Note: Descriptions are shown in the official language in which they were submitted.
WO 95117414 2 1 7 8 8 1 1 PCTNSg4/13687
-- 1
A2rrI-llEPA~rITI8 B VIRAI, or T _ lzoIIDE8
Field of Invention
The invention relates to oligonucleotide
5 compositions, rhArr--e~1tical compositions containing such
ol igrn~lrlFotidesl and their use for the prevention and
treatment of hepatitis B infection.
Ba-huLuu,,d of the Invention
me consequences of a viral infection depend upon
lO a number of factors, both viral and host. Those factors
which affect pathogenesis include the number of infecting
viral particles and their path to susceptible cells, the
speed of viral multiplication and spread, the effect of
the virus on cell functionf:, the host~s se~nr~ ry
15 rF~D~ .=F~ to the CFlll~lAr injury, and the immunologic and
nu.. ~ecific defenses of the host. In general, the
ef f ects of viral inf ection include acute and chronic
r.l;ni,r~ iC-~Ae:F.,,C, asymptomatic infections, induction of
various cancers, and chronic ~L OyL e:ssive neurological
20 disorders. Viruses are potent infectious pathoqF~ni r.
agents because virions ~ o.hlced in one cell can invade
other cells and thus cause a spreading infection.
Viruses cause important functional alterations of the
invaded cells, often resulting in death of the cells.
Hepatitis viruses constitute a major medical
problem throughout the world. Like the other hepatitis
viruses, the hepatitis B virus (HBV) produces a whole
.I e.:Llu.u of; 1 ln~ccFc, ranging from acute to chronic and
from subclinical or asymptomatic to fatal and fl~lm;n:-nt
30 P.pproximately 5% of the world's population, probably at
least 400 million people, are presently infected with the
hepatitis B virus (HBV). HBV presents a high risk of
acute fulminant hepatitis, as well as chronic liver
WO 9~/17414 PCr/US94/13687--
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disease, including cirrhosis, chronic active hepatitis,
and the eventual devs1~, L of primary hepatoc-~l 1..1 9r
carcinoma in individuals who remain chronic carriers of
the virus.
Therapeutic studies during the last ten years have
identified prom,ising drugs with antiviral effects,
i nr~ n~ the nucleotide analog adenine arabinoside (Ara-
A), its more soluble ~ , ste Ara-AMP, and
Interferon-alpha. Although effective in some patients,
10 treatment with such agents has been shown frequently to
result in only a transient response, or to have
significant toxicity. Accordingly, there is a continuing
need for methods and therapeutic agents to stop viral
replication and prevent the spread of the virus to
15 additional cells. However, this goal presents
ct~nci-lsrable diffioulties. A major problem is that of
inhibiting the virus without harming the host cells. The
A9n e of viral multirlic~tion on csl~ r genes
limits the points of differential attack. Even the
20 largest viruses have few h;o~-h~"~icsl r-9~ ti~nc that are
not duplicative of those of the host. Further, it is
only after extensive viral multiplication and C.9111l1;~r
alteration have O~,~;ULL~'d that viral infections become
evident. Therefore, the usual approach to control is
25 prophylaxis. Therapy in most cases is limited to
3ituations where the killing of some uninfected cells can
be tolerated if the damage is subsequently repaired.
Another important limitation of antiviral therapy
is the emergence of resistant mutants. In order to
30 minimize selection of such mutants, the principles valid
for treatment of bacterial infections are equally
applicable to viruses: adequate dosage, multi-drug
treatment, and avoiding therapy unless clearly indicated.
Therefore, because of the serious nature of viral
3 5 inf çction and the obstacles presented by the nature of
WO 95117414 2 1 7 8 8 1 I PCTIUS94/13687
-- 3 --
the infecting virus, there is an urgent need for methods
which control viral replication. A method which would be
applicable to RNA and DNA viruses would have widespread
applicability .
Synthetic antisense oligonucleotides have been
used as inhibitors of viral gene expression. Smith et
al., Proc. Natl. Acad. Sci. USA 83:2787-2791 (1986),
report antiviral activity of an oligo (nucleoside
methyl rhncrhnnAte) complementary to the splice junction
10 of herpes simplex virlls type I immediate early ~L~_ r.~.~As
4 and 5. See also Agris et a~., Inhibition of vt~c~ lAr
stomatitis virus protein synthesis and infection by
methylrhnsrh~n~tes, Biochem. 25, 6268-62~5 (1986);
7 i k et al ., Inhibition of Rous sarcoma virus
15 replication and cell transformation by a specific
nl i~orl~nyynucleotide, Proc. Natl. Acad. Sci. USA
75:280-284 (1978); 7r --nik et al., Inhibition of
replication and expression of human T-cell ly ' L~ u~ic
virus type III in cultured cells by ~ CJ~ synthetic
20 oligonucleotides complementary to viral RNA, Proc. Natl.
Acacl. Sci. USA 83, 4143-4146 (1986). Goodarzi et al., J.
Gen. Virol. 71:3021-3025 (1990), report inhibition of the
expression of the gene for hepatitis B virus surface
antigen by antisense ol ~-,o~ nYynucleotides directed at
25 the cap site of mRNA and regions of the translational
initiation site of the HBsAg gene. Off~.,D~ely~ et al.,
In vivo inhibition of duck hepatitis B virus replication
and gene expression by rhnsphc~rothioate modified
antisense oligodeoxynucleotides, ENBO J. 12: 1257-1262,
30 No. 3 (1993), report inhibition of duck hepatitis B virus
(DHBV) replication _y antisense oligdeoxynucleotides from
the pre-S/S-region, the polymerase region, and the pre-
C/C region. Particularly effective were an antisense
oligodeo~cynucleotide from the pre-S region and one from
35 the direct repeat II (DRII) region.
WO 95/17414 PCTIUS94/13687--
21 7881 1
-- 4 --
STTMM~T~Y OF THE INVENTION
The invention relates to antisense
ol 1 gnn~ ntides, preferably antisense
oli~cJde~yyllucleotides~ as antiviral agents against HBV;
5 rhA Lical compositions providing such antiviral
oli~on~ leotides; and methods for their use in inhibiting
HBV. Antisense oligonucleotide compositions
complementary to the HBV DR2 region completely block
viral transcription, antigen production, and replication.
lO Such antiviral oligonucleotides can be provided to the
target cell either ~ y~ cly as an antisense DNA or
RNA, or by insertion of a sense DNA sequence into an
expression vector capable o~ producing multiple copies of
the antisense oligonucleotides ~n~lngennTlcly within the
15 target cell.
The invention includes an olignn~leotide having
antiviral activity against hepatitis B virus (HBV),
consisting ~GG~nti~lly of a gequence subst:~nt;:~lly
complementary to a portion of plus (+) strand of the HBV
20 genome, which portion consists of the DR2 ll-mer (SEQ ID
NO: 44 ) plu8 0-6 nucleotides of 5 ' f lanking sequence and
0-30 nucleotides of 3' flanking sequence. Examples of
such oligonucleotides include those with the sequence:
5 ' -TGGGCGTTCACG-.lG-.lCGCCATGCAACGTGCAGAGGTGAAGCGAAG--3 '
25 (SEQ ID NO: 45),
S'--TGGGCGTTCA~ ,;,. c ~C~ATGCAACGTGCAGAGGTGAAGCGAAG--3'
(SEQ ID NO: 46),
5 '--~GGC~ cA~G/, ~ GC~:ATGCGACGTGCAGAGGTGAAGCGAAG--3 '
(SEQ ID NO: 47), or
5'--~.GG~ ~ACG~ cLc-:ATGCGACGTGCAGAGGTt:AA~ --
(S~Q ID NO: 48),
or a portion thereof, which portion is preferably at
least 15 nucleotides (and more preferably at least 18
nucleotides) in length. The oligonucleotide preferably
35 in~~ G the sPqn~nce 5'--ACGTGCAGAGGTGAAGCG--3' (SEQ ID
WO 95/17414 2 1 7 8 8 1 1 PCT/US94/13687
-- 5 --
NO: 21). Examples of olig~n~l~leotides of the invention
include the following:
5'--CAACGTGCAGAGGTGAAGCGA--3' tOLIGO HBV 10011; SEQ ID NO:
6) ;
5 5'--CGACGTGCAGAGGTGAAGCGA--3' (OLIGO CJP 114; SEQ ID NO:
10);
5'-ACGT~r~r:~t:GTGAAGCGA-3' (OLIGO CJP 140; SEQ ID NO: 24);
5'--CGACGTGCAGAGGTGAAGCG--3' (OLIGO CJP 150; SEQ ID NO:
20);
10 5'-GACGTGCAGAGGTGAAGCGA--3' (SEQ ID NO: 49);
5'--AACGTGCAGAGGTGAAGCGA--3' (SEQ ID NO: 50);
5'--GACGTGCAGAGGTGAAGCG--3' (SEQ ID NO: 51);
5'--AACGTGCAGAGGTGAAGCG--3' (SEQ ID NO: 52);
5'--CGTGCAGAGGTGAAGCGA--3' (OLIGO CJP 141: SEQ ID NO: 25);
15 5'--GTGCAGAGGTGAAGCGA--3' (OLIGO CJP 142: SEQ ID NO: 26);
5'--TGCAGAGGTAAGCGA-3 ' (OLIGO CJP 143 : SEQ ID NO: 27);
5'--GCAGAGGTGAAGCGA-3' (OLIGO CJP 144: SEQ ID NO: 28);
5'--CAGAGGTGAAGCGA--3' (OLIGO CJP 145: SEQ ID NO: 29);
5'--AGAGGTGAAGCGA--3' (OLIGO CJP 146: SEQ ID NO: 30);
20 5'--GAGGTGAAGCGA--3' (OLIGO CJP 147: SEQ ID NO: 31);
5'--AGGTGAAGCGA--3' (OLIGO CJP 148: SEQ ID NO: 32);
5'--CGACGTGCAGAGGTGAAGC--3' (OLIGO CJP 149: SEQ ID NO: 33);
5'--CGACGTGCAGAGGTGAAG--3' (OLIGO CJP 151: SEQ ID NO: 34);
5'--CGACGTGCAGAGGTGAA-3' (OLIGO CJP 152: SEQ ID NO: 35);
25 5'--CGACGTGCAGAGGTGA--3' (OLIGO CJP 153; SEQ ID NO: 36);
5'--CGACGTGCAGAGGTG--3' (OLIGO CJP 154: SEQ ID NO: 37);
5'--CGACGTGCAGAGGT--3' (OLIGO CJP 155: SEQ ID NO: 38);
5'--CGACGTGCAGAGG--3' (OLIGO CJP 156: SEQ ID NO: 39);
5'--CGACGTGCAGAG--3' (OLIGO CJP 157: SEQ ID NO: 40);
30 5'--AACGTGCAGAGGTGAAGCGA--3' (OLIGO CJP 158: SEQ ID NO:
41);
5 '--CGACGTGCAGAGGTGAAGCGAAG--3 ' (OLIGO CJP 159: SEQ ID
NO: 42); and
5'--CGACGTGCAGAGGTGAAGCGAA--3' (OLIGO CJP 160: SEQ ID NO:
35 43)-
WO95/17414 PCT/US94/13687
2~7881 1
-- 6 --
Preferred oligonucleotides of the invention
include those having 2 sequence consisting essentially of
one of the following:
5'--QACGTGQGAGGTGAAGCGA--3' (OLIGO HBV 10011; SEQ ID NO:
5 6);
5'--CGACGTGCAGAGGTGAAGCGA--3' (OLIGO CJP 114; SEQ ID NO:
10 ) ;
5'--ACGTGQGAGGTGAAGCGA--3' (OLIGO CJP 140; SEQ ID NO: 24);
5'--CGACGTGCAGAGGTGAAGCG--3' (OLIGO CJP 150; SEQ ID NO:
10 20);
5 '--GACGTGQGAGGTGAAGCGA-3 ' ( SEQ ID NO : 4 9 ~;
5'-AACGTGrAr~Ar-r7TGAAGCGA-3' (SEQ ID NO: 50);
5'-GACGTGQGAGGTGAAGCG-3 ' (SEQ ID NO: 51); or
5'--AACGTGQGAGGTGAAGCG--3' (SEQ ID NO: 52).
