Note: Descriptions are shown in the official language in which they were submitted.
CA 02326695 2000-10-26
WO 99/55847 PGT/US99/09027
1
DESCRIPTION
Enzymatic Nucleic Acid Treatment Of Diseases Or
Conditions Related To Hepatitis C Virus Infection
This patent application claims priority to Blatt et al., USSN (Not Yet
Assigned),
filed February 24, 1999, Blatt et al., USSN 60/100,842, filed September 18,
1998, and
McSwiggen et al., USSN 60/083,217 filed April 27, 1998, all of these earlier
applications
are entitled "ENZYMATIC NUCLEIC ACID TREATMENT OF DISEASES OR
CONDITIONS RELATED TO HEPATITIS C VIRUS INFECTION". Each of these
applications are hereby incorporated by reference herein in their entirety
including the
drawings.
Backa~round Of The Invention
This invention relates to methods and reagents for the treatment of diseases
or
conditions relating to the hepatitic C virus infection.
The following is a discussion of relevant art, none of which is admitted to be
prior
art to the present invention.
In 1989, the HCV was determined to be an RNA virus and was identified as the
causative agent of most non-A non-B viral Hepatitis (Choo et al., Science.
1989; 244:359-
362). Unlike retroviruses such as HIV, HCV does not go though a DNA
replication phase
and no integrated forms of the viral genome into the host chromosome have been
detected
2 0 (Houghton et al., Hepatology 1991;14:381-388). Rather, replication of the
coding (plus)
strand is mediated by the production of a replicative (minus) strand leading
to the
generation of several copies of plus strand HCV RNA. The genome consists of a
single,
large, open-reading frame that is translated into a polyprotein (Kato et al.,
FEBS Letters.
1991; 280: 325-328). This polyprotein subsequently undergoes post-
translational cleavage,
2 5 producing several viral proteins (Leinbach et al., Virology. 1994: 204:163-
169).
Examination of the 9.5-kilobase genome of HCV has demonstrated that the viral
nucleic acid can mutate at a high rate (Smith et al.,Mol. Evol. 1997 45:238-
246). This rate
of mutation has led to the evolution of several distinct genotypes of HCV that
share
approximately 70% sequence identity (Simmonds et al., J. Gen. Virol. 1994;75
:1053-1061}.
3 0 It is important to note that these sequences are evolutionarily quite
distant. For example,
the genetic identity between humans and primates such as the chimpanzee is
approximately 98%. In addition, it has been demonstrated that an HCV infection
in an
individual patient is composed of several distinct and evolving quasispecies
that have 98%
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
2
identity at the RNA level. Thus, the HCV genome is hypervariable and
continuously
changing. Although the HCV genome is hypervariable, there are 3 regions of the
genome
that are highly conserved. These conserved sequences occur in the 5' and 3'
non-coding
regions as well as the 5'-end of the core protein coding region and are
thought to be vital
for HCV RNA replication as well as translation of the HCV polyprotein. Thus,
therapeutic agents that target these conserved HCV genomic regions may have a
significant impact over a wide range of HCV genotypes. Moreover, it is
unlikely that drug
resistance will occur with ribozymes specific to conserved regions of the HCV
genome.
In contrast, therapeutic modalities that target inhibition of enzymes such as
the viral
proteases or helicase are likely to result in the selection for drug resistant
strains since the
RNA for these viral encoded enzymes is located in the hypervariable portion of
the HCV
genome.
After initial exposure to HCV, the patient will experience a transient rise in
liver
enzymes, which indicates that inflammatory processes are occurring (Alter et
al., IN:
Seeff LB, Lewis JH, eds. Current Perspectives in Hepatology. New York: Plenum
Medical
Book Co; 1989:83-89). This elevation in liver enzymes will occur at least 4
weeks after the
initial exposure and may last for up to two months (Farci et al., New England
Journal of
Medicine. 1991:325:98-104). Prior to the rise in liver enzymes, it is possible
to detect HCV
RNA in the patient's serum using RT-PCR analysis (Takahashi et al., American
Journal of
2 0 Gastroenterology. 1993:88:2:240-243). This stage of the disease is called
the acute stage and
usually goes undetected since 75% of patients with acute viral hepatitis from
HCV
infection are asymptomatic. The remaining 25% of these patients develop
jaundice or
other symptoms of hepatitis.
Acute HCV infection is a benign disease, however, and as many as 80% of acute
2 5 HCV patients progress to chronic liver disease as evidenced by persistent
elevation of
serum alanine aminotransferase (ALT) levels and by continual presence of
circulating
HCV RNA (Sherlock, Lancet 1992; 339:802). The natural progression of chronic
HCV
infection over a 10 to 20 year period leads to cirrhosis in 20to50% of
patients (Davis et al.,
Infectious Agents and Disease 1993;2:150:154) and progression of HCV infection
to
3 0 hepatocellular carcinoma has been well documented (Liang et al.,
Hepatology. 1993;
18:1326-1333; Tong et al., Western Journal of Medicine, 1994; Vol. I60, No. 2:
133-138).
There have been no studies that have determined sub-populations that are most
likely to
progress to cirrhosis and/or hepatocellular carcinoma, thus all patients have
equal risk of
progression.
3 5 It is important to note that the survival fox patients diagnosed with
hepatocellular
carcinoma is only 0.9 to 12.8 months from initial diagnosis (Takahashi et al.,
American
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
3
Journal of Gastroenterology. 1993:88:2:240-243). Treatment of hepatocellular
carcinoma
with chemotherapeutic agents has not proven effective and only 10% of patients
will
benefit from surgery due to extensive tumor invasion of the liver (Trinchet et
al., Presse
Medicine. 1994:23:831-833). Given the aggressive nature of primary
hepatocellular
carcinoma, the only viable treatment alternative to surgery is liver
transplantation
(Pichlmayr et al., Hepatology. 1994:20:33S-40S).
Upon progression to cirrhosis, patients with chronic HCV infection present
with
clinical features, which are common to clinical cirrhosis regardless of the
initial cause
(D'Amico et al., Digestive Diseases and Sciences. 1986;31:5: 468-475). These
clinical
features may include: bleeding esophageal varices, ascites, jaundice, and
encephalopathy
(Zakim D, Boyer TD. Hepatology a textbook of liver disease. Second Edition
Volume I. 1990
W.B. Saunders Company. Philadelphia). In the early stages of cirrhosis,
patients are
classified as compensated meaning that although liver tissue damage has
occurred, the
patient's liver is still able to detoxify metabolites in the blood-stream. In
addition, most
patients with compensated liver disease are asymptomatic and the minority with
symptoms
report only minor symptoms such as dyspepsia and weakness. In the later stages
of
cirrhosis, patients are classified as decompensated meaning that their ability
to detoxify
metabolites in the bloodstream is diminished and it is at this stage that the
clinical features
described above will present.
2 0 In 1986, D'Amico et al. described the clinical manifestations and survival
rates in
1155 patients with both alcoholic and viral associated cirrhosis (D'Amico
supra). Of the
1155 patients, 435 (37%) had compensated disease although 70% were
asymptomatic at
the beginning of the study. The remaining 720 patients (63%) had decompensated
liver
disease with 78% presenting with a history of ascites, 31% with jaundice, 17%
had
2 5 bleeding and 16% had encephalopathy. Hepatocellular carcinoma was observed
in six
(.5%) patients with compensated disease and in 30 (2.6%) patients with
decompensated
disease.
Over the course of six years, the patients with compensated cirrhosis
developed
clinical features of decompensated disease at a rate of 10% per year. In most
cases, ascites
3 0 was the first presentation of decompensation. In addition, hepatocellular
carcinoma
developed in 59 patients who initially presented with compensated disease by
the end of
the six-year study.
With respect to survival, the D'Amico study indicated that the five-year
survival
rate for all patients on the study was only 40%. The six-year survival rate
for the patients
35 who initially had compensated cirrhosis was 54% while the six-year survival
rate for
patients who initially presented with decompensated disease was only 21%.
There were
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
4
no significant differences in the survival rates between the patients who had.
alcoholic
cirrhosis and the patients with viral related cirrhosis. The major causes of
death for the
patients in the D'Amico study were liver failure in 49%; hepatocellular
carcinoma in 22%;
and, bleeding in 13% (D'Amico supra).
Chronic Hepatitis C is a slowly progressing inflammatory disease of the liver,
mediated by a virus (HCV) that can lead to cirrhosis, liver failure andlor
hepatocellular
carcinoma over a period of 10 to 20 years. In the US, it is estimated that
infection with
HCV accounts for 50,000 new cases of acute hepatitis in the United States each
year (NIH
Consensus Development Conference Statement on Management of Hepatitis C March
1997). The prevalence of HCV in the United States is estimated at 1.8% and the
CDC
places the number of chronically infected Americans at approximately 4.5
million people.
The CDC also estimates that up to 10,000 deaths per year are caused by chronic
HCV
infection. The prevalence of HCV in the United States is estimated at 1.8% and
the CDC
places the number of chronically infected Americans at approximately 4.5
million people.
The CDC also estimates that up to 10,000 deaths per year are caused by chronic
HCV
infection.
Numerous well controlled clinical trials using interferon (IFN-alpha) in the
treatment of chronic HCV infection have demonstrated that treatment three
times a week
results in lowering of serum ALT values in approximately SO% (range 40% to
70%) of
2 0 patients by the end of 6 months of therapy (Davis et al., New England
Journal of Medicine
1989; 321:1501-1506; Marcellin et al., Hepatology. 1991; 13:393-397; Tong et
al., Hepatology
1997:26:747-754; Tong et al., Hepatology 1997 26(6): 1640-1645). However,
following
cessation of interferon treatment, approximately SO% of the responding
patients relapsed,
resulting in a "durable" response rate as assessed by normalization of serum
ALT
2 5 concentrations of approximately 20 to 25%.
In recent years, direct measurement of the HCV RNA has become possible through
use of either the branched-DNA or Reverse Transcriptase Polymerase Chain
Reaction
(RT-PCR) analysis. In general, the RT-PCR methodology is more sensitive and
leads to
more accurate assessment of the clinical course (Tong et al., supra). Studies
that have
30 examined six months of type 1 interferon therapy using changes in HCV RNA
values as a
clinical endpoint have demonstrated that up to 35% of patients will have a
loss of HCV
RNA by the end of therapy (Marcellin et al., supra). However, as with the ALT
endpoint,
about SO% of the patients relapse six months following cessation of therapy
resulting in a
durable virologic response of only 12% (Marcellin er al., supra). Studies that
have
3 5 examined 48 weeks of therapy have demonstrated that the sustained
virological response
is up to 25% (1~1IH consensus statement: 1997). Thus, standard of care for
treatment of
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
chronic HCV infection with type 1 interferon is now 48 weeks of therapy using
changes in
HCV RNA concentrations as the primary assessment of efficacy (Hoofnagle et
al., New
England Journal of Medicine 1997; 336(5) 347-356).
Side effects resulting from treatment with type 1 interferons can be divided
into
5 four general categories, which include 1. Influenza-like symptoms; 2.
Neuropsychiatric; 3.
Laboratory abnormalities; and, 4. Miscellaneous (Dusheiko et al., Journal of
Viral Hepatitis.
1994:1:3-5}. Examples of influenza-like symptoms include; fatigue, fever;
myalgia;
malaise; appetite loss; tachycardia; rigors; headache and arthralgias. The
influenza-like
symptoms are usually short-lived and tend to abate after the first four weeks
of dosing
(Dushieko et al., supra). Neuropsychiatric side effects include: irritability,
apathy; mood
changes; insomnia; cognitive changes and depression. The most important of
these
neuropsychiatric side effects is depression and patients who have a history of
depression
should not be given type 1 interferon. Laboratory abnormalities include;
reduction in
myeloid cells including granulocytes, platelets and to a lesser extent red
blood cells.
These changes in blood cell counts rarely lead to any significant clinical
sequellae
(Dushieko et al., supra). In addition, increases in triglyceride
concentrations and
elevations in serum alanine and aspartate aminotransferase concentration have
been
observed. Finally, thyroid abnormalities have been reported. These thyroid
abnormalities
are usually reversible after cessation of interferon therapy and can be
controlled with
2 0 appropriate medication while on therapy. Miscellaneous side effects
include nausea;
diarrhea; abdominal and back pain; pruritus; alopecia; and rhinorrhea. In
general, most
side effects will abate after 4 to 8 weeks of therapy (Dushieko et al.,
supra).
Welch et al., Gene Therapy 1996 3(11): 994-1001 describe in vitro an in vivo
studies with two vector expressed hairpin ribozymes targeted against hepatitis
C virus.
Sakamoto et al., J. Clinical Investigation 1996 98(12):2720-2728 describe
intracellular cleavage of hepatitis C virus RNA and inhibition of viral
protein translation
by certain vector expressed hammerhead ribozymes.
Lieber et al., J. Virology 1996 70{12):8782-8791 describe elimination of
hepatitis
C virus RNA in infected human hepatocytes by adenovirus-mediated expression of
certain
3 0 hammerhead ribozymes.
Ohkawa et al., 1997, J. Hepatology, 27; 78-84, describe in vitro cleavage of
HCV
RNA and inhibition of viral protein translation using certain in vitro
transcribed
hammerhead ribozymes.
Barber et al., International PCT Publication No. WO 97/32018, describe the use
of
3 5 an adenovirus vector to express certain anti-hepatitis C virus hairpin
ribozymes.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
6
Kay et al., International PCT Publication No. WD 96/18419, describe certain
recombinant adenovirus vectors to express anti-HCV hammerhead ribozyme.
Yamada et al., Japanese Patent Application No. JP 07231784 describe a specific
poly-(L)-lysine conjugated hammerhead ribozyme targeted against HCV.
Draper, U.S. Patent No. 5,610,054, descibes enzymatic nucleic acid molecule
capable of inhibiting replication of HCV.
Alt et al., Hepatology 1995 22(3): 707-717, describe specific inhibition of
hepatitis
C viral gene expression by certain antisense phosphorothioate
oligodeoxynucleotides.
Summary Of The Invention
This invention relates to ribozymes, or enzymatic nucleic acid molecules,
directed
to cleave RNA species of hepatitis C virus (HCV) and/or encoded by the HCV. In
particular, applicant describes the selection and function of ribozymes
capable of
specifically cleaving HCV RNA. Such ribozymes may be used to treat diseases
associated
with HCV infection.
Due to the high sequence variability of the HCV genome, selection of ribozymes
for broad therapeutic applications would likely involve the conserved regions
of the
HCV genome. Specifically, the present invention describes hammerhead ribozymes
that
would cleave in the conserved regions of the HCV genome. A list of the thirty
hammerhead ribozymes derived from the conserved regions (5'- Non Coding Region
2 0 (NCR), 5'- end of core protein coding region, and 3'- NCR) of the HCV
genome is
shown in Table IV . In general, Applicant has found that enzymatic nucleic
acid
molecules that cleave sites located in the 5' end of the HCV genome would
block
translation while ribozymes that cleave sites located in the 3' end of the
genome would
block RNA replication. Approximately 50 HCV isolates have been identified and
a
sequence alignment of these isolates from genotypes la, lb, , 2a, 2b, 2c, 3a,
3b, 4a, 5a,
and 6 was performed. These alignments were used by the Applicant to identify
30
hammerhead ribozymes sites within regions highly conserved between genotypes.
Twenty three ribozyme sites were identified in regions of greatest homology
within the
conserved region.. Therefore, one ribozyme can be designed to cleave all the
different
isolates of HCV. According to the Applicant, ribozymes designed against
conserved
regions of various HCV isolates will enable efficient inhibition of HCV
replication in
diverse patient populations and may ensure the effectiveness of the ribozymes
against
HCV quasispecies which evolve due to mutations in the non-conserved regions of
the
HCV genome.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
7
By "inhibit" is meant that the activity of HCV or level of RNAs encoded by HCV
genome is reduced below that observed in the absence of the nucleic acid,
particularly,
inhibition with ribozymes preferably is below that level observed in the
presence of an
inactive RNA molecule able to bind to the same site on the mRNA, but unable to
cleave
that RNA.
By "enzymatic nucleic acid" it is meant a nucleic acid molecule capable of
catalyzing reactions including, but not limited to, site-specific cleavage
and/or ligation of
other nucleic acid molecules, cleavage of peptide and amide bonds, and traps-
splicing.
Such a molecule with endonuclease activity may have complementarity in a
substrate
binding region to a specified gene target, and also has an enzymatic activity
that
specifically cleaves RNA or DNA in that target. That is, the nucleic acid
molecule with
endonuclease activity is able to intramolecularly or intermolecularly cleave
RNA or DNA
and thereby inactivate a target RNA or DNA molecule. This complementarity
functions to
allow suffcient hybridization of the enzymatic RNA molecule to the target RNA
or DNA
to allow the cleavage to occur. 100% complementarity is preferred, but
complementarity
as low as 50-75% may also be useful in this invention. The nucleic acids may
be modified
at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid
is used
interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA,
catalytic
DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme,
2 0 endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA
enzyme. All of
these terminologies describe nucleic acid molecules with enzymatic activity.
The specific
enzymatic nucleic acid molecules described in the instant application are not
limiting in
the invention and those skilled in the art will recognize that all that is
important in an
enzymatic nucleic acid molecule of this invention is that it has a specific
substrate binding
2 5 site which is complementary to one or more of the target nucleic acid
regions, and that it
have nucleotide sequences within or surrounding that substrate binding site
which impart a
nucleic acid cleaving activity to the molecule.
By "enzymatic portion" or "catalytic domain" is meant that portion/region of
the
ribozyme essential for cleavage of a nucleic acid substrate (for example see
Figure 1 ).
3 0 By "substrate binding arm" or "substrate binding domain" is meant that
portion/region of a ribozyme which is complementary to (i.e., able to base-
pair with) a
portion of its substrate. Generally, such complementarity is I00%, but can be
less if
desired. For example, as few as 10 bases out of 14 may be base-paired. Such
arms are
shown generally in Figure 1 and 3. That is, these arms contain sequences
within a
3 5 ribozyme which are intended to bring ribozyme and target RNA together
through
complementary base-pairing interactions. The ribozyme of the invention may
have binding
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
8
arms that are contiguous or non-contiguous and may be of varying lengths. The
length of
the binding arms) are preferably greater than or equal to four nucleotides;
specifically 12-
100 nucleotides; more specifically 14-24 nucleotides long. If two binding arms
are
chosen, the design is such that the length of the binding arms are symmetrical
(i. e., each of
the binding arms is of the same length; e.g., five and five nucleotides, six
and six
nucleotides or seven and seven nucleotides long) or asymmetrical (i. e., the
binding arms
are of different length; e.g., six and three nucleotides; three and six
nucleotides long; four
and five nucleotides long; four and six nucleotides long; four and seven
nucleotides long;
and the like).
In one of the preferred embodiments of the inventions herein, the enzymatic
nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also
be
formed in the motif of a hepatitis d virus, group I intron, group II intron or
RNaseP RNA
(in association with an RNA guide sequence) or Neurospora VS RNA. Examples of
such
hammerhead motifs are described by Dreyfus, supra, Rossi et al. , 1992, AIDS
Research
and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257,
Hampel
and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53,
Haseloff and
Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18,
299; of the
hepatitis d virus motif is described by Perrotta and Been, 1992 Biochemistry
31, 16; of the
RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman,
1990,
2 0 Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835;
Neurospora VS RNA
ribozyme motif is described by Collies (Saville and Collies, 1990 Cell 61, 685-
696;
Saville and Collies, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collies
and Olive,
1993 Biochemistry 32, 2795-2799; Guo and Collies, 1995, EMBO. J. 14, 363);
Group II
introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels and
Pyle, 1995,
2 5 Biochemistry 34, 2965; Pyle et al., International PCT Publication No. WO
96/22689; of
the Group I intron by Cech et al., U.S. Patent 4,987,071; and of DNAzyme motif
by
Chartrand et al., 1995, Nucleic Acids Research 23, 4092; Santoro et al., 1997,
PNAS 94,
4262. These specific motifs are not limiting in the invention and those
skilled in the art
will recognize that all that is important in an enzymatic nucleic acid
molecule of this
3 0 invention is that it has a specific substrate binding site which is
complementary to one or
more of the target gene RNA regions, and that it have nucleotide sequences
within or
surrounding that substrate binding site which impart an RNA cleaving activity
to the
molecule.
By "equivalent" RNA to HCV is meant to include those naturally occurring RNA
35 molecules associated with HCV infection in various animals, including
human, rodent,
primate, rabbit and pig. The equivalent RNA sequence also includes in addition
to the
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
9
coding region, regions such as 5'-untranslated region, 3'-untranslated region,
introns,
intron-exon junction and the like.
By "complementarity" is meant a nucleic acid that can form hydrogen bonds)
with
another RNA sequence by either traditional Watson-Crick or other non-
traditional types
(for example, Hoogsteen type) of base-paired interactions.
In a preferred embodiment the invention provides a method for producing a
class
of enzymatic cleaving agents which exhibit a high degree of specificity for
the RNA of a
desired target. The enzymatic nucleic acid molecule is preferably targeted to
a highly
conserved sequence region of a target mRNAs encoding HCV proteins such that
specific
treatment of a disease or condition can be provided with either one or several
enzymatic
nucleic acids. Such enzymatic nucleic acid molecules can be delivered
exogenously to
specific cells as required. Alternatively, the ribozymes can be expressed from
DNA/RNA
vectors that are delivered to specific cells.
Such ribozymes are useful for the prevention of the diseases and conditions
discussed above, and any other diseases or conditions that are related to the
levels of HCV
activity in a cell or tissue.
By "related" is meant that the inhibition of HCV RNAs and thus reduction in
the
level respective viral activity will relieve to some extent the symptoms of
the disease or
condition.
2 0 In preferred embodiments, the ribozymes have binding arms which are
complementary to the target sequences in Tables IV-IX. Examples of such
ribozymes are
also shown in Tables IV-IX. Examples of such ribozymes consist essentially of
sequences defined in these Tables. Other sequences may be present which do not
interfere
with such cleavage.
2 5 By "consists essentially of is meant that the active ribozyme contains an
enzymatic center or core equivalent to those in the examples, and binding arms
able to
bind mRNA such that cleavage at the target site occurs. Other sequences may be
present
which do not interfere with such cleavage.
Thus, in a first aspect, the invention features ribozymes that inhibit gene
3 0 expression and/or viral replication. These chemically or enzymatically
synthesized RNA
molecules contain substrate binding domains that bind to accessible regions of
their target
mRNAs. The RNA molecules also contain domains that catalyze the cleavage of
RNA.
The RNA molecules are preferably ribozymes of the hammerhead or hairpin motif.
Upon
binding, the ribozymes cleave the target mRNAs, preventing translation and
protein
3 5 accumulation. In the absence of the expression of the target gene, HCV
gene expression
and/or replication is inhibited.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
In a preferred embodiment, ribozymes are added directly, or can be complexed
with cationic lipids, packaged within liposomes, or otherwise delivered to
target cells. The
nucleic acid or nucleic acid complexes can be locally administered to relevant
tissues ex
vivo, or in vivo through injection, infusion pump or stent, with or without
their
5 incorporation in biopolymers. In another preferred embodiment, the ribozyme
is
administered to the site of HCV activity (e.g., hepatocytes) in an appropriate
liposomal
vehicle.
In another aspect of the invention, ribozymes that cleave target molecules and
inhibit HCV activity are expressed from transcription units inserted into DNA
or RNA
10 vectors. The recombinant vectors are preferably DNA plasmids or viral
vectors. Ribozyme
expressing viral vectors could be constructed based on, but not limited to,
adeno-
associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the
recombinant vectors
capable of expressing the ribozymes are delivered as described above, and
persist in target
cells. Alternatively, viral vectors may be used that provide for transient
expression of
ribozymes. Such vectors might be repeatedly administered as necessary. Once
expressed,
the ribozymes cleave the target mRNA. Delivery of ribozyme expressing vectors
could be
systemic, such as by intravenous or intramuscular administration, by
administration to
target cells ex-planted from the patient followed by reintroduction into the
patient, or by
any other means that would allow for introduction into the desired target cell
(for a review
2 0 see Couture and Stinchcomb, 1996, TIG., 12, 510). In another aspect of the
invention,
ribozymes that cleave target molecules and inhibit viral replication are
expressed from
transcription units inserted into DNA, RNA, or viral vectors. Preferably, the
recombinant
vectors capable of expressing the ribozymes are locally delivered as described
above, and
transiently persist in smooth muscle cells. However, other mammalian cell
vectors that
2 5 direct the expression of RNA may be used for this purpose.
By "patient" is meant an organism which is a donor or recipient of explanted
cells
or the cells themselves. "Patient" also refers to an organism to which
enzymatic nucleic
acid molecules can be administered. Preferably, a patient is a mammal or
mammalian
cells. More preferably, a patient is a human or human cells.
3 0 By "vectors" is meant any nucleic acid- and/or viral-based technique used
to
deliver a desired nucleic acid.
These ribozymes, individually, or in combination or in conjunction with other
drugs, can be used to treat diseases or conditions discussed above. For
example, to treat a
disease or condition associated with HCV levels, the patient may be treated,
or other
3 5 appropriate cells may be treated, as is evident to those skilled in the
art.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
11
Other features and advantages of the invention will be apparent from the
following
description of the preferred embodiments thereof, and from the claims.
Description Of The Preferred Embodiments
The drawings will first briefly be described.
Drawings:
Figure 1 shows the secondary structure model for seven different classes of
enzymatic nucleic acid molecules. Arrow indicates the site of cleavage. -------
-- indicate
the target sequence. Lines interspersed with dots are meant to indicate
tertiary interactions.
- is meant to indicate base-paired interaction. Group I Intron: P1-P9.0
represent various
stem-loop structures (Cech et aL, 1994, Nature Struc. Bio., 1, 273). RNase P
(M1RNA):
EGS represents external guide sequence (Forster et al., 1990, Science, 249,
783; Pace et
al., 1990, J. Biol. Chem., 265, 3587). Group II Intron: 5'SS means 5' splice
site; 3'SS
means 3'-splice site; IBS means intron binding site; EBS means exon binding
site (Pyle et
al., 1994, Biochemistry, 33, 2716). VS RNA: I-VI are meant to indicate six
stem-loop
structures; shaded regions are meant to indicate tertiary interaction
(Collies, International
PCT Publication No. WO 96/19577). HDV Ribozyme: : I-IV are meant to indicate
four
stem-loop structures (Been et al., US Patent No. 5,625,047). Hammerhead
Ribozyme:
I-III are meant to indicate three stem-loop structures; stems I-III can be of
any length and
may be symmetrical or asymmetrical (IJsman et al., 1996, Curr. Op. Struct.
Bio., 1, 527).
