U.S. patent number 5,594,122 [Application Number 08/308,869] was granted by the patent office on 1997-01-14 for antisense oligonucleotides targeted against human immunodeficiency virus.
This patent grant is currently assigned to Genesys Pharma Inc.. Invention is credited to Albert D. Friesen.
United States Patent |
5,594,122 |
Friesen |
January 14, 1997 |
Antisense oligonucleotides targeted against human immunodeficiency
virus
Abstract
The present invention is directed to oligonucleotides comprising
nucleotide sequences sufficiently complementary to conserved
regions of human immunodeficiency virus genetic material such that
when bound to said region, the oligonucleotides effectively prevent
expression of the genetic material.
Inventors: |
Friesen; Albert D. (Winnipeg,
CA) |
Assignee: |
Genesys Pharma Inc. (Vancouver,
CA)
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Family
ID: |
22165010 |
Appl.
No.: |
08/308,869 |
Filed: |
September 19, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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81572 |
Jun 23, 1993 |
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Current U.S.
Class: |
536/24.5;
536/23.1; 435/91.1; 435/6.12 |
Current CPC
Class: |
C12N
15/1132 (20130101); A61P 31/12 (20180101); C12N
2310/315 (20130101) |
Current International
Class: |
C12N
15/11 (20060101); C07H 021/04 (); C12Q 001/68 ();
A61K 048/00 () |
Field of
Search: |
;435/6,172.3,320.1,91.1
;536/24.5 ;514/44 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0375408 |
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Jun 1990 |
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EP |
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0386563 |
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Sep 1990 |
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EP |
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WO89/08146 |
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Sep 1989 |
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WO |
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WO90/06934 |
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Jun 1990 |
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WO |
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WO92/02531 |
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Feb 1992 |
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WO |
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WO92/10590 |
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Jun 1992 |
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WO |
|
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Primary Examiner: Leguyader; John L.
Attorney, Agent or Firm: Morrison & Foerster
Parent Case Text
This application is a continuation of application Ser. No.
08/081,572, filed Jun. 23, 1993, now abandoned.
Claims
I claim:
1. An oligonucleotide having the nucleotide sequence complementary
to the human immunodeficiency virus nucleic acid sequence +1189 to
+1208.
2. A composition comprising a an oligonucleotide according to claim
1 and a physiologically acceptable carrier therefor.
3. The composition according to claim 2 further comprising a
physiologically effective amount of a liposome preparation.
4. The composition according to claim 3 wherein the liposome
preparation comprises lipofectin.
5. An oligonucleotide having the nucleotide sequence:
6. A composition comprising an oligonucleotide according to claim 5
and a physiologically acceptable carrier therefor.
7. The composition according to claim 6 further comprising a
physiologically effective amount of a liposome preparation.
8. The composition according to claim 7 wherein the liposome
preparation comprises lipofectin.
9. An oligonucleotide having the nucleotide sequence:
wherein s stands for a sulfur atom.
10. A composition comprising an oligonucleotide according to claim
9 and a physiologically acceptable carrier therefor.
11. The composition according to claim 10 further comprising a
liposome preparation.
12. The composition according to claim 11 wherein the liposome
preparation comprises lipofectin.
Description
FIELD OF INVENTION
This invention relates to oligonucleotide (ODN) based therapeutics,
particularly the treatment of infections of the human
immunodeficiency virus (HIV).
BACKGROUND OF THE INVENTION
The present invention relates to ODNs suitable for use in treatment
of HIV infected individuals by inhibition of replication of HIV in
infected cells.
HIV is responsible for the disease that has come to be known as
acquired immunodeficiency syndrome (AIDS). Although initially
recognized in 1981, no cure has yet been found for this inevitably
fatal disease. HIV is spread by a variety of means such as sexual
contact, infected blood or blood products and perinatally. Because
of the complexity of HIV infection and the paucity of effective
therapies, a great deal of effort has been expended in developing
methods for detecting, treating and preventing infection.
Diagnostic procedures have been developed for identifying infected
persons, blood and other biological products.
The HIV genome has been well characterized. Its approximately 10 kb
encode sequences containing regulatory segments for HIV replication
as well as the gag, pol and env genes coding for the core proteins,
the reverse transcriptase-protease-endonuclease, and the internal
and external envelope glycoproteins, respectively. HIV tends to
mutate at a high rate causing great genetic variation between
strains of the viruses and indeed between virus particles of a
single infected individual. There are a few "conserved" regions of
the HIV genome which tend not to mutate. These regions are presumed
to encode portions of proteins essential for virus function which
can thus withstand very few mutational events.
The HIV env gene encodes the glycoprotein, gp160, which is normally
processed by proteolytic cleavage to form gp120, the external viral
glycoprotein, and gp41, the viral transmembrane glycoprotein. The
gp120 remains associated with HIV virions by virtue of noncovalent
interactions with gp41. These noncovalent interactions are weak,
consequently most of the gp120 is released from cells and virions
in a soluble form.
