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BACKGROUND OF THE INVENTION
1. Field of the Invention:
The present invention relates to a method for inhibiting propagation of
virus and to an anti-viral agent. More particularly, the present invention
relates to a method for inhibiting propagation of virus wherein a specific
oligo- or polydeoxynucleotide is applied to the place infected by virus as
well as an anti-viral agent containing a specific oligo- or
polydeoxynucleotide.
2. Description of the Prior Art:
At the early stage of the remedy of diseases caused by virus, chemically
synthesized medicines and antibiotics were employed as anti-viral agents
to inhibit the propagation of virus. In the case of the chemically
synthesized medicines, however, their anti-viral spectrum is rather narrow
and harmful side-effects brought about by such medicines often come into
question. In the case of the antibiotics, on the other hand, there is a
detrimental drawback that development of new-type antibiotics are always
required in addition to efforts to reduce any harmful side-effect
manifested by antibiotics since virus becomes resistant to the antibiotics
used and finally becomes immune thereto with the lapse of time.
With the recent development of biotechnology, especially genetic
engineering, identification of the structural genes of virus has become a
great theme of research among those engaged in the study of
microorganisms. In 1977, S. C. Inglis et al. reported the in vitro
translation of cytoplasmic RNA extracted from chick embryo fibroblasts
infected with influenza A virus in a wheat germ cell-free
protein-synthesizing system, with the result that the synthesis of the
virus-specific polypeptide corresponding to the hybridized v-RNA segment
is reduced [Virology 78, 522-536 (1977)]. However, the experiments
referred to in this reference are carried out with a cell-free system
having no relation to the study of medicine. Such structural gene
identification is also reported by B. M. Paterson et al. in Proc. Natl.
Acad. Sci., U.S.A., 74, 4370-4374 (1977). The experiments by Paterson et
al. are also carried out with a cell-free system. In these two references,
what is used for inhibiting the synthesis of protein is an enormous
genetic structure such as RNA itself extracted from the influenza virus in
the firstly mentioned reference or rabbit .beta. globin clone P.beta.Gl in
the secondly mentioned reference. Such an enormous structure could not be
expected to inhibit the intracellular synthesis of protein.
In 1978, P. C. Zamecnik et al. reported that a tridecamer
oligodeoxynucleotide complementary to the reiterated 3'- and 5'-terminal
nucleotides of Rous sarcoma virus (RSV) is an efficient inhibitor of the
synthesis of protein specified by the viral RNA in wheat cell-free system
[Proc. Natl. Acad. Sci., U.S.A., 75, 285-288 (1978)] and in an in vitro
tissue culture system [ibid. 75, 280-284 (1978)]. Concerning the first
reference, the cell-free system is still used as before but some
improvement is recognized in respect of using a DNA with a smaller
molecular structure for inhibiting the translation of protein. In the
second reference, experiments are carried out using the tridecamer in a
tissue culture system for inhibiting replication of virus and
transformation of cells. The DNA used in these references for inhibiting
the replication and the transformation is selected from a region other
than a specific coding region and is not desirable. In the same year, N.
D. Hastie et al. also reported that hybridization of globin mRNA to its
corresponding cDNA was found to specifically inhibit translation of the
mRNA in a cell-free system [ibid. 75, 1217-1221 (1978)]. Novel in this
reference is only that a certain coding region of DNA is selected for
inhibition of the translation by hybridization of mRNA. However, the
experiments referred to therein were still carried out in a cell-free
system. There has been a demand for developing medicines based on this
theory since publication of these references in 1978. Hitherto, however,
there has been reported no reference in connection with a device for
applying this theory practically to experiments in vivo.
In 1983, a PCT patent application relating to oligonucleotide therapeutic
agent and methods of making same (Molecular Biosystems INC., Intl. Publn.
No. WO83/01451) was published. However, what is disclosed in this
application is a mere statement that (-)-strand DNA selected from a
certain coding region inhibited SV 40 transformed cell.
Although the expression "in vivo" is often used in Example 1 of this
reference, the descriptions referred to therein are, in view of the
context, apparently suggestive of the experiments in vitro cell culture
system. Even if such experiments in vitro were deemed as experiments in
vivo, they lack concrete conditions thereof and are nothing but a mere
mention of expectation of desirable effects. Anyway, this reference
suggests for the first time application of RNA-DNA hybridization technique
to a medicine but lacks detailed descriptions for substantiating utility
as medicines and is nothing more than a mere aggregation of the knowledges
manifested at that stage. Thus, what is taught in this patent application
involves nothing beyond the technical level disclosed in the references
described above.
Under the circumstances mentined above, there is a great demand for
providing an entirely new type of anti-viral agent by developing the
theory of inhibiting mRNA translation of virus on the basis of the RNA-DNA
hybridization technique to realize inhibition of the propagation of virus
in vivo.
BRIEF SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide a method
for inhibiting propagation of virus, which comprises delivering the
specific oligo- or polydeoxynucleotide to the place infected by the virus.