Hairpin Ribozyme: Helix 1, 4 and 5 can be of any length; Helix 2 is between 3
and 8
base-pairs long; Y is a pyrimidine; Helix 2 (H2) is provided with a least 4
base pairs (i.e.,
n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 or more
bases
(preferably 3 - 20 bases, i. e., m is from 1 - 20 or more). Helix 2 and helix
S may be
covalently linked by one or more bases (i.e., r is 1 base). Helix 1, 4 or 5
may also be
extended by 2 or more base pairs (e.g., 4 - 20 base pairs) to stabilize the
ribozyme
structure, and preferably is a protein binding site. In each instance, each N
and N'
independently is any normal or modified base and each dash represents a
potential base-
pairing interaction. These nucleotides may be modified at the sugar, base or
phosphate.
Complete base-pairing is not required in the helices, but is preferred. Helix
1 and 4 can be
3 0 of any size (i. e., o and p is each independently from 0 to any number,
e.g., 20) as long as
some base-pairing is maintained. Essential bases are shown as specific bases
in the
structure, but those in the art will recognize that one or more may be
modified chemically
(abasic, base, sugar and/or phosphate modifications) or replaced with another
base without
significant effect. Helix 4 can be fonmed from two separate molecules, i.e.,
without a
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
12
connecting loop. The connecting loop when present may be a ribonucleotide with
or
without modifications to its base, sugar or phosphate. "q" is 2 bases. The
connecting
loop can also be replaced with a non-nucleotide linker molecule. H refers to
bases A, U,
or C. Y refers to pyrimidine bases. " " refers to a covalent bond. (Burke et
al., 1996,
Nucleic Acids & Mol. Biol., 10, 129; Chowrira et al., US Patent No.
5,631,359).
Figure 2 is a graph displaying the ability of ribozymes targeting various
sites
within the conserved S' HCV UTR region to cleave the transcripts made from
several
genotypes.
Figure 3 is a schematic representation of the Dual Reporter System utilized to
demonstrate ribozyme mediated reduction of luciferase activity in cell
culture.
Figure 4 is a graph demonstrating the ability of ribozymes to reduce
luciferase
activity in OST-7 cells.
Figure 5 is a graph demonstrating the ability of ribozymes targeting sites
HCV.S-
313 and HCV.S-318, to reduce luciferase activity in OST-7 cells compared to
their
inactive controls.
Figure 6A is a bar graph demonstrating the effect of ribozyme treatment on HCV-
Polio virus (PV) replication. HeLa cells in 96-well plates were infected with
HCV-PV at a
multiplicity of infection (MOI) of 0.1. Virus inoculum was then replaced with
media
containing 5% serum and ribozyme or control (200nM), as indicated, complexed
to a
2 0 cationic lipid. After 24 hour cells were lysed 3 times by freeze/thaw and
virus was
quantified by plaque assay. Scrambled control (SAC), binding control (BAC), 3
P=S
ribozymes, and 4 P=S ribozymes are indicated. Plaque forming units (pfu)/ml
are shown
as the mean of triplicate samples + standard deviation (S.D.).
Figure 6B is a bar graph demonstrating the effect of ribozyme treatment on
wild
2 5 type PV replication. HeLa cells in 96-well plates were infected with wild
type PV at an
MOI = 0.05 for 30 minutes. All ribozymes contained 4P=S in (B). Plaque forming
units
(pfu)/ml are shown as the mean of triplicate samples + standard deviation
(S.D.).
Figure 7 is a schematic representation of various hammerhead ribozyme
constructs
targeted against HCV RNA.
3 0 Figure 8 is a graph demonstrating the effect of site 183 ribozyme
treatment on a
single round of HCV-PV infection. HeLa cells were infected with HCV-PV at an
MOI = 5
for 30 minutes prior to treatment with ribozymes or control. Cells were lysed
after 6, 7, or
8 hours and virus was quantified by plaque assay. Ribozyme binding arm/stem II
formats
(7/4, 7/3, 6/4, 6/3) and scrambled control (SAC, 7/4 format) are indicated.
All contasned
3 5 4P=S stabilization. Results in pfulml are shown as the median of duplicate
samples +
range.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
13
Figure 9 shows the secondary structure models of three ribozyme motifs
described
in this application.
Figure 10 shows the activity of anti-HCV ribozymes in combination with
Interferon. Results in pfu/ml are shown as the median of duplicate samples ~
range. BAC,
binding attenuated control molecule; IF, interferon; Rz, hammerhead ribozyme
targeted to
HCV site 183; pfu, plaque forming unit.
Ribozyes
Seven basic varieties of naturally-occurring enzymatic RNAs are known
presently.
In addition, several in vitro selection (evolution) strategies (Orgel, 1979,
Proc. R. Soc.
London, B 205, 435) have been used to evolve new nucleic acid catalysts
capable of
catalyzing cleavage and ligation of phosphodiester linkages (Joyce, 1989,
Gene, 82, 83-87;
Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American
267, 90-97;
Breaker et al., 1994, TIBTECH 12, 268; Bartel et al.,1993, Science 261:1411-
1418;
Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker,
1996,
Curr. Op. Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,
4262; Tang et
al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra; Long &Uhlenbeck,
1994,
supra; Ishizaka et al., 1995, supra; Vaish et al., 1997, Biochemistry 36,
6495; all of these
are incorporated by reference herein). Each can catalyze a series of reactions
including the
hydrolysis of phosphodiester bonds in traps (and thus can cleave other RNA
molecules)
2 0 under physiological conditions. Table I summarizes some of the
characteristics of some of
these ribozymes. In general, enzymatic nucleic acids act by first binding to a
target RNA.
Such binding occurs through the target binding portion of an enzymatic nucleic
acid which
is held in close proximity to an enzymatic portion of the molecule that acts
to cleave the
target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a
target
2 5 RNA through complementary base-pairing, and once bound to the correct
site, acts
enzymatically to cut the target RNA. Strategic cleavage of such a target RNA
will destroy
its ability to direct synthesis of an encoded protein. After an enzymatic
nucleic acid has
bound and cleaved its RNA target, it is released from that RNA to search for
another target
and can repeatedly bind and cleave new targets.
30 The enzymatic nature of a ribozyme is advantageous over other technologies,
since
the concentration of ribozyme necessary to affect a therapeutic treatment is
lower. This
advantage reflects the ability of the ribozyme to act enzymatically. Thus, a
single
ribozyme molecule is able to cleave many molecules of target RNA. In addition,
the
ribozyme is a highly specific inhibitor, with the specificity of inhibition
depending not
3 5 only on the base-pairing mechanism of binding to the target RNA, but also
on the
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
14
mechanism of target RNA cleavage. Single mismatches, or base-substitutions,
near the
site of cleavage can be chosen to completely eliminate catalytic activity of a
ribozyme.
Nucleic acid molecules having an endonuclease enzymatic activity are able to
repeatedly cleave other separate RNA molecules in a nucleotide base sequence-
specific
manner. Such enzymatic nucleic acid molecules can be targeted to virtually any
RNA
transcript, and efficient cleavage achieved in vitro (Zaug et al., 324, Nature
429 1986 ;
Uhlenbeck, 1987 Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA
8788, 1987;
Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and Gerlach, 334
Nature 585,
1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids
Research 1371,
1989; Chartrand et al., 1995, Nucleic Acids Research 23, 4092; Santoro et al.,
1997, PNAS
94, 4262).
Because of their sequence-specificity, traps-cleaving ribozymes show promise
as
therapeutic agents for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med.
Chem.
30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).
Ribozymes
can be designed to cleave specific RNA targets within the background of
cellular RNA.
Such a cleavage event renders the RNA non-functional and abrogates protein
expression
from that RNA. In this rnanner, synthesis of a protein associated with a
disease state can
be selectively inhibited.
Ribozymes that cleave the specified sites in HCV RNAs represent a novel
2 0 therapeutic approach to infection by the hepatitis C virus. Applicant
indicates that
ribozymes are able to inhibit the activity of HCV and that the catalytic
activity of the
ribozymes is required for their inhibitory effect. Those of ordinary skill in
the art will find
that it is clear from the examples described that other ribozymes that cleave
HCV RNAs
may be readily designed and are within the invention.
2 5 Tar eg t sites
Targets for useful ribozymes can be determined as disclosed in Draper et al.,
WO
93/23569; Sullivan et al., WO 93/23057; Thompson et al., WO 94/02595; Draper
et al.,
WO 95/04818; McSwiggen et al., US Patent No. 5,525,468 and hereby incorporated
by
reference herein in totality. Rather than repeat the guidance provided in
those documents
3 0 here, below are provided specific examples of such methods, not limiting
to those in the
art. Ribozyrnes to such targets are designed as described in those
applications and
synthesized to be tested in vitro and in vivo, as also described. Such
ribozymes can also be
optimized and delivered as described therein.
The sequence of HCV RNAs were screened for optimal ribozyme target sites using
35 a computer folding algorithm. Hammerhead or hairpin ribozyme cleavage sites
were
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
identified. These sites are shown in Tables IV-VIII (All sequences are S' to
3' in the
tables). The nucleotide base position is noted in the Tables as that site to
be cleaved by the
designated type of ribozyme. The nucleotide base position is noted in the
Tables as that
site to be cleaved by the designated type of ribozyme.
5 Because HCV RNAs are highly homologous in certain regions, some ribozyme
target sites are also homologous (see Table IV and VIII). In this case, a
single ribozyme
will target different classes of HCV RNA. The advantage of one ribozyme that
targets
several classes of HCV RNA is clear, especially in cases where one or more of
these
RNAs may contribute to the disease state.
10 Hammerhead or hairpin ribozymes were designed that could bind and were
individually analyzed by computer folding (Jaeger et al., 1989 Proc. Natl.
Acad. Sci. USA,
86, 7706) to assess whether the ribozyme sequences fold into the appropriate
secondary
structure. Those ribozymes with unfavorable intramolecular interactions
between the
binding arms and the catalytic core are eliminated from consideration. Varying
binding
15 arm lengths can be chosen to optimize activity. Generally, at least 5 bases
on each arm are
able to bind to, or otherwise interact with, the target RNA. Ribozymes of the
hammerhead
or hairpin motif were designed to anneal to various sites in the mRNA message.
The
binding arms are complementary to the target site sequences described above.
Riboz~rme Synthesis
2 0 Synthesis of nucleic acids greater than 100 nucleotides in length is
difficult using
automated methods, and the therapeutic cost of such molecules is prohibitive.
In this
invention, small nucleic acid motifs (e.g., hammerhead or the hairpin
ribozymes) are used
for exogenous delivery. The simple structure of these molecules increases the
ability of
the nucleic acid to invade targeted regions of the mRNA structure. However,
these
2 5 nucleic acid molecules can also be expressed within cells from eukaryotic
promoters (e.g.,
Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc.
Natl.
Acad. Sci. USA 83, 399; SullengerScanlon et al., 1991, Proc. Natl. Acad. Sci.
USA, 88,
10591-5; Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Dropulic et
al., 1992 J.
Virol, 66, 1432-41; Weerasinghe et al., 1991 J. Virol, 65, 5531-4; Ojwang et
al., 1992
30 Proc. Natl. Acad. Sci. USA 89, 10802-6; Chen et al., 1992 Nucleic Acids
Res., 20, 4581-
9; Sarver et al., 1990 Science 247, 1222-1225; Thompson et al., 1995 Nucleic
Acids Res.
23, 2259). Those skilled in the art realize that any nucleic acid can be
expressed in
eukaryotic cells from the appropriate DNA/RNA vector. The activity of such
nucleic
acids can be augmented by their release from the primary transcript by a
ribozyme (Draper
3 5 et al., PCT W093/23569, and Sullivan et al., PCT W094/02595, both hereby
incorporated
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
16
in their totality by reference herein; Ohkawa et al., 1992 Nucleic Acids Symp.
Ser., 27, 15-
6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993
Nucleic Acids
Res., 21, 3249-55; Chowrira et al., 1994 J. Biol. Chem. 269, 25856).
The ribozymes in the examples were chemically synthesized. The method of
synthesis used follows the procedure for normal RNA synthesis as described in
Usman et
al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990 Nucleic Acids
Res., 18,
5433; and Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684 and makes use
of
common nucleic acid protecting and coupling groups, such as dimethoxytrityl at
the S
end, and phosphoramidites at the 3'-end. Small scale synthesis were conducted
on a 394
Applied Biosystems, Inc. synthesizer using a modified 2.5 ~tmol scale protocol
with a 5
min coupling step for alkylsilyl protected nucleotides and 2.5 min coupling
step for 2'-O-
methylated nucleotides. Table II outlines the amounts, and the contact times,
of the
reagents used in the synthesis cycle. A 6.5-fold excess (163 pL of 0.1 M =
16.3 ~,mol) of
phosphoramidite and a 24-fold excess of S ethyl tetrazole (238 ~L of 0.25 M =
59.5 ~zmol)
relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average
coupling
yields on the 394 Applied Biosystems, Inc. synthesizer, determined by
colorimetric
quantitation of the trityl fractions, were 97.5-99%. Other oligonucleotide
synthesis
reagents for the 394 Applied Biosystems, Inc. synthesizer : detritylation
solution was 2%
TCA in methylene chloride (ABI); capping was performed with 16% N methyl
imidazole
2 0 in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI);
oxidation
solution was 16.9 mM I2, 49 mM pyridine, 9% water in THF (Millipore). B & J
Synthesis
Grade acetonitrile was used directly from the reagent bottle. S Ethyl
tetrazole solution
(0.25 M in acetonitrile) was made up from the solid obtained from American
International
Chemical, Inc.
2 5 Deprotection of the RNA was performed as follows. The polymer-bound
oligoribonucleotide, trityl-off, was transferred from the synthesis column to
a 4mL glass
screw top vial and suspended in a solution of methylamine (MA) at 65 °C
for 10 min.
After cooling to -20 °C, the supernatant was removed from the polymer
support. The
support was washed three times with 1.0 mL of EtOH:MeCN:H20/3:1:1, vortexed
and the
3 0 supernatant was then added to the first supernatant. The combined
supernatants,
containing the oligoribonucleotide, were dried to a white powder.
The base-deprotected oligoribonucleotide was resuspended in anhydrous
TEA~HF/NMP solution (250 pL of a solution of I.SmL N methylpyrrolidinone, 750
~L
TEA and 1.0 mL TEA~3HF to provide a 1.4M HF concentration) and heated to
65°C for
3 5 1.5 h. The resulting, fully deprotected, oligomer was quenched with 50 mM
TEAB (9
mL) prior to anion exchange desalting.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
17
For anion exchange desalting of the deprotected oligomer, the TEAB solution
was
loaded onto a Qiagen 500~ anion exchange cartridge (Qiagen Inc.) that was
prewashed
with 50 mM TEAB (10 mL). After washing the loaded cartridge with SO mM TEAB
(10
mL), the RNA was eluted with 2 M TEAB (10 mL) and dried down to a white
powder.
Inactive hammerhead ribozymes were synthesized by substituting switching the
order of GSA6 and substituting a U for A14(numbering from Hertel, K. J., et
al., 1992,
Nucleic Acids Res., 20, 3252). Inactive ribozymes were may also by synthesized
by
substituting a U for GS and a U for A14. In some cases, the sequence of the
substrate
binding arms were randomized while the overall base composition was
maintained.
The average stepwise coupling yields were >98% (Wincott et al., 1995 Nucleic
Acids Res. 23, 2677-2684).
Hairpin ribozymes are synthesized in two parts and annealed to reconstruct the
active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res., 20, 2835-2840).
Ribozymes are also synthesized from DNA templates using bacteriophage T7 RNA
polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180, 51 ).
Ribozymes are modified to enhance stability and/or enhance catalytic activity
by
modification with nuclease resistant groups, for example, 2'-amino, 2'-C-
allyl, 2'-flouro,
2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and
Cedergren,
1992 TIBS 17, 34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin
et al.,
2 0 1996 Biochemistry 6, 14090).
Ribozymes were purified by gel electrophoresis using general methods or are
purified by high pressure liquid chromatography (HPLC; See Stinchcomb et al.,
International PCT Publication No. WO 95/23225, the totality of which is hereby
incorporated herein by reference) and are resuspended in water.
2 5 The sequences of the ribozymes that are chemically synthesized, useful in
this
study, are shown in Tables IV-IX. Those in the art will recognize that these
sequences
are representative only of many more such sequences where the enzymatic
portion of the
ribozyme (all but the binding arms) is altered to affect activity. For
example, stem-loop II
sequence of hammerhead ribozymes can be altered (substitution, deletion,
and/or
3 0 insertion) to contain any sequences provided a minimum of two base-paired
stem structure
can form. Similarly, stem-loop IV sequence of hairpin ribozymes, can be
altered
(substitution, deletion, and/or insertion) to contain any sequence, provided a
minimum of
two base-paired stem structure can form. Preferably; no more than 200 bases
are inserted
at these locations. The sequences listed in Tables IV-IX may be formed of
3 5 ribonucleotides or other nucleotides or non-nucleotides. Such ribozymes
(which have
enzymatic activity) are equivalent to the ribozymes described specifically in
the Tables.
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
18
Optimizing Ribozvme A.ctivity
Catalytic activity of the ribozymes described in the instant invention can be
optimized as described by Draper et al., supra. 'The details will not be
repeated here, but
include altering the length of the ribozyme binding arms, or chemically
synthesizing
ribozymes with modifications (base, sugar and/or phosphate) that prevent their
degradation by serum ribonucleases and/or enhance their enzymatic activity
(see e.g.,
Eckstein et al., International Publication No. WO 92/07065; Perrault et al.,
1990 Nature
344, 565; Pieken et al., 1991 Science 253, 314; Usman aid Cedergren, 1992
Trends in
Biochem. Sci. 17, 334; Usman et al., International Publication No. WO
93/15187; and
Rossi et al., International Publication No. WO 91/03162; Sproat, US Patent No.
5,334,711; and Burgin et al., supra; all of these describe various chemical
modifications
that can be made to the base, phosphate and/or sugar moieties of enzymatic RNA
molecules). Modifications which enhance their efficacy in cells, and removal
of bases
from stem loop structures to shorten RNA synthesis times and reduce chemical
requirements are desired. (All these publications are hereby incorporated by
reference
herein).
There are several examples in the art describing sugar and phosphate
modifications
that can be introduced into enzymatic nucleic acid molecules without
significantly
effecting catalysis and with significant enhancement in their nuclease
stability and
2 0 efficacy. Ribozymes are modified to enhance stability and/or enhance
catalytic activity by
modification with nuclease resistant groups, for example, 2'-amino, 2'-C-
allyl, 2'-flouro,
2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and
Cedergren,
1992 TIBS 17, 34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin
et al.,
1996 Biochemistry 35, 14090). Sugar modification of enzymatic nucleic acid
molecules
2 5 have been extensively described in the art (see Eckstein et al.,
International Publication
PCT No. WO 92/07065; Perrault et al. Nature 1990, 344, 565-568; Pieken et al.
Science
1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-
339;
Usman et al. International Publication PCT No. WO 93/15187; Sproat, US Patent
No.
5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702; all of the
references are
3 0 hereby incorporated in their totality by reference herein). Such
publications describe
general methods and strategies to determine the location of incorporation of
sugar, base
and/or phosphate modifications and the like into ribozymes without inhibiting
catalysis,
and are incorporated by reference herein. In view of such teachings, similar
modifications
can be used as described herein to modify the nucleic acid catalysts of the
instant
3 5 invention.
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
19
Nucleic acid catalysts having chemical modifications which maintain or enhance
enzymatic activity are provided. Such nucleic acid is also generally more
resistant to
nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the
activity may not
be significantly lowered. As exemplified herein such ribozymes are useful in a
cell and/or
in vivo even if activity over all is reduced 10 fold (Burgin et al., 1996,
Biochemistry, 35,
14090). Such ribozymes herein are said to "maintain" the enzymatic activity on
all RNA
ribozyme.
Therapeutic ribozymes delivered exogenously must optimally be stable within
cells
until translation of the target RNA has been inhibited long enough to reduce
the levels of
the undesirable protein. This period of time varies between hours to days
depending upon
the disease state. Clearly, ribozymes must be resistant to nucleases in order
to function as
effective intracellular therapeutic agents. Improvements in the chemical
synthesis of RNA
(Wincott et al., 1995 Nucleic Acids Res. 23, 2677; incorporated by reference
herein) have
expanded the ability to modify ribozymes by introducing nucleotide
modifications to
enhance their nuclease stability as described above.
By "nucleotide" as used herein is as recognized in the art to include natural
bases
(standard), and modified bases well known in the art. Such bases are generally
located at
the 1' position of a sugar moiety. Nucleotide generally comprise a base, sugar
and a
phosphate group. The nucleotides can be unmodified or modified at the sugar,
phosphate
2 0 and/or base moiety, (also referred to interchangeably as nucleotide
analogs, modified
nucleotides, non-natural nucleotides, non-standard nucleotides and other ; see
for example,
Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No.
WO
92/07065; Usman et al., International PCT Publication No. WO 93/15187; all
hereby
incorporated by reference herein). There are several examples of modified
nucleic acid
2 5 bases known in the art and has recently been summarized by Limbach et al.,
1994, Nucleic
Acids Res. 22, 2183. Some of the non-limiting examples of base modifications
that can
be introduced into enzymatic nucleic acids without significantly effecting
their catalytic
activity include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,
pseudouracil, 2, 4,
6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,
30 5-alkylcytidines (e.g., S-methylcytidine), 5-alkyluridines (e.g.,
ribothymidine),
5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines
(e.g. 6-
methyluridine) and others (Burgin et al., 1996, Biochemistry, 35, 14090). By
"modified
bases" in this aspect is meant nucleotide bases other than adenine, guanine,
cytosine and
uracil at 1' position or their equivalents; such bases may be used within the
catalytic core
3 5 of the enzyme and/or in the substrate-binding regions.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
By "abasic" is meant sugar moieties lacking a base or having other chemical
groups in place of base at the 1' position.
By "unmodified nucleoside" is meant one of the bases adenine, cytosine,
guanine,
uracil joined to the 1' carbon of beta-D-ribo-furanose.
5 By "modified nucleoside" is meant any nucleotide base which contains a
modification in the chemical structure of an unmodified nucleotide base, sugar
and/or
phosphate.
Various modifications to ribozyme structure can be made to enhance the utility
of
ribozymes. Such modifications will enhance shelf life, half life in vitro,
stability, and ease
10 of introduction of such ribozymes to the target site, e.g., to enhance
penetration of cellular
membranes, and confer the ability to recognize and bind to targeted cells.
Administration of Ribozvmes
Sullivan et al., PCT WO 94/02595, describes the general methods for delivery
of
enzymatic RNA molecules . Ribozymes may be administered to cells by a variety
of
15 methods known to those familiar to the art, including, but not restricted
to, encapsulation
in liposomes, by iontophoresis, or by incorporation into other vehicles, such
as hydrogels,
cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For
some
indications, ribozymes may be directly delivered ex vivo to cells or tissues
with or without
the aforementioned vehicles. Alternatively, the RNA/vehicle combination is
locally
2 0 delivered by direct injection or by use of a catheter, infusion pump or
stent. Other routes
of delivery include, but are not limited to, intravascular, intramuscular,
subcutaneous or
joint injection, aerosol inhalation, oral (tablet or pill form), topical,
systemic, ocular,
intraperitoneal and/or intrathecal delivery. More detailed descriptions of
ribozyme
delivery and administration are provided in Sullivan et al., supra. and Draper
et al., PCT
W093/23569 which have been incorporated by reference herein.
The molecules of the instant invention can be used as pharmaceutical agents.
Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a
symptom to
some extent, preferably all of the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the invention can be administered
(e.g.,
3 0 RNA, DNA or protein) and introduced into a patient by any standard means,
with or
without stabilizers, buffers, and the like, to form a pharmaceutical
composition. When it
is desired to use a lipid or liposome delivery mechanism, standard protocols
for
formulation can be followed. The compositions of the present invention may
also be
formulated and used as tablets, capsules or elixirs for oral administration;
suppositories for
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
21
rectal administration; sterile solutions; suspensions for injectable
administration; and the
like.
The present invention also includes pharmaceutically acceptable formulations
of
the compounds described. These formulations include salts of the above
compounds, e.g.,
acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic
acid, and
benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or
formulation in a form suitable for administration, e.g., systemic
administration, into a cell
or patient, preferably a human. Suitable forms, in part, depend upon the use
or the route of
entry, for example oral, transdermal, or by injection. Such forms should not
prevent the
composition or formulation to reach a target cell (i.e., a cell to which the
negatively
charged polymer is desired to be delivered to). For example, pharmacological
compositions injected into the blood stream should be soluble. Other factors
a.re known in
the art, and include considerations such as toxicity and forms which prevent
the
composition or formulation from exerting its effect.
By "systemic administration" is meant in vivo systemic absorption or
accumulation of drugs in the blood stream followed by distribution throughout
the entire
body. Administration routes which lead to systemic absorption include, without
limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral,
intrapulmonary
2 0 and intramuscular. Each of these administration routes expose the desired
negatively
charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The
rate of entry
of a drug into the circulation has been shown to be a function of molecular
weight or size.
The use of a liposome or other drug carrier comprising the compounds of the
instant
invention can potentially localize the drug, for example, in certain tissue
types, such as the
2 5 tissues of the reticular endothelial system (RES). A liposome formulation
which can
facilitate the association of drug with the surface of cells, such as,
lymphocytes and
macrophages is also useful. This approach may provide enhanced delivery of the
drug to
target cells by taking advantage of the specificity of macrophage and
lymphocyte immune
recognition of abnormal cells, such as the HCV infected liver cells.
30 The invention also features the use of a composition comprising surface-
modified
liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-
circulating
liposomes or stealth liposomes). These formulations offer an method for
increasing the
accumulation of drugs in target tissues. This class of drug carriers resists
opsonization and
elimination by the mononuclear phagocytic system (MPS or RES}, thereby
enabling
3 5 longer blood circulation times and enhanced tissue exposure for the
encapsulated drug
(Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem. Pharm.
Bull. 1995,
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
22
43, 1005-1011). Such liposomes have been shown to accumulate selectively in
tumors,
presumably by extravasation and capture in the neovascularized target tissues
(Lasic et al.,
Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238,
86-90).
The long-circulating liposomes enhance the pharmacokinetics and
pharmacodynamics of
DNA and RNA, particularly compared to conventional cationic liposomes which
are
known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,
42, 24864-
24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et
al.,
International PCT Publication No. WO 96/10390; Holland et al., International
PCT
Publication No. WO 96/10392; all of these are incorporated by reference
herein). All of
these references are incorporated by reference herein.
In addition other cationic molecules may also be utilized to deliver the
molecules
of the present invention. For example, ribozymes may be conjugated to
glycosylated
poly(L-lysine) which has been shown to enhance localization of antisense
oligonucleotides
into the liver (Nakazono et al., 1996, Hepatology 23, 1297-1303; Nahato et
al., 1997,
Biochem Pharm. 53, 887-895). Glycosylated poly(L-lysine) may be covently
attached to
the enzymatic nucleic acid or be bound to enzymatic nucleic acid through
electrostatic
interaction.