Like most viruses, HIV often elicits the production of neutralizing
antibodies. Unlike many other viruses and other infectious agents
for which infection leads to protective immunity, however, HIV
specific antibodies are insufficient to halt the progression of the
disease. Therefore, in the case of HIV, a vaccine that elicits the
immunity of natural infection could prove to be ineffective. In
fact, vaccines prepared from the HIV protein gp160 appear to
provide little immunity to HIV infection although they elicit
neutralizing antibodies. The failure to produce an effective
anti-HIV vaccine has led to the prediction that an effective
vaccine will not be available until the end of the 1990's.
Therapeutic agents currently used in treatment of AIDS often cause
severe side-effects which preclude their use in many patients. It
would, thus, be useful to have alternative methods of treating and
preventing the disease that do not entail vaccination and currently
available pharmaceutical agents.
Recently, attempts have been made to moderate protein production
associated with viral infections by interfering with the mRNA
molecules that direct their synthesis. By interfering with the
production of proteins, it has been hoped to effect therapeutic
results with maximum effect and minimal side effects. It is the
general object of such a therapeutic approach to interfere with or
otherwise modulate gene expression leading to undesired protein
formation.
One method for inhibiting specific gene expression which is
believed to have promise is the "antisense" approach.
Single-stranded nucleic acid, primarily RNA, is the target molecule
for ODNs that are used to inhibit gene expression by an antisense
mechanism. A number of workers have reported such attempts: Stein
and Cohen (1988) Cancer Res., 48:2659-2668; Walder (1988) Genes
& Development, 2:502-504; Marcus-Sekura (1988) Anal. Biochem.,
172:289-295; Zon (1987) J. Pro. Chem., 6:131-145; Zon (1988) Pharm.
Res., 5:539-549; Van der Krol et al. (1988) Biotechniques,
6:958-973; and Loose-Mitchell (1988) TIPS, 9:45-47. Antisense ODNs
are postulated to exert an effect on target gene expression by
hybridizing with a complementary RNA sequence. The hybrid RNA-ODN
duplex appears to interfere with one or more aspects of RNA
metabolism including processing, translation and metabolic
turnover. Chemically modified ODNs have been used to enhance
nuclease stability and cell permeability.
Duplex DNA can be specifically recognized by oligomers based on a
recognizable nucleomonomer sequence. The motif termed "GT"
recognition has been described by Beal et al. (1992) Science,
251:1360-1363; Cooney et al. (1988) Science, 241:456-459; and Hogan
et al., EP Publication 375408. In the G-T motif, the ODN is
oriented antiparallel to the target purine-rich sequence and A-T
pairs are recognized by adenine or thymine residues and G-C pairs
by guanine residues.
Sequence-specific targeting of both single-stranded and duplex
target sequences has applications in diagnosis, analysis, and
therapy. Under some circumstances wherein such binding is to be
effected, it is advantageous to stabilize the resulting duplex or
triplex over long time periods.
Covalent crosslinking of the oligomer to the target provides one
approach to prolong stabilization. Sequence-specific recognition of
single-stranded DNA accompanied by covalent crosslinking has been
reported by several groups. For example, Vlassov et al. (1986) Nuc.
Acids Res., 14:4065-4076, describe covalent bonding of a
single-stranded DNA fragment with alkylating derivatives of
nucleomonomers complementary to target sequences. A report of
similar work by the same group is that by Knorre et al. (1985)
Biochimie, 67:785-789. It has also been shown that
sequence-specific cleavage of single-stranded DNA can be mediated
by incorporation of a modified nucleomonomer which is capable of
activating cleavage. Iverson and Dervan (1987) J. Am. Chem. Soc.,
109:1241-1243. Covalent crosslinking to a target nucleomonomer has
also been effected using an alkylating agent complementary to the
single-stranded target nucleomonomer sequence. Meyer et al. (1989)
J. Am. Chem. Soc., 111:8517-8519. Photoactivated crosslinking to
single-stranded ODNs mediated by psoralen has been disclosed. Lee
et al. (1988) Biochem., 27:3197-3203. Use of crosslinking in
triple-helix forming probes has also been disclosed. Horne et al.
(1990) J. Am. Chem. Soc., 112:2435-2437.
Use of N.sup.4,N.sup.4 -ethanocytosine as an alkylating agent to
crosslink to single-stranded and double-stranded oligomers has also
been described. Webb and Matteucci (1986) J. Am. Chem. Soc.,
108:2764-2765; (1986) Nuc. Acids Res., 14:7661-7674; and Shaw et
al. (1991) J. Am. Chem. Soc., 113:7765-7766. These papers also
describe the synthesis of ODNs containing derivatized cytosine. The
synthesis of oligomers containing N.sup.6,N.sup.6 -ethanoadenine
and the crosslinking properties of this residue in the context of
an ODN binding to a single-stranded DNA has been described.
Matteucci and Webb (1987) Tetrahedron Letters, 28:2469-2472.