It is another object of the present invention to provide a new type of an
anti-viral agent comprising a specific oligo- or polydeoxynucleotide.
It is still another object of the present invention to provide the use of
an oligo- or polydeoxynucleotide identical in DNA sequence with a portion
of the structure of DNA hybridizable to an mRNA generated on the
propagation of virus, the structure being capable of the hybriding to the
mRNA, for inhibiting the propagation of the virus.
Other objects, features and advantages of the present invention will become
apparent more fully by the following description.
Further, the present invention can more fully be understood from the
following description in conjunction with the accompanying drawings in
which:
FIG. 1 is a graph showing the result of experiments which demonstrate the
inhibitory effects of the oligodeoxynucleotide (the 20 A) on the
cytopathic effects of HSV-1.
FIG. 2 is a graph showing the result of experiments which demonstrate the
inhlitory effects of the oligodeoxynucleotide (the 20 A) on propagation of
virus.
FIG. 3 is a graph showing the effect of a certain combination of the
oligodeoxynucleotides for the inhibition of multiplication of virus and
the pathogenic effect thereof as compared with a single
oligodeoxynucleotide used therein.
FIG. 4 is a graph showing the result of experiments which were designed to
demonstrate the in vivo effectiveness (in a living animal) of the
anti-viral agent of this invention.
DETAILED DESCRIPTION OF THE INVENTION
The present inventor has found that when an oligodeoxynucleotide or
polydeoxynucleotide identical in DNA sequence with a portion of the
structure of DNA hybridizable to mRNA generated on the propagation of
virus is delivered to the place where the mRNA exists, the structure being
capable by hybridizing to the mRNA, the propagation of the virus is
effectively inhibited. By administering the oligo- and
polydeoxynucleotides to animals infected by a virus, the inventor has
succeeded in providing an anti-viral agent capable of effectively
inhibiting the propagation of virus in vivo. The present invention has
been accomplished on the basis of the above finding.
As described previously, the hybridization of viral or globin RNA to the
corresponding DNA for inhibition of the synthesis of protein was known
from the references mentioned above. However, the majority of experiments
relating thereto was carried out in a cell-free system except Zamecnik's
second reference (Proc. Natl. Acad. Sci., U.S.A., 75, 280-284) and
Molecular Biosystem Inc. reference (PCT, Intl. Publn. No. WO83/01451)
wherein inhibition of the propagation of virus in an in vitro cell culture
system is described. In the Zamecnik's in vitro experiments, however, DNA
is selected from a region other than a specific coding region for the
virus. By the term "coding region" is meant herein regions of viral genes
which code for (determine the amino acid sequence of) viral proteins and
which undergo the transcription by an RNA polymerase to produce mRNA. On
the other hand, the Molecular Biosystem Inc. reference lacks disclosures
on concrete conditions for the experiments. The present inventor has
carried out extensive researches and experiments both in vitro and in vivo
systems using specific oligodeoxynucleotides or polydeoxynucleotides
selected from a specific coding region and made it possible for the first
time to develop the theory of inhibiting the propagation of virus on the
basis of the RNA-DNA hybridization technique to a practically utilizable
level of therapy, thereby enabling the use of the oligodeoxynucleotides or
polydeoxynucleotides as a medicine for inhibiting the propagation of
virus. Thus, the present invention is quite different from the known arts
in respect of the two concepts; the selection of DNA from a specific
coding region which is hybridizable to mRNA of virus and the use of a
single stranded deoxynucleotide having a short chain length as the DNA
selected from the specific coding region, and is also distinguished by
developing the above concepts to a practically utilizable level by way of
abundant in vivo experiments.
In accordance with the present invention, therefore, there is provided a
method for inhibiting the propagation of virus which comprises delivering
one or more oligodeoxynucleotides or polydeoxynucleotides to a place where
an mRNA generated on the propagation of virus exists, characterized in
that the oligodeoxynucleotides or polydeoxynucleotides are identical in
DNA sequence with a portion of the structure of DNA hybridizable to the
mRNA, the structure beihg capable of hybridizing to the mRNA.
In accordance with the present invention, there is also provided an
anti-viral agent which comprises an effective amount of an
oligodeoxynucleotide or polydeoxynucleotide with a conventional liquid
vehicle or excipient, characterized in that the oligodeoxynucleotide or
polydeoxynucleotide is identical in DNA sequence with a portion of the
structure of DNA hybridizable to an mRNA generated on the propagation of
virus, the structure being capable of hybridizing to the mRNA.
The inventor first discovered that, when a synthetic RNA as mRNA is
translated in an in vitro protein synthesis system, the protein synthesis
is inhibited by an oligodeoxynucleotide which hybridizes to the synthetic
RNA. (See Example 25 described below).
Oligodeoxynucleotides which do not hybridize to the RNA did not inhibit the
protein synthesis in this system.