The present invention also includes compositions prepared for storage or
administration which include a pharmaceutically effective amount of the
desired
2 0 ~ compounds in a pharmaceutically acceptable carrier or diluent.
Acceptable carriers or
diluents for therapeutic use are well known in the pharmaceutical art, and are
described,
for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.
Gennaro edit. 1985) hereby incorporated by reference herein. For example,
preservatives,
stabilizers, dyes and flavoring agents may be provided. Id. at 1449. These
include sodium
2 5 benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition,
antioxidants and
suspending agents may be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit
the
occurrence, or treat (alleviate a symptom to some extent, preferably all of
the symptoms) a
disease state. The pharmaceutically effective dose depends on the type of
disease, the
3 0 composition used, the route of administration, the type of mammal being
treated, the
physical characteristics of the specific mammal under consideration,
concurrent
medication, and other factors which those skilled in the medical arts will
recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active
ingredients is administered dependent upon potency of the negatively charged
polymer.
3 5 Alternatively, the enzymatic nucleic acid molecules of the instant
invention can be
expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub,
1985 Science
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
23
229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci. USA 83, 399;
Scanlon et
al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992
Antisense
Res. Dev., 2, 3-15; Dropulic et al., 1992 J. Virol, 66, 1432-41; Weerasinghe
et al., 1991
J. Virol, 65, 5531-4; Ojwang et al., 1992 Proc. Natl. Acad. Sci. USA 89, 10802-
6; Chen
et al., 1992 Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science 247,
1222-1225;
Thompson et al., 1995 Nucleic Acids Res. 23, 2259; Good et al., 1997, Gene
Therapy, 4,
45; all of the references are hereby incorporated in their totality by
reference herein).
Those skilled in the art realize that any nucleic acid can be expressed in
eukaryotic cells
from the appropriate DNA/RNA vector. The activity of such nucleic acids can be
augmented by their release from the primary transcript by a ribozyme (Draper
et al., PCT
WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992 Nucleic
Acids
Symp. Ser., 27, IS-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30;
Ventura et al.,
1993 Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994 J. Biol. Chem.
269, 25856; all
of the references are hereby incorporated in their totality by reference
herein).
In another aspect of the invention, enzymatic nucleic acid molecules that
cleave
target molecules are expressed from transcription units (see for example
Couture et al.,
1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors
are
preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors
could be
constructed based on, but not limited to, adeno-associated virus, retrovirus,
adenovirus, or
2 0 alphavirus. Preferably, the recombinant vectors capable of expressing the
ribozymes are
delivered as described above, and persist in target cells. Alternatively,
viral vectors may be
used that provide for transient expression of ribozymes. Such vectors might be
repeatedly
administered as necessary. Once expressed, the ribozymes cleave the target
mRNA. The
active ribozyme contains an enzymatic center or core equivalent to those in
the examples,
2 5 and binding arms able to bind target nucleic acid molecules such that
cleavage at the target
site occurs. Other sequences may be present which do not interfere with such
cleavage.
Delivery of ribozyme expressing vectors could be systemic, such as by
intravenous or
intramuscular administration, by administration to target cells ex-planted
from the patient
followed by reintroduction into the patient, or by any other means that would
allow for
3 0 introduction into the desired target cell (for a review see Couture et
al., 1996, TIG., 12,
510).
In one aspect the invention features, an expression vector comprising nucleic
acid
sequence encoding at least one of the nucleic acid catalyst of the instant
invention is
disclosed. The nucleic acid sequence encoding the nucleic acid catalyst of the
instant
3 5 invention is operable linked in a manner which allows expression of that
nucleic acid
molecule.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
24
In another aspect the invention features, the expression vector comprises: a
transcription initiation region (e.g., eukaryotic pol I, II or III initiation
region); b) a
transcription termination region (e.g., eukaryotic pol I, II or III
termination region); c) a
gene encoding at least one of the nucleic acid catalyst of the instant
invention; and wherein
said gene is operably linked to said initiation region and said termination
region, in a
manner which allows expression and/or delivery of said nucleic acid molecule.
The vector
may optionally include an open reading frame (ORF) for a protein operably
linked on the
5' side or the 3'-side of the gene encoding the nucleic acid catalyst of the
invention; and/or
an intron (intervening sequences).
Transcription of the ribozyme sequences are driven from a promoter for
eukaryotic
RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III
(pol III).
Transcripts from pol II or pol III promoters will be expressed at high levels
in all cells; the
levels of a given pol II promoter in a given cell type will depend on the
nature of the gene
regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic
RNA
polymerase promoters are also used, providing that the prokaryotic RNA
polymerase
enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990 Proc.
Natl.
Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993 Nucleic Acids Res., 21, 2867-
72;
Lieber et al., 1993 Methods Enzymol., 217, 47-66; Zhou et al., 1990 Mol. Cell.
Biol., 10,
4529-37). Several investigators have demonstrated that ribozymes expressed
from such
2 0 promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992
Antisense
Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. U S A, 89,
10802-6; Chen
et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Natl. Acad.
Sci. U S A,
90, 6340-4; L'Huillier et al., 1992 EMBO J. 11, 4411-8; Lisziewicz et al.,
1993 Proc.
Natl. Acad. Sci. U. S A., 90, 8000-4; Thompson et al., 1995 Nucleic Acids Res.
23, 2259;
2 5 Sullenger & Cech, 1993, Science, 262, 1566). More specifically,
transcription units such
as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA
{tRNA) and adenovirus VA RNA are useful in generating high concentrations of
desired
RNA molecules such as ribozymes in cells (Thompson et al., supra; Couture and
Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res., 22, 2830;
Noonberg et
30 al., US Patent No. 5,624,803; Good et al., 1997, Gene Ther. 4, 45;
Beigelman et al.,
International PCT Publication No. WO 96/18736; all of these publications are
incorporated
by reference herein. The above ribozyme transcription units can be
incorporated into a
variety of vectors for introduction into mammalian cells, including but not
restricted to,
plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated
virus
3 5 vectors), or viral RNA vectors (such as retroviral or alphavirus vectors)
(for a review see
Couture and Stinchcomb, 1996, supra).
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
In yet another aspect the invention features an expression vector comprising
nucleic acid sequence encoding at least one of the catalytic nucleic acid
molecule of the
invention, in a manner which allows expression of that nucleic acid molecule.
The
expression vector comprises in one embodiment; a) a transcription initiation
region; b) a
5 transcription termination region; c) a gene encoding at least one said
nucleic acid
molecule; and wherein said gene is operably linked to said initiation region
and said
termination region, in a manner which allows expression and/or delivery of
said nucleic
acid molecule. In another preferred embodiment the expression vector
comprises: a) a
transcription initiation region; b) a transcription termination region; c) an
open reading
10 frame; d) a gene encoding at least one said nucleic acid molecule, wherein
said gene is
operably linked to the 3'-end of said open reading frame; and wherein said
gene is
operably linked to said initiation region, said open reading frame and said
termination
region, in a manner which allows expression and/or delivery of said nucleic
acid molecule.
In yet another embodiment the expression vector comprises: a) a transcription
initiation
15 region; b) a transcription termination region; c) an intron; d) a gene
encoding at least one
said nucleic acid molecule; and wherein said gene is operably linked to said
initiation
region, said intron and said termination region, in a manner which allows
expression
and/or delivery of said nucleic acid molecule. In another embodiment, the
expression
vector comprises: a) a transcription initiation region; b) a transcription
termination region;
2 0 c) an intron; d) an open reading frame; e) a gene encoding at least one
said nucleic acid
molecule, wherein said gene is operably linked to the 3'-end of said open
reading frame;
and wherein said gene is operably linked to said initiation region, said
intron, said open
reading frame and said termination region, in a manner which allows expression
and/or
delivery of said nucleic acid molecule.
2 5 Interferons
Type I interferons (IFN) are a class of natural cytokines that includes a
family of
greater than 25 IFN-a (Pests, 1986, Methods Enzymol. 119, 3-14) as well as IFN-
(3, and
IFN-w. Although evolutionarily derived from the same gene (Diaz et al., 1994,
Genomics
22, 540-552), there are many differences in the primary sequence of these
molecules,
3 0 implying an evolutionary divergence in biologic activity. All type I IFN
share a common
pattern of biologic effects that begin with binding of the IFN to the cell
surface receptor
(Pfeffer & Strulovici, 1992, Transmembrane secondary messengers for IFN-a/(3.
In:
Interferon. Principles and Medical Applications., S. Baron, D.H. Coopenhaver,
F.
Dianzani, W.R. Fleischmann Jr., T.K. Hughes Jr., G.R. Kimpel, D.W. Niesel,
G.J.
Stanton, and S.K. Tyring, eds. 151-160). Binding is followed by activation of
tyrosine
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
26
kinases, including the Janus tyrosine kinases and the STAT proteins, which
leads to the
production of several IFN-stimulated gene products (Johnson et al., 1994, Sci.
Am. 270,
68-75). The IFN-stimulated gene products are responsible for the pleotropic
biologic
effects of type I IFN, including antiviral, antiproliferative, and
immunomodulatory effects,
cytokine induction, and HLA class I and class II regulation (Pestka et al.,
1987, Annu. Rev.
Biochem 56, 727). Examples of IFN-stimulated gene products include 2-5-
oligoadenylate
synthetase (2-5 OAS), ~i2-microglobulin, neopterin, p68 kinases, and the Mx
protein
(Chebath & Revel, 1992, The 2-5 A system: 2-5 A synthetase, isospecies and
functions.
In: Interferon. Principles and Medical Applications. S. Baron, D.H.
Coopenhaver, F.
Dianzan, W.R. Jr. Fleischmann, T.K. Jr Hughes, G.R. Kimpel, D.W. Niesel, G.J.
Stanton,
and S.K. Tyring, eds., pp. 225-236; Samuel, 1992, The RNA-dependent P1/eIF-2a
protein
kinase. In: Interferon. Principles and Medical Applications. S. Baron, D.H.
Coopenhaver, F. Dianzani, W.R. Fleischmann Jr., T.K. Hughes Jr., G.R. Kimpel,
D.W.
Niesel, G.H. Stanton, and S.K. Tyring, eds. 237-250; Horisberger, 1992, MX
protein:
function and Mechanism of Action. In: Interferon. Principles and Medical
Applications.
S. Baron, D.H. Coopenhaver, F. Dianzani, W.R. Fleischmann Jr., T.K. Hughes
Jr., G.R.
Kimpel, D.W. Niesel, G.H. Stanton, and S.K. Tyring, eds. 215-224). Although
all type I
IFN have similar biologic effects, not all the activities are shared by each
type I IFN, and,
in many cases, the extent of activity varies quite substantially for each IFN
subtype (Fish
2 0 et al, 1989, J. Interferon Res. 9, 97-114; Ozes et al., 1992, J.
Interferon Res. 12, 55-59).
More specifically, investigations into the properties of different subtypes of
IFN-a and
molecular hybrids of IFN-a have shown differences in pharmacologic properties
(Rubinstein, 1987, J. Interferon Res. 7, 545-551). These pharmacologic
differences may
arise from as few as three amino acid residue changes (Lee et al., 1982,
Cancer Res. 42,
1312-1316).
Eighty-five to 166 amino acids are conserved in the known IFN-a subtypes.
Excluding the IFN-a pseudogenes, there are approximately 25 known distinct IFN-
a
subtypes. Pairwise comparisons of these nonallelic subtypes show primary
sequence
differences ranging from 2% to 23%. In addition to the naturally occurring
IFNs, a non-
3 0 natural recombinant type I interferon known as consensus interferon (CIFN)
has been
synthesized as a therapeutic compound (Tong et al., 1997, Hepatology 26, 747-
754).
Interferon is currently in use for at least 12 different indications including
infectious and autoimmune diseases and cancer (Borden, 1992, N. Engl. J. Med.
326,
1491-1492). For autoimmune diseases IFN has been utilized for treatment of
rheumatoid
3 5 arthritis, multiple sclerosis, and Crohn's disease. For treatment of
cancer IFN has been
used alone or in combination with a number of different compounds. Specific
types of
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
27
cancers for which IFN has been used include squamous cell carcinomas,
melanomas,
hypemephromas, hemangiomas, hairy cell leukemia, and Kaposi's sarcoma. In the
treatment of infectious diseases, IFNs increase the phagocytic activity of
macrophages and
cytotoxicity of lymphocytes and inhibits the propagation of cellular
pathogens. Specific
indications for which IFN has been used as treatment include: hepatitis B,
human
papillomavirus types 6 and 11 (i.e. genital warts) (Leventhal et al., 1991, N
Engl J Med
325, 613-617), chronic granulomatous disease, and hepatitis C virus.
Numerous well controlled clinical trials using IFN-alpha in the treatment of
chronic HCV infection have demonstrated that treatment three times a week
results in
lowering of serum ALT values in approximately 50% (range 40% to 70%) of
patients by
the end of 6 months of therapy (Davis et al., 1989, The new England Journal of
Medicine
321, 1501-1506; Marcellin et al., 1991, Hepatology 13, 393-397; Tong et al.,
1997,
Hepatology 26, 747-754; Tong et al., Hepatology 26, 1640-1645). However,
following
cessation of interferon treatment, approximately 50% of the responding
patients relapsed,
resulting in a "durable" response rate as assessed by normalization of serum
ALT
concentrations of approximately 20 to 25%. In addition, studies that have
examined six
months of type 1 interferon therapy using changes in HCV RNA values as a
clinical
endpoint have demonstrated that up to 35% of patients will have a loss of HCV
RNA by
the end of therapy (Tong et al., 1997, supra). However, as with the ALT
endpoint, about
2 0 50% of the patients relapse six months following cessation of therapy
resulting in a
durable virologic response of only 12% (23). Studies that have examined 48
weeks of
therapy have demonstrated that the sustained virological response is up to
25%.
Ribozymes in combination with IFN have the potential to improve the
effectiveness of treatment of HCV or any of the other indications discussed
above.
2 5 Ribozymes targeting RNAs associated with diseases such as infectious
diseases,
autoimmune disases, and cancer, can be used individually or in combination
with other
therapies such as IFN to achieve enhanced efficacy.
Examples
The following are non-limiting examples showing the selection, isolation,
3 0 synthesis and activity of enzymatic nucleic acids of the instant
invention.
The following examples demonstrate the selection of ribozymes that cleave HCV
RNA. The methods described herein represent a scheme by which ribozymes may be
derived that cleave other RNA targets required for HCV replication.
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
28
Example 1: Identification of Potential Ribozyme Cleavage Sites in HCV RNA
The sequence of HCV RNA was screened for accessible sites using a computer
folding algorithm. Regions of the mRNA that did not form secondary folding
structures
and contained potential hammerhead and/or hairpin ribozyme cleavage sites were
identified. The sequences of these cleavage sites are shown in tables IV-VIII.
Example 2: Selection of Ribozyme Cleava»e Sites in HCV RNA
To test whether the sites predicted by the computer-based RNA folding
algorithm
corresponded to accessible sites in HCV RNA, 20 hammerhead sites were selected
for
analysis. Ribozyme target sites were chosen by analyzing genornic sequences of
HCV
(Input Sequence = HPCJTA (Acc#D11168 & DO1I71)) and prioritizing the sites on
the
basis of folding. Hammerhead ribozymes were designed that could bind each
target (see Figure
1 ) and were individually analyzed by computer folding (Christoffersen et al.,
1994 J. Mol.
Struc. Theochem, 311, 273; Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA,
86, 7706) to
assess whether the ribozyme sequences fold into the appropriate secondary
structure.
Those ribozymes with unfavorable intramolecular interactions between the
binding arms
and the catalytic core were eliminated from consideration. As noted below,
varying
binding arm lengths can be chosen to optimize activity. Generally, at least S
bases on each
arm are able to bind to, or otherwise interact with, the target RNA.
Selection of ribozyme candidates was initiated by scanning for all hammerhead
cleavage sites in an HCV RNA sequence derived from a patient infected with HCV
genotype lb. The results of this sequence analysis are shown in Table III. As
seen by
Table III, 1300 hammerhead ribozyme sites were identified by this analysis.
Next, in
order to identify hammerhead ribozyme candidates that would cleave in the
conserved
regions of the HCV genome, a sequence alignment of approximately 50 HCV
isolates
from genotypes la, lb, 2a, 2b, 2c, 3a, 3b, 4a, Sa, and 6 was completed. Within
genotype
sites were identified that are in areas having the greatest sequence identity
between all
isolates examined. This analysis reduced the hammerhead ribozyme candidates to
about
23 (Table III).
Due to the high sequence variability of the HCV genome, selection of ribozymes
3 0 for broad therapeutic applications should probably involve the conserved
regions of the
HCV genome. A list of the thirty-hammerhead ribozymes derived from the
conserved
regions (5'- Non-Coding Region (NCR), 5'- end of core protein coding region,
and 3'-
NCR) of the HCV genome is shown in Table IV. In general, ribozymes targeted to
sites
located in the 5' terminal region of the HCV genome should block translation
while
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
29
ribozymes cleavage sites located in the 3' terminal region of the genome
should block
RNA replication.
Example 3: Chemical Synthesis and Purification of Riboz~mes
Ribozymes of the hammerhead or hairpin motif were designed to anneal to
various
sites in the RNA message. The binding arms are complementary to the target
site
sequences described above. The ribozymes were chemically synthesized. The
method of
synthesis used followed the procedure for normal RNA synthesis as described in
Usman et
al., (1987 J. Am. Chem. Soc., 109, 7845), Scaringe et al., (1990 Nucleic Acids
Res., 18,
5433) and Wincott et al., supra, and made use of common nucleic acid
protecting and
coupling groups, such as dimethoxytrityl at the S'-end, and phosphoramidites
at the 3'-end.
The average stepwise coupling yields were >98%.
Inactive hammerhead ribozymes were synthesized by substituting switching the
order of GSAb and substituting a U for A14 (numbering from Hertel et al., 1992
Nucleic
Acids Res., 20, 3252). Hairpin ribozymes were synthesized in two parts and
annealed to
reconstruct the active ribozyme (Chowrira and Burke, 1992 Nucleic Acids Res.,
20, 2835-
2840). Ribozymes were also synthesized from DNA templates using bacteriophage
T7
RNA polymerise (Milligan and Uhlenbeck, 1989, Methods Enzymol. l 80, 51 ).
Ribozymes were modified to enhance stability by modification with nuclease
resistant
groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H (for a
review see
2 0 Usman and Cedergren, 1992 TIBS 17, 34). Ribozymes were purified by gel
electrophoresis using general methods or were purified by high pressure liquid
chromatography (HPLC; See Wincott et al., supra; the totality of which is
hereby
incorporated herein by reference) and were resuspended in water. The sequences
of the
chemically synthesized ribozymes used in this study are shown below in Table
IV -IX.
2 5 Example 4: Ribozyme Cleavage of HCV RNA Tar;~et in vitro
Ribozymes targeted to the HCV are designed and synthesized as described above.
These ribozymes can be tested for cleavage activity in vitro, for example
using the
following procedure. The target sequences and the nucleotide location within
the HCV
are given in Table IV.
30 Cleavage Reactions: Full-length or partially full-length, internally-
labeled target
RNA for ribozyme cleavage assay is prepared by in vitro transcription in the
presence of
[a-32p] CTP, passed over a G SO Sephadex column by spin chromatography and
used as
substrate RNA without fiurther purification. Alternately, substrates are 5'-
32P-end labeled
using T4 polynucleotide kinase enzyme. Assays are performed by pre-warming a
2X
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
concentration of purified ribozyme in ribozyme cleavage buffer (50 mM Tris-
HC1, pH 7.5
at 37°C, 10 mM MgCl2) and the cleavage reaction was initiated by adding
the 2X
ribozyme mix to an equal volume of substrate RNA (maximum of 1-5 nlV1) that
was also
pre-warmed in cleavage buffer. As an initial screen, assays are carried out
for 1 hour at
5 37°C using a final concentration of either 40 nM or 1 mM ribozyme,
i.e., ribozyme excess.
The reaction is quenched by the addition of an equal volume of 95% formamide,
20 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol after which the sample is
heated to 95°C for 2 minutes, quick chilled and loaded onto a
denaturing polyacrylamide
gel. Substrate RNA and the specific RNA cleavage products generated by
ribozyme
10 cleavage are visualized on an autoradiograph of the gel. The percentage of
cleavage is
determined by Phosphor Imager~ quantitation of bands representing the intact
substrate
and the cleavage products.
Example 5: Ability of HCV Ribozymes to Cleave HCV RNA in patient serum.
Ribozymes targeting sites in HCV RNA were synthesized using modifications that
15 confer nuclease resistance (Beigelman, 1995, J. Biol. Chem. 270, 25702). It
has been well
documented that serum from chronic hepatitis C patients contains on average 3
x 106
copies/ml of HCV RNA. To further select ribozyme product candidates, the 30
HCV
specific ribozymes are characterized for HCV RNA cleavage activity utilizing
HCV
RNA isolated from the serum of genotype lb HCV patients. The best candidates
from
2 0 the HCV genotype lb screen will be screened against isolates from the wide
range of
HCV genotypes including la, lb, 2a, 2b, 2c, 3a, 3b, 4a, Sa, and 6. Therefore,
it is
possible to select ribozyme candidates for further development based on their
ability to
broadly cleave HCV RNA from a diverse range of HCV genotypes and quasispecies.
Example 6: Ribozyme Cleavaee of Conserved HCV RNA Tar~Zet Sites in vitro
2 5 There are three regions of the genome that are highly conserved, both
within a
genotype and across different genotypes. These conserved sequences occur in
the 5' and
3' non-coding regions (NCRs) as well as the 5'-end of the Core Protein coding
region.
These regions are thought to be important for HCV RNA replication and
translation.
Thus, therapeutic agents that target these conserved HCV genomic regions may
have a
3 0 significant impact over a wide range of HCV genotypes. The presence of
quasispecies,
and the potential for infection with more than one genotype makes this a
critical feature of
an elective therapy. Moreover, it is unlikely that drug resistance will occur,
since
mutations that have been suggested to lead to drug resistance typically do not
occur within
these highly conserved regions. In order to target multiple genotypes and
decrease the
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
31
chance of developing drug resistance, Applicant has designed ribozymes that
cleave in
regions of identity within the conserved regions discussed above.
Sequence alignments were performed for the S' NCR, the 5' end of the Core
Protein coding region, and the 3' NCR. For the 5' NCR, 34 different isolates
representing
genotypes la, lb, 2a, 2b, 2c, 3a, 3b, 4a, 4f, and Sa were aligned. The
alignments included
the sequences from nucleotide position 1 to nucleotide position 350 (1$
nucleotides
downstream of the initiator ATG colon), using the reported sequence "HPCK1 S
1" as the
reference for numbering. For the Core Protein coding region, 44 different
isolates
representing genotypes la, lb, 2a, 2b, 2c, 3a, 3b, 4a, 4c, 4f, Sa, and 6a were
aligned.
These alignments included 600 nucleotides, beginning 8 nucleotides upstream of
the
initiator ATG colon. As the reference for numbering, the reported sequence
"HPCCOPR"
was used, with the "C" eight nucleotides upstream of the initiator colon ATG
designated
as "1". For the 3' NCR region, 20 different isolates representing genotypes
lb, 2a, 2b, 3a,
and 3b were aligned. These alignments included sequences in the 3' terminal
235
nucleotides of the genome, with the reported sequence "D85516" used as the
reference for
numbering, and the 235' nucleotide from the 3' end designated as "1".
During analysis of the alignments of each region, each sequence was compared
to
the respective reference sequence (identified above), and regions of identity
across all
isolates were determined. All potential ribozyme sites were identified in the
reference
2 0 sequence. The highest priority for choosing ribozyme sites was that the
site should have
100% identity across all isolates aligned, at every position in both the
cleavage site and
binding arms. Ribozyme sites that met these criteria were chosen. In addition,
two
specific allowances were made as follows. 1) If a potential ribozyme site had
100%
sequence identity at all except one or two nucleotide positions, then the
actual nucleotide
2 5 at that position was examined in the isolates) that differed. If that
nucleotide was such
that a ribozyme designed to allow "G:U wobble" base-paring could function on
all the
isolates, then that site was chosen. 2) If a potential ribozyme site had 100%
sequence
identity at all except one or two nucleotide positions, then the genotype of
the isolate
which contained the differing nucleotides) was examined. If the genotype of
the isolate
30 that differed was of extremely rare prevalence, then that site was also
chosen.
Ribozyme sites identified and referred to below use the following
nomenclature:
"region of the genome in which the site exists" followed by "nucleotide
position 5' to the
cleavage site" (according to the reference sequence and numbering described
above). For
example, a ribozyme cleavage site at nucleotide position 67 in the 5' NCR is
designated
3 5 "5-67", and a ribozyme cleavage site at position 48 in the core coding
region is designated
"~$».
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/090Z7
32
A number of these ribozymes were screened in an in vitro HCV cleavage assay to
select appropriate ribozyme candidates for cell culture studies. The ribozymes
selected
for screening targeted the 5' UTR region that is necessary for HCV
translation. These
sites are all conserved among the 8 major HCV genotypes and 18 subtypes, and
have a
high degree of homology in every HCV isolate that was used in the analysis
described
above. HCV RNA of four different genotypes (lb, 2a, 4, and 5) were isolated
from human
patients and the 5' HCV UTR and 5' core region were amplified using RT-PCR.
Run-off
transcripts of the 5' HCV UTR region 0750 nt transcripts) were prepared from
the RT-
PCR products, which contained a T7 promoter, using the T7 Megascript
transcription kit
and the manufacturers protocol (Ambion, Inc.). Unincorporated nucleotides are
removed
by spin column filtration on Bio-Gel P-60 resin (Bio-Rad). The f ltered
transcript was 5'
end labeled with 32P using Polynucleotide Kinase (Boehringer/Mannheim) and
150~Ci/wl
Gamma-32P-ATP (NEN) using the enzyme manufacturer's protocol. The kinased
transcript is spin purified again to remove unincorporated Gamma-32P-ATP and
gel
purified on 5% polyacrylamide gel.
Ribozymes targeting various sites from table IV were selected and tested on
the 5'
HCV UTR transcript sequence to test the efficiency of RNA cleavage. 15
ribozymes were
synthesized as previously described (Wincott et al., supra).
Assays were performed by pre-warming a 2X (2 p,M ) concentration of purified
2 0 ribozyme in ribozyme cleavage buffer (SOmM TRIS pH 7.5, l OmM MgCl2, 10
units
RNase Inhibitor (BoehringerlMannheim), lOmM DTT, O.Spg tRNA) and the cleavage
reaction was initiated by adding the 2X ribozyme mix to an equal volume of
substrate
RNA (17.46 pmole final concentration) that was also pre-warmed in cleavage
buffer. The
assay was carried out for 24 hours at 3'7 C using a final concentration of 1
~M ribozyme,
2 5 i. e., ribozyme excess. The reaction was quenched by the addition of an
equal volume of
95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol
after
which the sample is heated to 95~C for 2 minutes, quick chilled and loaded
onto a
denaturing polyacrylamide gel. Substrate RNA and the specific RNA cleavage
products
generated by ribozyme cleavage are visualized on an autoradiograph of the gel.