The oligonucleotides of the invention can be used
in a method of preventing replication of HBV in a
n cell, which method ~ n ~ oc the step of
introducing into the cell an inhibitory amount of (a) the
oli~nnll~-l e~tide, or (b) an expression vector containing a
20 s~ e that is transcribed within the cell to generate
an oligoribonucleotide of the invention. Such an
expression vector would preferably include LL~I,s.;.iption
control sequences that permit it to be ~Les~ed within a
hepatocyte. The oli~n~ otide may be administered to
25 an animal in the form o~ n rh -eutical composition
consisting essentially of an amount of the
nl l~nnl~ otide effective to inhibit replication of HBV
in the liver cells of an animal, and a rh~ tically
acceptable càrrier.
It is understood that when the oligonucleotide of
the invention is a rihnnt~ otide, I'T" in each of the
se~ C set forth herein represents "U".
WO 95/17414 2 1 7 8 8 l 1 PCT/US94113687
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RRTT~T~ DT CCRTPTION OF ~TF DRAWINGS
Fig. l is a map of the HBV qenome, showing the
codinq organization of the four major open reading
frames, as well as the 3.5 kb ~L~ ;C RNA and the
5 2.1 kb sl~hq~nl i C RNA species. DR1 and DR2 are shown.
Fig. 2 is a lt:~Lc:s~..L~t.ion of the computer-
qenerated putative 6ec- l- y ~LLu- LuLe of the RNA
6~ n~e of HBV (HPBADW1~ (n.t.6. 1500 to n.t.s. 1700),
showing DR2 as part of a stem-loop .L.u.;Lu~.
DET~TT ~n DESCRIPTION
Human hepatiti6 B viru6 (HBV) is now reco~n i 7sd to
be a member of a family of animal viruses called
hepadnaviruses (hepatotropic DNA viruses). Human HBV i~
classified as a hepadnavirus type 1. Similar viruses
15 infect other animal species, in~ lin~ UJO~ , qround
and tree squirrels, Pekin duck, and heron, producing
acute and chronic hepatitis as well as hepatocc~l 1 ul ;~r
carcinoma. Full-length molecular clones of these
hepadnaviruses have been obtained and their nucleotide
20 sequences ~ tc~ni nPd. The coding orqanization of the
1 i~n viruses is now known to be virtually irlF~nt j~ ~l
to that of human HBV, while the avian viruses are more
diverqent in terms of nucleotide 6equence, biological
properties, and coding organization. The duck HBV (DHBV)
25 genome appear6 to lack an X region, and its core antigen
coding reqion is sub6tantially larqer that that of the
1 i;~ln viru8es. Ganem ~ Varmus, The Molecular Biology
of the Hepatiti6 B Viru6e6; Ann. Rev. Biochem. 56:651-93
(1987) .
Replication 6trateqy of the hepatiti6 B viru6 i6
discussed by Seeqer et al., Science 23Z:477-484 (1986);
Khudyakov et al., FEBS Letters 243:115-118 (1989); Will
et al., J. of Virol. 61:904-911 (1987); and Hir6ch et
al., Nature 344:552-555 (1990).
Wo 95117414 PCrlUS94113687--
2l7881 1 8 -
Infectious human HBV virions, called "Dane
particles", are 42 or 43 nm double-shelled particles
which include the outer coat of HBV surface antigen
(HBsAg) and the HBV core antigen (HBcAg), a basic
5 rhnsrhnrrotein of 21 kd. Within the HBV nucleocapsid
core is a pL~ ~ inAntly double-stranded, but partially
single stranded, DNA genome measuring 3200 base pairs, a
well aE~ an ,~nAny~ c DNA polymerase which directs
replication and repair of HBV DNA. The full-length
l0 strand of HBV DNA is complementary to the viral RNAs,
including the mE~NAs, and by convention is designated to
be of minus polarity. The shorter complementary strand
is designated the plus strand . While the 5 ' end of the
plus strand is fixed, the position of the 3' end is
15 variable, even within molecules of the same viral stock.
In the Pn-lngPnn11c polymerase reaction, the single-
stranded gap is repaired by the addition of nucleotides
to the 3 ' end of the plus strand DNA. A second a~y ~Ly
occurs at the 5 ' termini of the viral genome ' 8 two
20 strands: protein is covalently linked to the 5' end of
the minus strand, where~s an oligonucleotide is attached
to the 5 ' end of the plus strand.
Replication of HBV proceeds via reverse
transcription of an RNA int~ te using protein and
25 RNA primers for the generation of the first and second
DNA strands. Large sections of the genome are translated
in more than one reading frame. Within a given reading
frame, multiple proteins are expressed from overlapping
transcripts, using different in-phase initiator codons.
30 The resulting closely related gene products are
posttranslationally processed and assembled into a
variety of structures of differing function or
s--hcPl 1 1l1 ~r distribution.
Four major open reading frames (ORFs) encoded by
35 the HBV minus strand have been identified and
WO95117414 ~ 1 7 8 1 1 PCr/US94/13687
characterized: l) the pre-S and S gene, which code for
the HBsAg and several other les6 well characterized gene
- products; 2) the C gene, which codes for HBcAg and
HBeAg; 3) the P gene, which codes for the viral DNA
5 polymerase; and 4) the X gene, which codes for the
transactivating X protein, HBx, often oLseLv~:d in
patients with hepatoQc~ r carcinoma. tHBeAg results
from proteolytic cleavage of the p22 precore
int~ ~';Ate, and is secreted from the cell. It is found
lO in serum as a 17 kd protein. )
Four major steps are believed to be C1 ` L-l to
the replication of h~ad.,avirus genomes:
l. closing of the single-stranded gap by the
addition of nucleotides to the 3 ' end of the plus strand
15 DNA, to form covalently closed circular DNA (cccDNA)
within the nucleus of infected hepatocytes;
2. transcription of cccDNA by host RNA polymerase
to generate an RNA template of plus strand polarity for
reverse transcription, with encapsidation of the pre-
20 genomic RNA into viral cores;
3 . synthesis of the f irst (minus) strand of DNA
by copying pregenomic RNA, using a protein primer (this
step is termed core associated reverse LL~j~S~;L lption);
and
4. synthesis of the second (plus) strand of DNA
by copying the first DNA strand using an oligomer of
viral RNA as primer, to form the mature viral genomic
DNA. Amplif ication of the viral genome is believed to
occur during synthesis of pregenomic RNA from cccDNA.
HBV viral RNA serves as both the template for
synthesis of genomic DNA via reverse transcription and
the ~ q r RNA f or synthesis of certain viral
proteins. This is achieved by the synthesis of two
classes of viral RNA, genomic (3.5 kb in length,
35 containing the complete viral genetic information) and
WO 95117414 PCTNS94/13687 ~
2l 7881 1
-- 10 --
g~ F- iC (2.1 and 2.4 kb in length). all of these RNAs
are of plu8 strand polarity, unspliced, and
polyadenylated at a common 3' ~P~minllc. Within the HBV
genome are conserved cis-acting ~ . L~ that play
5 i Lal.~ roles in the life cycle of the virus. Chief
among these are ll-nucleotide "direct repeat" sequence
designated DR1 and DR2. Drl and DR2 are distinguished
from e~ch other by their positions in the genome, their
flF~nkin~ sequences, and their biological functions. DR1
10 and DR2 are located near the 5 ' and the 3 ' ends of the
HBV plus strand, respectively, and play critical roles in
the initiation of viral DNA synthesis. In the prPq~
RNA, there are two copies of DR1, found at the 3' and 5'
ends, respectively, and one copy of DR2 located near the
15 3 ' end.
The other major cu.,seL ~_d sequence is the element
TATAA~ (SEQ ID N0: 1) found within the 5' end of the core
antigen coding sequence, which forms part of the
cleavage/polyadenylation signal specifying the common 3 '
20 termini of viral mRNAs. For details of the 8LLU~:LULe: and
function of the DR1 and DR2 s~Tl~n-~c, see Ganem &
Varmus, The Molecular Biology of the Hepatitis B Viruses,
Ann. Rev. Biochem. 56:651-93 tl987), and Seeger, Ganem,
and Varmus, Science 232:477-484 ~1986), the teArhin~c of
25 which are hereby inCUL~ULClted by reference. See also
Figure 1, which sets forth a map of the HBV viral genome,
showing the organizational ~.L.UuLu~a of the four major
ûRFs and the ~Le~ i c and s~7~ RNA species.
Synthesis of the minus strand of viral DNA is
30 believed to begin with the DR1 sequence that resides
within the t~rminAl repeat region, R, found at both the
5' and 3' ends of the ~Ley~ i~ RNA. Initiation could
occur near either the 5 ' or the 3 ' end of the RNA
template, since DR1 resides within the R sequence that is
35 t-~rmin~lly repeated in l,Lec"~ ic RNA. As rliccllcced in
W0 95/17414 2 1 7 8 8 1 1 PCr~ss4/13687
-- 11 --
Ganem & Varmus, supr~, at 663-664, initiation at the 3 '
end would permit elongation without interruption across
the entire genomic sequence, ending with a second copy of
the 9 nucleotide sequence found between the DRl
5 initiation site and the 5 ' boundary of R, whereas
initiation at the 5' end would require transfer of the
growing minus strand DNA molecule to the 3 ' end of the
same or another pr~q~n ; r RNA molecule.
The initiation site for synthesis of plus strand
10 DNA occurs on a minus strand DNA template at the sequence
which is 1~ tary to DR2 (DR2 ' ) . A short oligomer
of viral RNA, a fragment of the pregenomic RNA ~ Lc~ted
by RNAse ~, is covalently linked to the DNA at this 6ite.
However, when the attached RNA was sequenced (Lien et al.
15 .J. Virol . 57:229-37 (1986) ), the ~yu~ctecl DR sequence was
found to be flanked by 6 nucleotides from the DRl region,
rather than the expected DR2 region . This Su~ea LS that
an oligomer containing DRl, from either the 5 ' or 3 ' copy
of R, bAcPpA;rs with the DR2' site in the DNA minus
20 strand, and serves as a primer for initiation of plu8
strand synthesis from that position. The fact that the
oligomer has a 5' cap ~LLU~:LUL~: is evidence that the plus
strand primer originates from the 5 ' end of the
preq~- i r RNA, although the reason for such a
25 ~ l;c~ated priming -niFm is not immediately clear.
Synthe6is of the plus strand reaches a roA~lhlorl~ at the
protein-linked 5' end o~ the minus strand template,
reguiring transfer of the partial plus strand to the 3 '
end of a minus strand template. For unknown reasons, the
30 plus strand is usually not extended to the full length of
the minus strand; instead, plus strands are ;n~ te
and hete~ cJ~- P J ~ in length, with open circles the
dominant form of virion DNA.
The nucleotide sequence of DR2 and f lanking
35 sequences both 5 ' and 3 ' to DR2 are highly ~;o~.s~ d
WO 95/17414 PCTIUS94113687 --
21 7881 1
-- 12 --
among various HBV HBsAg subtypes and strains. The
nucleotide sequence of the DR2 region of HBV HBsAg
subtype ADW was compared with corr~ponrl i n~ sequences
from a number of other strains or subtypes of HBV by
5 using s~ u~ a data available in GenBank. Sequences and
designations are as listed in GenBankD. See Table I
below, where the symbol ~': " indicates a nucleotide
identical to the CULL~ >~ nq position of HPBADW.
According to GenBank, DR2 of HPBADW (Hepatitis B Vlrus
10 Subtype ADW) consists of nucleotides 1592 through 1602,
having the 6equence TTCACCTCTGC (SEQ ID NO: 2). The
numbering listed in Table 1 is that of Hepatitis B Virus
Subtype ADW from GenBank. The nucleotide seuuen~es of
various hepatitis virus strains can be f ound in Okamoto
15 ~t al., J. Gen. Virol. 69:2575-2583 (1988), and through
GenBank. The t~ hin~ of these references are hereby
in.;UL ~UL c.ted by ref erence .
W0 95/17414 2 1 7 8 8 1 1 PCI/US94/13687
- 13 -
.. .. .. .. .. .. .. .. ..
O o o z o o o o o
a a a a ~ a a a a
14 ~3 ~ Id W ~ ~ U~ ~O
_ _ _ _ _ _ _ _
r7
~--~C ...............
_~ C~) ................
- .. .. .. ..
:: :: :: :: :: :: ::
:: :: :: :: :: :: ::
:: :: :: :: :: :: :: ::
.. .. .. .. .. .. .. ~
.. .. .. .. .. .. ..
.. .. .. .. .. .. .. ..