The inventor secondly discovered that oligodeoxynucleotides of the
(-)-strand sequence of .alpha.-globin or .beta.-globin DNA inhibit the
translation of the corresponding mRNA. (See Example 26 described below).
As a result of the experiment, it was found that .alpha.-globin DNA
specifically inhibits the translation of .alpha.-globin mRNA in the in
vitro system. Furthermore, it was found that the .alpha.-globin DNA
inhibits specifically the translation of .beta.-globin mRNA in this
system. In addition, when DNA which does not hybridize to the
.alpha.-globin mRNA was used, the production of .alpha.-globin was not
inhibited. In a similar manner, the .beta.-globin production was not
inhibited by DNA which does not hybridize to .beta.-globin mRNA. It was
further found that not only DNA with a long chain but also
oligodeoxynucleotides have a strong inhibitory effect in this system, due
to hybridization.
On the basis of these experiments, further experiments concerning herpes
simplex virus (HSV) infection were performed. When DNA or
oligodeoxynucleotides which can hybridize to the immediate early mRNA of
HSV were given to cells infected by this virus, it was found that the
number of syncytia (a plaque like hole which was made by HSV due to the
fusion of infected cells) caused by HSV was remarkably reduced.
In the above experiments, CaCl.sub.2 and Ca phosphate were used together
with the DNA to stimulate penetration of the DNA into cells. In separate
experiments, however, it was found that the oligodeoxynucleotide can be
taken up even without these calcium salts, and thus inhibits the
propagation of HSV and its cytopathic effects. (See Example 7 described
below).
From these experiments, it became clear that the minus strand DNA, which
hybridizes to an mRNA of HSV-1 inhibits syncytium formation by this virus.
Based on this observation, the inventor further applied such DNA to mice
infected with HSV and demonstrated that such DNA can be used as an
anti-viral agent in vivo for the treatment of viral diseases.
Noteworthy here is that only the negative strand DNA specific for the mRNA
of that particular viral gene is used as an effective component in the
agent and method of the present invention. Therefore, the biosynthesis of
cellular protein programmed by other mRNA's should not be influenced by
using the DNA mentioned above.
There are two groups of viruses; DNA viruses (herpes simplex virus, adeno
virus, vaccinia virus, etc.) and RNA viruses (influenza virus, rhino
virus, and polio virus, etc.). RNA viruses either carry single stranded
RNA or double stranded RNA as a genome. Single stranded RNA viruses are
classified into those carrying plus stranded RNA and those carrying minus
stranded RNA as their genome.
Viral proteins always have to be synthesized by translation of a viral mRNA
which is a plus stranded RNA. Viruses with a double stranded DNA genome
produce mRNA (plus strand) from their minus stranded DNA strand. As to the
double stranded RNA viruses, the mRNA (plus stranded) is produced from the
minus stranded RNA. The genome of plus stranded RNA viruses itself
functions as a mRNA while the minus stranded RNA viruses use their RNA as
templates for the production of plus stranded RNA (mRNA).
According to this invention, the translation of a viral mRNA and thus the
synthesis of viral proteins is, independent from the mechanism of the
synthesis of mRNA, inhibited by a hybridizing minus strand DNA. Although
only examples wherein herpes simplex virus (double stranded DNA virus) and
influenza virus (single stranded RNA virus) were used are given herein,
the present invention can widely be applied for inhibiting in general the
propagation of viruses. Thus, propagation of viruses such as influenza
virus, adeno virus, leukemia virus, dengue virus, rabies virus, hepatitis
virus, measles virus, encephalitis virus, parainfluenza virus, rhino
virus, yellow fever virus and EB virus can be inhibited by the method and
the agent of this invention.
Thus, the present invention can widely be utilized for the prevention or
treatment of various diseases of animals and plants caused by viruses. The
respective pharmaceutical compositions are thus very useful in various
fields.
According to the method of the present invention, the propagation of RNA or
DNA viruses and their cytopathic effects can be stopped without
influencing host cells. Further, infection of uninfected host cells can be
prevented by the method of this invention. It was found by the inventor
that oligodeoxynucleotides alone hybridizing to the immediate early mRNA
of herpes simplex virus have neither effect on normal uninfected baby
hamster kidney cells (BHK cells) nor effect on mice. Therefore, it was
concluded that the oligodeoxynucleotides have neither harmful effect on
normal cells and animals nor influence on the growth rate of these cells
at all.
The negative stranded deoxynucleotides used in this invention may be
obtained by enzymatic hydrolysis of DNA, denaturation and subsequent
separation by affinity chromatography. Alternatively, synthetic negative
stranded oligodeoxynucleotides can be used. Thus, it is evident that the
negative stranded deoxynucleotides used in the experiments described
herein can be obtained by a variety of methods.