The
3 0 percentage of cleavage is determined by Phosphor Imager~ quantitation of
bands
representing the intact substrate and the cleavage products.
Observed cleavage fragment sizes from the gels are correlated to predicted
fragment sizes by comparison to the RNA marker. The optical density of
expected
cleavage fragments are determined from the phosphorimage plates and ranked
from
3 5 highest density, indicating the most cleavage product, to lowest of each
genotype of HCV
transcript tested. The top 3 cleaving ribozymes (out of 15 ribozymes tested)
are given
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
33
ranking values of 5, the next 3 highest densities are given ranking values of
4, etc for
every genotype tested. The ranking values for each ribozyme are averaged
between the
genotypes tested. Individual and average ribozyme ranking values are graphed
and
compared. The results (figure 2) demonstrate that many of these tested
ribozymes are able
to to give high levels of cleavage regardless of genotype. In particular,
ribozymes targeting
site HCV.S-258, HCV.S-294, HCV.S-313 (Sakamoto et al., .J Clinical
Investigation 1996
98(12):2720-2728), and HCV.S-318 (table IV) appear to demonstrate a consistent
pattern
of RNA cleavage
Example 7:Inhibition of Luciferase Activity Using HCV Tar etin~~es in OST7
Cells
The capability of ribozymes to inhibit HCV RNA intracellularly was tested
using
a dual reporter system that utilizes both firefly and Renilla luciferase
(figure 3). The
ribozymes targeted to the 5' HCV UTR region, which when cleaved, would prevent
the
translation of the transcript into luciferase. OST-7 cells were plated at
12,500 cells per
well in black walled 96 well plates (Packard) in medium DMEM containing 10 %
fetal
bovine serum, 1 % pen/strep, and 1 % L-glutamine and incubated at 37°C
overnight. A
plasmid containing T7 promoter expressing 5' HCV UTR and firefly luciferase
(T7C1-
341 (Wang et al., 1993, J. of Virol. 67, 3338-3344)) was mixed with a pRLSV40
Renilla control plasmid (Promega Corporation) followed by ribozyme, and
cationic lipid
to make a SX concentration of the reagents (T7C1-341 (4 p,g/ml), pRLSV40
renilla
luciferase control (6 ~,g/ml), ribozyme (250 nM), transfection reagent
(28.S~g/ml).
The complex mixture was incubated at 37~C for 20 minutes. The media was
removed from the cells and 120 ~,1 of Opti-mem media was added to the well
followed by
p,l of the SX complex mixture. 150 ~l of Opti-mem was added to the wells
holding the
2 5 untreated cells. The complex mixture was incubated on OST-7 cells for 4
hours, lysed
with passive lysis buffer (Promega Corporation) and luminescent signals were
quantified
using the Dual Luciferase Assay Kit using the manufacturer's protocol (Promega
Corporation). The ribozyme sequences used are given in table IV. The ribozymes
used
were of the hammerhead motif. The hammerhead ribozymes were chemically
modified
3 0 such that the ribozyme consists of ribose residues at five positions (see
for example Figure
7); position 4 has either 2'-C-allyl or 2'-amino modification; position 7 has
either 2'-amino
modification or 2-O-methyl modification; the remaining nucleotide positions
contain 2'-O-
methyl substitutions; four nucleotides at the 5' terminus contains
phosphorothioate
substitutions. Additionally, the 3' end of the ribozyme includes a 3'-3'
linked inverted
abasic moiety (abasic deoxyribose; iH). The data (figure 4) is given as a
ratio between the
CA 02326695 2000-10-26
WO 99155847 PCT/US99/09027
34
firefly and Renilla luciferase fluorescence. All of the ribozymes targeting 5'
HCV UTR
were able to reduce firefly luciferase signal relative to renilla luciferase.
Example 9: Ribozyme Mediated Inhibition of Luciferase Activity Compared to its
Inactive
Control in OST-7 Cells
The dual reporter system described above was utilized to determine the level
of
reduction of luciferase activity mediated by a ribozyme compared to its
inactive control.
Ribozymes, having the chemical composition described in the previous example,
to sites
HCV 313 and 318 (table IV) and their inactive controls were synthesized as
above. The
inactive control has the same nucleotide base composition as the active
ribozyme but the
nucleotide sequence has been scrambled. The protocols utilized for tissue
culture and
the luciferase assay was exactly as given in example 8 except the ribozyme
concentration
in the SX complex mixture was 1 mM (final concentration on the cells was 200
nM).
The results are given in figure 5. The ribozyme targeting HCV.S-318 was able
to greatly reduce firefly luciferase activity compared to the untreated and
inactive
controls. The ribozyme targeting HCV.S-313 was able to slightly reduce firefly
luciferase activity compared to the inactive control.
Example 10: RibozYme Inhibition of Viral Replication
During HCV infection, viral RNA is present as a potential target for ribozyme
cleavage at several processes: uncoating, translation, RNA replication and
packaging.
2 0 Target RNA may be more or less accessible to ribozyme cleavage at any one
of these
steps. Although the association between the HCV initial ribosome entry site
(IRES) and
the translation apparatus is mimicked in the HCV 5'UTR/luciferase reporter
system
(example 9), these other viral processes are not represented in the OST7
system. The
resulting RNA/protein complexes associated with the target viral RNA are also
absent.
2 5 Moreover, these processes may be coupled in an HCV-infected cell which
could further
impact target RNA accessibility. Therefore, we tested whether ribozymes
designed to
cleave the HCV 5'UTR could effect a replicating viral system.
Recently, Lu and Wimmer characterized an HCV-poliovirus chimera in which the
poliovirus IRES was replaced by the IRES from HCV (Lu & Wimmer, 1996, Proc.
Natl.
30 Acad. Sci. USA. 93, 1412-1417). Poliovirus (PV) is a positive strand RNA
virus like
HCV, but unlike HCV is non-enveloped and replicates efficiently in cell
culture. The
HCV-PV chimera expresses a stable, small plaque phenotype relative to wild
type PV.
The following ribozymes were synthesized for the experiment (table VIII):
ribozyme targeting site 183 (3 5'-end phosphorothioate linkages), scrambled
control to site
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
183, ribozyme to site 318 (3 5'-end phosphorothioate linkages), ribozyme
targeting site
183 (4 5'-end phosphorothioate linkages), inactive ribozyme targeting site 183
(4 5'-end
phosphorothioate linkages). HeLa cells were infected with the HCV-PV chimera
for 30
minutes and immediately treated with ribozyme. HeLa cells were seeded in U-
bottom 96-
5 well plates at a density of 9000-10,000 cells/well and incubated at
37°C under S% C02 for
24 h. Transfection of ribozyme (200 nM) was achieved by mixing of l OX
ribozyme (2000
nM) and l OX of a cationic lipid (80 pg/ml) in DMEM (Gibco BRL) with 5% fetal
bovine
serum (FBS). Ribozyme/lipid complexes were allowed to incubate for 15 minutes
at 37°C
under 5% CO2. Medium was aspirated from cells and replaced with 80 ~ls of DMEM
10 (Gibco BRL) with 5% FBS serum, followed by the addition of 20 Pls of lOX
complexes.
Cells were incubated with complexes for 24 hours at 37°C under 5%
COz .
The yield of HCV-PV from treated cells (Fig. 6A) was quantified by plaque
assay.
The plaque assays were performed by diluting virus samples in serum-free DMEM
(Gibco
BRL) and applying 100 ~l to HeLa cell monolayers {~80% confluent) in 6-well
plates for
15 30 minutes. Infected monolayers were overlayed with 3 ml 1.2% agar (Sigma)
and
incubated at 37°C under 5% C02. Two - three days later the overlay was
removed,
monolayers were stained with 1.2% crystal violet, and plaque forming units
were counted.
The data is shown in figure 6A. Ribozymes to site 183 inhibited HCV-PV
replication by
>80% (P < 0.05) compared to the scrambled control (Fig. 6A, first two bars).
In addition,
2 0 3 or 4 phosphorothioate stabilization was equally effective (P < 0.05 vs.
control for each)
in inhibiting viral replication (compare 1 S~ and 4~' bar in Fig. 6A).
Ribozymes to the 318
site also had a statistically significant (P < 0.05), effect on viral
replication (compare 2"d
and Std bar in Fig. 6A).
To confirm that a ribozyme cleavage mechanism was responsible for the
inhibition
2 5 of HCV-PV replication observed, HCV-PV infected cells were treated with
ribozymes to
site 183 that maintained binding arm sequences but contained a mutation in the
catalytic
core to attenuate cleavage activity (Table I). Viral replication in these
cells was not
inhibited compared to cells treated with the scrambled control ribozyme (Fig.
6A, 4~' and
5'" bar), indicating that ribozyme cleavage activity was required for the
inhibition of HCV
30 PV replication observed. In addition, ribozymes targeting site 183 of the
HCV 5'UTR had
no effect on wild type PV replication (Fig. 6B). These data provide evidence
that the
ribozyme-mediated inhibition of HCV-PV replication was dependent upon the HCV
5'
UTR and not a general inhibition of PV replication.
Ribozymes to site 183 were also tested for the ability to inhibit HCV-PV
3 5 replication during a single infectious cycle in HeLa cells (Fig. 8). Cells
treated with
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
36
ribozyme to site 183 (7/4 format) produced significantly less virus than cells
treated with
the scrambled control (>80% inhibition at 8h post infection, P < 0.001 ).
Example 11: Shorteni_n~~of Ribozyme lengths.
All the ribozymes described in example 10 above contained 7 nucleotides on
each
binding arms and contained a 4 base-paired stem II element (7/4 format). For
pharmaceutical manufacture of a therapeutic ribozyme it is advantageous to
minimize
sequence length if possible. Thus ribozymes to site 183 were shortened by
removing the
outer most nucleotide from each binding arm such that the ribozyme has six
nucleotides in
each binding arm and the stem II region is four base-paired long (6/4 format);
removing
one base-pair (2 nucleotides) in stem II resulting in a 3 base-paired stem II
(7/3 format); or
removing one nucleotide from each binding arm and shortening the stem II by
one base-
pair (6/3 format). (See Figure 7 for a schematic representation of each of
these
ribozymes). Ribozymes in all tested formats gave significant inhibition of
viral replication
(Fig. 8) with the 7/4, 7/3 and 6/3 formats being almost identical at the 8h
timepoint (P <
0.001 across time course for all formats). The shortest ribozyme tested (6/3
format) was
slightly more efficacious (>90% inhibition, P < 0.001 ) than the 7/4 ribozyme
(~80%
inhibition, P < 0.001 ). The 6/3 ribozyme may have a greater ability to access
site 183 in
the HCV-PV chimera.
Example 12' Combination Therapy of HCV Ribozvmes and Interferon
HeLa cells (10,000 cells per well) were pre-treated with 12.5 Units/ml of
Interferon alpha in complete media {DMEM + 5% FBS) or pre-treated with
complete
media alone for 4 hours and then infected with HCV-PV at an MOI = 0.1 for 30
minutes.
The viral inoculum was then removed and 200 nM ribozyme targeted to HCV site
183
(Rz) or binding attenuated control, which has mutations in the catalytic core
of the
2 5 ribozyme that severely attenuates the activity of the ribozyme, (BAC) was
delivered using
cationic lipid in complete media for 24 hours. After 24 hours, the cells were
lysed three
times by freeze/thaw to release virus and virus was quantified by plaque
assay. Viral yield
is shown as mean plaque forming units per ml (pfu/ml) + SEM. The data is shown
in
figure 10.
Pre-treatment with interferon (IFI~ reduces the viral yield by 10'1 in control
treated cells (BAC+IFN versus BAC). Ribozyme treated cells produce 2 x 10'1
less virus
than control-treated cells (Rz versus BAC). The combination of Rz and IFN
treatment
results in a synergistic 4 x 10'2 reduction in viral yield (Rz+IFN versus
BAC). An additive
effect would result in only a 3 x 10'1 reduction (1 x 10'1 + 2 x 10'1).
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
37
Example 13: Inhibition of Hepatitis C virus Usin~other RibozYme Motifs
A number of varying ribozyme motifs (RPI motifs 1-3; Figure 9), were tested
for
their ability to inhibit HCV propagation in tissue culture. An example of RPI
motif I is
described in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120, while an
example
of RPI motif II is described in Ludwig & Sproat, international PCT Publication
No. WO
98/58058). RPI motif III. is a new ribozyme motif which applicant has recently
developed
and an example of this motif was tested herein.
OST7 cells were maintained in Dulbecco's modified Eagle's medium (GIBCO
BRL) supplemented with 10% fetal calf serum, L-glutamine (2mM) and
penicillinlstreptomycin. For transfections, OST7 cells were seeded in black-
walled 96-well
plates (Packard Instruments) at a density of 12,500 cells/well and incubated
at 37°C under
S% C02 for 24 hours. Co-transfection of target reporter HCVT7C (0.8 ~g/ml),
control
reporter pRLSV40, (1.2 p,g/ml) and ribozyme, SO-200 nM was achieved by the
following
method: a SX mixture of HCVT7C (4 p,g/ml), pRLSV40 (6 p,g/ml), ribozyme (250-
1000
nM) and cationic lipid (28.5 ~,g/ml) was made in 150 pls of OPTI-MEM (GIBCO
BRL)
minus serum. Reporter/ribozyme/lipid complexes were allowed to form for 20
minutes at
37°C under S% C02. Medium was aspirated from OST7 cells and replaced
with 120 p.ls of
OPTI-MEM (GIBCO BRL) minus serum, immediately followed by the addition of 30
p,ls
of SX reporter/ribozyme/lipid complexes. Cells were incubated with complexes
for 4
hours at 37°C under 5% C02 . Luciferase assay was performed as
described in example 7.
The data is summarized in table IX, with each motif's results listed along
with its control.
All of the ribozyme motifs were able to reduce the amount of HCV produced by
the cells
compared to the ribozymes not targeted to any HCV (irrelevant controls).
Cell Culture Assays
2 5 Although there have been reports of replication of HCV in cell culture
(see
below), these systems are difficult to replicate and have proven unreliable.
Therefore,
as was the case for development of other anti-HCV therapeutics such as
interferon and
ribavirin, after demonstration of safety in animal studies applicant can
proceed directly
into a clinical feasibility study.
Several recent reports have documented in vitro growth of HCV in human cell
lines (Mizutani et al., Biochem Biophys Res Commun 1996 227(3):822-826; Tagawa
et
al., Journal of Gasteroenterology and Hepatology 1995 10(5):523-527; Cribier
et al.,
Journal of General Virology 76(10):2485-2491; Seipp et al., Journal of General
Virology
1997 78(10)2467-2478; Iacovacci et al., Research Virology 1997 148(2):147-151;
Iocavacci et al., Hepatology 1997 26(5) 1328-1337; Ito et al., Journal of
General Virology
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
38
1996 77(5):1043-1054; Nakajima et al., Journal of Virology 1996 70{5):3325-
3329;
Mizutani et al., Journal of Virology 1996 70(10):7219-7223; Valli et al., Res
Virol 1995
146(4): 285-288; Kato et al., Biochem Biophys Res Comm 1995 206(3):863-869).
Replication of HCV has been demonstrated in both T and B cell lines as well as
cell lines
derived from human hepatocytes. Demonstration of replication was documented
using
either RT-PCR based assays or the b-DNA assay. It is important to note that
the most
recent publications regarding HCV cell cultures document replication for up to
6-months.
In addition to cell lines that can be infected with HCV, several groups have
reported the successful transformation of cell lines with cDNA clones of full-
length or
partial HCV genomes (Harada et al., Journal of General Virology 1995 76(5)1215-
1221;
Haramatsu et al., Journal of Viral Hepatitis 1997 45(1):61-67; Dash et al.,
American Journal of
Pathology 1997 151(2):363-373; Mizuno et al., Gasteroenterology 1995
109(6):1933-40; Yoo et
al., Journal Of Virology 1995 69(1):32-38).
Animal Models
The best characterized animal system for HCV infection is the chimpanzee.
Moreover, the chronic hepatitis that results from HCV infection in chimpanzees
and
humans is very similar. Although clinically relevant, the chimpanzee model
suffers from
several practical impediments that make use of this model difficult. These
include; high
cost, long incubation requirements and lack of sufficient quantities of
animals. Due to
2 0 these factors, a number of groups have attempted to develop rodent models
of chronic
hepatitis C infection. While direct infection has not been possible several
groups have
reported on the stable transfection of either portions or entire HCV genomes
into rodents
(Yamamoto et al., Hepatology 1995 22(3): 847-855; Galun et al., Journal of
Infectious Disease
1995 172(1):25-30; Koike et al., Journal of general Virology 1995 76(12)3031-
3038;
2 5 Pasquinelli et al., Hepatology 1997 25(3): 719-727; Hayashi et al.,
Princess Talcamatsu Symp
1995 25:1430149; Mariya K, Yotsuyanagi H, Shintani Y, Fujie H, Ishibashi K,
Matsuura Y,
Miyamura T, Koike K. Hepatitis C virus core protein induces hepatic steatosis
in transgenic
mice. Journal of General Virology 1997 78(7) 1527-1531; Takehara et al.,
Hepatology 1995
21(3):746-751; Kawamura et al., Hepatology 1997 25(4): 1014-1021). In
addition,
30 transplantation of HCV infected human liver into immunocompromised mice
results in
prolonged detection of HCV RNA in the animal's blood.
Dia~~nostic uses
Ribozymes of this invention may be used as diagnostic tools to examine genetic
drift and mutations within diseased cells or to detect the presence of HCV RNA
in a cell.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
39
The close relationship between ribozyme activity and the structure of the
target RNA
allows the detection of mutations in any region of the molecule, which alters
the base-
pairing and three-dimensional structure of the target RNA. By using multiple
ribozymes
described in this invention, one may map nucleotide changes, which are
important to RNA
structure and function in vitro, as well as in cells and tissues. Cleavage of
target RNAs
with ribozymes may be used to inhibit gene expression and define the role
(essentially) of
specified gene products in the progression of disease. In this manner, other
genetic targets
may be defined as important mediators of the disease. These experiments will
lead to
better treatment of the disease progression by affording the possibility of
combination
therapies (e.g., multiple ribozymes targeted to different genes, ribozymes
coupled with
known small molecule inhibitors, or intermittent treatment with combinations
of
ribozymes andlor other chemical or biological molecules). Other in vitro uses
of
ribozymes of this invention are well known in the art, and include detection
of the
presence of mRNAs associated with HCV related condition. Such RNA is detected
by
determining the presence of a cleavage product after treatment with a ribozyme
using
standard methodology.
In a specific example, ribozymes which can cleave only wild-type or mutant
forms
of the target RNA are used for the assay. The first ribozyme is used to
identify wild-type
RNA present in the sample and the second ribozyme will be used to identify
mutant RNA
2 0 in the sample. As reaction controls, synthetic substrates of both wild-
type and mutant
RNA will be cleaved by both ribozymes to demonstrate the relative ribozyme
efficiencies
in the reactions and the absence of cleavage of the "non-targeted" RNA
species. The
cleavage products from the synthetic substrates will also serve to generate
size markers for
the analysis of wild-type and mutant RNAs in the sample population. Thus each
analysis
2 5 will require two ribozymes, two substrates and one unknown sample which
will be
combined into six reactions. The presence of cleavage products will be
determined using
an RNase protection assay so that full-length and cleavage fragments of each
RNA can be
analyzed in one lane of a polyacrylamide gel. It is not absolutely required to
quantify the
results to gain insight into the expression of mutant RNAs and putative risk
of the desired
3 0 phenotypic changes in target cells. The expression of mRNA whose protein
product is
implicated in the development of the phenotype (i.e., HCV) is adequate to
establish risk.
If probes of comparable specific activity are used for both transcripts, then
a qualitative
comparison of RNA levels will be adequate and will decrease the cost of the
initial
diagnosis. Higher mutant form to wild-type ratios will be correlated with
higher risk
3 5 whether RNA levels are compared qualitatively or quantitatively.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
Additional Uses
Potential usefulness of sequence-specific enzymatic nucleic acid molecules of
the
instant invention might have many of the same applications for the study of
RNA that
DNA restriction endonucleases have for the study of DNA (Nathans et al.; 1975
Ann. Rev
5 Biochem. 44:273). For example, the pattern of restriction fragments could be
used to
establish sequence relationships between two related RNAs, and large RNAs
could be
specifically cleaved to fragments of a size more useful for study. The ability
to engineer
sequence specificity of the ribozyme is ideal for cleavage of RNAs of unknown
sequence.
Other embodiments are within the following claims.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
41
TABLEI
Characteristics of naturally occurring, riboz~mes
Group I Introns
~ Size: 150 to >1000 nucleotides.
~ Requires a U in the target sequence immediately 5' of the cleavage site.
~ Binds 4-6 nucleotides at the 5'-side of the cleavage site.
~ Reaction mechanism: attack by the 3'-OH of guanosine to generate cleavage
products with 3'-OH and 5'-guanosine.
~ Additional protein cofactors required in some cases to help folding and
maintainance of the active structure.
~ Over 300 known members of this class. Found as an intervening sequence in
Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4,
blue-green algae,
and others.
~ Major structural features largely established through phylogenetic
comparisons,
mutagenesis, and biochemical studies [1,2].
Complete kinetic framework established for one ribozyme [3,4~5~6~.
789
~ Studies of ribozyme folding and substrate docking underway [ , , ].
' Michel, Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol.
(1994), I(1), 5-7.
Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic identification
of group I intron
cores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17.
Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the
Tetrahymena thermophila
ribozyme. 1. Kinetic description of the reaction of an RNA substrate
complementary to the active site.
Biochemistry (1990), 29(44), 10159-71.
Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage by the
Tetrahymena thermophila
ribozyme. 2. Kinetic description of the reaction of an RNA substrate that
forms a mismatch at the active
site. Biochemistry (1990), 29(44), 10172-80.
Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the Tetrahymena
Ribozyme Reveal an
Unconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5), 1560-70.
Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H.. A mechanistic
framework for the
second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry
(1996), 35(2), 648-58.
Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H..
Thermodynamic and activation
parameters for binding of a pyrene-labeled substrate by the Tetrahymena
ribozyme: docking is not diffusion-
controlled and is driven by a favorable entropy change. Biochemistry (1995),
34(44), 14394-9.
Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence of chemical
modification reveals
slow steps in the folding of a group I ribozyme. Biochemistry (1995), 34(19),
6504-12.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
42
Chemical modification investigation of important residues well established
(lo,t tj.
~ The small (4-6 nt) binding site may make this ribozyme too non-specific for
targeted RNA cleavage, however, the Tetrahymena group I intron has been used
to repair a
"defective" (3-galactosidase message by the ligation of new (i-galactosidase
sequences onto the
defective message (t2].
RNAse P RNA (M1 RNAI
~ Size: 290 to 400 nucleotides.
~ RNA portion of a ubiquitous ribonucleoprotein enzyme.
~ Cleaves tRNA precursors to form mature tRNA [t3].
~ Reaction mechanism: possible attack by M2+-OH to generate cleavage products
with 3'-OH and 5'-phosphate.
~ RNAse P is found throughout the prokaryotes and eukaryotes. The RNA subunit
has been sequenced from bacteria, yeast, rodents, and primates.
~ Recruitment of endogenous RNAse P for therapeutic applications is possible
through hybridization of an External Guide Sequence (EGS) to the target RNA
(t4~ts~
~ Important phosphate and 2' OH contacts recently identified (t6,1']
Zarrinkar, Patrick P.; Williamson, James R.. The P9.1-P9.2 peripheral
extension helps guide folding
of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8.
'° Strobel, Scott A.; Cech, Thomas R.. Minor groove recognition of the
conserved G.cntdot.U pair at
the Tetrahymena ribozyme reaction site. Science (Washington, D. C.) (1995),
267(5198), 675-9.
1' Strobel, Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved
G.cntdot.U Pair at the
Cleavage Site of the Tetrahymena Ribozyme Contributes to 5'-Splice Site
Selection and Transition State
Stabilization. Biochemistry (1996), 35(4), 1201-11.
12. Sullenger, Bruce A.; Cech, Thomas R.. Ribozyme-mediated repair of
defective mRNA by targeted
traps-splicing. Nature (London) (1994), 371(6498), 619-22.
". Robertson, H.D.; Altman, S.; Smith, J.D. J. Biol. Chem., 247, 5243-5251
(1972).
'". Forster, Anthony C.; Altman, Sidney. External guide sequences for an RNA
enzyme. Science
(Washington, D. C., 1883-) (1990), 249(4970), 783-6.
'3. Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by human
RNase P. Proc. Natl.
Acad. Sci. USA (1992) 89, 8006-10.
'6 Harris, Michael E.; Pace, Norman R.. Identification of phosphates involved
in catalysis by the
ribozyme RNase P RNA. RNA (1995), 1(2), 210-18.
Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary interactions in RNA:
2'-hydroxyl-base
contacts between the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U. S. A.
(1995), 92(26), 12510-
14.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
43
Group II Introns
~ Size: >1000 nucleotides.
Trans cleavage of target RNAs recently demonstrated (tg,t9].
~ Sequence requirements not fully determined.
~ Reaction mechanism: 2'-OH of an internal adenosine generates cleavage
products
with 3'-OH and a "lariat" RNA containing a 3'-5' and a 2'-5' branch point.
~ Only natural ribozyme with demonstrated participation in DNA cleavage (zo~zy
~
addition to RNA cleavage and ligation.
Major structural features largely established through phylogenetic comparisons
Z 0 (22~.
Important 2' OH contacts beginning to be identified (z3~
Kinetic framework under development (24]
Neurospora VS RNA
Size: 144 nucleotides.
~ Trans cleavage of hairpin target RNAs recently demonstrated (2s].
~ Sequence requirements not fully determined.
'g Pyle, Anna Marie; Green, Justin B.. Building a Kinetic Framework for Group
II Intron Ribozyme
Activity: Quantitadon of Interdomain Binding and Reaction Rate. 'Biochemistry
(1994), 33(9), 2716-25.
" Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a Group II Intron
into a New Multiple-
Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of
Reaction Mechanism and
StructurelFunction Relationships. Biochemistry (1995), 34(9), 2965-77.
zo Z~erl Steven' Guo Huatao~ Eskes Robert Yan Jian; Penman Phili S.;
Lambowitz, Alan
Y> > > > > > g> > P
M.. A group II intron RNA is a catalytic component of a DNA endonuclease
involved in intron mobility.
Cell (Cambridge, Mass.) (1995), 83(4), 529-38.
z' Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle,
Anna Mane. Group II intron
nbozymes that cleave DNA and RNA linkages with similar effciency, and lack
contacts with substrate 2'-
hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70.