In a recent paper, sequence-specific binding of an octathymidylate
conjugated to a photoactivatable crosslinking agent to both
single-stranded and double-stranded DNA is described. Praseuth et
al. (1988) Proc. Natl. Acad. Sci. (USA), 85:1349-1353. In addition,
targeting duplex DNA with an alkylating agent linked through a
5'-phosphate of an ODN has been described. Vlassov et al. (1988)
Gene 313-322; and Fedorova et al. (1988) FEBS Lett.,
228:273-276.
In effecting binding to obtain a triplex, to provide for instances
wherein purine residues are concentrated on one chain of the target
and then on the opposite chain, oligomers of inverted polarity can
be provided. By "inverted polarity" is meant that the oligomer
contains tandem sequences which have opposite polarity, i.e., one
having polarity 5'.fwdarw.3' followed by another with polarity
3'.fwdarw.5' or vice versa. This implies that these sequences are
joined by linkages which can be thought of as effectively a 3'--3'
internucleoside junction (however the linkage is accomplished), or
effectively a 5'--5' internucleoside junction. Such oligomers have
been suggested as by-products of reactions to obtain cyclic ODNs.
Capobionco et al. (1990) Nuc. Acids Res., 18:2661-2669.
Compositions of "parallel-stranded DNA" designed to form hairpins
secured with AT linkages using either a 3'--3' inversion or a
5'--5' inversion have been synthesized. van de Sande et al. (1988)
Science, 241:551-557. In addition, triple helix formation using
oligomers which contain 3'--3' linkages have been described. Horne
and Dervan (1990) J. Am. Chem. Soc., 112:2435-2437; and Froehler et
al. (1992) Biochem., 31:1603-1609.
The use of triple helix (or triplex) complexes as a means for
inhibition of the expression of target gene expression has been
previously adduced (International Application No. PCT/US89/05769).
Triple helix structures have been shown to interfere with target
gene expression (International Application No. PCT/US91/09321; and
Young et al. (1991) Proc. Natl. Acad. Sci., 88:10023-10026),
demonstrating the feasibility of this approach.
Various modifications have been found to be suitable for use in
ODNs. Oligomers containing 5-propynyl modified pyrimidines have
been described. Froehler et al. (1992) Tetrahedron Letters,
33:5307-5310. 2'-Deoxy-7-deazaadenosine and
2'-deoxy-7-deazaguanosine have been incorporated into ODNs and
assessed for binding to the complementary DNA sequences. Thermal
denaturation analysis (Tm) has shown that these substitutions
modestly decrease the Tm of the duplex when these analogs are
substituted for 2'-deoxyadenosine and 2'-deoxyguanosine. Seela and
Kehne (1987) Biochem., 26:2232-2238; and Seela and Driller (1986)
Nuc. Acids Res., 14:2319-2332. It has also been shown that ODNs
which alternate 2'-deoxy-7-deaza-adenosine and -thymidine can have
a slightly enhanced duplex Tm over ODNs containing
2'-deoxy-adenosine and -thymidine. Seela and Kehne (1985) Biochem.,
24:7556-7561.
2',3'-dideoxydeazapurine nucleosides have been used as chain
terminators for DNA sequencing. 7-propargyl amino linkers are used
for incorporation of fluorescent dyes into the nucleoside
triphosphates
DNA synthesis via amidite and hydrogen phosphonate chemistries has
been described. U.S. Pat. Nos. 4,725,677; 4,415,732; 4,458,066; and
4,959,463.
Prior attempts at antisense inhibition of HIV have focused on
inhibition of the synthesis of some particular viral protein
thought to be essential to the success of the infection and to RNAs
which are believed to have important biological function. It has
now been found that inhibition of viral gene expression and
replication can be more efficiently achieved by targeting the
conserved sites of the viral RNAs that signal the synthesis of
conserved HIV proteins, particularly the p24 core antigen
protein.
SUMMARY OF THE INVENTION
The present invention is directed to ODNs comprising nucleotide
sequences sufficiently complementary to conserved regions of human
immunodeficiency virus genetic material such that when bound to
said region, the ODNs effectively prevent expression of the genetic
material.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an autoradiograph of a SDS-PAGE showing in vivo synthesis
of HIV proteins and their breakdown products. FIG. 1 is described
in Example 3.
FIG. 2 is an autoradiograph of a SDS-PAGE showing significant
inhibition of expression of the HIV proteins by an antisense ODN
directed against the rev sequence. FIG. 2 is described in Example
4.
FIG. 3 is an autoradiograph of a SDS-PAGE showing significant
inhibition of expression of HIV proteins by antisense ODN directed
against the first splice donor site of the HIV-1 genome. FIG. 3 is
discussed in Example 5.
FIG. 4 is an autoradiograph of a SDS-PAGE showing the effects of
different concentrations of antisense ODNs on HIV-gene product
synthesis. FIG. 4 is discussed in Example 6.
FIG. 5 is an autoradiograph of a SDS-PAGE showing the concentration
dependent inhibitory effects of ODN GPI-2A on p24 expression in HIV
infected cells.
FIG. 6 is a bar graph showing the concentration dependent
inhibitory effects of ODN GPI-2A on p24 expression in HIV infected
cells.