In the cloning of RNA virus gene, the reverse transcriptase may be used. In
some cases, viral DNA present in infected cells and originated from the
RNA virus genome can be used. As a cloning vehicle, for example, lambda
phage (Charon phage) or plasmid pBR 322 can be used as vector. For
obtaining a single stranded (-) DNA, M 13 phage will be used as vector.
According to the present invention, the cloned DNA of coding region of any
part of the total viral genomes may be used. However, early or immediate
early genes whose expression is observed in early stages of infection are
particularly desirable for the prevention of virus propagation and the
concomitant cytopathic effects.
A recombinant M 13 phage containing a negative stranded viral DNA or a
recombinant DNA vector containing the double stranded viral DNA can be
produced by growing large quantities of E. coli harboring these vectors or
E. coli infected with the M 13 phage. Vectors containing the viral genes
were isolated and subjected to restriction endonuclease for excision of
the viral genes. The mixture of the excised viral genes and the vector DNA
was then subjected to agarose gel electrophoresis or sephadex column
chromatography for separating the virus gene DNA from the vector DNA. The
DNA molecules thus obtained were shortened by partial digestion with DNA
restriction endonuclease or ultrasonic treatment. The chain length of the
viral DNA fragments was adjusted to anywhere between 9 and 100 nucleotides
by isolating these DNA strands by gel electrophoresis and sephadex
chromatography.
It is to be noted that only the negative strand of viral DNA is effective
for the inhibition of the viral protein synthesis. When the DNA of vectors
are such as pBR 322 or lambda phage as the replicating form of M 13 phage
it is a double stranded DNA. Therefore, this double stranded DNA has to be
converted to a single stranded DNA followed by isolation of negative
stranded DNA. For this purpose, various methods can be used. For example,
DNA is denatured by heating to 100.degree. C. for 10 minutes and rapidly
cooled.
From a mixture of the separated negative and positive strand DNA, the
negative stranded DNA may be isolated, for example, by affinity
chromatography. For this purpose, the positive stranded DNA is first
prepared by cloning into M 13 phage. This is then conjugated to a support
material for chromatographic use. A mixture of negative and positive
strand DNA is then passed through the conjugated positive DNA column. The
negative strand DNA is bound to this column while the positive strand DNA
passes through the column. The bound negative strand DNA is then eluted
from the column.
In an alternative method for obtaining the negative strand DNA, it is
synthesized chemically in the form of oligo- or polydeoxynucleotides by a
proper synthetic method in organic chemistry.
In case of the herpes simplex virus, for example, one of the immediate
early DNA genes, Vmw 175, has a double strand consisting of a plus DNA
(5'-ATG.GCG.TCG.GAG . . . ) and a minus DNA (3'-TAC.CGC.AGC.CTC . . . ).
Only the minus DNA having a sequence beginning with TAC can hybridize to
an mRNA of Vmw 175. Thus, any of the DNA which is identical in a partial
structure with the minus DNA strand has to be selected and synthesized as
an oligodeoxynucleotide. The influenza virus genome includes the
hemagglutinin gene and NS (non-structural) genes. Since the hemagglutinin
gene of different serotypes of influenza virus vary in their RNA sequence,
the sequence of a single stranded DNA which hybridizes to the mRNA of the
NS genes is preferred for the purpose of the present invention. (-)Strand
DNA hybridizing to the mRNA from the NS genes is more desirable than that
hybridizing to the mRNA from the hemagglutinin gene because the
hemagglutinin gene mutates very frequently and the (-) strand DNA
hybridizing to the mRNA from the hemagglutinin gene of a single strain of
influenza virus may not crosshybridize to the mRNA from the hemagglutinin
gene of another serotype strain of influenza virus.
Further explanation for the NS-1 gene of influenza virus is given, for
example, by Lamb et al., [Cell 21 47 (1980)]. The DNA sequence which
hybridizes to an mRNA from NS-1 gene is 5'- . . .
ACT.TGA.CAC.AGT.GTT.GGA.ATC.CAT . . . -3'. Therefore, the whole or a part
of this sequence may be selected and synthesized.
The present invention can also be applied to viruses related to in the
field of poultry and livestock industries, such as Rous Sarcoma Virus. For
example, the inhibition of expression of one of the genes gag., pol. and
env. of this virus should inhibit the propagation of this virus. If, for
example, the inhibition of the expression of the gag. gene by a
hybridizing DNA molecule is desired, an oligo- or polydeoxynucleotide
having a part of the sequence 5'- . . . AAT.CAT.CTT.TAT.GAC.GGC.TTC . . .
-3' can be selected and synthesized, taking into consideration the RNA
sequence of P 19 which is a part of the gag. gene.
It is evident from the foregoing explanations, that in principle, for the
inhibition of virus propagation by a single stranded minus strand DNA,
effective single stranded DNA should be selected, which hybridizes to an
mRNA of this virus.