Michel, Francois; Ferat, Jean Luc. Structure and activities of group II
introns. Annu. Rev. Biochem.
(1995), 64, 435-61.
Abramovitz, Dana L.; Fnedman, Richard A.; Pyle, Anna Mane. Catalytic role of
2'-hydroxyl
groups within a group II intron active site. Science (Washington, D. C.)
(1996), 271(5254), 1410-13.
24 D~iels, Danette L.; Michels, William J., Jr.; Pyle, Anna Marie. Two
competing pathways for self
splicing by group II introns: a quantitative analysis of in vitro reaction
rates and products. J. Mol. Biol.
(1996), 256(1), 31-49.
'~ Guo, Hans C. T.; Collins, Richard A.. Effcient trans-cleavage of a stem-
loop RNA substrate by a
nbozyme derived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
44
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage
products with 2',3'-cyclic phosphate and 5'-OH ends.
~ Binding sites and structural requirements not fully determined.
~ Only 1 known member of this class. Found in Neurospora VS RNA.
Hammerhead Ribozyme
(see text for references)
~ Size: ~13 to 40 nucleotides.
~ Requires the target sequence UH immediately 5' of the cleavage site.
~ Binds a variable number nucleotides on both sides of the cleavage site.
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage
products with 2',3'-cyclic phosphate and 5'-OH ends.
~ 14 known members of this class. Found in a number of plant pathogens
(virusoids) that use RNA as the infectious agent.
~
Essential structural features largely defined, including 2 crystal structures
~2b~27~
~ Minimal ligation activity demonstrated (for engineering through in vitro
selection)
~xa~
Complete kinetic framework established for two or more ribozymes (29].
~ Chemical modification investigation of important residues well established
(30]
Hairpin Ribozyme
2 0 ~ Size: ~50 nucleotides.
~ Requires the target sequence GUC immediately 3' of the cleavage site.
~ Binds 4-6 nucleotides at the 5'-side of the cleavage site and a variable
number to
the 3'-side of the cleavage site.
z6 Scott, W.G., Finch, J.T., Aaron,K. The crystal structure of an all RNA
hammerhead
ribozyme:Aproposed mechanism for RNA catalytic cleavage. Cell, (1995), 81, 991-
1002.
Z' McKay, Structure and function of the hammerhead ribozyme: an unfinished
story. RNA, (1996), 2,
395-403.
Long, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead ribozymes. US
Patent No.
5,633,133.
29 Hertel, K.J., Herschlag, D., Uhlenbeck, O. A kinetic and thermodynamic
framework for the
hammerhead ribozyme reaction. Biochemistry, ( 1994) 33, 3374-3385.Beigehnan,
L., et al., Chemical
modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-
25708.
'° Beigelman, L., et al., Chemical modifications of hammerhead
ribozymes. J. Biol. Chem., (1995)
270, 25702-25708.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage
products with 2',3'-cyclic phosphate and 5'-OH ends.
3 known members of this class. Found in three plant pathogen (satellite RNAs
of
the tobacco ringspot virus, arabis mosaic virus and chicory yellow mottle
virus) which uses RNA
5 as the infectious agent.
~ Essential structural features largely defined [3132 33 34]
~ Ligation activity (in addition to cleavage activity) makes ribozyme amenable
to
engineering through in vitro selection [3s]
~ Complete kinetic framework established for one ribozyme [36].
10 ~ Chemical modification investigation of important residues begun [3~,~8].
Hepatitis Delta Virus (HDV~ Ribozyme
Size: ~60 nucleotides.
Trans cleavage of target RNAs demonstrated [39].
3' Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip. 'Hairpin'
catalytic RNA model:
evidence for helixes and sequence requirement for substrate RNA. Nucleic Acids
Res. (1990), 18(2), 299-
304.
'x Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.. Novel
guanosine requirement for
catalysis by the hairpin ribozyme. Nature (London) (1991), 354(6351), 320-2.
33 Beizal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M.; Butcher,
Samuel E.; Burke, John
M.. Essential nucleotide sequences and secondary structure elements of the
hairpin ribozyme. EMBO J.
(1993), 12(6), 2567-73.
Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.; Butcher, Samuel
E.. Substrate
selection rules for the hairpin ribozyme determined by in vitro selection,
mutation, and analysis of
mismatched substrates. Genes Dev. (1993), 7(1), 130-8.
's Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. In vitro
selection of active hairpin
ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes
Dev. (1992), 6(I), 129-34.
'6 Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics of
Intermolecular Catalysis by
Hairpin Ribozymes. Biochemistry (1995), 34(48), 15813-28.
3' Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait, Michael 1.. Purine
Functional Groups in
Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of
RNA. Biochemistry { 1995),
34(12), 4068-76.
'$ Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim;
Sorensen, Ulrik S.;
Gait, Michael J.. Base and sugar requirements for RNA cleavage of essential
nucleoside residues in internal
loop B of the hairpin ribozyme: implications for secondary structure. Nucleic
Acids Res. (1996), 24(4), 573-
81.
39 penotta, Anne T.; Been, Michael D.. Cleavage of oligoribonucleotides by a
ribozyme derived from
the hepatitis .delta. virus RNA sequence. Biochemistry (1992), 31(1), 16-21.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
46
~ Binding sites and structural requirements not fully determined, although no
sequences 5' of cleavage site are required. Folded ribozyme contains a
pseudoknot structure [~~].
~ Reaction mechanism: attack by 2'-OH 5' to the scissile bond to generate
cleavage
products with 2',3'-cyclic phosphate and 5'-OH ends.
~ Only 2 known members of this class. Found in human HDV.
~ Circular fore of HDV is active and shows increased nuclease stability [~1]
°° Perrotta, Anne T.; Been, Michael D.. A pseudoknot-like
structure required for effcient self
cleavage of hepatitis delta virus RNA. Nature (London) (1991), 350(6317), 434-
6.
Puttaraju, M.; Perrotta, Anne T.; Been, Michael D.. A circular traps-acting
hepatitis delta virus
ribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.
CA 02326695 2000-10-26
WO 99/55847 PC'T/US99/09027
47
Table II: 2.5 ~.mol RNA Synthesis G~cle
Reagent Equivalents Amount Wait
Time*
Phosphoramidites 6.5 163 wL 2.
S
S Ethyl Tetrazole 23.8 238 p,L 2.5
Acetic Anhydride 100 233 ~,L S
sec
N Methyl Imidazole 186 233 p.L S
sec
TCA 83.2 1.73 mL 21
sec
Iodine 8.0 1.18 mL 45
sec
Acetonitrile NA 6.67 mL NA
2 0 * Wait time does not include contact time during delivery.
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
48
Table III: Ribozyme Selection Characteristics
Characteristic Number
HCV Genome Length 9436 kb
All Hammerhead Cleavage Sites 1300
*
Conserved Region Hammerhead 23
Cleavage Sites **
HCV Genotype lb was the prototype strain
** Based on sequence alignments from HCV genotype la, lb,
lc,2a,2b,2c3a,3b,4a,4c,4f,Sa,and6a
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
49
Table IV: Hammerhead Ribozymes Derived from Conserved Regions of the HCV
Genome
Name Substrate Ribozyme Sequence
r NcR
HCV.S-50CUACUGU C UUCACGC GCGUGM CUGAUGAGGCCGUUAGGCCGM
ACAGUAG
HCV.S-67MAGCGU C UAGCCAU AUGGCUA CUGAUGAGGCCGUUAGGCCGAA
ACGCUUU
HCV.Sfi9AGCGUCU A GCCAUGG CCAUGGC CUGAUGAGGCCGUUAGGCCGAA
AGACGCU
HCV.S-92UGAGUGU C GUGCAGC GCUGCAC CUGAUGAGGCCGUUAGGCCGAA
ACACUCA
HCV.S-130GAGCCAU A GUGGUCU AGACCAC CUGAUGAGGCCGUUAGGCCGAA
AUGGCUC
HCV.S-136UAGUGGU C UGCGGAA UUCCGCA CUGAUGAGGCCGUUAGGCCGAA
ACCACUA
Z O HCV.S-153GGUGAGU A CACCGGA UCCGGUG CUGAUGAGGCCGUUAGGCCGAA
ACUCACC
HCV.S-180ACCGGGU C CUUUCUU AAGAAAG CUGAUGAGGCCGUUAGGCCGAA
ACCCGGU
HCV.S-183GCGUCCU U UCUUGGA UCCAAGA CUGAUGAGGCCGUUAGGCCGAA
AGGACCC
HCV.S-184GGUCCUU U CUUGGAU AUCCMG CUGAUGAGGCCGUUAGGCCGM
AAGGACC
HCV.S-258GUUGGGU C GCGAMG CUUUCGC CUGAUGAGGCCGUUAGGCCGAA
ACCCMC
HCV.S-270AAGGCCU U GUGGUAC GUACCAC CUGAUGAGGCCGUUAGGCCGAA
AGGCCUU
Z 5 HCV.S-294GGGUGCU U GCGAGUG CACUCGC CUGAUGAGGCCGUUAGGCCGM
AGCACCC
HCV.S-313GGGAGGU C UCGUAGA UCUACGA CUGAUGAGGCCGUUAGGCCGAA
ACCUCCC
HCV.S-31SGAGGUCU C GUAGACC GGUCUAC CUGAUGAGGCCGUUAGGCCGM
AGACCUC
HCV.S-318GUCUCGU A GACCGUG CACGGUC CUGAUGAGGCCGUUAGGCCGM
ACGAGAC
Con Rc~on
HCV.C-30UAMCCU C AMGAM UUUCUUU CUGAUGAGGCCGUUAGGCCGM
AGGUUUA
2 O HCV.C-48CAMCGU A ACACCM UUGGUGU CUGAUGAGGCCGUUAGGCCGM
ACGUUUG
HCV.C-60CMCCGU C GCCCACA UGUGGGC CUGAUGAGGCCGUUAGGCCGM
ACGGUUG
HCV.C-175GAGCGGU C ACMCCU AGGUUGU CUGAUGAGGCCGUUAGGCCGM
ACCGCUC
HCV.C-374GUAAGGU C AUCGAUA UAUCGAU CUGAUGAGGCCGUUAGGCCGAA
ACCUUAC
3' NCR
HCV.3-118UUUUUUU U UUUUUUU MAAAM CUGAUGAGGCCGUUAGGCCGM
25 AAAAMA
HCV.3-145GGUGGCU C CAUCUUA UAAGAUG CUGAUGAGGCCGUUAGGCCGM
AGCCACC
HCV.3-149GCUCCAU C UUAGCCC GGGCUAA CUGAUGAGGCCGUUAGGCCGM
AUGGAGC
HCV.3-151UCCAUCU U AGCCCUA UAGGGCU CUGAUGAGGCCGUUAGGCCGM
AGAUGGA
HCV.3-152CCAUCUU A GCCCUAG CUAGGGC CUGAUGAGGCCGUUAGGCCGM
MGAUGG
HCV.3-158UAGCCCU A GUCACGG CCGUGAC CUGAUGAGGCCGUUAGGCCGAA
AGGGCUA
NCV.3-161CCCUAGU C ACGGCUA UAGCCGU CUGAUGAGGCCGUUACGCCGM
30 ACUAGGG
HCV.3168CACGGCU A GCUGUGA UCACAGC CUGAUGAGGCCGUUAGGCCGM
AGCCGUG
HCV.3-18lGMAGGU C CGUGAGC GCUCACG CUGAUGAGGCCGUUAGGCCGAA
ACCUUUC
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
Table V: HCV Hammerhead Ribozyme and Target Sequence
No. Name Nt. Hammerhead Substrate
S Pos. Ribozyme
1 HCV-27 27 UAUGGUGCUGAUGAGXCGAA AGUGUCGCGACACUC CACCAUA
2 HCV-114114 GGUCCUGCUGAUGAGXCGAA AGGCUGCGCAGCCUC CAGGACC
3 HCV-128128 CUCCCGGCUGAUGAGXCGAA AGGGGGGCCCCCCUC CCGGGAG
4 HCV-148148 UUCCGCACUGAUGAGXCGAA ACCACUAUAGUGGUC UGCGGAA
lO 5 HCV-165165 UCCGGUGCUGAUGAGXCGAA ACUCACCGGUGAGUA CACCGGA
6 HCV-175175 UCCUGGCCUGAUGAGXCGAA AUUCCGGCCGGAAUU GCCAGGA
7 HCV-199199 UUGAUCCCUGAUGAGXCGAA AGAAAGGCCUUUCUU GGAUCAA
8 HCV-213213 AGGCAUUCUGAUGAGXCGAA AGCGGGUACCCGCUC
AAUGCCU
IS 9 HCV-252252 ACUCGGCCUGAUGAGXCGAA AGCAGUCGACUGCUA GCCGAGU
10 HCV-260260 CCAACACCUGAUGAGXCGAA ACUCGGCGCCGAGUA GUGUUGG
11 HCV-265265 GCGACCCCUGAUGAGXCGAA ACACUACGUAGUGUU GGGUCGC
12 HCV-270270 CUUUCGCCUGAUGAGXCGAA ACCCAACGUUGGGUC GCGAAAG
13 HCV-288288 CAGGCAGCUGAUGAGXCGAA ACCACAAUUGUGGUA CUGCCUG
20
14 HCV-298298 AGCACCCCUGAUGAGXCGAA AUCAGGCGCCUGAUA GGGUGCU
15 HCV-306306 CACUCGCCUGAUGAGXCGAA AGCACCCGGGUGCUU GCGAGUG
16 HCV-325325 UCUACGACUGAUGAGXCGAA ACCUCCCGGGAGGUC UCGUAGA
17 HCV-327327 GGUCUACCUGAUGAGXCGAA AGACCUCGAGGUCUC GUAGACC
2S 18 HCV-330330 CACGGUCCUGAUGAGXCGAA ACGAGACGUCUCGUA GACCGUG
19 HCV-407407 GGAACUUCUGAUGAGXCGAA ACGUCCUAGGACGUC
AAGUUCC
20 HCV-412412 GCCCGGGCUGAUGAGXCGAA ACUUGACGUCAAGUU CCCGGGC
21 HCV-413413 CGCCCGGCUGAUGAGXCGAA AACUUGAUCAAGUUC CCGGGCG
3O 22 HCV-426426 ACGAUCUCUGAUGAGXCGAA ACCACCGCGGUGGUC AGAUCGU
23 HCV-972472 CACACCCCUGAUGAGXCGAA ACGUGGGCCCACGUU GGGUGUG
24 HCV-489489 GUCUUCCCUGAUGAGXCGAA AGUCGCGCGCGACUA GGAAGAC
25 HCV-498498 CGUUCGGCUGAUGAGXCGAA AGUCUUCGAAGACUU CCGAACG
26 HCV-499499 CCGUUCGCUGAUGAGXCGAA AAGUCUUAAGACUUC CGAACGG
27 HCV-508508 AGGUUGCCUGAUGAGXCGAA ACCGUUCGAACGGUC GCAACCU
CA 02326695 2000-10-26
WO 99/55847 PCTNS991090Z7
S1
No. Name Nt. Hammerhead Substrate
Pos.Ribozyme
28 HCV-534534 UUGGGGA CUGAUGAGX CGAA AGGUUGUACAACCU AUCCCCAA
29 HCV-536536 CCUUGGG CUGAUGAGX CGAA AUAGGUUAACCUAU CCCCAAGG
S
30 HCV-546546 GGUCGGC CUGAUGAGX CGAA AGCCUUGCAAGGCU CGCCGACC
31 HCV-561561 CAGGCCC CUGAUGAGX CGAA ACCCUCGCGAGGGU AGGGCCUG
32 HCV-573573 CCAGGCU CUGAUGAGX CGAA AGCCCAGCUGGGCU CAGCCUGG
33 HCV-583583 CCAAGGG CUGAUGAGX CGAA ACCCAGGCCUGGGU ACCCUUGG
IO 39 HCV-588588 AGGGGCC CUGAUGAGX CGAA AGGGUACGUACCCU UGGCCCCU
35 HCV-596596 UGCCAUA CUGAUGAGX CGAA AGGGGCCGGCCCCU CUAUGGCA
36 HCV-598598 AUUGCCA CUGAUGAGX CGAA AGAGGGGCCCCUCU AUGGCAAU
37 HCV-632632 GUGACAG CUGAUGAGX CGAA AGCCAUCGAUGGCU CCUGUCAC
38 HCV-637637 GCGGGGU CUGAUGAGX CGAA ACAGGAGCUCCUGU CACCCCGC
1S
39 HCV-649649 AGGCCGG CUGAUGAGX CGAA AGCCGCGCGCGGCU CCCGGCCU
40 HCV-657657 CCCCAAC CUGAUGAGX CGAA AGGCCGGCCGGCCU AGUUGGGG
41 HCV-660660 GGGCCCC CUGAUGAGX CGAA ACUAGGCGCCUAGU UGGGGCCC
42 HCV-696696 UUACCCA X CGAA AUUGCGCGCGCAAU CUGGGUAA
20 CUGAUGAG
43 HCV-707707 UAUCGAU CUGAUGAGX CGAA ACCUUACGUAAGGU CAUCGAUA
44 HCV-710710 GGGUAUC CUGAUGAGX CGAA AUGACCUAGGUCAU CGAUACCC
45 HCV-714714 GUGAGGG CUGAUGAGX CGAA AUCGAUGCAUCGAU ACCCUCAC
96 HCV-730730 GUCGGCG CUGAUGAGX CGAA AGCCGCAUGCGGCU UCGCCGAC
2S 47 HCV-731731 GGUCGGC CUGAUGAGX CGAA AAGCCGCGCGGCUU CGCCGACC
48 HCV-748748 CGGAAUG CUGAUGAGX CGAA ACCCCAUAUGGGGU ACAUUCCG
99 HCV-752752 CGAGCGG CUGAUGAGX CGAA AUGUACCGGUACAU UCCGCUCG
50 HCV-753753 ACGAGCG CUGAUGAGX CGAA AAUGUACGUACAUU CCGCUCGU
O 51 HCV-758758 CGCCGAC CUGAUGAGX CGAA AGCGGAAUUCCGCU CGUCGGCG
52 HCV-761761 GGGCGCC CUGAUGAGX CGAA ACGAGCGCGCUCGU CGGCGCCC
53 HCV-7737?3 CGCCCCC CUGAUGAGX CGAA AGGGGGGCCCCCCU AGGGGGCG
54 HCV-806806 GAACCCG CUGAUGAGX CGAA ACACCAUAUGGUGU CCGGGUUC
55 HCV-812812 CCUCCAG CUGAUGAGX CGAA ACCCGGAUCCGGGU UCUGGAGG
56 HCV-813813 UCCUCCA CUGAUGAGX CGAA AACCCGGCCGGGUU CUGGAGGA
57 HCV-832832 UGUUGCG CUGAUGAGX CGAA AGUUCACGUGAACU ACGCAACA
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
52
No. Name Nt. Hammerhead Substrate
Pos. Ribozyme
58 HCV-847847 ACCGGGCCUGAUGAGXCGAA AGUUCCCGGGAACUUGCCCGGU
59 HCV-855855 AAAGAGCCUGAUGAGXCGAA ACCGGGCGCCCGGUUGCUCUUU
60 HCV-859859 AGAGAAA XCGAA AGCAACCGGUUGCUCUUUCUCU
CUGAUGAG
61 HCV-982982 UGCCUCACUGAUGAGXCGAA ACACAAUAUUGUGUAUGAGGCA
62 HCV-10011001 UAUGCAUCUGAUGAGXCGAA AUCAUGCGCAUGAUCAUGCAUA
63 HCV-10221022 CGCAGGGCUGAUGAGXCGAA ACGCACCGGUGCGUACCCUGCG
IO 64 HCV-10311031 UCUCCCGCUGAUGAGXCGAA ACGCAGGCCUGCGUUCGGGAGA
65 HCV-10321032 UUCUCCCCUGAUGAGXCGAA AACGCAGCUGCGUUCGGGAGAA
66 HCV-10981048 ACAACGGCUGAUGAGXCGAA AGGCGUUAACGCCUCCCGUUGU
67 HCV-10531053 ACCCAACCUGAUGAGXCGAA ACGGGAGCUCCCGUUGUUGGGU
68 HCV-10561056 GCUACCCCUGAUGAGXCGAA ACAACGGCCGUUGUUGGGUAGC
1$
69 HCV-10611061 UGAGCGCCUGAUGAGXCGAA ACCCAACGUUGGGUAGCGCUCA
70 HCV-11271127 GCAAGUCCUGAUGAGXCGAA ACGUGGCGCCACGUCGACUUGC
71 HCV-11321132 AACGAGCCUGAUGAGXCGAA AGUCGACGUCGACUUGCUCGUU
72 HCV-11361136 CCCCAACCUGAUGAGXCGAA AGCAAGUACUUGCUCGUUGGGG
20
73 HCV-11391139 CCGCCCCCUGAUGAGXCGAA ACGAGCAUGCUCGUUGGGGCGG
74 HCV-11531153 GGAACAGCUGAUGAGXCGAA AAGCGGCGCCGCUUUCUGUUCC
75 HCV-11541159 CGGAACACUGAUGAGXCGAA AAAGCGGCCGCUUUCUGUUCCG
76 HCV-11581158 AUGGCGGCUGAUGAGXCGAA ACAGAAAUUUCUGUUCCGCCAU
2S 77 HCV-11591159 CAUGGCGCUGAUGAGXCGAA AACAGAAUUCUGUUCCGCCAUG
78 HCV-11681168 CCCCACGCUGAUGAGXCGAA ACAUGGCGCCAUGUACGUGGGG
79 HCV-11891189 GAAAACGCUGAUGAGXCGAA AUCCGCAUGCGGAUCCGUUUUC
80 HCV-11931193 CGAGGAA XCGAA ACGGAUCGAUCCGUUUUCCUCG
CUGAUGAG
30 81 HCV-11991199 ACGAGGACUGAUGAGXCGAA AACGGAUAUCCGUUUUCCUCGU
82 HCV-11951195 GACGAGGCUGAUGAGXCGAA AAACGGAUCCGUUUUCCUCGUC
83 HCV-11961196 AGACGAGCUGAUGAGXCGAA AAAACGGCCGUUUUCCUCGUCU
84 HCV-12801280 GACCUGA XCGAA ACAUGGCGCCAUGUAUCAGGUC
CUGAUGAG