:: :: :: :: :: :: :: ::
.. .. .. .. .. .. .. .. _ _
_I ...... ........ 3
3l ~ ~ =
~ ~ ., ., .. m m
r, N , 3 U~ ~ V :~
O_ ~ .. . .. . . ,. . ~ ~ ~ C ~
:: :: :: :: :: :: :: :: 8 ~ 3 E
N . .. . .. ~ -- K -- ~ --
.. . m @ o @ @ @ ~ m
.. ~ ~D Id 5
:: :: ' :: :: :: - ~ r - ~ - 0
. . 8 . . ' . . "
................ : : : : _: . : V
:: :: :: :: :: :: ::
l , :: :: :: :: :: :: :: :: ;
,_.
,,
U~ .
- 3 3 ~ r~ ~7 3
= T' ~ 2 U~
r~ ~ r~
In O U) O
. , N
WO95/17414 2 1 788 1 1 PCT/US94/13687 --
-- 14 --
It has been flPtc~rminp~l that the antiviral
antisense oligomers of the invention, which are
dLy to a region of H8V plu5 strand DNA
comprising DR2; at least 3 and pref erably at least 4
5 ~ u..~eLv~d 5' flanking nucleotides; and 0-30, preferably
0-20, more preferably 0-lO, and most preferably 4-6 3'
flanking nucleotides, can totally block HBV replication.
"Antisense" is a term that means complementary to the
sense (or plus) strand. An antisense o1;~nn~clPntide
lO interacts in a sequence-sppri~ic manner with a rP11t~1Ar
nucleic acid target containing a sequence ~ ary
to the Ant;CPnce molecule. The ol iqn"~ leotides of the
invention are ~ l dry to the DR2 region of HBV plus
strand DNA and to the ~:ULL ~ iing region of HBV RNA
(which is also of plus strand polarity), and thus
interact with these regions, thereby inhibiting HBV viral
replication. The interaction of such ol ;~n~l~ lP~tides
with their compl ~ry or ~L-3Ct:~U~ sequences mny
result from hybridization interactions, or through other
-ni ~ nc which nre not yet fully understood. The
therapeutic applications of antisense ol i ~on~n1 c-otides
are described, e.g., in the following revlew articles:
Le Doan et al ., Antisense ol i ~nl1~ Potides as Potential
Antiviral and Ant i cAnn~r Agents, Bul l . C~ncer 76: 849--852
(1989~; Dolnick, BJ, Antisense Agents in Pharmacology,
Biochom. Pharmacol. 40:671-675 (1990); and Crooke, Annu.
Rev ph 7. Toxicol. 32:329-76 (1992).
The invention relates to a composition of
matter consisting essentially of an antiviral
3 o n1 i~lnll-~l Pctide, preferably an oligodeoxynucleotide,
having a nucleotide sP~Iu-~ ~æ substantially l 8dLy
to a portion of the plus str~nd of an HBV genome
comprising DR2 plus certain 5' and, optionally, 3'
flanking sequences, preferably both 5' and 3' flanking
35 sequences. Such oligon~lr-l Potides will also be
21 7881 1
Wo 9~117414 PCTIUS94/13687
-- 15 --
complementary to the f ULL~ ~"~ infl RNAs (e.g, - g-^n70r
RNA or genomic RNA), which are of plu5 strand polarity
Because the antisense ol i f3"n--rl ontides are substantially
compl LCILY to the DR2 regiûn of HBV, they are capable
5 of hybridizing to the HBV plus strand under physiological
conditions. Antisense oligonucleotides of the inventiûn
have been shown to be capable of completely inhibiting
HBV replication. Accordingly, the inventiûn also
relates to methods of inhibiting HBV replication in cells
10 cûntaining HBV, incluc~ing methods of preventing HBV
infection in an animal exposed tû HBV, and methods of
treating an animal infected with HBV; such animals
include, for example, humans and other primates, such as
rhi -n700c. The invention also relates to
15 pharmaceutical compositions for use in preventing HBV
infection in an animal exposed to HBV, or treating an
animal infected with BV. Preferred are such
rhArr--~o-ltical compositions formulated for parenteral
administration. Such rhAr~~^e~lti~Al cûmpositiûns will
20 contain an effective antiviral amount ûf an
nl ;gflnl~rlootide of the invention and a rhAr---~eutically
acceptable carrier.
The invention also relates to a composition of
matter consisting essentially of at least one antiviral
25 antisense oligonucleotide substantially cûmpl y to
a portion of the plus strand of an HBV genome comprising
DR2 plus 5' and/or 3' flanking se~r~onre~, preferably
including residues compl~ LaLy to all or substAnt;Ally
all of nucleotides 1588 thrûugh 1606, preferably all or
30 substantially all of nucleotides 1588 through 1608, of
HBV subtype ADW tHPBVADW), or CULL--I'n~'flinfJ nucleotides
of other HBV strains. CULL~ "'fl~nf3 se~lunnroc: for
strains of HBV ûther than BV subtype ADW, e.g., the
strains listed in Table I, infra, as well as other
35 strains of HBV that have been and will in the future be
,
WO 95/17414 PCT/US94/13687
217881 1
-- 16 --
icolated and sequenced, can be detorTnin~d by those of
ordinary skill in the art by Al i~nin~ sequences for
homology, e.g., by using an available database such as
GenBank .
Antiviral ol i g~n~ leotides of the invention can be
supplied to a target cell either e-Luy~ usly as DNA or
RNA, or endogenously, by supplying a DNA sequence from
which the desired ol i~nn~ Qtide may be transcribed by
the target cell. In the latter case, the DNA to be
l0 expressed may be supplied to the target cell, preferably
a hepatocyte, as a recombinant nucleic acid (e.g., a DNA
molecule) comprising a DR2 sequence and f lanking
oligonucleotides, wherein expression of said DNA is
capable of inhibiting viral replication. This nucleic
15 acid molecule is characterized in that it (a) is capable
of being replicated in a hepatocyte under conditions that
normally prevail in the hepatocyte, and (b) is
transcribed in a hepatocyte to produce an oligonucleotide
substantially complementary to a portion of the plus
20 strand of a hepatitis B viral genome consisting of DR2
and ~' and 3' fl~nl-inq sequences. The se~ue..~e
.s _L ibed into the antiviral oligonucleotide is
preferably operably linked to a cell-specific promoter to
direct expression in the hepatocyte. The invention also
25 i n~ a method for inhibiting hepatitis B virus
replication in a cell by introducing the oligonucleotide
molecule itself directly into the cell, or by ill~L odu~. ing
into the cell a nucleic acid which is transcribed within
the cell to produce multiple copies of the antiviral
30 oligonucleotide as an oligoribonucleotide.
D~rRTPTIQN OF SPECIFIC FMR~nTMP'NTS
Methods and oligonucleotide compositions are
provided for the inhibition of viral replication.
WO95/17414 2 1 788 1 l PCr/US94/13687
-- 17 --
The antisense oligonucleotide itself may be
provided ~xuut:nuusly to a host cell inf ected with the
virus or susceptible to viral infection. Another
approach, however, is to provide for expression of the
5 antiviral ol i ~on~1~ l eotide in the host cell . In such a
method, a DNA transcribable into the antisense
oli~on~ leotide of the invention is inuur~uLt.Led into an
expression vector ' ~ am from, and operatively linked
to, a suitable promoter which provides for tissue
lO specif ic or general expression . To treat or prevent
viral hepatitis, the DNA of the invention may be placed
'- LLe alu from a liver specific promoter, in order to
induce expression by hepatocytes in the liver; however,
it may not be n~r~ S:~ry to use a liver-specific promoter,
15 since expression of the antisense olig~n~cl~otides in
non-liver cells should be harmless to the cells. The DNA
with the ~ u~L iate regulatory regions is provided in
proper orientation to allow for expression. Methods for
constructing such expression vectors are known in the
20 art. See in particular, M~lecql7~r Cloning, A Laboratory
Manual, Sambrook et al., eds., Cold Spring Harbor
Laboratory, 2nd Edition, Cold Spring Harbor, NY (1989).
A wide variety of transcriptional regulatory
sequences may be employed. The signals may be derived
25 from viral sources, such as adenovirus, bovine papilloma
virus, simian virus, or the like, where the regulatory
signals are associated with a particular gene which has a
high level of expression. Alternatively, ~1 Lt!-~ from
l i An expression ~Ludu~ such as actin, col l A~n,
30 myosin, etc., may be employed.
The expres6ion of the HBV DNA in eukaryotic hosts
reSluires the use of eukaryotic regulatory regions. Such
regions will, in general, include a promoter region
sufficient to direct the initiation of antisense RNA
35 synthesis. Typical promoters include the promoter of the
WO 95/17414 PCT/US94113687 ~ -
2178~
-- 18 --
mouse metallofh;~nino I gene (Hammer, D. et al., ,J. Mol.
Appl. Gen. 1:273-288 (1982) ); the Tk promoter of herpes
virus (McXnight, S., Cell 31:355-365 (1982) ); the SV40
early promoter (Benoist et al., Nature 290:304-310
5 (1981) ); and the like. Other useful promoters include
liver specific promoters such as albumin, alpha-
fetoprotein, alpha-l-antitrypsin, retinol-binding
protein, asialoglycoprotein receptor, and viral promoters
and onh~nt~ors such as those of cyt~ virus; herpes
10 simplex I and II viruses; hepatitis A, B, and C viruses,
and Rous sarcoma virus (RSV) (Fang, X.J. et al.,
~epatology 10:781-787 (1989) ) . Such liver-spo~1fic
promoters are expected to be particularly useful when a
DNA sequence of the invention is placed in a vector which
15 is capable of transforming hepatocytes. The gene is
placed ~ ~ ~Leam from a suitable promoter which provides
for tissuQ specific or general expression. The DNA will
be ~L-~s~;Libed to produce RNA which will hybridize with
its target RNA, thereby inhibiting viral replication.
20 One particular vector useful for this task would be one
based on an adenoviral system, such as described by
Morsey ôt al., Abstract SZ 109, "Efficient Adenoviral
Gene Transduction in Human and Mouse Hepatocytes In V~ tro
and in Mouse Liver In Vivo", in J. of C'~o77~7~r
25 Riochomictry, S~rl 17E, Keystone Symposia on
Molecular and ~olll~lAr Biology, March 29-April 25, 1993,
or alternatively, a retroviral vector such as LNL6, a
derivative of the Moloney murine leukemia virus, as
described in R~conhorg et al., N. Eng. J. Med. 323, No.
30 9:570-578 (1990). Gene transfer into h~ o-;y~es using a
defective Herpes Simplex viral vector is described by Lu
et al., Abstract, page 66, Abstracts of the lIeeting on
Gene Therapy, Sept. 22-26, 1992, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York. This latter
35 approach is oRpe~iAlly valuable for introducing gene8
WO 95/17414 2 1 7 8 8 1 I PCTNS94/13687
into non-dividing hepatocytes, since HSV-l does not
require genomic integration for expression. The DNA and
the requisite regulatory elements may also be i~L~Lduced
into hepatocytes using an asialoglycoprotein carrier
5 system as described in Wu et al ., Biother~py 3: 87-95
(1991) .
The desired viral DNA and operably linked promoter
may be i,,L--,duced into a recipient cell either as a non-
replicating DNA or RNA --lec~le, which may be a linear
10 molecule or, more preferably, a closed covalent circular
molecule. Since such molecules are i n -~r~hl~ of
au~- replication, the expression of the desired
receptor molecule occurs through the transient expression
of the illLL ~Iduced sequ-n~~e . Where more long-term
15 expression is desired, the sequence may be integrated
into the host ~1,1 ~ . Alternatively, the i~L~duced
sequence may be inuuL~u-~-ted into a plasmid or viral
vector capable of autnn ~ replication in the recipient
host; such vectors include the cDNA expression vectors
20 described by Okayama, H., Mol. Cell . Rio. 3:280 tl983),
and others. Viral vectors include retrovirus vectors as
taught in W089/07136 (specifically for expression in
h~=~ato~;y Les) and the references cited therein.
In the methods of the invention, either the
25 i~nt;~ n~e oligon~ leotide itself, or a DNA which is
LLc.ns~LLbable into the antisense RNA of the invention, is
i-ll.L~duced into the cells of an animal surfering from the
viral disease.
As used herein, "substantially complementary"
30 means that an antisense ol ignn~lcl~-ntide of the invention
is capable of hybridizing with its RNA or DNA target
under physiological conditions. HBV nucleotide seqn~n~e
numbering herein is made with ref erence to the numbering
of Hepatitis B Virus (Subtype ADW), according to GenBank.