In general, the term "oligodeoxynucleotide" stands for a molecule having a
smaller number of deoxynucleotide units while the term
"polydeoxynucleotide" for a molecule having a larger number or
deoxynucleotide units. In this invention, for example, the number of
nucleotide units of the oligodeoxynucleotides is usually up to 25. Thus,
deoxynucleotides having more than 25 deoxynucleotide units may be
designated as "polydeoxynucleotides" in this invention.
The anti-viral agents of the present invention containing the oligo- and/or
polydeoxynucleotide with a suitable liquid vehicle or excipient and
optionally an auxiliary additive or additives are prepared in the form of
liquid preparations, injections, suppositories, etc. according to known
methods. On prescription, the desired oligodeoxynucleotides and
polydeoxynucleotides can be used singly or in combination with other
pharmacologically active ingredients. In this case, it is obvious that
these other ingredients should not adversely affect the antiviral activity
of the oligo- and polydeoxy-nucleotides.
The oligo- and polydeoxynucleotides can be processed, if desired, to
externally applicable preparations such as cream, ointment, liquid and
plaster by mixing the oligo- and polydeoxynucleotide as the active
ingredient with a suitable inert vehicle or excipient.
The liquid vehicles or excipients as well as the optional auxiliary
additives used in the anti-viral agents of the present invention are
conventional ones and commercially available.
Illustrative of the liquid vehicles, excipients and auxiliary additives
useful for the preparation of the anti-viral agents are, for example,
distilled water for injection use, physiological saline, an aqueous
solution of dextrose, vegetable oils for injection use, propylene glycol,
polyethylene glycol and benzyl alcohol (for injections and liquid
preparations); vaselin, vegetable oil, animal fat and polyethylene glycol
(for externally applicable preparations); and isotonic agents, dissolution
promoting agents, stabilizers, colorants, antiseptic agents, soothing
agents and the like additives (as usual auxiliary additives to
pharmaceutical preparations). In case of the injection preparations, the
active ingredient may desirably be dissolved in sterilized liquid
vehicles. It is evident that the above mentioned various liquid vehicles,
excipients and auxiliary additives should not inhibit nor disturb the
anti-viral action of the oligo- and polydeoxy-nucleotides.
The oligo- and polydeoxynucleotides of the present invention are processed
with the vehicles or excipients referred to above and optionally together
with auxiliary additives referred to above according to known methods.
Injection preparations and suppositories may usually contain 1-10 mg of
the oligo- and polydeoxynucleotides per ampoule or capsule. For human
patients, the daily dose is about 0.1-1,000 mg, preferably 1-100 mg (from
10-20 .mu.g/kg to 1000-2000 .mu.g/kg body weight). However, the particular
dose for each patient depends on a wide range of factors, for example, on
the effectiveness of the particular oligo- or polydeoxynucleotide used, on
the age, weight, general state of health, sex, on the diet, on the time
and mode of administration, on the rate of elimination, combination with
other medicaments jointly used and severity of the particular diseases to
which the therapy is applied.
The anti-viral agents of the present invention are administered
intravenously or applied directly to the part of the body of a patient
infected by virus so that the oligo- or polydeoxynucleotide may
effectively be delivered to the infected part.
The present invention will now be illustrated in more detail by the
following examples and referential examples.
Examples 1-5 relate to the preparation of the oligo- and
polydeoxynucleotides used in the present invention.
EXAMPLE 1
Herpes simplex virus DNA was isolated by phenol-chloroform extraction. The
isolated DNA was digested by the restriction enzyme BamHI.
The digested DNA was then mixed in the presence of T 4 phage ligase with
pBR 322 plasmid DNA previously treated with BamHI.
The resulting mixture containing ligated plasmids was given to E. coli and
the bacteria which contains the recombinant plasmid was selected by
incubating them on agar plates containing ampicillin to form colonies and
screening them for tetracycline sensitivity. Among the tetracycline
sensitive colonies, those bacteria having the herpes simplex virus DNA
were selected by the filter hybridization method with .sup.32 P labelled
herpes viral DNA as a probe. To obtain the recombinant plasmid DNA,
2.times.10.sup.6 -10.sup.7 E. coli cells containing the recombinant
plasmid were placed in 2 ml TSB (Tryptose Soy Broth).
The inoculated medium was shaken in a 20 ml test tube at 37.degree. C. for
about 15 hours until the optical absorption (Optical density: OD) of the
culture medium at 540 nm reached 0.5 (OD.sub.540 =0.5). At this point
chloramphenicol was added to a final concentration of 100 .mu.g/ml and
incubation was continued for 15 hours.
The bacteria suspension was then centrifuged and the pellet was resuspended
in a buffer containing 25% sucrose and 50 nM tris HCl (pH 8.0). The
suspension was left standing at 0.degree. C. for 5 minutes. To this
suspension, 4 ml of Triton X 100 was added and the resulting viscous
solution was centrifuged for 30 minutes at 0.degree. C. at 30,000 g. To
the supernatant of this solution (8 ml), 8 g of CsCl, 1 ml of a solution
of ethidium bromide (5 .mu.g ethidium bromide/ml) were added. The mixture
was further centrifuged for 48 hours at 100,000 g. The crude plasmid
fraction obtained after this centrifugation was mixed with 2 volumes of
isopropyl alcohol, and left standing for a few minutes. The upper layer
was removed and the solution containing DNA was dialized against 0.1M tris
HCl and 10 mM EDTA (pH 8.0) overnight.