DETAILED DESCRIPTION OF THE INVENTION
Several conserved sites within HIV RNA have now been found to be
effective targets for the inhibition of expression of viral gene
products by antisense ODNs and their analogues. The inhibition is
based on the capacity to block certain functions during viral
replication as measured by production of p24. The clinical
importance of p24, a cleavage product of p55, is evidenced by the
fact that serum levels of antibody to p24 antigen of HIV provide
evidence of the effectiveness of immune response to the virus as
well as serving as a marker of free virus in the serum of patients
with advanced stage AIDS. Goedert et al. (1989) N. Engl. J. Med.,
321:114.
According to the present invention, 20mer/15mer sequences were
designed and employed as anti-HIV chemotherapeutic agents. The
mechanism of action of antisense chemotherapeutics may be solely
due to binding to the mRNA or DNA so as to prevent translation or
transcription, respectively. The mechanism of action may also be
due to activation of RNase H and subsequent degradation of the
RNA.
The sequences are conserved in at least two different HIV isolates,
and, therefore the antisense ODNs are effective agents against a
wide variety of HIV strains.
The sequences were synthesized based on the phosphoramidite
chemistry of ODN synthesis on Applied Biosystems model 380D
automated DNA synthesizer. They were purified using ODN
purification cartridges and/or HPLC.
In therapeutic applications, the ODNs are utilized in a manner
appropriate for treatment of a variety of conditions by inhibiting
expression of the target genetic regions. For such therapy, the
ODNs, alone or in combination can be formulated for a variety of
modes of administration, including systemic, topical or localized
administration. Techniques and formulations generally can be found
in Remington's Pharmaceutical Sciences, Mack Publishing Co.,
Easton, Pa., latest edition. The ODN active ingredient is generally
combined with a pharmaceutically acceptable carrier such as a
diluent or excipient which can include fillers, extenders, binders,
wetting agents, disintegrants, surface-active agents, or
lubricants, depending on the nature of the mode of administration
and dosage forms. Typical dosage forms include tablets, powders,
liquid preparations including suspensions, emulsions and solutions,
granules, capsules and suppositories, as well as liquid
preparations for injections, including liposome preparations.
For systemic administration, injection is preferred, including
intramuscular, intravenous, intraperitoneal, and subcutaneous. For
injection, the ODNs of the invention are formulated in liquid
solutions, preferably in physiologically compatible buffers such as
Hank's solution or Ringer's solution. In addition, the ODNs can be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included. Dosages that can
be used for systemic administration preferably range from about
0.01 mg/Kg to 50 mg/Kg administered once or twice per day. However,
different dosing schedules can be utilized depending on (i) the
potency of an individual ODN at inhibiting the activity of its
target DNA or RNA, (ii) the severity or extent of the pathological
disease state, or (iii) the pharmacokinetic behavior of a given
ODN.
Systemic administration can also be by transmucosal or transdermal
means, or the compounds can be administered orally. For
transmucosal or transdermal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art, and include, for
example, bile salts and fusidic acid derivatives for transmucosal
administration. In addition, enhancers can be used to facilitate
permeation. Transmucosal administration can be through use of nasal
sprays, for example, or suppositories. For oral administration, the
ODNs are formulated into conventional oral administration forms
such as capsules, tablets, and tonics.
For topical administration, the ODNs of the invention are
formulated into ointments, salves, gels, or creams, as is generally
known in the art. Formulation of the invention oligomers for ocular
indications is based on standard compositions known in the art.
In addition to use in therapy, the ODNs of the invention can be
used as diagnostic reagents to detect the presence or absence of
the target nucleic acid sequences to which they specifically bind.
The enhanced binding affinity of the invention ODNs is an advantage
for their use as primers and probes. Diagnostic tests can be
conducted by hybridization through either double or triple helix
formation which is then detected by conventional means. For
example, the ODNs can be labeled using radioactive, fluorescent, or
chromogenic labels and the presence of label bound to solid support
detected. Alternatively, the presence of a double or triple helix
can be detected by antibodies which specifically recognize these
forms.
The use of ODNs containing the invention substitute linkages as
diagnostic agents by triple helix formation is advantageous since
triple helices form under mild conditions and the assays can thus
be carried out without subjecting test specimens to harsh
conditions. Diagnostic assays based on detection of RNA often
require isolation of RNA from samples or organisms grown in the
laboratory, which is laborious and time consuming, as RNA is
extremely sensitive to ubiquitous nucleases.
The ODN probes can also incorporate additional modifications such
as modified sugars and/or substitute linkages that render the ODN
especially nuclease stable, and would thus be useful for assays
conducted in the presence of cell or tissue extracts which normally
contain nuclease activity. ODNs containing terminal modifications
often retain their capacity to bind to complementary sequences
without loss of specificity. Uhlmann et al. (1990) Chem. Rev.,
90:543-584. As set forth above, the invention probes can also
contain linkers that permit specific binding to alternate DNA
strands by incorporating a linker that permits such binding.
Froehler et al. (1992) Biochem., 31:1603-1609; and Horne et al.
(1990) J. Am. Chem. Soc., 112:2435-2437.