85 HCV-12821282 GUGACCUCUGAUGAGXCGAA AUACAUGCAUGUAUCAGGUCAC
86 HCV-12871287 AUGCGGUCUGAUGAGXCGAA ACCUGAUAUCAGGUCACCGCAU
87 HCV-13731373 UAUCCACCUGAUGAGXCGAA ACAGCUUAAGCUGUCGUGGAUA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
53
No. Name Nt. Hammerhead Substrate
Pos. Ribozyme
88 HCV-13801380 GCCACCACUGAUGAGXCGAA AUCCACGCGUGGAUAUGGUGGC
89 HCV-14061406 CCGCUAGCUGAUGAGXCGAA ACUCCCCGGGGAGUCCUAGCGG
90 HCV-14091409 GGCCCGCCUGAUGAGXCGAA AGGACUCGAGUCCUAGCGGGCC
91 HCV-14181418 AGUAGGCCUGAUGAGXCGAA AGGCCCGCGGGCCUUGCCUACU
92 HCV-14231423 GGAAUAGCUGAUGAGXCGAA AGGCAAGCUUGCCUACUAUUCC
93 HCV-19261426 CAUGGAACUGAUGAGXCGAA AGUAGGCGCCUACUAUUCCAUG
IO 94 HCV-14281428 ACCAUGGCUGAUGAGXCGAA AUAGUAGCUACUAUUCCAUGGU
95 HCV-14291429 CACCAUGCUGAUGAGXCGAA AAUAGUAUACUAUUCCAUGGUG
96 HCV-17271727 ACUUGUCCUGAUGAGXCGAA AUGGAGCGCUCCAUCGACAAGU
97 HCV-17351735 CUGAGCGCUGAUGAGXCGAA ACUUGUCGACAAGUUCGCUCAG
98 HCV-17361736 CCUGAGCCUGAUGAGXCGAA AACUUGUACAAGUUCGCUCAGG
15
99 HCV-17901740 CAUCCCUCUGAUGAGXCGAA AGCGAACGUUCGCUCAGGGAUG
100 HCV-17571757 UAUAGGUCUGAUGAGXCGAA AUGGGGCGCCCCAUCACCUAUA
101 HCV-17621762 CUCGGUACUGAUGAGXCGAA AGGUGAUAUCACCUAUACCGAG
102 ACV-17951795 CCAGCAGCUGAUGAGXCGAA AAGGCCUAGGCCUUACUGCUGG
ZO
103 HCV-18061806 GGUGCGUCUGAUGAGXCGAA AUGCCAGCUGGCAUUACGCACC
104 HCV-18071807 AGGUGCGCUGAUGAGXCGAA AAUGCCAUGGCAUUACGCACCU
105 HCV-18151815 CACUGCCCUGAUGAGXCGAA AGGUGCGCGCACCUCGGCAGUG
106 HCV-18271827 GGUACGACUGAUGAGXCGAA ACCACACGUGUGGUAUCGUACC
ZS 107 HCV-18291829 CAGGUACCUGAUGAGXCGAA AUACCACGUGGUAUCGUACCUG
108 HCV-18321832 ACGCAGGCUGAUGAGXCGAA ACGAUACGUAUCGUACCUGCGU
109 HCV-18401840 CACCUGCCUGAUGAGXCGAA ACGCAGGCCUGCGUCGCAGGUG
110 HCV-18591854 UACACUGCUGAUGAGXCGAA ACCACACGUGUGGUCCAGUGUA
3O 111 HCV-18831883 CCACUACCUGAUGAGXCGAA ACAGGGCGCCCUGUUGUAGUGG
112 HCV-18861886 UCCCCACCUGAUGAGXCGAA ACAACAGCUGUUGUAGUGGGGA
113 HCV-19021902 CCGGACCCUGAUGAGXCGAA AUCGGUCGACCGAUCGGUCCGG
114 HCV-19061906 GGCACCGCUGAUGAGXCGAA ACCGAUCGAUCGGUCCGGUGCC
115 HCV-19171917 UUAUACGCUGAUGAGXCGAA AGGGGCAUGCCCCUACGUAUAA
116 HCV-19211921 CCAGUUACUGAUGAGXCGAA ACGUAGGCCUACGUAUAACUGG
117 HCV-19231923 CCCCAGUCUGAUGAGXCGAA AUACGUAUACGUAUAACUGGGG
CA 02326695 2000-10-26
WO 99/55$47 PCT/US99/09027
S4
No. Name Nt. Hammerhead Substrate
Pos.Ribozyme
118 HCV-19901990ACAGCCA XCGAA ACCAGUUAACUGGU UUGGCUGU
CUGAUGAG
119 HCV-19911991UACAGCC CUGAUGAGXCGAA AACCAGUACUGGUU UGGCUGUA
120 HCV-19981998AUCCAUG CUGAUGAGXCGAA ACAGCCAUGGCUGU ACAUGGAU
121 HCV-20432043UUGCACG CUGAUGAGXCGAA AGGGCCCGGGCCCU CCGUGCAA
122 HCV-20542054CCCCCCC CUGAUGAGXCGAA AUGUUGCGCAACAU CGGGGGGG
123 HCV-20632063GGUUGCC CUGAUGAGXCGAA ACCCCCCGGGGGGU C
GGCAACC
IO 124 HCV-20722072UCAAGGU CUGAUGAGXCGAA AGGUUGCGCAACCU CACCUUGA
125 HCV-20772077GCAGGUC CUGAUGAGXCGAA AGGUGAGCUCACCU UGACCUGC
126 HCV-21212121UUUGUGU CUGAUGAGXCGAA AGUGGCCGGCCACU UACACAAA
127 HCV-21222122UUUUGUG CUGAUGAGXCGAA AAGUGGCGCCACUU ACACAAAA
128 HCV-21372137UGGCCCC CUGAUGAGXCGAA AGCCACAUGUGGCU CGGGGCCA
IS
129 HCV-21492199AGGUGUU CUGAUGAGXCGAA ACCAUGGCCAUGGU U
AACACCU
130 HCV-21502150UAGGUGU CUGAUGAGXCGAA AACCAUGCAUGGUU AACACCUA
131 HCV-22192219CCUUAAA XCGAA AUGGUAAUUACCAU CUUUAAGG
CUGAUGAG
132 HCV-22212221AACCUUA CUGAUGAGXCGAA AGAUGGUACCAUCU UUAAGGUU
ZO
133 HCV-22612261CAGCACU CUGAUGAGXCGAA AGCCUGUACAGGCU UAGUGCUG
134 HCV-22622262GCAGCAC CUGAUGAGXCGAA AAGCCUGCAGGCUU AGUGCUGC
135 HCV-22952295AGGUCGC CUGAUGAGXCGAA ACGCUCUAGAGCGU UGCGACCU
136 HCV-23202320GAGCUCC CUGAUGAGXCGAA AUCUGUCGACAGAU CGGAGCUC
ZS 137 HCV-23272327GCGGGCU CUGAUGAGXCGAA AGCUCCGCGGAGCU CAGCCCGC
138 HCV-23442344UGUCGUG CUGAUGAGXCGAA ACAGCAGCUGCUGU CCACGACA
139 HCV-24172417UCUGAUG CUGAUGAGXCGAA AGGUGGAUCCACCU CCAUCAGA
140 HCV-24212421AUGUUCU CUGAUGAGXCGAA AUGGAGGCCUCCAU CAGAACAU
3O 141 HCV-24292429CGUCCAC CUGAUGAGXCGAA AUGUUCUAGAACAU CGUGGACG
192 HCV-25342534AGGCACA CUGAUGAGXCGAA ACGCGCGCGCGCGU CUGUGCCU
143 HCV-25852585GGUUCUC CUGAUGAGXCGAA AGGGCGGCCGCCCU AGAGAACC
144 HCV-26002600CGUUGAG CUGAUGAGXCGAA ACCACCAUGGUGGU CCUCAACG
145 HCV-26032603CCGCGUU CUGAUGAGXCGAA AGGACCAUGGUCCU C
AACGCGG
146 HCV-26712671CUUGAUG CUGAUGAGXCGAA ACCAGGCGCCUGGU ACAUCAAG
197 HCV-26752675UGCCCUU CUGAUGAGXCGAA AUGUACCGGUACAU C
AAGGGCA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
SS
No. Name Nt. hammerhead Substrate
Pos. Ribozyme
148 HCV-26902690 CCCCAGGCUGAUGAGXCGAA ACCAGCCGGCUGGUC CCUGGGG
149 HCV-27042704 CAGAGCA XCGAA AUGCCGCGCGGCAUA UGCUCUG
CUGAUGAG
150 HCV-27092709 CCGUACACUGAUGAGXCGAA AGCAUAUAUAUGCUC UGUACGG
151 HCV-27132713 CACGCCGCUGAUGAGXCGAA ACAGAGCGCUCUGUA CGGCGUG
152 HCV-2738273$ CCAGCAGCUGAUGAGXCGAA AGCAGGAUCCUGCUC CUGCUGG
153 HCV-27632763 AUGGCGUCUGAUGAGXCGAA AGCCCGUACGGGCUU ACGCCAU
IO 154 HCV-27642764 CAUGGCGCUGAUGAGXCGAA AAGCCCGCGGGCUUA CGCCAUG
155 HCV-28782878 GUAUUGUCUGAUGAGXCGAA ACCACCAUGGUGGUU ACAAUAC
156 HCV-28792879 AGUAUUGCUGAUGAGXCGAA AACCACCGGUGGUUA
CAAUACU
157 HCV-28842884 GAUAAAGCUGAUGAGXCGAA AUUGUAAUUACAAUA CUUUAUC
158 HCV-28872887 GGUGAUACUGAUGAGXCGAA AGUAUUGCAAUACUU UAUCACC
15
159 HCV-28882888 UGGUGAUCUGAUGAGXCGAA AAGUAUUAAUACUUU AUCACCA
160 HCV-29102910 ACGCACACUGAUGAGXCGAA AUGCGCCGGCGCAUU UGUGCGU
161 HCV-29112911 CACGCACCUGAUGAGXCGAA AAUGCGCGCGCAUUU GUGCGUG
162 HCV-29242929 GAGGGGGCUGAUGAGXCGAA ACCCACAUGUGGGUC CCCCCUC
ZO
163 HCV-29312931 ACAUUGACUGAUGAGXCGAA AGGGGGGCCCCCCUC UCAAUGU
164 HCV-29332933 GGACAUUCUGAUGAGXCGAA AGAGGGGCCCCUCUC
AAUGUCC
165 HCV-29392939 CCCCCCGCUGAUGAGXCGAA ACAUUGAUCAAUGUC CGGGGGG
166 HCV-29582958 AGGAUGACUGAUGAGXCGAA AGCAUCGCGAUGCUA UCAUCCU
ZS 167 HCV-29602960 GGAGGAUCUGAUGAGXCGAA AUAGCAUAUGCUAUC AUCCUCC
168 HCV-29632963 UGAGGAGCUGAUGAGXCGAA AUGAUAGCUAUCAUC CUCCUCA
169 HCV-29662966 AUGUGAGCUGAUGAGXCGAA AGGAUGAUCAUCCUC CUCACAU
170 HCV-29692969 CACAUGUCUGAUGAGXCGAA AGGAGGAUCCUCCUC ACAUGUG
3O 171 HCV-30593059 UCGCAGUCUGAUGAGXCGAA AUGGCAGCUGCCAUA ACUGCGA
172 HCV-31383138 UGGACGUCUGAUGAGXCGAA AUGGCCUAGGCCAUU ACGUCCA
1?3 HCV-31393139 UUGGACGCUGAUGAGXCGAA AAUGGCCGGCCAUUA CGUCCAA
174 HCV-31433143 CCAUUUGCUGAUGAGXCGAA ACGUAAUAUUACGUC CAAAUGG
175 HCV-31593154 CUUCAUGCUGAUGAGXCGAA AGGCCAUAUGGCCUU CAUGAAG
176 HCV-31553155 GCUUCAUCUGAUGAGXCGAA AAGGCCAUGGCCUUC AUGAAGC
177 HCV-32093209 AAUCCUGCUGAUGAGXCGAA AGCGGGGCCCCGCUA CAGGAUU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
56
No. Name Nt. Hammerhead Substrate
Pos.Ribozyme
178 HCV-32163216UGGGCCC CUGAUGAGX CGAA AUCCUGUACAGGAU UGGGCCCA
179 HCV-32333233GGUCUCG CUGAUGAGX CGAA AGGCCCGCGGGCCU ACGAGACC
180 HCV-32423242CCACCGC CUGAUGAGX CGAA AGGUCUCGAGACCU UGCGGUGG
181 HCV-32633263AGAAGAC CUGAUGAGX CGAA ACGGGCUAGCCCGU CGUCUUCU
182 HCV-32663266CAGAGAA X CGAA ACGACGGCCGUCGU CUUCUCUG
CUGAUGAG
183 HCV-32683268GUCAGAG CUGAUGAGX CGAA AGACGACGUCGUCU UCUCUGAC
lO 184 HCV-32903290AGGUGAU CUGAUGAGX CGAA AUCUUGGCCAAGAU CAUCACCU
185 HCV-32933293CCCAGGU CUGAUGAGX CGAA AUGAUCUAGAUCAU CACCUGGG
186 HCV-33293329CCAAGAU CUGAUGAGX CGAA AUGUCCCGGGACAU CAUCUUGG
187 HCV-33323332GUCCCAA X CGAA AUGAUGUACAUCAU CUUGGGAC
CUGAUGAG
188 HCV-33343334CAGUCCC CUGAUGAGX CGAA AGAUGAUAUCAUCU UGGGACUG
15
189 HCV-33473347GGGCGGA CUGAUGAGX CGAA ACGGGCAUGCCCGU CUCCGCCC
190 HCV-33993349UCGGGCG CUGAUGAGX CGAA AGACGGGCCCGUCU CCGCCCGA
191 HCV-33713371CCAGAAG CUGAUGAGX CGAA AUCUCCCGGGAGAU ACUUCUGG
192 HCV-34163416GGGCAAG CUGAUGAGX CGAA AGUCGCCGGCGACU CCUUGCCC
20
193 HCV-34193419UGGGGGC CUGAUGAGX CGAA AGGAGUCGACUCCU UGCCCCCA
199 HCV-34283428AGGCCGU CUGAUGAGX CGAA AUGGGGGCCCCCAU CACGGCCU
195 HCV-34823482GGCCUGU CUGAUGAGX CGAA AGGCUAGCUAGCCU CACAGGCC
196 HCV-35183518CCACUUG CUGAUGAGX CGAA ACCUCCCGGGAGGU UCAAGUGG
2S 197 HCV-35193519ACCACUU CUGAUGAGX CGAA AACCUCCGGAGGUU C
AAGUGGU
198 HCV-35273527CGGUGGA CUGAUGAGX CGAA ACCACUUAAGUGGU UUCCACCG
199 HCV-35283528GCGGUGG CUGAUGAGX CGAA AACCACUAGUGGUU UCCACCGC
200 HCV-35293529UGCGGUG CUGAUGAGX CGAA AAACCACGUGGUUU CCACCGCA
201 HCV-35763576ACGGUCC CUGAUGAGX CGAA ACACACAUGUGUGU UGGACCGU
202 HCV-36013601GGUCUUU CUGAUGAGX CGAA AGCCGGCGCCGGCU C
AAAGACC
203 HCV-36113611GGCCGGC CUGAUGAGX CGAA AGGGUCUAGACCCU AGCCGGCC
204 HCV-36843684GCCCCGG CUGAUGAGX CGAA AGGCGCAUGCGCCU CCCGGGGC
205 HCV-36963696GUAAGGG CUGAUGAGX CGAA ACGCGCCGGCGCGU UCCCUUAC
206 HCV-36973697UGUAAGG CUGAUGAGX CGAA AACGCGCGCGCGUU CCCUUACA
207 HCV-37013701AUGGUGU CUGAUGAGX CGAA AGGGAACGUUCCCU UACACCAU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
$7
No.Name Nt. Hammerhead Substrate
Pos.Ribozyme
208HCV-37023702CAUGGUG CUGAUGAGX CGAA AAGGGAAUUCCCUUA CACCAUG
209HCV-37293724GAGGUCC CUGAUGAGX CGAA AGCUACCGGUAGCUC GGACCUC
210HCV-37313731CCAGAUA CUGAUGAGX CGAA AGGUCCGCGGACCUC UAUCUGG
211HCV-37333733GACCAGA CUGAUGAGX CGAA AGAGGUCGACCUCUA UCUGGUC
212HCV-37353735GUGACCA CUGAUGAGX CGAA AUAGAGGCCUCUAUC UGGUCAC
213HCV-37403790GUCUCGU CUGAUGAGX CGAA ACCAGAUAUCUGGUC ACGAGAC
lO 214HCV-37613761GCACCGG CUGAUGAGX CGAA AUGACGUACGUCAUU CCGGUGC
215HCV-37623762CGCACCG CUGAUGAGX CGAA AAUGACGCGUCAUUC CGGUGCG
216HCV-37863786CUCCCCC CUGAUGAGX CGAA ACCGUCAUGACGGUC GGGGGAG
217HCV-37973797GGGACAG CUGAUGAGX CGAA AGGCUCCGGAGCCUA CUGUCCC
218HCV-38023802UCUGGGG CUGAUGAGX CGAA ACAGUAGCUACUGUC CCCCAGA
1$
219HCV-38353835GCCACCC CUGAUGAGX CGAA AAGAGCCGGCUCUUC GGGUGGC
220HCV-38513851AAGGGCA CUGAUGAGX CGAA AGCAGUGCACUGCUC UGCCCUU
221HCV-38583858UGCCCCG CUGAUGAGX CGAA AGGGCAGCUGCCCUU CGGGGCA
222HCV-38593859GUGCCCC CUGAUGAGX CGAA AAGGGCAUGCCCUUC GGGGCAC
ZO
223HCV-38723872AGAUGCC CUGAUGAGX CGAA ACAGCGUACGCUGUA GGCAUCU
224HCV-38783878CCCGGAA X CGAA AUGCCUAUAGGCAUC UUCCGGG
CUGAUGAG
225HCV-38803880AGCCCGG CUGAUGAGX CGAA AGAUGCCGGCAUCUU CCGGGCU
226HCV-38813881CAGCCCG CUGAUGAGX CGAA AAGAUGCGCAUCUUC CGGGCUG
2$ 227HCV-39083908CCUUCGC CUGAUGAGX CGAA ACCCCCCGGGGGGUU GCGAAGG
228HCV-90564056GGCACUU CUGAUGAGX CGAA AGUGCUCGAGCACUA
AAGUGCC
229HCV-40724072GGCUGCG CUGAUGAGX CGAA ACGCAGCGCUGCGUA CGCAGCC
230HCV-40874087UACCUUG CUGAUGAGX CGAA ACCCUUGCAAGGGUA CAAGGUA
3O 231HCV-41159115UGGCGGC CUGAUGAGX CGAA ACAGAUGCAUCUGUU GCCGCCA
232HCV-41759175CAGUUCU CUGAUGAGX CGAA AUGUUGGCCAACAUC AGAACUG
233HCV-41874187UGGUCCU CUGAUGAGX CGAA ACCCCAGCUGGGGUA AGGACCA
234HCV-42284228CUUACCA CUGAUGAGX CGAA AGGUGGAUCCACCUA UGGUAAG
235HCV-92334233AGGAACU CUGAUGAGX CGAA ACCAUAGCUAUGGUA AGUUCCU
236HCV-42374237GGCAAGG CUGAUGAGX CGAA ACUUACCGGUAAGUU CCUUGCC
237HCV-42389238CGGCAAG CUGAUGAGX CGAA AACUUACGUAAGUUC CUUGCCG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
58
No. Name Nt. Hammerhead Substrate
Pos. Ribozyme
238 HCV-42414241 CGUCGGCCUGAUGAGXCGAA AGGAACUAGUUCCUUGCCGACG
239 HCV-42809280 CACAUAUCUGAUGAGXCGAA AUGAUAUAUAUCAUAAUAUGUG
240 HCV-42839283 CAUCACACUGAUGAGXCGAA AUUAUGAUCAUAAUAUGUGAUG
241 HCV-43374337 GGUCCAGCUGAUGAGXCGAA ACUGUGCGCACAGUCCUGGACC
242 HCV-43704370 GCACGACCUGAUGAGXCGAA AGCCGCGCGCGGCUCGUCGUGC
243 HCV-43734373 CGAGCACCUGAUGAGXCGAA ACGAGCCGGCUCGUCGUGCUCG
IO 299 HCV-43794379 CGGUGGCCUGAUGAGXCGAA AGCACGAUCGUGCUCGCCACCG
295 HCV-49254925 UCCUCAA XCGAA AUUUGGGCCCAAAUAUUGAGGA
CUGAUGAG
246 HCV-49494444 AGUGUUGCUGAUGAGXCGAA ACAGAGCGCUCUGUCCAACACU
247 HCV-44609460 AGAAGGGCUGAUGAGXCGAA AUCUCUCGAGAGAUCCCCUUCU
298 HCV-44814481 CGAGGGGCUGAUGAGXCGAA AUGGCCUAGGCCAUCCCCCUCG
1$
249 HCV-44874487 UGGCCUCCUGAUGAGXCGAA AGGGGGAUCCCCCUCGAGGCCA
250 HCV-49964996 CCCCCUUCUGAUGAGXCGAA AUGGCCUAGGCCAUC
AAGGGGG
251 HCV-95284528 CUUCUUGCUGAUGAGXCGAA AGUGGCAUGCCACUCCAAGAAG
252 HCV-45774577 CGGCAUUCUGAUGAGXCGAA AUUCCGAUCGGAAUC
2O AAUGCCG
253 HCV-45864586 AAUACGCCUGAUGAGXCGAA ACGGCAUAUGCCGUAGCGUAUU
254 HCV-95914591 CCGGUAA XCGAA ACGCUACGUAGCGUAUUACCGG
CUGAUGAG
255 HCV-45934593 CCCCGGUCUGAUGAGXCGAA AUACGCUAGCGUAUUACCGGGG
256 HCV-45944594 ACCCCGGCUGAUGAGXCGAA AAUACGCGCGUAUUACCGGGGU
ZS 257 HCV-46164616 UCGGUAUCUGAUGAGXCGAA ACGGACAUGUCCGUCAUACCGA
258 HCV-46194619 UAGUCGGCUGAUGAGXCGAA AUGACGGCCGUCAUACCGACUA
259 HCV-46264626 UCUCCGCCUGAUGAGXCGAA AGUCGGUACCGACUAGCGGAGA
260 HCV-46729672 ACCGGUGCUGAUGAGXCGAA AGCCCGUACGGGCUACACCGGU
261 HCV-46974697 UGCAGUCCUGAUGAGXCGAA AUCACCGCGGUGAUCGACUGCA
262 HCV-47894789 UGAGCGCCUGAUGAGXCGAA ACACCGCGCGGUGUCGCGCUCA
263 HCV-97954795 CCGUUGUCUGAUGAGXCGAA AGCGCGAUCGCGCUCACAACGG
264 HCV-49204920 UCAUACCCUGAUGAGXCGAA AGCACAGCUGUGCUUGGUAUGA
265 HCV-49244924 GAGCUCACUGAUGAGXCGAA ACCAAGCGCUUGGUAUGAGCUC
266 HCV-49314931 CGGGCGUCUGAUGAGXCGAA AGCUCAUAUGAGCUCACGCCCG
267 HCV-49474947 CUGACUGCUGAUGAGXCGAA AGUCUCAUGAGACUACAGUCAG
CA 02326695 2000-10-26
WO 99/55847 PCTlUS99/09027
$9
No. Name Nt. Hammerhead Substrate
Pos.Ribozyme
268 HCV-49524952GCAACCU CUGAUGAGXCGAA ACUGUAGCUACAGU CAGGUUGC
269 HCV-49574957AGCCCGC CUGAUGAGXCGAA ACCUGACGUCAGGU UGCGGGCU
$
270 HCV-49659965UUCAGGU CUGAUGAGXCGAA AGCCCGCGCGGGCU UACCUGAA
271 HCV-49664966AUUCAGG CUGAUGAGXCGAA AAGCCCGCGGGCUU ACCUGAAU
272 HCV-49794974CCUGGUG CUGAUGAGXCGAA AUUCAGGCCUGAAU ACACCAGG
273 HCV-49894984GACGGGC CUGAUGAGXCGAA ACCCUGGCCAGGGU UGCCCGUC
IO 279 HCV-99919991CCUGGCA CUGAUGAGXCGAA ACGGGCAUGCCCGU CUGCCAGG
275 HCV-50045009AACUCCA CUGAUGAGXCGAA AUGGUCCGGACCAU CUGGAGUU
276 HCV-51025102GGUAUGC CUGAUGAGXCGAA ACCAGGUACCUGGU AGCAUACC
277 HCV-51075107GGCUUGG CUGAUGAGXCGAA AUGCUACGUAGCAU ACCAAGCC
278 HCV-51335133GGAGCCU CUGAUGAGXCGAA AGCCCUGCAGGGCU CAGGCUCC
1$
279 HCV-52185218UAGCCUA CUGAUGAGXCGAA ACAGCAGCUGCUGU AUAGGCUA
280 HCV-52205220CCUAGCC CUGAUGAGXCGAA AUACAGCGCUGUAU AGGCUAGG
281 HCV-53065306UAGUGAC CUGAUGAGXCGAA ACCUCCAUGGAGGU CGUCACUA
282 HCV-53095309UGCUAGU CUGAUGAGXCGAA ACGACCUAGGUCGU CACUAGCA
2O
283 HCV-53135313CAGGUGC CUGAUGAGXCGAA AGUGACGCGUCACU AGCACCUG
284 HCV-53305330CUCCGCC CUGAUGAGXCGAA ACCAGCAUGCUGGU AGGCGGAG
285 HCV-53395339CUGCAAG CUGAUGAGXCGAA ACUCCGCGCGGAGU CCUUGCAG
286 HCV-53425342GAGCUGC CUGAUGAGXCGAA AGGACUCGAGUCCU UGCAGCUC
2S 287 HCV-53595359CAGGCAA XCGAA AUGCGGCGCCGCAU AUUGCCUG
CUGAUGAG
288 HCV-53615361GUCAGGC CUGAUGAGXCGAA AUAUGCGCGCAUAU UGCCUGAC
289 HCV-53765376ACCACAC CUGAUGAGXCGAA ACCGGUUAACCGGU AGUGUGGU
290 HCV-53995399ACAAAAU CUGAUGAGXCGAA AUCCUACGUAGGAU CAUUUUGU
291 HCV-54235923CGGGAAC CUGAUGAGXCGAA ACAGCCGCGGCUGU UGUUCCCG
292 HCV-54265426UGUCGGG CUGAUGAGXCGAA ACAACAGCUGUUGU UCCCGACA
293 HCV-54275427CUGUCGG CUGAUGAGXCGAA AACAACAUGUUGUU CCCGACAG
294 HCV-55245524CUGCUUG CUGAUGAGXCGAA ACUGCUCGAGCAGU UCAAGCAG
295 HCV-55255525UCUGCUU CUGAUGAGXCGAA AACUGCUAGCAGUU C
AAGCAGA
296 HCV-558355$3ACCACGG CUGAUGAGXCGAA AGCAGCGCGCUGCU CCCGUGGU
297 HCV-55965596CCACCUG CUGAUGAGXCGAA ACUCCACGUGGAGU CCAGGUGG
CA 02326695 2000-10-26
WO 99/55$47 PCT/US99/09027
No.Name Nt. Hammerhead Substrate
Pos.Ribozyme
298HCV-56125612AGGCCUC CUGAUGAGX CGAA AGGGCCCGGGCCCUU GAGGCCU
299HCV-56205620UGCCCAG CUGAUGAGX CGAA AGGCCUCGAGGCCUU CUGGGCA
300HCV-56215621UUGCCCA X CGAA AAGGCCUAGGCCUUC UGGGCAA
CUGAUGAG
301HCV-56745674AGUGGAU CUGAUGAGX CGAA AGCCUGCGCAGGCUU AUCCACU
302HCV-56755675GAGUGGA CUGAUGAGX CGAA AAGCCUGCAGGCUUA UCCACUC
' 303HCV-57675767GAUGUUG CUGAUGAGX CGAA ACAGGAGCUCCUGUU CAACAUC
IO 304HCV-57685768AGAUGUU CUGAUGAGX CGAA AACAGGAUCCUGUUC
AACAUCU