The dialized solution was concentrated and the solution containing 200
.mu.g DNA was treated with an excess amount of restriction enzyme (BamHI).
The DNA digest was separated by agarose electrophoresis and the DNA of
herpes simplex virus was isolated from the gel by the electroelution
method. The DNA was further digested partially by DNase and heated to
100.degree. C. for 10 minutes followed by rapid cooling. Thus, DNA having
chain lengths of 9 to 100 oligodeoxynucleotides were obtained.
EXAMPLE 2
The replicative form of M 13 phage DNA (double stranded DNA) was cut with
BamHI. This digested M 13 DNA was mixed with herpes simplex virus DNA
which had been digested by BamHI and isolated as described above. They
were ligated with T 4 phage ligase. E. coli were transfected with the
ligated DNA in the presence of CaCl.sub.2 The transfected bacteria were
plated on agar plates using a top agar which contained IPTG
(isopropyl-.beta.-D-thio-galactopyranoside) an X-gal (an indicator).
Recombinant M 13 phages containing herpes simplex virus DNA form colorless
phage plaques within 24 hours. M 13 phages without insertion form blue
plaques. Therefore, the colorless phage plaques were chosen. To select
recombinant M 13 phage which contains (-) strand HSV DNA, phages were
checked for their capacity to hybridize with labelled (+) HSV DNA which is
chemically synthesized. In this manner were selected M 13 phages
containing a part of the (-) strand of HSV DNA were selected. The desired
recombinant M13 phage was increased in quantity and a single stranded DNA
was isolated from this phage. For further purification of the single
stranded HSV DNA, M 13 phage DNA can be removed from this mixture by
restriction enzyme digestion in the presence of certain
oligodeoxynucleotides. Alternatively the DNA mixture, without purification
can be partially digested with DNase and oligodeoxynucleotides having
chain lengths between 9 and 100 can be isolated.
EXAMPLE 3
A DNA synthesizer (Applied Biosystem Company) was used to synthesize minus
strand DNA of the immediate-early gene (Vmw 175 or ICP 4) of herpes
simplex virus. In this example, the DNA sequence
3'-CGC.AGC.CTC.TTG.TTC.GTC.GC-5' was synthesized which hybridizes with
mRNA of Vmw 175. This deoxynucleotide is eicosamer and is referred to
hereinafter simply as the 20 A.
EXAMPLE 4
A DNA synthesizer (Applied Biosystem Company) was used to synthesize minus
strand DNA of the immediate-early gene (Vmw 12) of herpes simplex virus.
In this example, the DNA sequence 3'-GCA.CCC.GGG.ACC.TTT.ACC.GC-5' was
synthesized which hybridizes with mRNA of Vmw 12. This deoxynucleotide is
eicosamer and is referred to hereinafter simply as the 20 B.
EXAMPLE 5
A DNA synthesizer (Applied Biosystem Company) was used to synthesize minus
strand DNA of the NS 1 gene of influenza virus. In this example, the DNA
sequence 3'-CTA.AGT.TTG.TGA.CAC.AGT.TCA-5' was synthesized which
hybridizes with mRNA of NS 1. This deoxynucleotide is heneicosamer and is
referred to hereinafter simply as the 20 C.
EXAMPLE 6
This example relates to an experiment by which it is confirmed that the
oligodeoxynucleotide inhibits the synthesis of virus protein in vitro.
Table 1 illustrates the inhibitory effect of the 20 A on Vmw 175 formation
and the corresponding cytopathic effect (Syncytium Formation) of HSV 1.
TABLE 1
______________________________________
Relative amounts
Relative Cytopathic
The 20 A/well
of Vmw 175 effects (Syncytium)
______________________________________
-- 1 1
33.6 .mu.g 0.48 0.35
______________________________________
BHK cells (1.5.times.10.sup.5) were plated in each well (1.5 cm in
diameter) of a Limbro dish and MEM with 10% calf serum was added. The
cells were then incubated in a 5% CO.sub.2 atmosphere at 36.degree. C.
Subsequently, the cells were infected with 5 moi of HSV-1 in the presence
or absence of the 20 A. After 5 hours, the medium was removed. The cells
were then contacted for 1 hour with .sup.35 S-methionine (100
.mu.Ci/.mu.l) and the 20 A disrupted, and proteins were analyzed by
polyacrylamide gel electrophoresis followed by autoradiography. For
evaluation of the relative cytopathic effect, experimental conditions were
identical to the above except that the numbe of syncytia was counted 13
hours after the infection. No decrease of cellular protein synthesis was
observed under these conditions.