35 cuLL--lJu~ n~ sequences for HBV strains and subtypes,
WO9S/17414 2 1 7 8 8 t I PCT/US94113687
-- 20 --
other than HBV subtype ADW, e.g., the strains listed in
Table l, infra, as well a6 other strains of HBV that have
been and will in the future be isolated and sequenced,
can be det~i n~ by those of ordinary skill in the art
5 by aligning sequences for homology using an available
database such as GenBank. This allows for selection of
5~ c specif~r for non-human r~-mTA~l iAn species or for
human HBV strains prevalent in particular populations or
y~ Lc~Lhic areas. Alternatively, ~ C.LL- cl,.. llng c~ncF~n-uc
10 or ;u~seLved sequences having broader /~rPl ir~Ahi 1 ity may
be readily detl rminPd by comparing CULL~ L .1~1in~
se~ue~ es from multiple HBV strains. It is contemplated
that such cuLL~ in~ sequences are f1-nr~ionAl
equivalents of the Se~"ue~1Ce for ADW and ADWl.
The methods and oligonucleotide compositions of
the present invention can be utilized to prevent viral
infection as well as to treat viral infections. The
compositions comprising vectors containing nucleic acids
transcribable into the anti-HBV oligonucleotides of the
20 invention may be administered to prevent a virus
inf ection or to combat the virus once it has entered the
host .
As used herein, "consisting essentially of" has
its usual meaning, i.e., that one or more compositions of
25 matter of the invention may be used together, either in
admixture or ~ in~d in a single lec~le, with other
materials that do not alter the essential nature of the
invention. For example, while the antisense
ol i ~ m1rleotide sequences of the invention are essential
30 to the invention, it is contemplated that they may be
used in admixture or in rh~Tnic~l combination with one or
more other materials, including other oligonl~rl~otides
antisense to other portions of HBV RNA; materials that
increase the biological stability of the
35 olig~n~lrleotides; or materials that increase their
WO95117414 21 7 8 R 1 1 pcrlus94ll3687
ability to pen~LL~Le selectively their hepatocyte target
cells, and reach and hybridize their target RNA.
- Furth. ~, it is r~co~ni7~cl that ol i~nnllrl~otides may
be modified to achieve greater stability, inr~ 1ng
5 b~rl~hnn~ modif ications such as rhns~hnrothioates,
methylll~n~lh~ ~ -tes, r~ ,dithioates,
E~ n_l h- ~ ~a idates, phosphate esters, and other
modifications as described in Uhlman and Peyman,
Antisense Olignnllrleotides: A New Therapeutic Prinr1rle.
10 Chemical Reviews 90 (4) :544-584 (1990), at 546-560, the
t~Arhing of which is hereby inCoL~)uLa-ted hy reference.
All such modif ications are contemplated eguivalents of
the antisense oligonucleotides of the invention.
The following ~liccllcclnn provides eYamples of the
15 kinds of modifications that may be employed, but those of
skill in the art will readily rC~co~ni 7e others. For
eYample, the antisense ol ignnllrleotides may be provided
in stAhi 1 i ~ form, e.g., with phosphotriester 1 ~nkA~c-c,
or by hlorkin~ against ~Ynnllrl~Ace attack with
20 methylrh~,7lk~ cter linkA~-~c, with 3' deoYythymidine,
as a phenylisourea derivative, or by linking other
molecules such as Ami nnA~ridine or polylysine to the 3 '
end of the olig~n~lrl~otide. See e.g., AntirAnr~r
Research 10:1169-118Z, at 1171-2 (1990), the t~Arhin~ of
25 which is incoL~,Lc.ted herein by reference. For antisense
oligonucleotides supplied ~ g~ u51y~ increased
selectivity for hepatocytes may be achieved by linking
antisense oligonucleotides of the invention to natural
ligands such as ASOR (ACiAln~r~- coid) or to synthetic
30 ligands that will bind to the hepatic asialoglycoprotein
(ASGP) receptor. See e.g., BiochemistrY 29, No. 43
(1990), Spiess, "The Asialoglycoprotein Receptor: A Model
for Endocytic Transport Receptors". See also Wu and Wu,
J. B~ol.Chem. 267. No. 18:12436-12439 (1992), reporting
35 inhibition of HBV viral gene eYpression and replication
Wo 95/17414 PCT/U594/13687--
21 7881 ~
-- 22 --
in HepG2 cells by a 21-mer oligonucleotide complementary
to the H}3V polyadenylation slgnal. The nl i,~ was
complexed to a (poly)L-lysine-asialouL-_ ~ id ~u1~juu~te
that targets the asialoglycoprotein receptor Or
5 hepatocytes . In another ` ' i - L, ribozymes Day be
targeted by linking to an r~ J-m~ tide o~ the
invention, since there are a number of ribozyme target
cleavage sites in the DR2 region of the viral RNA. See
Q.g., Von 1'- i c7~e~ r F, Blum HE, Wands JR, ThreQ
10 ribozymes LLc.r,s~,Libed from a ~:ingle DNA template
efficiently cleave hepatitis B virus y.~ RNA,
8~ochem. Biophys. Res. Commun. 189:743-748 (1992). The
~rhin~ of the foregoing references is inc..~.~,Lc.ted
herein by reference.
In general, a high efficiency, cell-specific
delivery system for in vivo th_Lc~ u~ic use may utilize a
number of approaches, in ll~in-, the following: l)
specif ic delivery through hepatocyte specif ic r . .a ~Lu-
mediated process such as the asialoglycoprotein ~e~ LuL,
20 as rl;~ --ccecl abovQ; 2) delivery of antisense
gl~g9~ ryl1ucleotides in 1 ir with or without
specific targeting with - ~~lonA1 antiho~ c directQd
against specific cell surface receptors; 3) rQtrovirus-
mediated transfer of DNA expressing the antisense
25 ~;u~-~LLuuL of interest; and 4) direct targeting to cells
of antisense oligodeoxynucleotides following c~..juycltion
to ~lon~l antiho~ c that are speci~ic for cell
surface receptors that function in a L~ LUL~ ted
endocytotic process; 5) specific delivery to hepatocyte_
30 via a replication-defective HBV vector. To modify
hepadnaviral infection in vivo, a hepato~yLc ~ecific
delivery system whereby substantially all hepatocytes are
provided with an effective amount of the antisense
construct will probably be required.
WO 95/17414 2 1 7 8 8 ~ ~ PCTiUS94/13687
-- 23 --
The antisense compositions of the invention may be
administered as individual therapeutic agents or in
combination with other therapeutic agents. They can be
admini6tered alone, but are generally administered with a
5 pharmaceutically acceptable carrier selected on the basis
of the chosen route of administration and standard
rhA -outical practice. The dosage administered will of
course vary ~opont3in~ upon known pharr- -' inotic/
rhArr-~-odynamic characteristics of the particular agent
l0 and its mode and rout~ of administration, as well as the
age, weight, and health (inr~ l;n~ renal and hepatic
function) of the recipient, the nature and extent of
disease, kind of cu..;uLL~ therapy, frequency and
duration of L.eai , and the er~ect desired. Usually a
15 daily dose of active ingredient can be about 0 . l to l00
mg per kilogram of body weight. Ordinarily 0 . 5 to 50,
and preferably l to l0 mg per kg of body weight per day
given in divided doses or in sustained release form
(inrl~ ;n~ sustained i11LLav~l~u~ls infusion) will be
20 effective to achieve the desired ef~ects.
Dosage forms suitable for internal administration
generally contain about l milligram to about 500
milligrams of active ingredient per unit. The active
ingredient will ordinarily be present in an amount of5 about 0.5 to 95~ by weight o~ the total pharr--o-1t~
~ar ~tion. It is oYrected that the antisense
ol i ~nn--nl ootide compositions of the invention mày be
administered parenterally (e.g., i1-LLav~11uusly,
preferably by i11LL~v.,huus infusion). For parenteral
30 administration, the compositions will be formulated as a
sterile, non ~y uy~:nic solution, suspension, or 1~ n.
The preparations may be supplied as a liquid formulation
or lyophilized powder to be diluted with a
rhArr~~eutically acceptable, sterile, nU.1 ~y-o~nic
35 parenteral vehicle of suitable tonicity, e.g., water for
.
Wo 9S/174~4 PCr/US94113687 `
2~ 7881 ~ 24 -
injection, normal saline, or a suitable sugar-containing
vehicle , e . g ., D5W , D5 / 0 . 4 5 , D5 / 0 . 2 , or a vehicle
containing mannitol, dextrose, or lactose. Suitable
rhA~-~e~ltical carriers, a6 well a6 rh~ eutlcal
5 necessities for use in rh~rr ~eutical formulations, are
described in ~emington~s ph~ tiC~ S~tF~n~-~c, a
standard reference text in this field, and in the USP/NF.
The oligonucleotides or their derivatives may also
be administered in 1 i, - or microspheres
10 (microparticles) . Methods for preparing 1 ~ and
microspheres f or administration to a patient are known to
those 6killed in the art. U. S . Pat. No. 4, 789, 734
describes methods f or ~n -~rs~ ting biological materials
in 1 i roc~ - . The material is dissolved in an aqueoUc
15 solut1on; the appropriate rhnCrholirids and lipids added,
along with surfactants, if required; and the material
dialyzed or sonicated, as n~ c~ . A review of known
methods is provided by G. Gregoriadis, Chapter 14,
~T ;r - -", Drug carriers in ~iology and M~i~inF~, pp.
20 287-341 (An~ Tnin Press, 1979). Microspheres formed of
polymers are well known to those skilled in the art, and
can be tailored for passage through the gastrointestinal
trAct directly into the bloodstream. Alternatively, the
nl il; ~ or their derivatives can be in ~oL~JuLclted into
25 mi~:Lo~ ras, and implanted for slow release over a
period of time . See, e. g ., U . S . Patents Nos . 4, 925, 673
and 3,62S,214.
The following examples are offered by way of
illustration and not by way of limitation.
3 0 E~aliPL~ I
~rr~n~:fection of HuH 7 cells
HuH 7 hepatoma CQllS (Cancer Research 42: 3858-3863
(1982) ) were seeded into 6 well plates (35mm/well) and
grown to 70-90% confluency in Dlllhecco's Minimal
21 7881 1
WO 95117414 PCTIUS94/13687
-- 25 --
Essential Medium supplemented with 10% fetal bovine
serum. Cells were transfected according to a
modification of the ~.va~luLe described by Chen and
Okayama, Nol.Cell. Biol. 7:2745-2752 (1987). [The
5 modiflcation in this yLvceduLc~ was to incubate the cells
in an ai ,l ~ of 5% CO2 throughout the experiment, as
opposed to incubating the cells in a lower C02 Clt. ,' ~s
during the actual transfection step. ]
Cells were transfected in duplicate with 35 to 50 ng
10 replicatiu.. _ _tent HBV plasmid ~ oll,.LLu. ~ corltAin;n~
HBV DNA sequences thead-to-tail dimer of HBV DNA ~BsAg
subtype adw in pGEM-72f (+) (Promega) ], which was obtained
from J. Wands, MGH, Boston [see Blum et al., J. Virol.
65(4) :1836-1842 (1991) ]. Cell supernatants were
15 harvested post-transfection on a daily basis for up to
6 days, and cells refed daily with 2mls of medium/well.
The Du~L..at~nts were stored at 4 C until assayed for
the presence of HBsAg using the Auszyme M~noClonAl
Diagnostic Kit, a v ially available enzyme
20 i - y from Abbott LabvLc~)Lies, North Chicago, IL.