305HCV-58015801GAGGAGC CUGAUGAGX CGAA AGUUGAGCUCAACUC GCUCCUC
306HCV-58055805CUGGGAG CUGAUGAGX CGAA AGCGAGUACUCGCUC CUCCCAG
307HCV-58215821GAAGGCC CUGAUGAGX CGAA AAGCAGCGCUGCUUC GGCCUUC
308HCV-58275827GCCCACG CUGAUGAGX CGAA AGGCCGAUCGGCCUU CGUGGGC
IS
309HCV-58285828CGCCCAC CUGAUGAGX CGAA AAGGCCGCGGCCUUC GUGGGCG
310HCV-58435843CACCGGC CUGAUGAGX CGAA AUGCCGGCCGGCAUU GCCGGUG
311HCV-58585858UGCUGCC CUGAUGAGX CGAA AUGGCCGCGGCCAUU GGCAGCA
312HCV-58675867CAAGGCC CUGAUGAGX CGAA AUGCUGCGCAGCAUA GGCCUUG
20
313HCV-58735873CCUUCCC CUGAUGAGX CGAA AGGCCUAUAGGCCUU GGGAAGG
319HCV-59055905CGCUCCA CUGAUGAGX CGAA AGCCCGCGCGGGCUA UGGAGCG
315HCV-59305930AAGCCAC CUGAUGAGX CGAA AGUGCACGUGCACUC GUGGCUU
316HCV-59375937ACCUUAA X CGAA AGCCACGCGUGGCUU UUAAGGU
CUGAUGAG
2S 317HCV-59385938GACCUUA CUGAUGAGX CGAA AAGCCACGUGGCUUU UAAGGUC
318HCV-59395939UGACCUU CUGAUGAGX CGAA AAAGCCAUGGCUUUU
AAGGUCA
319HCV-59405940AUGACCU CUGAUGAGX CGAA AAAAGCCGGCUUUUA AGGUCAU
320HCV-59455945CGCUCAU CUGAUGAGX CGAA ACCUUAAUUAAGGUC AUGAGCG
3O 321HCV-59655965CUCGGCG CUGAUGAGX CGAA AGGGCGCGCGCCCUC CGCCGAG
322HCV-59$15981GCAAGUU CUGAUGAGX CGAA ACCAGGUACCUGGUU
AACUUGC
323HCV-59825982AGCAAGU CUGAUGAGX CGAA AACCAGGCCUGGUUA ACUUGCU
329HCV-59905990UGGCAGG CUGAUGAGX CGAA AGCAAGUACUUGCUC CCUGCCA
325HCV-60046004GCCGGGG CUGAUGAGX CGAA AGAGGAUAUCCUCUC CCCCGGC
326HCV-60206020CCCCGAC CUGAUGAGX CGAA ACCAGGGCCCUGGUC GUCGGGG
327HCV-60236023CGACCCC CUGAUGAGX CGAA ACGACCAUGGUCGUC GGGGUCG
CA 02326695 2000-10-26
WO 99/5584? PCT/US99/09027
61
No. Name Nt. Hammerhead Substrate
Pos.Ribozyme
328 HCV-60296029CACACAC CUGAUGAGX CGAA ACCCCGAUCGGGGU CGUGUGUG
329 HCV-60446094GACGCAG CUGAUGAGX CGAA AUUGCUGCAGCAAU CCUGCGUC
S
330 HCV-60516051ACGUGCC CUGAUGAGX CGAA ACGCAGGCCUGCGU C
GGCACGU
331 HCV-61066106CGAAGCG CUGAUGAGX CGAA ACGCUAUAUAGCGU UCGCUUCG
332 HCV-61076107GCGAAGC CUGAUGAGX CGAA AACGCUAUAGCGUU CGCUUCGC
333 HCV-61116111CCCCGCG CUGAUGAGX CGAA AGCGAACGUUCGCU UCGCGGGG
j~ 334 HCV-64136413UUUGCAU CUGAUGAGX CGAA AUGCCGUACGGCAU CAUGCAAA
335 HCV-65746574CCUGGAA X CGAA AGUUCGGCCGAACU AUUCCAGG
CUGAUGAG
336 HCV-65766576GCCCUGG CUGAUGAGX CGAA AUAGUUCGAACUAU UCCAGGGC
337 HCV-65776577CGCCCUG CUGAUGAGX CGAA AAUAGUUAACUAUU CCAGGGCG
IS 338 HCV-66376637GUAGUGG CUGAUGAGX CGAA AGUCCCCGGGGACU UCCACUAC
339 HCV-66386638CGUAGUG CUGAUGAGX CGAA AAGUCCCGGGACUU CCACUACG
340 HCV-66936643CGUCACG CUGAUGAGX CGAA AGUGGAAUUCCACU ACGUGACG
341 HCV-66716671GGCAUUU CUGAUGAGX CGAA ACGUUGUACAACGU A
AAAUGCC
342 HCV-67036703GGUGAAG CUGAUGAGX CGAA AUUCGGGCCCGAAU UCUUCACC
20
343 HCV-67096704CGGUGAA X CGAA AAUUCGGCCGAAUU CUUCACCG
CUGAUGAG
344 HCV-67066706UUCGGUG CUGAUGAGX CGAA AGAAUUCGAAUUCU UCACCGAA
345 HCV-67076707AUUCGGU CUGAUGAGX CGAA AAGAAUUAAUUCUU CACCGAAU
346 HCV-67156715CCCGUCC CUGAUGAGX CGAA AUUCGGUACCGAAU UGGACGGG
2S 347 HCV-67306730CCUGUGC CUGAUGAGX CGAA ACCGCACGUGCGGU UGCACAGG
348 HCV-67396739CGGAGCG CUGAUGAGX CGAA ACCUGUGCACAGGU ACGCUCCG
349 HCV-67446744CACGCCG CUGAUGAGX CGAA AGGGUACGUACGCU CCGGCGUG
350 HCV-67596759CGUAGGA CUGAUGAGX CGAA AGGUCUGCAGACCU CUCCUACG
351 HCV-67616761CCCGUAG CUGAUGAGX CGAA AGAGGUCGACCUCU CCUACGGG
352 HCV-67646764CCUCCCG CUGAUGAGX CGAA AGGAGAGCUCUCCU ACGGGAGG
353 HCV-67766776GGAAUGU CUGAUGAGX CGAA ACAUCCUAGGAUGU CACAUUCC
354 HCV-67826782CGACCUG CUGAUGAGX CGAA AAUGUGAUCACAUU CCAGGUCG
355 HCV-67886788UGAGCCC CUGAUGAGX CGAA ACCUGGAUCCAGGU CGGGCUCA
356 HCV-67946799AUUGGUU CUGAUGAGX CGAA AGCCCGAUCGGGCU C
AACCAAU
357 HCV-68026802AACCAGG CUGAUGAGX CGAA AUUGGUUAACCAAU ACCUGGUU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
62
No.Name Nt. Hammerhead Substrate
Pos.Ribozyme
358HCV-68096809GUGACCC CUGAUGAGX CGAA ACCAGGUACCUGGUU GGGUCAC
359HCV-68196814GAGCUGU CUGAUGAGX CGAA ACCCAACGUUGGGUC ACAGCUC
S
360HCV-68216821CGCAUGG CUGAUGAGX CGAA AGCUGUGCACAGCUC CCAUGCG
361HCV-69066906GCCAGCC CUGAUGAGX CGAA ACGUUUAUAAACGUA GGCUGGC
362HCV-69226922GGGGGGA CUGAUGAGX CGAA ACCCCCUAGGGGGUC UCCCCCC
363HCV-69246924GAGGGGG CUGAUGAGX CGAA AGACCCCGGGGUCUC CCCCCUC
364HCV-69316931GGCCAAG CUGAUGAGX CGAA AGGGGGGCCCCCCUC CUUGGCC
365HCV-69346934GCUGGCC CUGAUGAGX CGAA AGGAGGGCCCUCCUU GGCCAGC
366HCV-69436993AGCUGAA X CGAA AGCUGGCGCCAGCUC UUCAGCU
CUGAUGAG
367HCV-69586958CGCAGAC CUGAUGAGX CGAA AUUGGCUAGCCAAUU GUCUGCG
368HCV-69616961AGGCGCA CUGAUGAGX CGAA ACAAUUGCAAUUGUC UGCGCCU
1S
369HCV-70347034GCCACAG CUGAUGAG7CCGAA AGGUUGGCCAACCUC CUGUGGC
370HCV-71187118CCGCUCG CUGAUGAGX CGAA AGCGGGUACCCGCUU CGAGCGG
371HCV-71197119UCCGCUC CUGAUGAGX CGAA AAGCGGGCCCGCUUC GAGCGGA
372HCV-71457145CAACGGA CUGAUGAGX CGAA ACUUCCCGGGAAGUA UCCGUUG
20
373HCV-71957195UAUGGGC CUGAUGAGX CGAA ACGCGGGCCCGCGUU GCCCAUA
374HCV-72027202GUGCCCA CUGAUGAGX CGAA AUGGGCAUGCCCAUA UGGGCAC
375HCV-72187218GGGUUGU CUGAUGAGX CGAA AUCCGGGCCCGGAUU ACAACCC
376HCV-72197219AGGGUUG CUGAUGAGX CGAA AAUCCGGCCGGAUUA CAACCCU
2S 377HCV-72347234GGACUCU CUGAUGAGX CGAA ACAGUGGCCACUGUU AGAGUCC
378HCV-72357235AGGACUC CUGAUGAGX CGAA AACAGUGCACUGUUA GAGUCCU
379HCV-72517251UAGUCCG CUGAUGAGX CGAA ACUUUUCGAAAAGUC CGGACUA
380HCV-72587258AGGGACG CUGAUGAGX CGAA AGUCCGGCCGGACUA CGUCCCU
3O 381HCV-72627262CCGGAGG CUGAUGAGX CGAA ACGUAGUACUACGUC CCUCCGG
382HCV-72667266ACCGCCG CUGAUGAGX CGAA AGGGACGCGUCCCUC CGGCGGU
383HCV-72887288AGGCGGC CUGAUGAGX CGAA AUGGGCAUGCCCAUU GCCGCCU
389HCV-72967296CCCGUGG CUGAUGAGX CGAA AGGCGGCGCCGCCUA CCACGGG
385HCV-73547359CACGGUG CUGAUGAGX CGAA ACUCUGUACAGAGUC CACCGUG
386HCV-73867386GUCUUAG CUGAUGAGX CGAA AGCCAGCGCUGGCUA CUAAGAC
387HCV-73897389AAAGUCU CUGAUGAGX CGAA AGUAGCCGGCUACUA AGACUUU
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
63
No. Name Nt. Hammerhead Substrate
Pos.Ribozyme
388 HCV-73957395CUGCCGA CUGAUGAGXCGAA AGUCUUAUAAGACU UUCGGCAG
389 HCV-73967396GCUGCCG CUGAUGAGXCGAA AAGUCUUAAGACUU UCGGCAGC
390 HCV-73977397AGCUGCC CUGAUGAGXCGAA AAAGUCUAGACUUU CGGCAGCU
391 HCV-74117411GGCCGAC CUGAUGAGXCGAA AUCCGGAUCCGGAU CGUCGGCC
392 HCV-74147919AACGGCC CUGAUGAGXCGAA ACGAUCCGGAUCGU CGGCCGUU
393 HCV-74217421CGCUGUC CUGAUGAGXCGAA ACGGCCGCGGCCGU UGACAGCG
IO 394 HCV-74987498CAUGGAG CUGAUGAGXCGAA AGUACGAUCGUACU CCUCCAUG
395 HCV-75017501GGGCAUG CUGAUGAGXCGAA AGGAGUAUACUCCU CCAUGCCC
396 HCV-75197514CCCCCUC CUGAUGAGXCGAA AGGGGGGCCCCCCU UGAGGGGG
397 HCV-75397539UCGCUGA CUGAUGAGXCGAA AUCAGGGCCCUGAU CUCAGCGA
398 HCV-75417591CGUCGCU CUGAUGAGXCGAA AGAUCAGCUGAUCU CAGCGACG
IS
399 HCV-75527552AGACCAA XCGAA ACCCGUCGACGGGU CUUGGUCU
CUGAUGAG
400 HCV-75547554GUAGACC CUGAUGAGXCGAA AGACCCGCGGGUCU UGGUCUAC
401 HCV-75587558CACGGUA CUGAUGAGXCGAA ACCAAGAUCUUGGU CUACCGUG
402 HCV-75607560CUCACGG CUGAUGAGXCGAA AGACCAAUUGGUCU ACCGUGAG
ZO
403 HCV-75897589AGCAGAC CUGAUGAGXCGAA AUGUCGUACGACAU CGUCUGCU
404 HCV-75927592AGCAGCA CUGAUGAGXCGAA ACGAUGUACAUCGU CUGCUGCU
405 HCV-76007600GGACAUU CUGAUGAGXCGAA AGCAGCAUGCUGCU C
AAUGUCC
906 HCV-76067606UGUGUAG CUGAUGAGXCGAA ACAUUGAUCAAUGU CCUACACA
ZS 407 HCV-76677667ACGCGUU CUGAUGAGXCGAA AUGGGCAUGCCCAU C
AACGCGU
408 HCV-77237723ACUGCGG CUGAUGAGXCGAA AUGUUGUACAACAU CCCGCAGU
409 HCV-77757775CGUCCAG CUGAUGAGXCGAA ACUUGCAUGCAAGU CCUGGACG
410 HCV-77897789GUCCCGG CUGAUGAGXCGAA AGUGGUCGACCACU ACCGGGAC
3O 411 HCV-78397839AGAAGUU CUGAUGAGXCGAA AGCCUUAUAAGGCU A
AACUUCU
412 HCV-78977897CUACGGA CUGAUGAGXCGAA AGAAGUUAACUUCU AUCCGUAG
413 HCV-78497849UUCUACG CUGAUGAGXCGAA AUAGAAGCUUCUAU CCGUAGAA
414 HCV-?8537853CUUCUUC CUGAUGAGXCGAA ACGGAUAUAUCCGU AGAAGAAG
415 HCV-78947899AAAUUUA CUGAUGAGXCGAA AUUUGGCGCCAAAU CUAAAUUU
416 HCV-78967896CCAAAUU CUGAUGAGXCGAA AGAUUUGCAAAUCU A
AAUUUGG
417 HCV-79007900AUAGCCA CUGAUGAGXCGAA AUUUAGAUCUAAAU UUGGCUAU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
64
No.Name Nt. I3ammerhead Substrate
Pos.Ribozyme
418HCV-79017901CAUAGCCCUGAUGAGX CGAA AAUUUAGCUAAAUUU GGCUAUG
419HCV-79067906UGCCCCACUGAUGAGX CGAA AGCCAAAUUUGGCUA UGGGGCA
S 420HCV-79557955CGGAGCGCUGAUGAGX CGAA AUGUGGUACCACAUC CGCUCCG
921HCV-79607960CCACACGCUGAUGAGX CGAA AGCGGAUAUCCGCUC CGUGUGG
422HCV-8075$075AUACGAUCUGAUGAGX CGAA AGGCGAGCUCGCCUU AUCGUAU
423HCV-80768076AAUACGACUGAUGAGX CGAA AAGGCGAUCGCCUUA UCGUAUU
lO 929HCV-80788078GGAAUACCUGAUGAGX CGAA AUAAGGCGCCUUAUC GUAUUCC
925HCV-81708170GAAUCCGCUGAUGAGX CGAA ACGAGGAUCCUCGUA CGGAUUC
426HCV-81768176GUACUGGCUGAUGAGX CGAA AUCCGUAUACGGAUU CCAGUAC
427HCV-81828182AGGAGAGCUGAUGAGX CGAA ACUGGAAUUCCAGUA CUCUCCU
428HCV-81878187UGCCCAGCUGAUGAGX CGAA AGAGUACGUACUCUC CUGGGCA
1S
429HCV-82018201GGAACUCCUGAUGAGX CGAA ACCCGCUAGCGGGUU GAGUUCC
930HCV-82068206CACCAGGCUGAUGAGX CGAA ACUCAACGUUGAGUU CCUGGUG
431HCV-82078207UCACCAGCUGAUGAGX CGAA AACUCAAUUGAGUUC CUGGUGA
432HCV-82278227UUUCUUUCUGAUGAGX CGAA AUUUCCAUGGAAAUC
ZO AAAGAAA
q33HCV-83578357GCGACUUCUGAUGAGX CGAA AUGGCCUAGGCCAUA
AAGUCGC
434HCV-83628362CGUGAGCCUGAUGAGX CGAA ACUUUAUAUAAAGUC GCUCACG
435HCV-83668366GCUCCGUCUGAUGAGX CGAA AGCGACUAGUCGCUC ACGGAGC
436HCV-83788378CGAUGUACUGAUGAGX CGAA AGCCGCUAGCGGCUC UACAUCG
ZS 437HCV-83808380CCCGAUGCUGAUGAGX CGAA AGAGCCGCGGCUCUA
CAUCGGG
438HCV-83848389GGCCCCCCUGAUGAGX CGAA AUGUAGAUCUACAUC GGGGGCC
439HCV-89248429CGGCGAUCUGAUGAGX CGAA ACCGCAGCUGCGGUU AUCGCCG
490HCV-84258425CCGGCGA X CGAA AACCGCAUGCGGUUA UCGCCGG
CUGAUGAG
3O 441HCV-84278427CACCGGCCUGAUGAGX CGAA AUAACCGCGGUUAUC GCCGGUG
442HCV-84608460CCGCAGCCUGAUGAGX CGAA AGUCGUCGACGACUA GCUGCGG
443HCV-85088508GCAGCUCCUGAUGAGX CGAA ACAGGCCGGCCUGUC GAGCUGC
444HCV-85228522AGUCCUGCUGAUGAGX CGAA AGCUUUGCAAAGCUC CAGGACU
445HCV-85408540CGUUCACCUGAUGAGX CGAA AGCAUCGCGAUGCUC GUGAACG
446HCV-85588558UAACGACCUGAUGAGX CGAA AGGUCGUACGACCUU GUCGUUA
497HCV-85618561AGAUAACCUGAUGAGX CGAA ACAAGGUACCUUGUC GUUAUCU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/090Z7
6S
No. Name Nt. Hammerhead Substrate
Pos.Ribozyme
448 HCV-85648564CACAGAU CUGAUGAGX CGAA ACGACAAUUGUCGU UAUCUGUG
449 HCV-86388638GGGGGCA CUGAUGAGX CGAA AGUACCUAGGUACU CUGCCCCC
S 450 HCV-86718671CAAGUCG CUGAUGAGX CGAA AUUCUGGCCAGAAU ACGACUUG
451 HCV-86988698GUUGGAG CUGAUGAGX CGAA AGCAUGAUCAUGCU CCUCCAAC
452 HCV-87018701CACGUUG CUGAUGAGX CGAA AGGAGCAUGCUCCU CCAACGUG
453 HCV-87288728UUUGCCG CUGAUGAGX CGAA AUGCGUCGACGCAU CCGGCAAA
IO 454 HCV-87748779CCCGUGC CUGAUGAGX CGAA AGGGGGGCCCCCCU UGCACGGG
455 HCV-88428842GGGCGCA CUGAUGAGX CGAA ACAUGAUAUCAUGU AUGCGCCC
456 HCV-88548854UGCCCAU CUGAUGAGX CGAA AGGUGGGCCCACCU UAUGGGCA
457 HCV-88558855UUGCCCA CUGAUGAGX CGAA AAGGUGGCCACCUU AUGGGCAA
458 HCV-88718871GUCAUCA CUGAUGAGX CGAA AAUCAUCGAUGAUU UUGAUGAC
IS
459 HCV-88808880AAGAAGU CUGAUGAGX CGAA AGUCAUCGAUGACU CACUUCUU
460 HCV-89318931AUCUGAC CUGAUGAGX CGAA AUCCAGGCCUGGAU UGUCAGAU
461 HCV-89348934UAGAUCU CUGAUGAGX CGAA ACAAUCCGGAUUGU CAGAUCUA
462 HCV-89398939CCCCGUA CUGAUGAGX CGAA AUCUGACGUCAGAU CUACGGGG
20
463 HCV-89418941GGCCCCG CUGAUGAGX CGAA AGAUCUGCAGAUCU ACGGGGCC
469 HCV-90659065GUUUCCU CUGAUGAGX CGAA AGGCAUGCAUGCCU CAGGAAAC
465 HCV-90749074GUACCCC CUGAUGAGX CGAA AGUUUCCGGAAACU UGGGGUAC
966 HCV-90809080AGGGCGG CUGAUGAGX CGAA ACCCCAAUUGGGGU ACCGCCCU
2S 467 HCV-90889088GACUCGC CUGAUGAGX CGAA AGGGCGGCCGCCCU UGCGAGUC
468 HCV-90959095GUCUCCA X CGAA ACUCGCAUGCGAGU CUGGAGAC
CUGAUGAG
469 HCV-91199119UAGCGCG CUGAUGAGX CGAA ACACUUCGAAGUGU CCGCGCUA
970 HCV-91269126AGUAGCC CUGAUGAGX CGAA AGCGCGGCCGCGCU AGGCUACU
30 471 HCV-91319131GGGACAG CUGAUGAGX CGAA AGCCUAGCUAGGCU ACUGUCCC
472 HCV-91369136CCCUUGG CUGAUGAGX CGAA ACAGUAGCUACUGU CCCAAGGG
473 HCV-92269226CAGCUGG CUGAUGAGX CGAA ACGCGGCGCCGCGU CCCAGCUG
474 HCV-92389238GCUGGAC CUGAUGAGX CGAA AGUCCAGCUGGACU UGUCCAGC
475 HCV-92419241CCAGCUG CUGAUGAGX CGAA ACAAGUCGACUUGU C
CAGCUGG
976 HCV-92509250AGCAACG CUGAUGAGX CGAA ACCAGCUAGCUGGU UCGUUGCU
977 HCV-92519251CAGCAAC CUGAUGAGX CGAA AACCAGCGCUGGUU CGUUGCUG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
66
No.Name Nt. Hammerhead Substrate
Pos.Ribozyme
478HCV-92549254AACCAGCCUGAUGAGX CGAA ACGAACCGGUUCGUU GCUGGUU
479HCV-92789278UGUGAUACUGAUGAGX CGAA AUGUCUCGAGACAUA UAUCACA
S 480HCV-92809280GCUGUGACUGAUGAGX CGAA AUAUGUCGACAUAUA UCACAGC
481HCV-92829282AGGCUGUCUGAUGAGX CGAA AUAUAUGCAUAUAUC ACAGCCU
982HCV-92929292GGCACGACUGAUGAGX CGAA ACAGGCUAGCCUGUC UCGUGCC
483HCV-93269326GUAGGAGCUGAUGAGX CGAA AGGCACCGGUGCCUA CUCCUAC
1~ 484HCV-93299329AAAGUAGCUGAUGAGX CGAA AGUAGGCGCCUACUC CUACUUU
485HCV-93329332CGGAAAGCUGAUGAGX CGAA AGGAGUAUACUCCUA CUUUCCG
486HCV-93359335CUACGGACUGAUGAGX CGAA AGUAGGAUCCUACUU UCCGUAG
487HCV-93369336CCUACGGCUGAUGAGX CGAA AAGUAGGCCUACUUU CCGUAGG
488HCV-93379337CCCUACGCUGAUGAGX CGAA AAAGUAGCUACUUUC CGUAGGG
1S
989HCV-93419341CUACCCCCUGAUGAGX CGAA ACGGAAAUUUCCGUA GGGGUAG
490HCV-93479347AGAUGCCCUGAUGAGX CGAA ACCCCUAUAGGGGUA GGCAUCU
491HCV-93539353GCAGGUACUGAUGAGX CGAA AUGCCUAUAGGCAUC UACCUGC
492HCV-93559355GAGCAGGCUGAUGAGX CGAA AGAUGCCGGCAUCUA CCUGCUC
20
493HCV-93629362GGUUGGGCUGAUGAGX CGAA AGCAGGUACCUGCUC CCCAACC
494HCV-93859385GAGUGAUCUGAUGAGX CGAA AGCUCCCGGGAGCUA AUCACUC
495HCV-93889388CUGGAGUCUGAUGAGX CGAA AUUAGCUAGCUAAUC ACUCCAG
496HCV-93929392UGGCCUGCUGAUGAGX CGAA AGUGAUUAAUCACUC CAGGCCA
2S 497HCV-94029402GAUGGCCCUGAUGAGX CGAA AUUGGCCGGCCAAUA GGCCAUC
Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992
Nucleic Acids Res. 20:
3252). The length of stem II may be 2 base-pairs.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
67
Table VI: Additional HCV Hammerhead (HIS Ribozyme and Target Sequence
Pos. Ribozyme Substrate
S 14 CGCCCCC CUGAUGAG X CGAA CCCCGAU U GGGGGCG
AUCGGGG
34 AGUGAUC CUGAUGAG X CGAA CCACCAU A GAUCACU
AUGGUGG
38 GGGGAGU CUGAUGAG X CGAA CAUAGAU C ACUCCCC
AUCUAUG
42 CACAGGG CUGAUGAG X CGAA GAUCACU C CCCUGUG
AGUGAUC
57 AAGACAG CUGAUGAG X CGAA AGGAACU A CUGUCUU
l~ AGUUCCU
62 GCGUGAA CUGAUGAG X CGAA CUACUGU C UUCACGC
ACAGUAG
64 CUGCGUG CUGAUGAG X CGAA ACUGUCU U CACGCAG
AGACAGU
65 UCUGCGU CUGAUGAG X CGAA CUGUCUU C ACGCAGA
AAGACAG
79 AUGGCUA CUGAUGAG X CGAA AAAGCGU C UAGCCAU
IS ACGCUUU
81 CCAUGGC CUGAUGAG X CGAA AGCGUCU A GCCAUGG
AGACGCU
92 UCAUACU CUGAUGAG X CGAA AUGGCGU U AGUAUGA
ACGCCAU
93 CUCAUAC CUGAUGAG X CGAA UGGCGUU A GUAUGAG
AACGCCA
96 ACACUCA CUGAUGAG X CGAA CGUUAGU A UGAGUGU
ACUAACG
109 GCUGCAC CUGAUGAG X CGAA UGAGUGU C GUGCAGC
ACACUCA
142 AGACCAC CUGAUGAG X CGAA GAGCCAU A GUGGUCU
AUGGCUC
192 AAGAAAG CUGAUGAG X CGAA ACCGGGU C CUUUCUU
ACCCGGU
195 UCCAAGA CUGAUGAG X CGAA GGGUCCU U UCUUGGA
AGGACCC
ZS 196 AUCCAAG CUGAUGAG X CGAA GGUCCUU U GUUGGAU
AAGGACC
197 GAUCCAA CUGAUGAG X CGAA GUCCUUU C UUGGAUC
AAAGGAC
204 GCGGGUU CUGAUGAG X CGAA CUUGGAU C AACCCGC
AUCCAAG
227 ACGCCCA CUGAUGAG X CGAA UGGAGAU U UGGGCGU
AUCUCCA
228 CACGCCC CUGAUGAG X CGAA GGAGAUU U GGGCGUG
30 AAUCUCC
282 GUACCAC CUGAUGAG X CGAA AAGGCCU U GUGGUAC
AGGCCUU
359 GGUUUAG CUGAUGAG X CGAA CACGAAU C CUAAACC
AUUCGUG
357 UGAGGUU CUGAUGAG X CGAA GAAUCCU A AACCUCA
AGGAUUC
363 UUUCUUU CUGAUGAG X CGAA UAAACCU C AAAGAAA
AGGUUUA
381 UAGGUGU CUGAUGAG X CGAA CAAACGU A ACACCUA
ACGUUUG
388 GCGGCGG CUGAUGAG X CGAA AACACCU A CCGCCGC
AGGUGUU
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