EXAMPLE 7
This example relates to an experiment by which it is confirmed that the
oligodeoxynucleotide is taken up by cells.
This experiment indicates that the single stranded oligodeoxynucleotide 20
A is indeed permeable into cultured cells. BHK cells (baby hamster kidney
cells; 4.4.times.10.sup.5 cells) were infected with 7.6.times.10.sup.4 PFU
of HSV-1/ml. Control cells were not infected but were given the same
amount of the culture medium. The cells were incubated for 3 hours in a
CO.sub.2 incubator at 37.degree. C. After the incubation, the cells were
exposed to 7.66 p moles of the .sup.32 P labelled 20 A. After the exposure
to the labelled 20 A, the culture medium was removed and the cells were
washed twice with 0.3 ml of PBS.
The cells were then left for 30 minutes at 36.degree. C. in a solution
containing 0.1 M NaCl, 33 .mu.M ZnCl.sub.2, 3360 units of nuclease S1 and
33.3 mM sodium acetate buffer (pH 4.5).
Subsequently, the cells were washed twice with the buffer described above
but without nuclease S1. The washed cells were disrupted in 0.3 ml of a
solution containing 10 mM EDTA, 0.6 % sodium dodecyl sulfate, and 10 mM
tris-HCl buffer (pH 7.5). At this point, TCA-insoluble, nuclease
S1-sensitive, radioactive 20 A was measured. It was found that at least
4.68.times.10.sup.3 molecules of the 20 A had been taken up per cell
during the exposure period of 8 hours. The actual amount is probably 10
times higher than this value because of the rapid hydrolysis of .sup.32 P
terminal phosphates.
The following Example 8 illustrates that the 20 A has an inhibitory effect
on the propagation of virus.
EXAMPLE 8
Approximately 1.5.times.10.sup.5 BHK cells were plated per each well (1.5
cm in diameter) of a Limbro dish. These cells were incubated in 5%
CO.sub.2 at 36.degree. C. for 48 hours and infected with HSV-1
(7.6.times.10.sup.4 PFU/ml) in 160 .mu.l of MEM. The infection was carried
out for 3 hours at 36.degree. C. in 5% CO.sub.2. After the infection the
20 A was applied to the infected cells. After 8 hours exposure, the medium
was changed to normal MEM with 10% calf serum. At 15 hours after the
infection, the culture fluid was harvested and virus titer was measured.
The results are shown in Table 2 below.
TABLE 2
______________________________________
Amount of the 20 A/well
Relative amount of virus
______________________________________
0 100
0.112 .mu.g 45
0.224 .mu.g 35
______________________________________
Examples 9-13 relate to experiments by which it is confirmed that the
oligodeoxynucleotide inhibits destruction of animal cells by virus in
vitro.
EXAMPLE9
(a) Approximately 1.5.times.10.sup.5 BHK cells were placed in each well
(1.5 cm in diameter) of a Limbro dish for cell culture. The cells were
incubated for 47 hours at 36.degree. C. in 5% CO.sub.2 and then infected
with 160 .mu.l of HSV-1 (7.6.times.10.sup.4 PFU/ml) for 3 hours at
36.degree. C. To these infected cells, a complex of calcium phosphate and
the 20 A in various amount was added. Ca.sup.++ concentration of the media
varied from 1.36 mM to 24.8 mM depending on the concentration of the 20 A.
The complex was prepared as described by Ruddle's group (Loiter et al,
Proc. Nat. Acad. Sci. 79 422, 1982). The infected cells were exposed to
the 20 A for 8 hours. The culture medium was then removed and replaced
with a normal culture medium without the 20 A and the cells were grown for
additional 13 hours.
(b) The control cells were treated identically except for the addition of
the 20 A (Ca-phosphate control).
(c) As an additional control, an infected culture was prepared which was
treated with neither Ca-phosphate nor the 20 A (virus control). The tissue
culture cells prepared above (a), (b), (c), were fixed, stained and the
number of syncytia was counted.
The result of this experiment is shown in FIG. 1, wherein the axis X
(abscissa) stands for the amount of DNA in terms of .mu.g added to each
well (i.e. ".mu.g DNA/well") and the axis Y (ordinate) for the number of
syncytia in terms of percentage (i.e. "% number of syncytia") formed in a
cell culture as compared with the value obtained in the case of the
experiment (c) for "virus control", the value in this case being set
always as 100 and wherein a plot drawn by a solid line (a) ( ) shows the
result obtained with the experiment (a) while a plot drawn by a dotted
line (b) ( ) shows the result obtained with the experiment (b). As shown
in FIG. 1, the syncytium formation was remarkably inhibited by the 20 A.
Calcium phosphate or calcium chloride did not influence the number of the
syncytia.