Incorporation of base analogs into probes that also contain
covalent crosslinking agents has the potential to increase
sensitivity and reduce background in diagnostic or detection
assays. In addition, the use of crosslinking agents will permit
novel assay modifications such as (1) the use of the crosslink to
increase probe discrimination, (2) incorporation of a denaturing
wash step to reduce background and (3) carrying out hybridization
and crosslinking at or near the melting temperature of the hybrid
to reduce secondary structure in the target and to increase probe
specificity. Modifications of hybridization conditions have been
previously described. Gamper et al. (1986) Nuc. Acids Res.,
14:9943.
ODNs of the invention are suitable for use in diagnostic assays
that employ methods wherein either the oligomer or nucleic acid to
be detected are covalently attached to a solid support as described
in U.S. Pat. No. 4,775,619. The ODNs are also suitable for use in
diagnostic assays that rely on polymerase chain reaction (PCR)
techniques to amplify target sequences according to methods
described, for instance, in European Patent Publication No. 0 393
744. ODNs of the invention containing a 3' terminus that can serve
as a primer are compatible with polymerases used in PCR methods
such as the Taq or Vent.TM. (New England Biolabs) polymerase. ODNs
of the invention can thus be utilized as primers in PCR
protocols.
The ODNs are useful as primers that are discrete sequences or as
primers with a random sequence. Random sequence primers can be
generally about 6, 7, or 8 nucleomonomers in length. Such primers
can be used in various nucleic acid amplification protocols (PCR,
ligase chain reaction, etc.) or in cloning protocols. The
substitute linkages of the invention generally do not interfere
with the capacity of the ODN to function as a primer. ODNs of the
invention having 2'-modifications at sites other than the 3'
terminal residue, other modifications that render the ODN RNase H
incompetent or otherwise nuclease stable can be advantageously used
as probes or primers for RNA or DNA sequences in cellular extracts
or other solutions that contain nucleases. Thus, the ODNs can be
used in protocols for amplifying nucleic acid in a sample by mixing
the ODN with a sample containing target nucleic acid, followed by
hybridization of the ODN with the target nucleic acid and
amplifying the target nucleic acid by PCR, LCR or other suitable
methods.
The ODNs derivatized to chelating agents such as EDTA, DTPA or
analogs of 1,2-diaminocyclohexane acetic acid can be utilized in
various in vitro diagnostic assays as described in, for instance,
U.S. Pat. Nos. 4,772,548, 4,707,440 and 4,707,352. Alternatively,
ODNs of the invention can be derivatized with crosslinking agents
such as 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine or
5-(3-(4-bromobutyramido)prop-1-yl)-2'-deoxyuridine and used in
various assay methods or kits as described in, for instance,
International Publication No. WO 90/14353.
In addition to the foregoing uses, the ability of the oligomers to
inhibit gene expression can be verified in in vitro systems by
measuring the levels of expression in subject cells or in
recombinant systems, by any suitable method. Graessmann et al.
(1991) Nuc. Acids Res., 19:53-59. In the present case, levels of
p24 have been measured as indicative of virus replication.
All references cited herein are incorporated herein by reference in
their entirety.
The first embodiment of the present invention is an ODN
complementary to the region between the 5' long terminal repeat
(LTR) and the first initiation codon (AUG) of the gag gene. This
region contains highly conserved sequences required for efficient
viral RNA packaging. Klotman and Wong-Staal (1991) in: The Human
Retroviruses by Gallo & Jay, eds. Acad. Press. The antisense
ODN is referred to as "anti-gag." The ODN is of sufficient length
and complementarity to inhibit expression of the gag gene. The
complementary site is from bases +262 to +281 as numbered according
to Ratner et al. (1985) Nature, 313:277-283. In a preferred
embodiment the anti-gag ODN has the specific sequence:
The second embodiment of the present invention is an ODN
complementary to the sequence immediately downstream of the major
splice acceptor site but upstream of the AUG initiation codon of
the tat gene (3' of nucleotide 5358). Translation of this
transcript is essential for efficient viral gene expression and
replication. The antisense ODN is referred to as "anti-gag-pol."
The ODN is of sufficient length and complementarity to inhibit
expression of the gag-pol gene. The complementary site is from
bases +5399 to +5418 as numbered according to Ratner et al. (1985).
In a preferred embodiment the anti-gag-pol ODN has the
sequence:
The third embodiment of the present invention is an ODN
complementary to the rev gene which is involved in the regulated
expression of HIV structural genes. Feinberg et al. (1986) Cell,
46:807; and Sodroski et al. (1986) Nature, 321:412. It has
previously been observed that cytoplasmic RNAs that encode the
virion structural proteins gag, pol and env are not found in the
absence of a functional rev gene product. Sodroski et al. (1986)
Nature, 321:412-417; Knight et al. (1987) Science, 236:837-840;
Malim et al. (1988) Nature, 335:181-183; and Hadzopoulou-Cladaras
et al. (1989) J. Virol., 63:1265-1274. rev mutants of HIV-1 are
incapable of inducing the synthesis of the viral structural
proteins and are therefore replication defective. Sadaie et al.