HBsAg is a marker for viral replication, and the ability
of ol i~n~ leotide to block HBsAg pro~ cti-~n indicates
inhibition of viral replication.
oli~onucleotide8
25 The following ol i~ y............. ucleotides were
synth~ci ~e:l in a Milligen Biosearch 8750 DNA synth~ r,
using ~c~ ethyl-rhosrh~lramidite syntheses (~retr~hedron
Lett 22: 1859-1862 (1981) ):
Oliao ID~ }Isv lOOll
3 O Do~cr i .~ t r~n DR2 ~ntL~nn~
~: S' CAI~ CGT GCA GAG GTG AAG CGA 3' ~SEQ ID NO: 6)
WO 95/17414 PCT/US94/13687
2178811
-- 26 --
Al nA Tn~ IIBV 10012
De~crir~t; A ~ DR2 cramble of ant$sem~e
Seauence: 5' AGC GAA GTG AGG ACG TGC AAC 3' (SEQ ID N0: 7)
Ol$ao ID~ CP 10053
5 Descrl~tion DR1 ant$sense
Soauonce: 5 ' TTA GGC AGA GGT G AA Ar~A GTT 3 ' (SEQ ID N0: 8)
O~$ao IDt CP 10052
DesCrl~t$on DR1 scramb1e of antisense
seauenco: S' ATC GGA GGA TGG TTA AAT GAA 3' (SEQ ID N0: 9)
10 Puri~ication of Oli~vdeo~Y..~cleotides.
Ant;~-n~:e and 5-.~L '~ 01 i~o~c~oxynucleotides were
purified after NH40H de~ L (55C, 6 hours) and NAP 25
column tPhA~--iA) desalting with 0.1 M NaHC03 by rever6e
phase HPLC (trityl on, TEAA 0.1 M, pH 7.25/acetonitrile
15 gradient) . The r~ u~ yll~cleotides were lyorh i l 10~,
deblocked with 1 M acetic acid for 1 hour, neutralized
with 1.0 M NaHC03, passed through a NAP 10 column, and
then lyophilized to dryness.
Results
HuH 7 hepatoma cells transfected wit_ 35-50 ng of
pla~:mid containing HTD HBV subtype adw2 genome, and
treated with 8 ~Lg/well of the DR2 antisense
oli~rn~rleotide HBV 10011 (SEQ ID N0: 6) at the time of
transfection, produced significantly less BsAg over the
25 six day test period than did control ~Le-.-are~;~ed cells
not treated with an anti6ense oli~nv~rl~otlde. In
contrast, the DR1 antisense ~ n~rleotide CP 10053 (SEQ
ID N0: 8 ) did not cause a detectable decrease in the
5- lAtion of HBsAg, compared to control supernatants
30 ~rom transfected cells untreated with oligrn~rl-~otide.
The two scrambled antisense oli~-~n~cleotides, HBV 10012
WO 9Sl17414 2 1 7 8 8 1 1 PCTIUS94/13687
-- 27 --
(SEQ ID NO: 7) and CP 10052 (SEQ ID NO: 9) slightly
decreased the amount of HBsAg present in ~-u~e...ata..~
The obs~- v~d difference in the activity of DRl and DR2
antisense oli~nr~llr]eotides is surprising cnnR;~ring that
5 DR1 and DR2 share 11 identical nucleotides and both play
a critical role in HBV replication. Furth~ e:, DRl
appears to be involved at an earlier step in HBV
replication, since synthesis of the first DNA strand is
believed to be initiated within DRl.
It is thought that the DR2 antisense
oligon~rl Pntide may be acting at any of several poCci hl~
levels . It may bind to and have an antisense ef fect on
the HBV 3.5 kb ~l~g~ ;c RNA, the HBV 2.1 kb mRNA and/or
the HBV 2 . 4 kb mRNA. The DR2 R-~q~ nre is present in both
15 the polymerase and the X protein ORFs. The antiviral
olignn~rl ~otides might also bind to the
oligori hn~ rleotide primer generated during HBV
replication, thereby preventing oligori hnmlrl eotide
priming at the DR2 site on minus strand DNA and
20 inhibiting plus strand DNA synthesis. It should be
re~o~n i 7~1, however, that ol i; ~ of the invention are
also complementary to and are capable of hybridizing with
plus strand HBV DNA containing DR2. While the nucleotide
sequence of this region is highly CO~I86 L v~d amoung
25 different HBV strains, a finding which suggests that
nucleotide sequence and its function in viral replication
may be important, secnn~lAry and tertiary LLLu-_LuL~:s may
also be important with respect to interaction with
protein. The invention as claimed, however, is not
30 predicated on any particular qniFr or ~ni,
through which the DR2 ol i~nnllrleotides act to block HBV
replication .
DRl and DR2 share 11 identical nucleotides, but
have dif f erent f lanking s~qu~nr~q . Flanking regions of
35 both DRl and DR2 are also conserved among various species
Wo 95/17414 PCr/US94/13687
21 7881 1 - 28 -
and strains of HBV, indicating the importance of these
regions in HBV DNA replication. Noreover, flanking
soTlonre~ of DR2 of many human and w_,o~ HBVs contain
a two-fold ~y y, making the majority of DR2 sequence
5 and the 5 ' -f lanking region of DR2 into a single--stranded
loop. A putative secondary l.L-u-;Lu~e of the DR2 region
o~ HBV DNA is shown in Fig. 2, using a subopti_al RNA
folding program (see Jaeger, JA, Turner, DH, and Zuker,
M, Proc. Natl. Ac~d. scl. USA 86:7706 (1989) ) . However,
10 8ecnn~l~ry ~-L-u~i~u~ e evaluation of the two DRl sequences
in the ~ ; r RNA suggested that one of the DRl
sequences might also be found as a single stranded loop.
Nevertheless, as described above, DR1 antisense did not
decrease HBsAg acc~ tion.
The ~.u~.osed -- ~niom of action of antisense
ol; rJonllrl eotides requires hybridization of an
ol i ~nm~r~ ontide to its complementary sequence in the RNA
target. Therefore, for an antisense oligonucleotide to be
effective, the _ 1~ Lary target sequence must be
20 available for hybridization. In most cases, target mRNA is
not single-stranded random coil, but rather contains
sor~n-l5~ry and tertiary ~,LLu~Lu.cs. Target RNA :-L-u- Lu-~ has
been shown to affect the affinity and rate of
nl i~nnl~rleotide hybridization, as well as the efficacy of
25 antisense oligonllrl~ntides [See Yoon, R. Turner, D.H., and
Tinco, I, Jr., J. Mol. E~iol. 99:507 (1975); Freier, S.M.
and Tinoco, I., Jr., Rirrhomictry 14:3310 (1975);
rrhlonhorll, O.C., J. Molec. Biol 65:25 (1972); Herschlag D.
and Cech, T.R., Riochomi~:try 29:10159 (1990); and Fedor, M.
3 0 J . and Uhlenbeck . O . C ., Proc . Natl . Acad . scl . U . S . A .
87:1668 (1990) ] . Thus, when designing antisense
oligon~~rlontides it may be helpful to r~nn~ o~ mRNA
structure and the potential influence of this ~-~u~,Lu~: on
oligonucleotide hybridization. For example, it was recently
35 reported that the tightest binding of antisense
W095/17414 2 1 788 ~ 1 PCT/US94/13687
-- 29 --
n~ 1eotides occurs at target sites for which
disruption of the target stucture was minimal (see Lima,
W . F ., Monia , R . P ., Ecker , D . J . and Freier , S . N .
Bioche~istry 31:12055 (1992) ) . Thus, cnnci-9~ation of
5 the target seC~n~:~ry nLLU~LULe ~uyye~Ln that single-
stranded regions should be ~ ct~d over double ..L.~ ed
regions. However, not all singlQ --LL~I.ded regions
exhibit nLL -;LUL~8 favorable to hybridization (e.g., the
single-stranded portions of loops). It was reported that
10 oligon~cleotides ~ 1~ Lary to the 5 ' side of the
single-stranded loop Or kras RNA may exhibit tighter
binding affinity ~ - ed to the ol i~nllrl~otides
complementary to the 3 ' side of the loop (Lima et al .,
supra). However, tat and tar regions of HIV mRNA, which
15 also contain a single-stranded loop, did not exhibit any
preferential binding to the 3 ' side of the loop tEcker et
al., Science 257:958-961 (1992) ] .
Based on the current state of knowledge, target
sites for antisense attack must still be det--rmin~A
20 exper~r ~1 ly, although the ~L~ser.Oe of a single-
stranded loop in a region of mRNA may indicate to a
logical point for research. However, studies of putative
RNA ~c~n~lAry nLL~ UL~ may provide insight into the
results that have been obtained experimentally.
The oligonucleotides were tested for nnn~;rQ~i fiC
cytotoxicity by means of the N~rT assay. ~he assay is
t on the reduction of the tetrazolium salt NTT
( 3 - ( 4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyl-2~-tetrazolium
bromide) by the mito 1.~ ial l.ydL~yel~ase of viable cells
30 to form a blue formazan product (D. Gerlier et al., J.
T ~1. Methods 94:57-63 (1986); D.S. Heo et al., Cancer
~ç~ 50:3681-3690 (lsso) ) . This assay measures cell
respiration and the amount of formazan ~L~ ced is
proportional to the num~er of living cells in culture.
35 HuH 7 cells incubated in the ~L~sel,~.e of an
WO 95117414 2 l 7 8 ~3 l l PCr/US94/13687
-- 30 --
oligonucleotide of the invention or a scrambed control
exhibited a reading in the MTT assay similar to that
observed with HuH 7 cells incubated without addition of
oligonllrlentide. Thus, the decrease in HBsAg expression
5 observed with the oligonucleotides of the invention
appears to ~e a specif ic inhibition, not a general
cytotoxic effect of the ol irJQnllcleotide
lssalsP},13 2
In order to elucidate further the structure-
10 activity relatinnRhirs of antisense olignnl~r~eotides
~ 1 ~ Ary to plus strand HBV DNA in the DR2 region, a
number of additional olignucleotides were syntheslzed and
tested for antiviral activity in the manner described
above .
15 OliaonucleQtides
The following oligodeoxynucleotides were syn~hP~i ~od in a
Milligen Biosearch 8750 DNA synthPci z-~r, using
asialoethyl-rhnsrhnramidite syntheses (TQtr7hedron Lett
22: 1859-1862 (1981) ):
20 Oliao IDJ~ CJP 114
Descri~tion DR2 antisense
Seauence: 5 ' CGA CGT GCA GAG GTG AAG CGA 3 ' ( SEQ ID N0:
10)
Oliqo ID~ CJP 113
25 Descril~tion DR2 antisense
Seauence: 5' TGC AGA GGT GAA GC 3' (SEQ ID N0: 11)
0~ ~ ~o In~ CJP 112
Descri~tion DR2 PnticPnRP
Seauence: 5 ' TGA AGC GAA GTG CA 3 ' ( SEQ ID N0: 12 )
217~81 1
WO 95/17414 PcrluS94113687
-- 31 --
Oliao ID~ CJP lll
Descril~tion DR2 antisense
Seauence: 5' GAG GTG AAG CGA AG 3' (SEQ ID NO: 13)
Oliqo ID~ CJP ll0
5 DescriPtion DR2 antisense
Seauence: 5' GGT GAA GCG A 3' (SEQ ID NO: 14)
Oliao Tn~ CJP l09
Descri~tion DR2 antisense
Seauence: 5' CGT GCA GAG GTGAAG CGA AGT 3' (SEQ ID NO:
l0 15 )
Oliao ID# CJP 108
Descrintion DR2 antisense
Seauence: 5' CGA CGT GCA GAG GTG AAG CGA AGT 3' (SEQ ID
NO: 16)
15 Oliao IDJ~ CJP lOl
Descri~tion DR2 antisense
Seauence: 5 ' GGT GAA GCG A 3 ' ( SEQ ID NO: 17 )
Oligo ID~ CJP l00
Desc~i~tion DR2 antisense
20 Seauence: 5' CGA CGT GCA GA 3' (SEQ ID NO: 18)
RESULTS
Since DR2 antisense oligodeoxynucleotide HBV l00ll
(5' CAA CGT GCA GAG GTG AAG CGA 3'; SEQ ID NO: 6) e~chibited
strong anti-EIBV activity, a 6eries of other
25 oli~u~ev--y--ucleotides complementary to the DR2 region of
plus strand HBV DNA were 5ynt-hc~ci~ and tested in the
manner described above. Originally, a series of DR2
antisense oligonucleotides was ~l~Ci ~n-~d ~ased on the
putative sec~n~ ry structure of the DR2 loop region (Flgure
Wo 95/17414 PCT/US94/13687
2 1 7 8~
-- 32 --
1), with the rationale being to target antisense
olignn~r~ ntides to the putative single-stranded region
of the DR2 loop, which may be favorable for hybridization
in a manner similar to H-ras mRNA (Lima, 1992). The
5 antiviral effect of each of these ol iqnnllcl~otides i5
shown in Table 2, and tliCCIICC~-7 below.
