TAAA~
~L~
DNA CNP. ATPa~e
0005 -3-------+lS
0006 +5-~ +22 69%
0020 +12-------+29 18S
0008 +23-------+40 17~
0023 +41-------+58 96%
0059 +53-------+~6 18% ._ _
HEL:tCASE/PRIMASE
0051 -15-~ +3
0024 +4------+21 7816
0014 ~22--------+39 10~
00;!2 . +40-------~57 58S
~ .
SPLICE JUNCTION
0057 -13------+5 32%
S~2
DNA BINDING PRoTEIN
003Y +4-~ 21 10%
~3Q ` '
DNA PO~YMERASE
0040 . +3-------+20 55%
3ZL~L2
DNA SYNTHESIS PROTEIN
0025 -6-------+12 1~
001~ +4-------+21 3~t
0018 +22--------+39
0026 ~40-------~58 93
01Sl +59--------+76
44
HELIC~5EJP~I~ASE
002~ +3-------+20 8~%
0038 +21--------+38 - 98~
0035 +39-------+S6 66%
SUBSTITUTE SHEET
, .. ..
.. . , ~ ... .
. ` , : :: - . :
- ., , . ~ .. :, : ~, .
`~ . . ,- .
,: .: , ;
WO ~2/03051 PCT/US91/057~6
23
TA~L~ IV
~ REDUCTION
OLIGOMER OF HSV-l
NO. GENESEQUEN~ CONC. VTR PLAQUE
0002 UL85'-C~-Ç9Q-~9~-ÇÇ~-TC~-CAC-3' 100~M 35%
0002 UL85'-ç~-çg~-~9~-çça-59~-ç~_-3' 200~M 63
0002 UL85l-cTa-~99-~g~--ça-~çQ-ça~-3l 100uM 49
0002 UL85l-cTa-cGa~9~-ccA~ -cAc-3l 200~M 76~
0002 UL85'-3Q-Ç~ -CC~-~CG-CAC-3' 200~M 29%
0002 UL8- 5'-C~3-CG~-TGT-ÇÇa-~Ç~-C~C-3~ 200~M 23%
0004 UL85'-9a~ Q5-GaT-GTC-Ça~-3' 200~M 32%
A, C, G or T ~ Pho~phat~ dlest~r linkage
~, ~, 9 or ~ ~ Mothylpho~phonate linkago
~A~E~ ' '
A. ~ aEY~
01i~o~or ~O. 99n~ U Qn~ ~Q~h~
~1) 0021 U~3 ~32t)
82%
0028 U~8 (62%)
~2) 0013 UL8
0046 UL8 99
0047 U~8
B. ~
QUL99~ 9~ Q~n~ Toaoth~r
0013 U~8
67%
0019 ULS2
a'`JQ~'T~ T
.. .. ~. . `
` . ; ~ `.~ . . `. ~. . .
`. `