(1988) Science, 239:910. rev is, therefore, said to be important in
governing the transition from the expression of the early
regulatory genes to that of the late structural genes. Greene
(1991) in Mechanisms of Disease. Ed. by F. Epstein. The antisense
ODN is referred to as "anti-rev." The ODN is of sufficient length
and complementarity to inhibit expression of the rev gene. The
complementary site is from bases +5552 to +5566 as numbered
according to Ratner et al. (1985). In a preferred embodiment the
anti-rev ODN has the following sequence:
The fourth embodiment of the present invention is an ODN
complementary to the region within the second splice acceptor site.
This region contains highly conserved sequences required for
efficient viral RNA packaging. Klotman and Wong-Staal (1991) in:
The Human Retroviruses by Gallo & Jay, eds. Acad. Press. The
antisense ODN is referred to as "GPI-2A." The ODN is of sufficient
length and complementarity to inhibit expression of the gag gene.
The complementary site is from bases +1189 to +1208 as numbered
according to Ratner et al. (1985). In a preferred embodiment, the
GPI-2A ODN has the specific sequence:
In a further preferred embodiment, the ODNs were chemically
modified by substitution of the naturally occurring oxygen of the
phosphodiester backbone with sulfur to form the corresponding
phosphorothioate derivatives of the oligomers. The positions of the
sulfur are as shown below.
Anti-gag: 5' C'CG'CC'CC'TC'GC'CTC'TTG'CC'G 3' (SEQ ID NO:4);
Anti-gagpol: 5' G'GC'TC'CA'TTTC'TTG'CTC'TC'C 3' (SEQ ID NO:5);
Anti-rev: 5' C'CG'C'TTCTTC'C'TGC'C 3' (SEQ ID NO:6); and
GPI-2A: 5' G'GTTC'TTTTG'GTCC'TTG'TC'T 3' (SEQ ID NO:7).
In accordance with the present invention, methods of modulating the
expression of the p24 protein are provided. The targeted RNA, or
cells containing it, are treated with the ODN analogs which bind to
specific regions of the RNA coding for the HIV p24 core structural
protein. The RNA targeted sites include regions involved in the
mechanism of expression of the HIV p24 core structural protein.
The following examples are intended to illustrate, but not to
limit, the invention. Efforts have been made to insure accuracy
with respect to numbers used (e.g., amounts, temperatures, etc.),
but some experimental errors and deviations should be taken into
account. Unless indicated otherwise, parts are parts by weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
EXAMPLE 1
Cell Culture
To determine the effect of antisense oligomer on viral gene
expression, B4.14 cells, provided by Dr. David Rekosh, Microbiology
Department, University of Virginia, were seeded at a cell density
of 5,000-12,000 cells per well in 24-well/35 mm plastic tissue
culture plates and were maintained in Iscove's Modified Dulbecco's
Medium with 10% calf serum, 50 .mu.g/ml gentamycin and 200 .mu.g/ml
hygromycin B at 37.degree. C. in a humidified incubator with 5%
CO.sub.2 for a few hours. Subsequently, the incubated cells were
washed and incubated under the same conditions with medium
containing the indicated concentrations of ODN and 10% serum heat
inactivated to reduce serum nuclease activity. The same oligomer
sequences, but with switched polarity were used as controls.
EXAMPLE 2
Viral Antigen Assay
Cells cultured as described in Example 1 were labeled with 75 to
150 .mu.Ci/ml [.sup.35 S]-methionine (70% L-Methionine/15%
L-Cysteine) in the presence of methionine-free medium containing
29.2 mg/100 ml glutamine, 50 .mu.g/ml gentamycin, 200 .mu.g/ml
hygromycin B, 10% heat inactivated fetal calf serum plus the
desired concentration of oligomer. The [.sup.35 S]-methionine
concentration was 185 MBq and the specific activity was 1057
Ci/mmole). Labeled samples were subsequently washed with phosphate
buffered saline (PBS) and resuspended in 200 .mu.l lysis buffer
comprised of 50 mM Tris, pH 7.2; 150 mM NaCl; 5 mM EDTA; 1%
Triton-100; 0.2% Deoxycholic acid.
Culture medium containing labeled virus was treated with 10% Triton
X-100 to a 1% final concentration to disrupt virus particles. The
samples were preabsorbed with protein A-Sepharose beads for 30 min.
at 4.degree. C. [.sup.35 S]-methionine-labeled viral proteins were
then immunoprecipitated for 2 hours using protein A-Sepharose beads
and 2.5 .mu.l/sample of polyclonal rabbit antiserum directed
against HIV-1 p25/24, obtained from the National Institute of
Allergy and Infectious Diseases (AIDS Research and Reference
Reagent Program). The antibodies were obtained from National
Institute of Allergy & Infectious Disease (AIDS Research &
Reference Reagent Program) and MicroGeneSys, Inc.
The resulting pellets were washed 4 times with lysis buffer, once
with lysis buffer containing 500 mM NaCl and finally once with TNE
buffer comprised of 10 mM Tris, pH 7.2; 25 mM NaCl; 1 mM EDTA.