HBV lO011 is a 21-mer consisting of nucleotides
complementary to the entire eleven-nucleotide DR2 as well
as four 5' flanking nucleotides and six 3' flanking
10 nucleotides. The oligodeoxynucleotide designated CJP 114
is a 21-mer differing from HBV 10011 only with respect
to a single nucleotide at n.t.n. 1607 (numbering relative
to HPBV ADW from GenBank). Comparing the DR2 region of
various HBV strains using GenBank revealed a nucleotide
15 change from T in HBV ADW to C in HBV ADN1 and HBV ADRCG.
CJP 114, which contained a G rather than an A at n.t.n.
1607, is complementary to the plus stand of the HBV ADWl
strain. Although the assay for inhibition of HBV
replication employed in these experiments used an HBV
20 ADW2 strain, which has a T at n.t.n. 1607, CJP 114
nevertheless blocked replication of HBV ADW2.
Oligodeoxynucleotide CJP 108 is a 24--mer,
differing from CJP 114 in that it has an additional three
nucleotides complementary to the DR2 5' flanking region.
25 Surprisingly, CJP 108 does not inhibit HBV replication.
Similarly, CJP 109, a 21-mer lacking (relative to
CJP 108) three nucleotides from the DR2 3' fl; nl~;n~
region, does not inhibit HBV replication.
01 ignnl~r-leotide CJP 126, having the sequence of HBV 10011
30 with a blo~ n~ group at the 3 ' end, also exhibited
decre~sed antiviral activity. This provides further
evidence that f lanking sequences or groups at the 3 ' -end
of the HBV 10011 sequence may be detrimental to antiviral
actlvity .
WO95/17414 2l 881 l PCr~S94113687
It appears that nucleotide& compl l clr y to the
entire DR2 region as well as several nucleotides from
both the 5' and 3 ' flanking regions are required for
antiviral activity. Oli~;deu~.yllucleotide CJP 113, a 14-mer
5 containing nucleotides _ 1~ Lary to the entire DR2
but only two nucleotides from the 5' flanking region and
a single nucleotide from the 3' flanking region, did not
block HBV replication. CJP 111, containing nucleotides
1- Lary to eight of eleven of the DR2 nucleotides
10 and six nucleotides from the 5' flanking region, did not
inhibit HBV replication. CJP 101 and CJP 110, both 10-
mers complementary to 6iX of the eleven DR2 nucleotides
and f ive 5 ' f lanking nucleotides, did not inhibit HBV
replication. CJP 112, a 14-mer containing nucleotides
15 complementary to four of eleven of the DR2 nucleotides
plus ten nucleotides from the 5' flanking region, did not
inhibit HBV replication. CJP 100, an 11-mer
compl~ Ly to five of the eleven DR2 nucleotides and
5iX 3 ' f lanking nucleotides, did not inhibit H~3V
20 replication.
Surprisingly, antisense oligon~ eotides
to hybridize to a putative loop region of the DR2 region
(CJP 111 and CJP 110) and antisense oligonucleotides
designed to hybridize to a putative loop and either 5 ' or
25 3~ flanking region (CJP 113, CJP 109, CJP 108, CJP 112)
did not exhibit antiviral activity. However, HBV 10011
and CJP 114, which contain a single nucleotide
difference, did exhibit antiviral activity. This
suggests that two nucleotides at the 5 ' end of antisense
30 Ol i~r~n~l~ leotides HVB 10011 and CJP 114 may not be
important for antiviral activity. To test this
hypothesis, ollg~nl~lPotide CJP 140, itlPntic~l to HBV
10011 and CJP 114 except that the two 5 ' nucleotides of
the latter were deleted, was synthesized and tested. CJP
35 140 was found to inhibit _ let~Ply HVB replication.
W095117414 PCTIUS94113687--
21 7881 1 34 _
Accordingly, it appears that more than ~our 5' flanking
nucleotides are l~nnf.--~C~c::~ry for full antiviral activity.
While up to twelve, preferably only up to six, 5'
flanking nucleotides may be included, oligomers having
5 only up to f our 5 ' f lanking nucleotides are more
preferred for ease of synthesis and rh~r~--e~tical
delivery .
To further elucidate these :>~LU- LUL~ ~ctivity
relati-~nchirc, ol;~nm~lP~tide HBV 1018 (SEQ ID N0: 19),
10 which is an 11-mer, 1~ Lary to the DP~2 sequence
alone, without either 3' or 5' flanking se~ c, was
syn~h~ci 7ed and tested. It failed to inhibit HBV
replication .
Oligon~lc~entide CJP 150 (SEQ ID N0: 20), which is
15 identical to CJP 114 except for a single nucleotide
deletion at the 3 ' end, reduced the amount of HBsAg in
transfected cell supernatants to 59i of levels found in
supernatant6 of cells transfected with the HBV plasmid
DNA alone. Since Ql;gnn~ leotide CJP 150 exhibits a
20 slightly decreased antiviral activity relative to
ol ig~n~ otides HBV 10011 and CJP 114, it appears that
the A residue complementary to nucleotide 1588 of the HBV
genome may be n ~ ~cs~ry for full antiviral activity. A
modif ied oligonucleotide having the s~ e 5 ' -
25 ACGTGCAGAGGTGAAGCG-3' (SEQ ID N0: 21) would also be
expected to have antiviral activity.
Thus, only oligonucleotides targeted to a narrow
region of DR2 sequence exhibit antiviral activity. These
experimental results appear in Table 2 below. There
30 appears to be no apparent correlation between the
putative secnn~l Iry structure (Fig. 1) and antiviral
activity obs~. v.:d experimentally. However, not only RNA
structure but also its interaction with proteins may be
important f or antiviral activity . This must be
3 5 determined experimentally .
Wo95/174~4 2, 7 8 8 1 1 PCT/US9~113687
PrPl iminAry evidence indicates that the
oligon~rl~otides of the invention exhibit speci 1~i c
antiviral activity against HBV since these
oligonucleotides are non-toxic in a mito~ ial enzyme
5 assay and there is no evidence, by gross morphology, of
toxicity to hepatocytes in culture.
W09S/17414 PCT/US~4/13687--
- 36 -
21 7881 1
_ _ _ ~ ~ ~ ~ _ _ _ _ _ _
N 0 _ O .:r O tll r~ D N 0 ~` 0
.. .. .. .. .. .. .. .. .. .. .. .. .. ..
2 ~ z; ~2: z z z ~ Z Z
a a a a a a a ~ a a a a a a
H H H H H H H H H H H H H H
~ ~ a ~ a a ~ ~ ~ CY a ~ a ~
r~
¢ ~ U 3 ~¢
~, . . .
-- -- ' ' ' . . , ' ~ rS
...,,,,, . .¢
,s ,s ,s , ., ,s
u~ 3 , rS
..U~ . '
~- a
N .. H
0 -- o _ _ _ _ _ _ ~ O
_ _ O ~ --I
Q ~0 H ~'
Gl C~ O
-- z o ~r o o ~ 0 ~ ~ o
~1 o In o ~1 0 0 ~1 _~ O O
In O U~ O
W095/17414 2 ~ i 8~ 1 ~ PCTiUS94/l3687
-- 37 --
The experimental results presented in Table 2
indicate that a region comprised of DR2 plu8 f lanking
SDq~Qn~c is i~ UL ~ t for the normal function of E18V.
Oligonucleotides HBV 10011 and CJP 114, both of which map
5 to nucleotide positions 1588 to 1609 inclusive of the
hepatitis B genome (numbering relative to HPBV ADW from
GenBank), prevent the A< l ~tion of detectable levels
of BsAg in cell supernatant. HBV 10011 and CJP 114
differ from each other only at position 1607, with BV
10 10011 containing an A and CJP 114 containing a G. This
suggests that basepairing between the antiviral
oligr~n~ eQtide and the viral nucleic acid at position
1607 may not be n~C~ssAry for the antiviral effect seen
experimentally. Accordingly, an ol iq~nl~le~tide with the
15 s~q~ nl~e 3' AGCGAAGTGGAGACGTGCA 5' (OLIGO CJP 140; SEQ ID
NO: 24), was synth~C; 7~d and found to have an antiviral
effect.
Antiviral ol ~gorlurleotides CJP 101, which
l,OLL~DlJU~ s to positions 1588 to 1598, does not inhibit
20 HBV surface antigen expression. This indicates that some
or all of the nucleotides mapping from positions 1599 to
1609 are essential for an antiviral effect.
ol iqr~n-~ ltide CJP 100, which cu-. ~D~o~lds to position
1599 to 1609, has no effect on the A~ - lAtion of
25 Hepatitis B surface antigen. This indicates that some or
all of the nucleotides uuL~e~y~ n~ to posltions 1588 to
1597 are also critically important for the inhibition
effected by oligonucleotides HBV 10011 and CJP 114.
Oligonucleotide CJP 113 .;~L~ ~u,.ds to positions 1590 to
30 1603 and is not active as an inhibitor of Hepatitis B
replication. This indicates that all or some of the
nucleotides mapping at positions 1588, 1589, 1604, 1605,
1606, 1607, 1608, and 1609 are required for antiviral
activity. ûligonucleotide CJP 108 is j~l~ntjcAl to CJP
35 114, except that CJP 108 contains an additional three
Wo 95/17414 PCr/US94~136~7
2 1 788 1 1
-- 38 --
nucleotides at the 3 ' end; however, CJP 108 does not
exhibit antiviral activity. One or more oi~ the extra
three 3 ' nucleotidcs appear to abolish the antiviral
activity . Oligonucleotide CJP 109 also ~ n~ 8 the s~me
5 three 3' t~nm~nAl nucleotides as does CJP 108, and CJP
109 is also inactive in the assay. Similarly,
oligonucleotides CJP 112 and CJP 111 exhibit no antiviral
activity in the assay. Both contain a 3 ' nucleotide or
nucleotides which may abolish activity, as well as
10 lacking important 5' sequences present in HBV 10011 and
CJP 114.
Consistent with the foregoing, the following DR2
oliqnn~ otide8 (shown 5' to 3~) would also be expected
to exhibit anti-HBV activity:
15 OLIGO CJP 141: CGTGCAGAGGTGAAGCGA (SEQ ID NO: 25);
OLIGO CJP 142: GTGCAGAGGTGAAGCGA (SEQ ID NO: 26);
OLIGO CJP 143: TGCAGAGGTAAGCGA (SEQ ID NO: 27);
OLIGO CJP 144: GCAGAGGTGAAGCGA (SEQ ID NO: 28);
OLIGO CJP 145: CAGAGGTGAAGCGA (SEQ ID NO: 29);
20 OLIGO CJP 146: AGAGGTGAAGCGA (SEQ ID NO: 30);
OLIGO CJP 147: GAGGTGAAGCGA (SEQ ID NO: 31);
OLIGO CJP 148: AGGTGAAGCGA (SEQ ID NO: 32);
OLIGO CJP 149: CGACGTGCAGAGGTGAAGC (SEQ ID NO: 33);
OLIGO CJP 150: CGACGTGCAGAGGTGAAGCG (SEQ ID NO: 20);
25 OLIGO CJP 151: CGACGTGCAGAGGTGAAG (SEQ ID NO: 34);
OLIGO CJP 152 : CGACGTGCAGAGGTGAA (SEQ ID NO: 35);
OLIGO CJP 153; CGACGTGCAGAGGTGA (SEQ ID NO: 36);
OLIGO CJP 154 : CGACGTGCAGAGGTG (SEQ ID NO: 37);
OLIGO CJP 155: CGACGTGCAGAGGT (SEQ ID NO: 38);
30 OLIGO c~rP 156: CGACGTGCAGAGG (SEQ ID NO: 39);
OLIGO CJP 157: CGACGTGCAGAG (SEQ ID NO: 40);
OLIGO CJP 158: AACGT~rAr-Ar-r,TGAAGCGA (SEQ ID NO: 41);
OLIGO CJP 159: CGACGTGCAGAGGTr-~Ar-Cr-~Ar- (SEQ ID NO: 42);
OLIGO CJP 160: CGACGTGCAGAGGTGAAGCGAA (SEQ ID NO: 43).
W0 95/17414 2 1 7 8 8 1 I Pcr/uss4ll3687
-- 39 --
Additional active sequences may be determined based on
the results of experiments carried out to delineate the
precise 3' and 5' boundaries of activity, and then
selecting common nucleotide sequenceG.
All publications and patent applications mentioned
in the specif ication are indicative of the level of skill
of those skilled in the art to which this invention
pertains. All publications and patent applications are
incu ~ ~u~ ~.ted herein by ref erence to the same extent as if
10 each individual publication or patent application were
specifically and individually stated to be inCUL~lL~t.ed
by ref erence .