EXAMPLE 10
(a) Approximately 1.5.times.10.sup.5 BHK cells were placed per well (1.5 cm
in diameter) of a Limbro dish. The cells were incubated at 36.degree. C.
for 47 hours under 5% CO.sub.2. To these cells were added 160 .mu.l of
HSV-1 (7.6.times.10.sup.4 PFU/ml) and infections were allowed for 3 hours
at 36.degree. C. To these infected cells various amounts of the 20 A were
added in the form of a calcium chloride complex in the final Ca.sup.++
concentration of 4.55 mM. The treatment was continued for 8 hours. The
cells were then further incubated for 14 hours.
(b) In addition, an identical culture as described above (a) was prepared
except that an oligodeoxynucleotide which has no relation with HSV-1
sequence 5'-CAC.GAC.AGA.GGG.CGA.-3' was prepared and incubated. All other
conditions were identical with the case of (a).
(c) A similar culture in which the 20 A was left out was prepared and
incubated under the same condition as above. This is referred to herein as
"virus control".
The cells described above in (a), (b) and (C) were fixed, stained and the
number of syncytia was counted. The results of this experiment are shown
in FIG. 2, wherein the axis X (abscissa) stands for the amount of DNA in
terms of .mu.g added to each well (i.e. ".mu.g DNA/well") and the axis Y
(ordinate) for the number of syncytia in terms of percentage (i.e. "%
number of syncytia") formed in a cell culture as compared with the value
obtained in the case of the experiment (c) for "virus control", the value
in this case being set always as 100 and wherein a plot drawn by a solid
line (a) ( ) shows the result obtained with the experiment (a) while a
plot drawn by a dotted line (b) ( ) shows the result obtained with the
experiment (b).
As shown in FIG. 2, the 20 A has an inhibitory effect on the number of
syncytia produced by HSV-1. From this experiment, it can be concluded that
the 20 A inhibits the syncytium formation in the presence of 4.55 mM
calcium ion. Oligodeoxynucleotides which do not have any relation with the
known DNA sequences of herpes simplex virus did not have any inhibitory
effect. It became clear from this experiment that the minus strand of
herpes simplex virus DNA specifically inhibits the cytopathic effect of
herpes simplex virus.
EXAMPLE 11
In Example 9 the effect achieved by using the synthetic eicosamer 20 A
related to the minus strand of HSV-DNA is illustrated. This experiment
relates to effectiveness of cloned herpes simplex DNA. Using the pBR 322
vector containing HSV-1 DNA (1.45 megadalton, obtained by digestion of
HSV-1 DNA with BamHI), it was possible to inhibit syncytium formation by
HSV-1. In this experiment, HSV-1 DNA was not excised from the recombinant
plasmid. The denatured and partially digested recombinant pBR 322
containing HSV-1 DNA (0.16 .mu.g) was placed in each well of the Limbro
dish as in the Example 9. The effect was examined as described in Example
9 and an inhibition of syncytium formation was observed. As a control for
this experiment, DNA of pBR 322 alone was used. In this control
experiment, no inhibition was found.
EXAMPLE 12
This example illustrates a synergistic effect of a mixture of the
oligodeoxynucleotides.
Approximately 1.5.times.10.sup.5 BHK cells were plated in each well (1.5 cm
in diameter) of a Limbro dish. The cells were incubated in 5% CO.sub.2 at
36.degree. C. for 47 to 48 hours and infected with HSV-1
(7.6.times.10.sup.4 PFU/ml) in 160 .mu.l of MEM. Infection was carried out
for 3 hours at 36.degree. C. in 5% CO.sub.2. After the infection, the 20 A
alone or in mixture with the 20 B (224:1) was dissolved in a solvent and
0.3 ml of this solution was applied to the cells in the presence of 4.55
mM calcium ions. The exposure of these cells to the 20 A and the 20 B was
carried out for 8 hours.
On the other hand, the cells were allowed to receive the identical
treatments to those described above except for the absence of the 20 A and
the 20 B. This is referred to herein as virus control.
The cells were fixed, stained and the number of syncytia was counted. The
results of these experiments are shown in FIG. 3 wherein the axis X
(abscissa) stands for the amount of DNA in terms of .mu.g added to each
well (i.e. ".mu.g DNA/well") and the axis Y (ordinate) for the number of
syncytia in terms of percentage (i.e. "% number of syncytia") formed in a
cell culture as compared with the value obtained in the case of "virus
control", the value in this case being set always as 100 and wherein a
plot drawn by a solid line designated as "20 A" ( ) shows the result
obtained with the experiment using the 20 A alone while a plot drawn by a
solid line designated as "20 A+20 B (224:1)" ( ) shows the result
obtained with the experiment using a combination of the 20 A and the 20 B
in the ratio of 224:1. As shown in FIG. 3, when the mixture of the 20 A
and the 20 B was used, the observed in vitro effect was stronger as
compared with the case of using the 20 A alone. From this result, it is
concluded that the conjoint use of minus stranded DNA corresponding to a
portion having different DNA sequence of the minus stranded D | | |