Samples were then resuspended in 20-30 .mu.l 2X SDS sample buffer,
boiled for 5-10 min, applied to a 12.5% SDS polyacrylamide gel
electrophoresis and then analyzed by electrophoresis, according to
the method described by Laemmli (1970) Nature, 227:680-685. The
results obtained are listed in Table 1 and in FIGS. 1-3. Percent
inhibition is determined by densitometric analysis of the
autoradiography. The first two ODNs (anti-gag and anti-tat) were
phosphorothioate derivatives [sulfurization on alternate bases].
Inhibition was observed at ODN concentrations of 5 .mu.M assayed
after 3 days incubation with the oligomer. The third oligomer was a
15-mer phosphodiester derivative. Observed inhibition was at
oligomer concentration of 200 .mu.g/ml assayed after 6 days
incubation with the oligomer.
TABLE 1 ______________________________________ Preliminary
Observation Inhibition of viral protein synthesis by antisense
oligomer in B4.14 cells Com- Sequence 5'-3' plementary Func- %
inhi- (SEQ ID NO: 1) Site tion bition
______________________________________ CCGCCCCTCGCCTCTTGCCG 262-281
Splice 30 (SEQ ID NO: 2) Donor GGCTCCATTTCTTGCTCTCC 5399-5418 tat
30 (SEQ ID NO: 3) initi- ator CCGCTTCTTCCTGCC 5552-5566 Rev 40
______________________________________
EXAMPLE 3
FIG. 1 is an autoradiograph of a SDS-PAGE showing in vivo synthesis
of HIV-1 viral proteins and their breakdown products. The following
samples were run. Two hundred .mu.l of CMT3 [wild-type (left)] and
B4.14 [transfected line (right)] cell lysates following metabolic
labeling with [.sup.35 S]-methionine were immunoprecipitated with
rabbit serum against p24 viral antigen as described above. The
positions of the viral proteins (p160; p55 and p24) are clearly
visible in the B4.14 cell lysate but not in control cell line CMT3
cell lysate.
EXAMPLE 4
FIG. 2 is an autoradiograph of a SDS-PAGE showing a significant
inhibition of expression of HIV proteins by the antisense ODN
directed to the rev sequence. The following experiment was
performed. Two hundred .mu.l of B4.14 [transfected line] cell
lysates following 3 days treatment with antisense [AS]; and sense,
the inverse complement of the antisense oligomer [S]; and
subsequent [.sup.35 S]-methionine labeling were immunoprecipitated
with rabbit serum directed against p24 viral antigen as described
above. Equal amounts of protein were loaded on each lane.
EXAMPLE 5
FIG. 3 is an autoradiograph of a SDS-PAGE showing a significant
inhibition of expression of HIV proteins by the antisense ODN
directed to the first splice site donor of the HIV-1 genome. The
following experiment was performed. Two hundred .mu.l of B4.14
[transfected cell line] cell lysates/medium following 6 days
treatment with antisense [AS]; sense, the inverse complement of the
antisense ODN [S]; and control [B4.14] cell lysate only]; and
subsequent .sup.35 S-methionine labeling were immunoprecipitated
with rabbit serum directed against p24 viral antigen as described
above. Equal amounts of protein were added in each lane.
EXAMPLE 6
The Effects of Different Concentrations of the Antisense ODNs
To determine whether there was a dose relationship of the antisense
ODNs on HIV gene expression, the following experiment was
performed.
The cells were cultured as described in Example 1 and incubated
overnight with different concentrations of ODNs directed against
the first splice donor site in the presence of 5 .mu.g/ml
Lipofectin and 1% heat-inactivated fetal calf serum. The medium was
subsequently replaced with fresh medium containing 10%
heat-inactivated serum. ODN was then added and incubated for 7
days. Western blot analysis was performed with rabbit polyclonal
antibody directed against HIV p24/55 proteins.
Following SDS-polyacrylamide electrophoresis, cellular proteins
were electrophoretically transferred to Immobilon membrane
(Schleicher and Schuell) as follows. An Immobilon membrane was
placed in methanol in a clean dish, washed several times in
deionized distilled water and soaked in western transfer buffer
(60.6 g Tris-HCl; 288 g glycine; 4 l methanol and distilled water
to 20 l). The apparatus used is the Bio-Rad Trans-Blot cell, used
according to the manufacturer's instructions. Western blot analysis
was performed using the Vectastain ABC kit (Alkaline Phosphatase
Rapid IgG) (Vector Laboratories) according to the manufacturer's
instructions.
The results are shown in FIG. 4 where it can be seen that 0.5 and 1
.mu.M antisense ODN are equally effective at preventing p24
synthesis.
EXAMPLE 27
The Effects of Different Concentrations of the ODN GPI-2A
To determine the ability of the ODN GPI-2A to inhibit expression of
p24 in HIV infected cells, the following experiments were
performed.