Although the f oregoing invention has been
described in some detail by way of illustration and
15 example for purposes of clarity of understanding, it will
be apparent to those of skill in the art that certain
changes and modif ications may be practiced within the
scope of the ~rpPnrlPfl claims.
WO 9S/17414 PCT/13S94113687 ~
2~ 788~ 1
-- 40 --
~NCIS LISTINO
( 1 ) a~ ilAL
(1) APPLI~ Coney, Le~lie R.
P~chuk, C~therLne J.
Yoon, 2~yongge~n
(lL) T~Trl~ OF INVlNTION: ANTI--HEPATI~IS B YIRaL
nT, ~ TIDEs
(111) NUHBER OF __ ~ 52
( ~ ) w ~ ADDRESS ~
~A) ~ I Fi8h 6~ rh~r~ n
~B) STR~SET: 225 Fr~nklln street
C~ CITY: Boston
~D~ STAT15: Y - L' e
~IS I COU~TRY: U.S.A.
[F~ Z~P: 02110-2804
(- ) COMPUTER READABLE FOR~5:
(A) ~OEDIUH TYP15: 3.5" Di~kette, 1.44 Mb
(B) COF;E~UTER: IBM PS/2 Model 50Z or 55SX
(C) ~. SYSTEH~ MS-DOS (Verslon 5.0)
(D) SO~WARE~ WordPeriect ~Version 5.1)
~L) CURRENT APPLICATION DATa:
(A) APPLICATION N~MBISR:
(B) FILINO DA$1s:
(C) crJ~b,-~F
(- iL) PRIOR APPLICATION llATA:
(A) APPLICATION NUHB~SRI 08/172,538
(B) FILING DATI5~ 23 Dec~er 1993
(-~111) ARIIEY/AOENT Lnr~ :
(A) NAME: FrAser, JanLs ~C.
(~) NUNBER: 34,819
(C) h~ /DOOEET NUHBl!R: 06120/002WOl
( L~ rrT ~
(A) ~ISLEPBONE: (61~) 542--5070
(B~ T~FAS: ~617) 542--8906
( C ) 'Iq!-T .F~ 2 0 015 4
(2) _ FOR SISQUENOE l'~LOn NUMBISR: 1:
(1) 8EQUENOE rT~.-~. . _ " ,, ~
(A) L~NOTII: 6
(B) TYPIS: nucleic ~cid
(C) : ~Lngle
(D) TOPOLOOY: lLne~r
(~ci) ~Q_CD L ~.~ : SEQ ID NO: l:
W095117414 2 1 7 8 8 1 1 PCTIU594/13687
-- 41 --
TATAAA 6
(2) ~ FOR S15QUENOE l'~lUn NaMBERt 2:
( ~ ) DISQUEIIC~
(A) I~N~Tpl ll
(B) 2~PBt nucleLc acLd
(C) I I l~Lngle
(D) TOPOLO~;Ys line~r
(~L) 815QUISNC15 r S SEQ ID NO: 2:
TTQCCTCTG C ll
(2) _ FOR SEQUENCE ~I~rn~r NUMBER: 3:
(L) SBQUI511OE r~ ~rDrl:~rTc~c
(A) FNOT~: 62
(B) 2rD3s: nucleic ncid
~C) : ~ingl(t
(D) TOPOEOGY: llne~r
(~cL) SEQUENCI5 I~.D~- : SEQ ~D NO: ~:
I.u~ .,u~ ,u C..~ CTTQCCTCT GQCGTTGQ ~Grr..rr~r r~ Tr~.~r 60
Q 62
~2) . FOR Dl!~QUENOE r NOMBER~ 4:
(L) S15QUl!NCIS ~- ._.~.".. ,,_
(A) TFNoTn: 62
(B) 2YDE: nucleLc acid
(c) : s inglo
(D) 20POLOOY: lLnenr
~L) 515QIII~NCI!: _ : SEQ ID NO: 4:
.OE~.n,l.~ GTOQCTTCG CTTQCCTCT GQCGTTGQ Trr~r~re~r rGTr~ ^rr 60
62
(2) lnr FOR SEQUENCE IDENT~F~CATION NUMBER: 5:
) S15QUISNCE ~ ;D:
~a) LEN~: 62
B) 2YP35: nucleic ~cid
~C) : siingle
~D) 20POLO~Y: linenr
~0 95/17414 PCTIUS94/13687
2 1 7;~8 l ~ 42 ~
(l~i) BEQUE'ACE l~ro~ SEQ ID NO: 5:
~CCr~'~rr~'r GTGCACTTCG CTTCACCTCT GCACGTCGQ Tr'`~ ^r~' CGTGAACGCC 60
62
(2) ~hr~ FOR 82QUENC~ cA~F AUMB2R: 6:
(i) Sl/Q0EVCE rP'~ ,5S
(A) ~NGTE~2 21
(B) ~YPES nuclelc ac~d
(C) ~ ~ ~lngle
(D) TOPOLOGY: llne~r
(~Ci) SEQUENOE l~r,~ SEQ ID NO: 6:
r.~ TC~ AGGTGAZ~GCG A 21
~2) ~Ar~ ~ FOR 8EQUENCE ~L~r~ r~ A h~ERI 7:
(i) 8EQUENCE rP~
(a) I~l!UaT~: 21
(1~) TSPE: nucleic acid
(c) _ : ~ingle
(D) ~OPOLOGY: linear
(:cl) 81SQUE~ACE Srow~r : 8EQ ID NO: 7:
r~ rr~ GGACGTGQA C 21
(2) . FOR SISQUENCE ~L~r~ A~mEa: 8:
(1) 515QU~:NOE ~-v~
I~CNGTIl: 21
~I~) ~PE: nuclelc ~cld
~C) : lngle
(D) ~OPOI~OGY: llnear
(:Ci) S15QUENOE ~r~o~ : SEQ ID I~O: 8:
TTAGGQGAG GTGAI~AAAGT T 21
(2) FOR 815QTII~NCE L~rn~-r~ hUMB15R: 9:
~1) SlCQl~DiCE r-- . -.. ~ .. ~
(~) Ll!31GTII: 21
( 1~ ) l~YP15: nuc le lc r c ld
~C) : lngle
(D) 'rOPOLOGY llnoar
~ WO 9S/17414 2 ~ 7 8 8 ~ ~ PCT/US94113687
-- 43 --
~Icl) 8DQUENCE l~ SEQ ID NO: 9:
ATCGaAGaAT GGTTAAATGA A 21
(2~ rOR SEQUENCE IDrllTIrIC~ ON NlIMBER: 10:
(i~ SEQUENCr ~'~'~
(A) LDNOTII: 21
(n) ~rYPE: nucleic ae$d
(C) : s$ngle
(D) TOPOLOGY: linoar
(:~1) 8EQUENCD ~ : SEQ Il) NO: 10:
CGACGTGCAG AGaTGAAGCG A 21
(2) . FOR ôl!~QUENCE l.. ~.~r NUUBER: 11:
($) 8rQUENeE r-~-o~
(A) T T~Na~: 14
(B) ~PE: nueloLe ae$d
(c) ~ : $ngle
(D) ~roPOLoaY: linear
(~cL) SEQUENCE L~ : SEQ ID NO: 11:
TaCAGAGaTG AAaC 14
(2) . ~ FOR 8EQUENOE lL.~.~ NUI~SR: 12:
(i) 8EQUENer ~ . . r~
(A) T rNoT~ 1~
(B) lqrPE: nueleie aeld
(C) : Oingle
(D) ~rOPOLOaY: llnoar
(~ ) 8EQUENCE L.~.oon~l SEQ ID NO: 12:
T"''-~'"~ TaQ 14
(2) FOR 8EQUENCE r NUIIBER: 13:
( ~ ) SDQUENC~ - ...._ . ~.... _
(A) LENaq~: 14
(B) lqrP~: nueleie oeld
(C) : n$ngle
(D) TOPOLOaY: l$near
WO9S/17414 PCT/US94/13687 ~
21 7881 1
-- 44 --
(:ci~ 8DQUISNCD l~ SEQ ID NO: 13:
C'`--~T""''--" GAAG 14
~2) _ FOR SDQUENCI! lLl~n~r_._ NUUBER: 14:
(1) g~:QUlSNCD ~ 7
(A) LDNGTI~: 10
(1~) TYPE~ nucl~lc z~cid
(C) ~ lngl~
(D) TOPOI.O~;Y: llnellr
815QUENC15 ~ t ôEQ ID NO: 14:
GGTGAAGCGA 1 0
( 2, FOR 8EQUENCIS ~ r ~ u NUMI~ER: 15:
(i~ 8DQUENCE r~..o~
(A~ LDNG~I: 21
rYPE: nucleLc cld
(C~ s ffingle
(D~ TOPOLOGY: lLne~r
(sl) 8DQUENOE t SEQ ID NO: lS:
CGTGCAGAGG l'r'`~ --'`"-- T 21
(2~ . ~OR 815QUENCE IDDNTIFICA~ION NUU--DER: 16
(1) 815QUDNCD r~v.o~.,, " "~
( A ~ LDNOT~I 2 4
(B) TYP~z nuclelc 2:cid
(C) ~ fflngl~
(D) q~OPOI.o~Y line~r
(~d~ ~sDQUENCE 1~ : SEQ ID NO: 16:
rr.s~rGTC~v~ AGGTGAAGCG AAGT 24
(2~ FOR SEQUENC~ r~ ON NIIMI~ER: 17:
(i.) 8DQUDNCD r~
(A) L~sGTl{: 10
(1~) lYP15: nucleic ~cLd
(C) : sLngle
(D) TOPOLOI~Y: linaar
(~ci) SEQUENCD l~ SEQ ID NO: 17:
~--Tr~ 10
~WO 9S/17414 2 1 7 8 8 1 1 PCT/US94/13687
-- 45 --
(2) FOR 8EQUENCE ~ r~IluA AUM8ER~ 18:
($) 515QTnNOE rv~ Y~. ~
(~) ~:NGTR: 11
~8) ~YP~S nuclelc acid
(C) slngle
(D) ~OPOLOOY lLnoar
(ICL) 8EQUENOE . t SEQ ID NO: 18
c~.--rTr--~- A 11
(2) ~Arl rOR SEQUENOE ~ ~A~r1~LUA AUM8ER: 19
(L) 8~QUENOE ~ ~Tr--t
(a) LISNGTR~ 11
(B) q~YPls nuclelc acid
(C) lngle
(D) ~OPOLOOY: lLne~r
(~ ) 8EQUENOE u~ : S~Q ID NO: 19:
TC'--~--'-- G 11
(2) . FOR SEQUENCE ~I~r~A~F ~f8ER: 20:
(1) 8~QUENCE r~ ~:.l~OT~Tr~
(A) LI NGTB ~ 20
(B) ~CYP15 nuclelc ACLd
(c) ~ ~ lngle
( D ) TOPOLOOY: 1 lnear
(~CL) slsQuENOE ~ SEQ ID NO: 20:
~_r. ~-qr~~ r~-r,5~~~~~ 20
(2) _ FOR 8EQUrNOE LUCA~r AUM8ER: 21:
( L ) 8EQ~NCD rr. o " - , _, ~, " _
(A) LENO~: 18
(~) TYPI5 nuclelc ~cld
(C) ~lngle
(D) TOPOLOOY: llneelr
(~L) 8EQUENOE : SEQ ~D NO: 21:
ACGTGCAGAG GTGAAGCG 18
(2) _ FOR 8~QUENC~ r_ AUM8ZR: 22
( L ) 8~5QUENOE r .. o ~
WO 95117414 CTIU ~
2 1 7~ P S94113687
~ 46 ~
(a) LoNa~EI 32
(B) TYPE~ nucluLc ~cid
(C) : singlo
(D) TOPOLOGY: linear
SEQUENCE L~ ~ s SEQ ID NO: 22:
TGCACTTCCC TTCACCTCTG QCGTTGCAT GG 32
(2) FOR SEQ21ENCIS 1 S~ r NU~BER: 23:
( 1) 8EQUENOE rl ~
(A) T~NGT'': 32
(I~) TYPEs nucleic ~cid
(C) : 8ingle
(D) TOPOLOGY~ line~r
(~cl) S--QUENCE l~ ~ : SEQ ID NO: 23:
TGCACTTCGC TTCACCTCTG CACGTCGCAT GG 32
(2) OR 8EQUENOE I' NU118ER~ 24s
( 1 ) SEQU--NCE ~ ~ 2
(A) LENGT~: 19
(B) TYPE: nucleic acid
(C) 1 2~i ngle
(D) TOPOLOGY: line~lr
(~-L) SEQ~2ENCE DESCRIPTION: SEQ ID NO: 2C:
ACGTGCAGAG GTGAAGCGA 19
~2) . FOR SEQUENCo rL~ JA N21MBEX: 25:
(i) SEQ2~ENOE ~~
(A) LoNGTIl 18
(B) TYPE: nucleic Acid
(C) : ~ingle
(D) TOPOLOGY: linear
(:Ci) ôEQUENOE l~ : SEQ ID NO: 25:
t~cTr--~ ---- TGAAGCGA 18
(2) ~OR SEQUENCE ~I~OA~lr' NlIMBER: 26:
(1) ôlSQUENOE r ~ T2 ~T~
( A) LENGTII: 17
(B) TYPE: nucleic acid
(C) L : single
(D) TOPOLOGY: linear
~0 95/17414 2 1 7 8 8 1 1 PCT/13S94/13687
-- 47 --
(~i) 8EQUENCIS ~ SEQ ID NO: Z6:
GTGCAGAGGT GA~GCGA 17
(2) . FOR SEQaENOE 1~ ~UISBER: 27:
( L ) SEQllENCE r~ T ~
(a) I.ENG~EI: lS
(B) lYPE: nucle$c acid
(c) I ~ Lngle
(D) TOPOEOGY: llnear
(~ci) 8EQUENOE L.~.;A-~__ SEQ ID NO: 27:
T~ T~ AGCGA 15
(Z) . FOR SEQUENCE ~ r _ __ NUMBER: 28:
( $ ) SISQUENCE ~I~ r - , . ... _ l r~ .. . _
(A) I.l!NGl~: 15
(B) ~YPEt nuclelc acid
(C) 2 ~lngle
(D) ~OPOI,OGY: lLnear
(~1) 815QUENCE ,__ ~ SEQ ID NO: 2~:
T---~ AGCGA 15
(2) . rOR SEQUENOE ~ NUMBERs 29:
(i) SISQ111!31OE ~ T!~- " _~
(A) ~ENGlq~: 14
(B) l'YPE: r~uclelc acld
(C) : ~lngle
( D ~ I~OPOI.OGY: 1 lnear
<:ci) S15QU15NOE : SEQ ID NO: 29:
CAGAGGTGBA GCGA 14
( 2 ) . FOR SEQUENCD ~ r NUMBER: 3 0:
(i) SISQUENOE ~"~--- .. r
- (A) LISN~TEl: 13
(B) ~YPIS: nucl~ic acLd
(C) t ~lngle
(D) TOPO~OOY~ l~near
WO 9S/17414 PCT/US94/13687--
217881 l
-- 48 --
(:ci) SEQUEIIOE l~ un SEQ ID NO: 30:
P-~--T-''~- CGA 13
(2) ~1~,, FOR SEQUI!NCE ~l~n r~ un NUM_ER: 31:
( 1 ) 8EQUENOE r~
(A~ LENGTI{: 12
(Il) ~I!YPE2 nucleic ~cid
(C) s s~ingle
(D) TOPOIOOY~ llne~lr
(~i) 815QU~CE u~ ~ : SEQ ID NO: 31:
GAGG~GAAGC GA 12
(2) . FOR SEQUENOE ~Un NU~4BER: 32:
(i) SISQUENOE rl~..os~. . ....~. " _
(a) LEN<~: 11
(_) 'rYPE: nucleLo ~cid
(C) ~ : ~ingle
( D ) TOPOLOGY: l Lnear
(~L) 815QUENOE r SEQ ID NO: 32:
AGGTGAAGCG A 11
(2) . FOR SEQUENC~ NUM_ER: 33:
(i) 815QUENOE, ~
(A) LlSNalqls 19
(B) TlrPI!:: nucleLc llcld
(C) : aLnglo
(D) !I!OPoLoaY ~ line-~r
(~i) 815QUD~OE L.~ SEQ ID NO: 33:
CGACG~GCAG ~ 7.__ l9
(Z) FOR SEQUENCE 11~ ~ ~Un NUU_ER: 34:
( i) SEQIIENCE r~
(A) IJ!:NGTIl: 18
(_) lqrPE: nucleic ~cid
(C) 8~ alngle
(D) q~OPOLOGY: lLne~r
(:ci) 815QUENCl L~ : SEQ ID NO: 34:
rr~ ~ AGGTGAAG 18
21 7881 1
WO 95117414 PCTIUS94/13687
-- 49 ~
~2) 'h~ FOR SEQU~5NCE IDENTIFICATION NUMBER: 35:
(1) SEQUENOE r~..o~. ,"_T~,, " _1
(A) LENG~ l 7
(B) TYPE: nuclelc acid
(C) : 81ngle
(D) ~OPOLOGY: linear
(~cl) 815QUENOE ~ DW1~ : SEQ ID NO: 35:
rr~ 3TC~ ' AGGTGAA 17
(2) Lr~rl FOR SEQUENCE ILI~.1r~ - NUMBER: 36:
(1~ SEQUENOE r~o~
(A) LENCTI~ 16
(B) Tr.D15 nucleic acLd
(C) I : flingle
(D) TOPOLOGY: lLne~lr
(~cL) SEQUENCE r : SEQ ID NO: 36:
CGACGTGQC AGGTGA 16
~2) . ~OR SISQUENCE l~ r NU~IBER: 37:
(1) SISQUISNCE rl~
(A) LENG~ 15
(B) Tr~D15 nucleLc acLd
(C~ ~ : 13Lngle
(D) ~OPOI.OOY: line~r
(~Ci) 8EQUENCE Ll:.D~ SEQ ID NO: 37:
CGACGTGCAG AGGTG lS
(2) FO.~ SEQUENOE ~ r NU~lBERs 38:
( i ) S15QUENC~ r~
(A) LENGT~l 14
(-D) Tr.DE: nucleLc acid
(C) ~ t oLngl~e
(D) q!OPO~y lLn~ulr
(~cL) S15QUENCE ~D~L1 I SEQ ID NO: 38:
CGACGTGCAG AGGT l 4
(Z) FOR SEQUENOE ~ 1r NUIIBER: 39:
(1) SEQUENCE r~o-. ,__,~., " _
WO 9S/17414 PCTNS94/13687 `
217881 1
-- 50 --
(A) LENaTB: 13
(B) TYPE~ nuclelc acLd
(c) ~ I ~lngle
(D) TOPOBOGY: llnear
(~c$) SEQUENOE r~ SEQ ID NO: 39:
CGACGTGQG AGG 13
(2) . FOR 8EQUENCE lJcm~F~ NUMBER: 40:
) 5~5QUIsNcE rv~
(A) T~NGTEI: 12
(B~ TYPE: nuclelc acid
(C) ~ slnqle
(D) TOPOLOI~r linear
(~ci) 815QUENCE l~iD~lt : SEQ ~D NO: 40:
CGACGTGQG AG 12
(2) ~nr~ FOR SEQUENOE IDISNTIFICATION NUMBER: 41:
(1) 815QU~SNCE rv~ ",, " _
(A) IJSNGTIl~ 20
(B) TYPE: nuclcLc acid
(C) ~ s ~lngle
(D) TOPOLoaYI llnear
(~i) 8EQUENOE : SEQ ~D NO: 41:
p ~ rGTGr 1` ^ ~ GGTGAAGCGA 2 0
(2) . FOIR SEQUENOE l~.~.lrl~ NUMBER: 42:
( i ) S~QUENOE r ..
(A~ LENGTB: 2 3
(B) TYPE: nuclelc llcid
(C) : slngle
(D) TOPOLOGY: linear
(~i) 8EQUENCE L~rD~lt_ : SEQ ID NO: 42:
rr~r~Tc^~" AGGTGAAGCG AAG 23
(2) lnr~ -- FOR SEQUENCE ~ F_ NUNBER: 43:
(i) 8EQUENOE r~
( A) LBNGTB: 22
(B) TYPE: nucleic acld
(C) ' : single
(D) TOPO~OGY: llnear
(~i) Sl!:QUENOE : SEQ ~D NO: 43:
CGACGTGQG AGGTGAAGCG AA 2Z
217881 ~
~0 9S/17414 PCTNS94/13687
-- 51 --
(2) -- FOR 8EQUENCE ~ r ~Un NUMBER: 44
(~ ) SEQUENCE r ~ ~TcrTr tt
(A) LENaT~ 201
(8) l~YPE~ nucleic ~cid
(C) z slngle
(D) ~rOPOLOGY~ line~r
(~i) 8~5QUENOE D r~UnS SEQ ID NO: 44:
r CCTCCGACQ Ccr~r~r u. ~ .. C GCGGACTCCC ~ 60
Tu~ rrr ~rrTG TGCACTTCGC -~Cu.~ QCGTCGQT ~ . .. n~rr7lr~ 120
r.T ~ r rc- ~rr~ rcrT GCCQAaaTC TTGQTAAGA GGACTCTTCG ACTTTQGQ 180
ATGTQACGA rcr- rT-r~ -- G 201
(2) . FOR 8EQUENOE ~l~ln~LFL~_~LUn NUMBER~ 45:
(1) SEQUENOE ~r l~
(A) LENOT.~ 47
(B) TYPE~ nucleic ~cid
(C) s ~~ : slngle
(D) ~rOPOLOGYs line~r
(:ci) SEQUENOE ~CD~A~LUn SEQ ID NO: 45:
1~U~ ~C CATGCAACGT GCAGACaTGA AGCGAAG 47
(2) . FOR SEQUENOE L~n~lr NUMBER: 46:
(~ ) SEQUENOE r~
(A) LENaTI~: 47
(B) ~rYPE: nucleic ~cid
(C) : ninyle
(D) ~ropoLoGy: line~r
(~i) SEQUENOE ~I D A~I ~U SEQ ID NO: 46:
.u QTGQACGT GCAGACGTaA AGCGAAG 47
(2) ~h.. FOR 8EQUENOE r NUIIBER: 47:
( i ) 815QUENOE
LENGT~II: 47
(B) ~rYPE: nucleic ~Cid
(C) t tiingle
(D) TOPOLoay: lineAr
(~i) 8EQU~NOE l~D ALL SEQ ID NO: 47:
L~ll~ u~-~;- 1U- C CATGCGACGT GCAGAGGTGA AGCGAAG 47
(2) . ~ FOR 8EQUENCE IDENTIFICATION NUUBER: 48:
.
WO 95/17414 PCT/US94/13687--
2 1 78~ i ~
-- 52 ~
(1) 815QUlSNOE ro~
~A) I~aTEl~ 47
(s) TYP15: nucleio ~cid
~C) ~ ~Lngle
(D~ ToPoLoaY. lino-r
8BQUENOE l~ SEQ ID NO: 4a
r''rrC""~T --^I~ ^_Tr ~ AGCGAAa 47
(2) . FOR 815QU15NCB ~ mER: 49
(1) 815QUBNCE . ~ T~
~A) IJ!:NGll~: 20
) TYE nucleic ~cid
~C) ~ Isingle
(D~ ToPoLoaY line~r
(~cl) 8EQUENCB ~ : SEQ ID NO: 49:
Gr-r~ r^~ zo
(2) _ FOR 8BQUISNCB Llo~ll!' NUNElBRs 50:
(i) 8~QUENCB rr~
(A) ~IGTII: 20
~s) TrPl/~ nucleic ~cid
) ingle
) ~roPoLoaYt l$ne~r
~$) li~QUlSNCE : SEQ rD NO: 50:
Tr~ 20
~2) . FOR 8BQUENOE ~S~.15~ NI~BR: 51:
$ ) 8~:QIIISNCB ~Tl~ o~ -.. ~
(A) ~15NaTEI: 19
(B) TYP~5: nucl~ic ~cld
s$ngl-
(D) TOPOLoaY l$ne~:r
(~cl) 815QUENCE L.~ U: SEQ ID NO: 51:
.. ~,Tr~ GGTGAAGCG 19
(2) . OR 8EQlllSNOE ~ UI~ NUMBI!:R: 52:
( 1 ) 8lsQulsNcB ~
~A) ~NaT~: 19
~s) TYPB: nucls$c cld
~C) single
~D) TOPOLOaY line~r
~ s~l ) 81!:QUE~OE ~ : SEQ ID NO: 5 2:
AACGTGCAGA GaTGAAGCG 19