Cells were incubated overnight with 0.1, 0.5 and 1.0 .mu.M of the
ODN in the presence of 1% heat-inactivated fetal calf serum. The
serum concentration was subsequently raised to 10% and incubated
for 3 days. About 3.times.10.sup.7 cpm/probe was immunoprecipitated
using rabbit polyclonal antibody directed against p24/25 viral
proteins as described above. The lane marked control had the sense
strand, the inverse complement of the antisense oligomer, added to
the cells rather than the sense strand. The autoradiograph in FIG.
5 shows that there was a dose-dependent inhibition of the HIV viral
core antigen, among others.
The autoradiograph was then subjected to densitometry analysis. The
results, presented in FIG. 6, indicate that at 1.0 .mu.M, the ODN
inhibited about 50% of the p24 synthesis.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 8 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:1: CCGCCCCTCGCCTCTTGCCG20 (2) INFORMATION
FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGCTCCATTTCTTGCTCTCC20 (2) INFORMATION FOR SEQ ID NO:3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:3: CCGCTTCTTCCTGCC15 (2)
INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY: misc.sub.--
feature (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "This
position is Cs wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY:
misc.sub.-- feature (B) LOCATION: 3 (D) OTHER INFORMATION: /note=
"This position is Cs wherein s is sulfur." (ix) FEATURE: (A)
NAME/KEY: misc.sub.-- feature (B) LOCATION: 5 (D) OTHER
INFORMATION: /note= "This position is Cs wherein s is sulfur." (ix)
FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 7 (D)
OTHER INFORMATION: /note= "This position is Cs wherein s is
sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B)
LOCATION: 9 (D) OTHER INFORMATION: /note= "This position is Cs
wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.--
feature (B) LOCATION: 11 (D) OTHER INFORMATION: /note= "This
position is Cs wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY:
misc.sub.-- feature (B) LOCATION: 14 (D) OTHER INFORMATION: /note=
"This position is Cs wherein s is sulfur." (ix) FEATURE: (A)
NAME/KEY: misc.sub.-- feature (B) LOCATION: 17 (D) OTHER
INFORMATION: /note= "This position is Cs wherein s is sulfur." (ix)
FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 19 (D)
OTHER INFORMATION: /note= "This position is Cs wherein s is
sulfur." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
NCNCNCNTNGNCTNTTNCNG20 (2) INFORMATION FOR SEQ ID NO:5: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix)
FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 1 (D)
OTHER INFORMATION: /note= "This position is Cs wherein s is
sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B)
LOCATION: 3 (D) OTHER INFORMATION: /note= "This position is Cs
wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.--
feature (B) LOCATION: 5 (D) OTHER INFORMATION: /note= "This
position is Cs wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY:
misc.sub.-- feature (B) LOCATION: 7 (D) OTHER INFORMATION: /note=
"This position is Cs wherein s is sulfur." (ix) FEATURE: (A)
NAME/KEY: misc.sub.-- feature (B) LOCATION: 11 (D) OTHER
INFORMATION: /note= "This position is Cs wherein s is sulfur." (ix)
FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 14 (D)
OTHER INFORMATION: /note= "This position is Cs wherein s is
sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B)
LOCATION: 17 (D) OTHER INFORMATION: /note= "This position is Cs
wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.--
feature (B) LOCATION: 19 (D) OTHER INFORMATION: /note= "This
position is Cs wherein s is sulfur." (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:5: NGNTNCNTTTNTTNCTNTNC20 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix)
FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 1 (D)
OTHER INFORMATION: /note= "This position is Cs wherein s is
sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B)
LOCATION: 3 (D) OTHER INFORMATION: /note= "This position is Cs
wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.--
feature (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "This
position is Cs wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY:
misc.sub.-- feature (B) LOCATION: 10 (D) OTHER INFORMATION: /note=
"This position is Cs wherein s is sulfur." (ix) FEATURE: (A)
NAME/KEY: misc.sub.-- feature (B) LOCATION: 11 (D) OTHER
INFORMATION: /note= "This position is Cs wherein s is sulfur." (ix)
FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 14 (D)
OTHER INFORMATION: /note= "This position is Cs wherein s is
sulfur." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: NCNNTTCTTNNTGNC15
(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY: misc.sub.--
feature (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "This
position is Cs wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY:
misc.sub.-- feature (B) LOCATION: 5 (D) OTHER INFORMATION: /note=
"This position is Cs wherein s is sulfur." (ix) FEATURE: (A)
NAME/KEY: misc.sub.-- feature (B) LOCATION: 10 (D) OTHER
INFORMATION: /note= "This position is Cs wherein s is sulfur." (ix)
FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 14 (D)
OTHER INFORMATION: /note= "This position is Cs wherein s is
sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B)
LOCATION: 17 (D) OTHER INFORMATION: /note= "This position is Cs
wherein s is sulfur." (ix) FEATURE: (A) NAME/KEY: misc.sub.--
feature (B) LOCATION: 19 (D) OTHER INFORMATION: /note= "This
position is Cs wherein s is sulfur." (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:7: NGTTNTTTTNGTCNTTNTNT20 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:8: GGTTCTTTTGGTCCTTGTCT20
__________________________________________________________________________
* * * * *