68
Pos. Ribozyme Substrate
431 CACCAAC CUGAUGAG X CGAA GUCAGAU C GUUGGUG
AUCUGAC
439 CUCCACC CUGAUGAG X CGAA AGAUCGU U GGUGGAG
ACGAUCU
S 443 ACACGUA CUGAUGAG X CGAA GUGGAGU U UACGUGU
ACUCCAC
449 AACACGU CUGAUGAG X CGAA UGGAGUU U ACGUGUU
AACUCCA
945 CAACACG CUGAUGAG X CGAA GGAGUUU A CGUGUUG
AAACUCC
451 GCGCGGC CUGAUGAG X CGAA UACGUGU U GCCGCGC
ACACGUA
516 CUUCCAC CUGAUGAG X CGAA GCAACCU C GUGGAAG
AGGUUGC
688 AUUGCGC CUGAUGAG X CGAA CGGAGGU C GCGCAAU
ACCUCCG
702 AUGACCU CUGAUGAG X CGAA UCUGGGU A AGGUCAU
ACCCAGA
719 CGCACGU CUGAUGAG X CGAA AUACCCU C ACGUGCG
AGGGUAU
790 ACCCCAU CUGAUGAG X CGAA CCGACCU C AUGGGGU
AGGUCGG
IS 861 AUAGAGA CUGAUGAG X CGAA UUGCUCU U UCUCUAU
AGAGCAA
862 GAUAGAG CUGAUGAG X CGAA UGCUCUU U CUCUAUC
AAGAGCA
863 AGAUAGA CUGAUGAG X CGAA GCUCUUU C UCUAUCU
AAAGAGC
865 GAAGAUA CUGAUGAG X CGAA UCUUUCU C UAUCUUC
AGAAAGA
2O 867 AGGAAGA CUGAUGAG X CGAA UUUCUCU A UCUUCCU
AGAGAAA
869 AGAGGAA CUGAUGAG X CGAA UCUCUAU C UUCCUCU
AUAGAGA
871 CAAGAGG CUGAUGAG X CGAA UCUAUCU U CCUCUUG
AGAUAGA
872 CCAAGAG CUGAUGAG X CGAA CUAUCUU C CUCUUGG
AAGAUAG
875 GGGCCAA CUGAUGAG X CGAA UCUUCCU C UUGGCCC
25 AGGAAGA
877 CAGGGCC CUGAUGAG X CGAA UUCCUCU U GGCCCUG
AGAGGAA
889 CAAACAG CUGAUGAG X CGAA CUGCUGU C CUGUUUG
ACAGCAG
894 AUGGUCA CUGAUGAG X CGAA GUCCUGU U UGACCAU
ACAGGAC
895 GAUGGUC CUGAUGAG X CGAA UCCUGUU U GACCAUC
3O AACAGGA
902 AAGCUGG CUGAUGAG X CGAA UGACCAU C CCAGCUU
AUGGUCA
909 UAAGCGG CUGAUGAG X CGAA CCCAGCU U CCGCUUA
AGCUGGG
910 AUAAGCG CUGAUGAG X CGAA CCAGCUU C CGCUUAU
AAGCUGG
915 ACCUGAU CUGAUGAG X CGAA UUCCGCU U AUCAGGU
AGCGGAA
916 CACCUGA CUGAUGAG X CGAA UCCGCUU A UCAGGUG
AAGCGGA
918 CGCACCU CUGAUGAG X CGAA CGCUUAU C AGGUGCG
AUAAGCG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
69
Pos. Ribozyme Substrate
934 CAGCCCG CUGAUGAG X CGAA AACGCAU C CGGGCUG
AUGCGUU
943 GACAUGG CUGAUGAG X CGAA GGGCUGU A CCAUGUC
ACAGCCC
950 CAUUCGU CUGAUGAG X CGAA ACCAUGU C ACGAAUG
ACAUGGU
964 UGAGUUG CUGAUGAG X CGAA GACUGCU C CAACUCA
AGCAGUC
970 AAUGCUU CUGAUGAG X CGAA UCCAACU C AAGCAUU
AGUUGGA
977 CAUACAC CUGAUGAG X CGAA CAAGCAU U GUGUAUG
AUGCUUG
1008 CCGGGGG CUGAUGAG X CGAA CAUGCAU A CCCCCGG
IO AUGCAUG
1067 UGGGAGU CUGAUGAG X CGAA UAGCGCU C ACUCCCA
AGCGCUA
1071 AGCGUGG CUGAUGAG X CGAA GCUCACU C CCACGCU
AGUGAGC
1079 UGGCCGC CUGAUGAG X CGAA CCACGCU C GCGGCCA
AGCGUGG
1100 UAGUGGG CUGAUGAG X CGAA CCAGCAU C CCCACUA
AUGCUGG
IS 1107 AUUGUCG CUGAUGAG X CGAA CCCCACU A CGACAAU
AGUGGGG
1115 GGCGUCG CUGAUGAG X CGAA CGACAAU A CGACGCC
AUUGUCG
1152 GAACAGA CUGAUGAG X CGAA GGCCGCU U UCUGUUC
AGCGGCC
1181 AUCCGCA CUGAUGAG X CGAA GGGACCU C UGCGGAU
AGGUCCC
2O 1199 GGGAGAC CUGAUGAG X CGAA UUUUCCU C GUCUCCC
AGGAAAA
1202 ACUGGGA CUGAUGAG X CGAA UCCUCGU C UCCCAGU
ACGAGGA
1204 CAACUGG CUGAUGAG X CGAA CUCGUCU C CCAGUUG
AGACGAG
1210 GGUGAAC CUGAUGAG X CGAA UCCCAGU U GUUCACC
ACUGGGA
1213 GAAGGUG CUGAUGAG X CGAA CAGUUGU U CACCUUC
25 ACAACUG
1214 AGAAGGU CUGAUGAG X CGAA AGUUGUU C ACCUUCU
AACAACU
1219 AGGCGAG CUGAUGAG X CGAA UUCACCU U CUCGCCU
AGGUGAA
1220 GAGGCGA CUGAUGAG X CGAA UCACCUU C UCGCCUC
AAGGUGA
1222 GCGAGGC CUGAUGAG X CGAA ACCUUCU C GCCUCGC
AGAAGGU
3O 1227 UACCGGC CUGAUGAG X CGAA CUCGCCU C GCCGGUA
AGGCGAG
1239 UGUCUCA CUGAUGAG X CGAA CGCCGGU A UGAGACA
ACCGGCG
1299 AGUCCUG CUGAUGAG X CGAA AGACAGU A CAGGACU
ACUGUCU
1257 AUUGAGC CUGAUGAG X CGAA CUGCAAU U GCUCAAU
AUUGCAG
1261 AUAGAUU CUGAUGAG X CGAA AAUUGCU C AAUCUAU
AGCAAUU
1265 CGGGAUA CUGAUGAG X CGAA GCUCAAU C UAUCCCG
AUUGAGC
CA 02326695 2000-10-26
WO 99/SS847 PCT/US99/09027
Pos. Ribozyme Substrate
1267 GCCGGGACUGAUGAGX CGAA AGAUUGAUCAAUCUA UCCCGGC
1269 UGGCCGGCUGAUGAGX CGAA AUAGAUUAAUCUAUC CCGGCCA
1299 AUAUCCCCUGAUGAGX CGAA AGCCAUGCAUGGCUU GGGAUAU
1305 AUCAUCACUGAUGAGX CGAA AUCCCAAUUGGGAUA UGAUGAU
1321 UGUAGGCCUGAUGAGX CGAA ACCAGUUAACUGGUC GCCUACA
1326 GCUGUUGCUGAUGAGX CGAA AGGCGACGUCGCCUA CAACAGC
1337 ACACCACCUGAUGAGX CGAA AGGGCUGCAGCCCUA GUGGUGU
10
1395 UAACUGCCUGAUGAGX CGAA ACACCACGUGGUGUC GCAGUUA
1351 CCGGAGUCUGAUGAGX CGAA ACUGCGAUCGCAGUU ACUCCGG
1352 UCCGGAGCUGAUGAGX CGAA AACUGCGCGCAGUUA CUCCGGA
1355 GGAUCCGCUGAUGAGX CGAA AGUAACUAGUUACUC CGGAUCC
IS 1361 CUUGUGGCUGAUGAGX CGAA AUCCGGAUCCGGAUC CCACAAG
1449 AAGACCUCUGAUGAGX CGAA AGCCCAGCUGGGCUA AGGUCUU
1454 CAAUCAA X CGAA ACCUUAGCUAAGGUC UUGAUUG
CUGAUGAG
1456 CACAAUCCUGAUGAGX CGAA AGACCUUAAGGUCUU GAUUGUG
2O 1460 ACAUCACCUGAUGAGX CGAA AUCAAGAUCUUGAUU GUGAUGU
1468 AAAGAGUCUGAUGAGX CGAA ACAUCACGUGAUGUU ACUCUUU
1969 CAAAGAGCUGAUGAGX CGAA AACAUCAUGAUGUUA CUCUUUG
1472 CGGCAAA X CGAA AGUAACAUGUUACUC UUUGCCG
CUGAUGAG
1474 GCCGGCACUGAUGAGX CGAA AGAGUAAUUACUCUU UGCCGGC
25
1475 CGCCGGCCUGAUGAGX CGAA AAGAGUAUACUCUUU GCCGGCG
1484 CCCCGUCCUGAUGAGX CGAA ACGCCGGCCGGCGUU GACGGGG
1493 UGUAAGUCUGAUGAGX CGAA ACCCCGUACGGGGUC ACUUACA
1997 GUCGUGUCUGAUGAGX CGAA AGUGACCGGUCACUU ACACGAC
3O 1498 UGUCGUGCUGAUGAGX CGAA AAGUGACGUCACUUA
CACGACA
1513 AGCUUGCCUGAUGAGX CGAA ACCCCCCGGGGGGUC GCAAGCU
1521 GUGUGGCCUGAUGAGX CGAA AGCUUGCGCAAGCUC GCCACAC
1538 AGGACGUCUGAUGAGX CGAA ACGCUCUAGAGCGUC ACGUCCU
1593 GAAGAAGCUGAUGAGX CGAA ACGUGACGUCACGUC CUUCUUC
1596 GGUGAAGCUGAUGAGX CGAA AGGACGUACGUCCUU CUUCACC
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
71
Pos. Ribozyme Substrate
1597 GGGUGAA CUGAUGAG X CGAA CGUCCUU C UUCACCC
AAGGACG
1549 UUGGGUG CUGAUGAG X CGAA UCCUUCU U CACCCAA
AGAAGGA
S 1550 CUUGGGU CUGAUGAG X CGAA CCUUCUU C ACCCAAG
AAGAAGG
1574 UGAGCUG CUGAUGAG X CGAA AGAGAAU C CAGCUCA
AUUCUCU
1580 UGUUUAU CUGAUGAG X CGAA UCCAGCU C AUAAACA
AGCUGGA
1583 UGGUGUU CUGAUGAG X CGAA AGCUCAU A AACACCA
AUGAGCU
1607 UCCUGUU CUGAUGAG X CGAA GGCACAU C AACAGGA
1~ AUGUGCC
1636 GUUGAGG CUGAUGAG X CGAA AAUGAAU C CCUCAAC
AUUCAUU
1640 CGGUGUU CUGAUGAG X CGAA AAUCCCU C AACACCG
AGGGAUU
1651 GGCAAAG CUGAUGAG X CGAA ACCGGGU U CUUUGCC
ACCCGGU
1652 CGGCAAA CUGAUGAG X CGAA CCGGGUU C UUUGCCG
AACCCGG
IS 1659 UGCGGCA CUGAUGAG X CGAA GGGUUCU U UGCCGCA
AGAACCC
1655 GUGCGGC CUGAUGAG X CGAA GGUUCUU U GCCGCAC
AAGAACC
1666 UGCGUAG CUGAUGAG X CGAA GCACUGU U CUACGCA
ACAGUGC
1667 GUGCGUA CUGAUGAG X CGAA CACUGUU C UACGCAC
AACAGUG
2O 1669 GUGUGCG CUGAUGAG X CGAA CUGUUCU A CGCACAC
AGAACAG
1681 CGAGUUG CUGAUGAG X CGAA CACAAGU U CAACUCG
ACUUGUG
1682 ACGAGUU CUGAUGAG X CGAA ACAAGUU C AACUCGU
AACUUGU
1687 UCCGGAC CUGAUGAG X CGAA UUCAACU C GUCCGGA
AGUUGAA
1690 GCAUCCG CUGAUGAG X CGAA AACUCGU C CGGAUGC
25 ACGAGUU
1723 GUCGAUG CUGAUGAG X CGAA UGCAGCU C CAUCGAC
AGCUGCA
1764 GGCUCGG CUGAUGAG X CGAA CACCUAU A CCGAGCC
AUAGGUG
1773 AGGUCCC CUGAUGAG X CGAA CGAGCCU A GGGACCU
AGGCUCG
1785 GGCCUCU CUGAUGAG X CGAA CCUGGAU C AGAGGCC
3O AUCCAGG
1794 CAGCAGU CUGAUGAG X CGAA GAGGCCU U ACUGCUG
AGGCCUC
1861 GAAACAG CUGAUGAG X CGAA CCAGUGU A CUGUUUC
ACACUGG
1866 GGGGUGA CUGAUGAG X CGAA GUACUGU U UCACCCC
ACAGUAC
1867 UGGGGUG CUGAUGAG X CGAA UACUGUU U CACCCCA
AACAGUA
1868 UUGGGGU CUGAUGAG X CGAA ACUGUUU C ACCCCAA
AAACAGU
1955 UGUUGAG CUGAUGAG X CGAA UGCUGCU U CUCAACA
AGCAGCA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
72
Pos. Ribozyme Substrate
1956 UUGUUGA CUGAUGAG X CGAA GCUGCUU C UCAACAA
AAGCAGC
1958 UGUUGUU CUGAUGAG X CGAA UGCUUCU C AACAACA
AGAAGCA
2020 CUUGGUG CUGAUGAG X CGAA ACUGGGU U CACCAAG
ACCCAGU
2021 UCUUGGU CUGAUGAG X CGAA CUGGGUU C ACCAAGA
AACCCAG
2094 CGAAAGC CUGAUGAG X CGAA CACGGAU U GCUUUCG
AUCCGUG
2098 CUUCCGA CUGAUGAG X CGAA GAUUGCU U UCGGAAG
AGCAAUC
2099 GCUUCCG CUGAUGAG X CGAA AUUGCUU U CGGAAGC
1~ AAGCAAU
2100 UGCUUCC CUGAUGAG X CGAA UUGCUUU C GGAAGCA
AAAGCAA
2157 AUACACC CUGAUGAG X CGAA AACACCU A GGUGUAU
AGGUGUU
2163 UCAACUA CUGAUGAG X CGAA UAGGUGU A UAGUUGA
ACACCUA
2165 AGUCAAC CUGAUGAG X CGAA GGUGUAU A GUUGACU
AUACACC
IS 2168 GGUAGUC CUGAUGAG X CGAA GUAUAGU U GACUACC
ACUAUAC
2173 GUAUGGG CUGAUGAG X CGAA GUUGACU A CCCAUAC
AGUCAAC
2179 GAGCCUG CUGAUGAG X CGAA UACCCAU A CAGGCUC
AUGGGUA
2186 AGUGCCA CUGAUGAG X CGAA ACAGGCU C UGGCACU
AGCCUGU
2O 2194 GCAGGGG CUGAUGAG X CGAA UGGCACU A CCCCUGC
AGUGCCA
2207 UAAAGUU CUGAUGAG X CGAA GCACUGU C AACUUUA
ACAGUGC
2212 GAUGGUA CUGAUGAG X CGAA GUCAACU U UACCAUC
AGUUGAC
2213 AGAUGGU CUGAUGAG X CGAA UCAACUU U ACCAUCU
AAGUUGA
2214 AAGAUGG CUGAUGAG X CGAA CAACUUU A CCAUCUU
25 AAAGUUG
2222 UAACCUU CUGAUGAG X CGAA CCAUCUU U AAGGUUA
AAGAUGG
2223 CUAACCU CUGAUGAG X CGAA CAUCUUU A AGGUUAG
AAAGAUG
2228 ACAUCCU CUGAUGAG X CGAA UUAAGGU U AGGAUGU
ACCUUAA
2229 UACAUCC CUGAUGAG X CGAA UAAGGUU A GGAUGUA
AACCUUA
3O 2236 CCCCACA CUGAUGAG X CGAA AGGAUGU A UGUGGGG
ACAUCCU
2283 UCUCCUC CUGAUGAG X CGAA CUGGACU C GAGGAGA
AGUCCAG
2366 AACAGGG CUGAUGAG X CGAA AGACACU U CCCUGUU
AGUGUCU
2367 GAACAGG CUGAUGAG X CGAA GACACUU C CCUGUUC
AAGUGUC
2373 GUGAAGG CUGAUGAG X CGAA UCCCUGU U CCUUCAC
ACAGGGA
2374 GGUGAAG CUGAUGAG X CGAA CCCUGUU C CUUCACC
AACAGGG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
73
Pos. Ribozyme Substrate
2377 GGUGGUG CUGAUGAG X CGAA UGUUCCU U CACCACC
AGGAACA
2378 GGGUGGU CUGAUGAG X CGAA GUUCCUU C ACCACCC
AAGGAAC
S 2387 GAGCCGG CUGAUGAG X CGAA CCACCCU A CCGGCUC
AGGGUGG
2394 GUGGACA CUGAUGAG X CGAA ACCGGCU C UGUCCAC
AGCCGGU
2398 ACCAGUG CUGAUGAG X CGAA GCUCUGU C CACUGGU
ACAGAGC
2406 UGGAUCA CUGAUGAG X CGAA CACUGGU U UGAUCCA
ACCAGUG
2907 GUGGAUC CUGAUGAG X CGAA ACUGGUU U GAUCCAC
IU AACCAGU
2411 GGAGGUG CUGAUGAG X CGAA GUUUGAU C CACCUCC
AUCAAAC
2493 GUACAGG CUGAUGAG X CGAA GUGCAGU A CCUGUAC
ACUGCAC
2449 UAUACCG CUGAUGAG X CGAA UACCUGU A CGGUAUA
ACAGGUA
2454 GACCCUA CUGAUGAG X CGAA GUACGGU A UAGGGUC
ACCGUAC
IS 2956 CUGACCC CUGAUGAG X CGAA ACGGUAU A GGGUCAG
AUACCGU
2461 AACCGCU CUGAUGAG X CGAA AUAGGGU C AGCGGUU
ACCCUAU
2468 AGGAGAC CUGAUGAG X CGAA CAGCGGU U GUCUCCU
ACCGCUG
2471 CAAAGGA CUGAUGAG X CGAA CGGUUGU C UCCUUUG
ACAACCG
2O 2473 CACAAAG CUGAUGAG X CGAA GUUGUCU C CUUUGUG
AGACAAC
2476 GAUCACA CUGAUGAG X CGAA GUCUCCU U UGUGAUC
AGGAGAC
2477 UGAUCAC CUGAUGAG X CGAA UCUCCUU U GUGAUCA
AAGGAGA
2483 CCCAUUU CUGAUGAG X CGAA UUGUGAU C AAAUGGG
AUCACAA
2499 CACGAUA CUGAUGAG X CGAA UGGGAGU A UAUCGUG
2S ACUCCCA
2496 AACACGA CUGAUGAG X CGAA GGAGUAU A UCGUGUU
AUACUCC
2498 GCAACAC CUGAUGAG X CGAA AGUAUAU C GUGUUGC
AUAUACU
2503 GAAAAGC CUGAUGAG X CGAA AUCGUGU U GCUUUUC
ACACGAU
2507 GAAGGAA CUGAUGAG X CGAA UGUUGCU U UUCCUUC
3O AGCAACA
2508 AGAAGGA CUGAUGAG X CGAA GUUGCUU U UCCUUCU
AAGCAAC
2509 GAGAAGG CUGAUGAG X CGAA UUGCUUU U CCUUCUC
AAAGCAA
2510 GGAGAAG CUGAUGAG X CGAA UGCUUUU C CUUCUCC
AAAAGCA
2513 CCAGGAG CUGAUGAG X CGAA UUUUCCU U CUCCUGG
AGGAAAA
2514 GCCAGGA CUGAUGAG X CGAA UUUCCUU C UCCUGGC
AAGGAAA
2516 CCGCCAG CUGAUGAG X CGAA UCCUUCU C CUGGCGG
AGAAGGA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/090Z7
74
Pos. Ribozyme Substrate
2545 CAUCCACCUGAUGAGXCGAA AGCAGGCGCCUGCUUGUGGAUG
2569 CCUGGGCCUGAUGAGXCGAA AUCAGCAUGCUGAUAGCCCAGG
S 2619 GGCCAGGCUGAUGAGXCGAA ACGCCGCGCGGCGUCCCUGGCC
2636 AGGAGAGCUGAUGAGXCGAA AUGCCAUAUGGCAUUCUCUCCU
2637 AAGGAGACUGAUGAGXCGAA AAUGCCAUGGCAUUCUCUCCUU
2639 GGAAGGACUGAUGAGXCGAA AGAAUGCGCAUUCUCUCCUUCC
2641 AAGGAAGCUGAUGAGXCGAA AGAGAAUAUUCUCUCCUUCCUU
1~
2694 CACAAGGCUGAUGAGXCGAA AGGAGAGCUCUCCUUCCUUGUG
2645 ACACAAGCUGAUGAGXCGAA AAGGAGAUCUCCUUCCUUGUGU
2648 AAAACACCUGAUGAGXCGAA AGGAAGGCCUUCCUUGUGUUUU
2653 ACAGAAA XCGAA ACACAAGCUUGUGUUUUUCUGU
CUGAUGAG
IS 2654 CACAGAA XCGAA AACACAAUUGUGUUUUUCUGUG
CUGAUGAG
2655 GCACAGACUGAUGAGXCGAA AAACACAUGUGUUUUUCUGUGC
2656 GGCACAGCUGAUGAGXCGAA AAAACACGUGUUUUUCUGUGCC
2657 CGGCACACUGAUGAGXCGAA AAAAACAUGUUUUUCUGUGCCG
2O 2732 GGAGCAGCUGAUGAGXCGAA AGCAGCGCGCUGCUCCUGCUCC
2749 UGGUGGUCUGAUGAGXCGAA ACGCCAGCUGGCGUUACCACCA
2750 GUGGUGGCUGAUGAGXCGAA AACGCCAUGGCGUUACCACCAC
2791 UCCACACCUGAUGAGXCGAA AUGCAGCGCUGCAUCGUGUGGA
2807 CUACAAA XCGAA ACCACCCGGGUGGUUUUUGUAG
25 CUGAUGAG
2808 CCUACAA XCGAA AACCACCGGUGGUUUUUGUAGG
CUGAUGAG
2809 ACCUACACUGAUGAGXCGAA AAACCACGUGGUUUUUGUAGGU
2810 GACCUACCUGAUGAGXCGAA AAAACCAUGGUUUUUGUAGGUC
2813 UUAGACCCUGAUGAGXCGAA ACAAAAAUUUUUGUAGGUCUAA
3O
2817 AGUAUUACUGAUGAGXCGAA ACCUACAUGUAGGUCUAAUACU
2819 AGAGUAUCUGAUGAGXCGAA AGACCUAUAGGUCUAAUACUCU
2822 UCAAGAGCUGAUGAGXCGAA AUUAGACGUCUAAUACUCUUGA
2825 AGGUCAA XCGAA AGUAUUAUAAUACUCUUGACCU
CUGAUGAG
2827 CAAGGUCCUGAUGAGXCGAA AGAGUAUAUACUCUUGACCUUG
2833 UGGUGACCUGAUGAGXCGAA AGGUCAAUUGACCUUGUCACCA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
Pos. Ribozyme Substrate
2836 GUGUGGU CUGAUGAG X CGAA ACCUUGU C ACCACAC
ACAAGGU
2845 CACUUUG CUGAUGAG X CGAA CCACACU A CAAAGUG
AGUGUGG
2854 GGCGAGG CUGAUGAG X CGAA AAAGUGU U CCUCGCC
ACACUUU
2855 UGGCGAG CUGAUGAG X CGAA AAGUGUU C CUCGCCA
AACACUU
2858 GCCUGGC CUGAUGAG X CGAA UGUUCCU C GCCAGGC
AGGAACA
2867 ACCAUAU CUGAUGAG X CGAA CCAGGCU C AUAUGGU
AGCCUGG
2870 ACCACCA CUGAUGAG X CGAA GGCUCAU A UGGUGGU
1~ AUGAGCC
2889 CUGGUGA CUGAUGAG X CGAA AUACUUU A UCACCAG
AAAGUAU
2891 CCCUGGU CUGAUGAG X CGAA ACUUUAU C ACCAGGG
AUAAAGU
2993 CAAAGAU CUGAUGAG X CGAA CAGAGCU A AUCUUUG
AGCUCUG
2996 UGUCAAA CUGAUGAG X CGAA AGCUAAU C UUUGACA
AUUAGCU
1S 2998 AAUGUCA CUGAUGAG X CGAA CUAAUCU U UGACAUU
AGAUUAG
2999 UAAUGUC CUGAUGAG X CGAA UAAUCUU U GACAUUA
AAGAUUA
3005 GUUUGGU CUGAUGAG X CGAA UUGACAU U ACCAAAC
AUGUCAA
3006 AGUUUGG CUGAUGAG X CGAA UGACAUU A CCAAACU
AAUGUCA
2O 3019 CGAGCAG CUGAUGAG X CGAA CCAAACU C CUGCUCG
AGUUUGG
3020 GAAUGGC CUGAUGAG X CGAA UCCUGCU C GCCAUUC
AGCAGGA
3026 GACCGAG CUGAUGAG X CGAA UCGCCAU U CUCGGUC
AUGGCGA
3027 GGACCGA CUGAUGAG X CGAA CGCCAUU C UCGGUCC
AAUGGCG
3029 GCGGACC CUGAUGAG X CGAA CCAUUCU C GGUCCGC
25 AGAAUGG
3033 AUGAGCG CUGAUGAG X CGAA UCUCGGU C CGCUCAU
ACCGAGA
3038 GCACCAU CUGAUGAG X CGAA GUCCGCU C AUGGUGC
AGCGGAC
3047 CAGCCUG CUGAUGAG X CGAA UGGUGCU C CAGGCUG
AGCACCA
3073 UACAAAG CUGAUGAG X CGAA AUGCCGU A CUUUGUA
ACGGCAU
3O 3076 GCGUACA CUGAUGAG X CGAA CCGUACU U UGUACGC
AGUACGG
3077 CGCGUAC CUGAUGAG X CGAA CGUACUU U GUACGCG
AAGUACG
3080 GAGCGCG CUGAUGAG X CGAA ACUUUGU A CGCGCUC
ACAAAGU
3087 AGCCCCU CUGAUGAG X CGAA ACGCGCU C AGGGGCU
AGCGCGU
3095 CACGAAU CUGAUGAG X CGAA AGGGGCU U AUUCGUG
AGCCCCU
3096 GCACGAA CUGAUGAG X CGAA GGGGCUU A UUCGUGC
AAGCCCC
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
76
Pos. Ribozyme Substrate
3098 AUGCACG CUGAUGAG X CGAA GGCUUAU U CGUGCAU
AUAAGCC
3099 CAUGCAC CUGAUGAG X CGAA GCUUAUU C GUGCAUG
AAUAAGC
S 3112 CCGCACC CUGAUGAG X CGAA UGCAUGU U GGUGCGG
ACAUGCA
3125 CUCCGGC CUGAUGAG X CGAA GGAAAGU A GCCGGAG
ACUUUCC
380 ACGUACG CUGAUGAG X CGAA GACAGGU A CGUACGU
ACCUGUC
3184 AUAGACG CUGAUGAG X CGAA GGUACGU A CGUCUAU
ACGUACC
3188 GGUCAUA CUGAUGAG X CGAA CGUACGU C UAUGACC
1~ ACGUACG
3190 AUGGUCA CUGAUGAG X CGAA UACGUCU A UGACCAU
AGACGUA
3198 GGGGUAA CUGAUGAG X CGAA UGACCAU C UUACCCC
AUGGUCA
3200 GCGGGGU CUGAUGAG X CGAA ACCAUCU U ACCCCGC
AGAUGGU
3201 AGCGGGG CUGAUGAG X CGAA CCAUCUU A CCCCGCU
AAGAUGG
IS 3259 CGGGCUC CUGAUGAG X CGAA UGGCAGU A GAGCCCG
ACUGCCA
3269 UGUCAGA CUGAUGAG X CGAA UCGUCUU C UCUGACA
AAGACGA
3271 CAUGUCA CUGAUGAG X CGAA GUCUUCU C UGACAUG
AGAAGAC
3374 GUCCCAG CUGAUGAG X CGAA AGAUACU U CUGGGAC
AGUAUCU
2O 3375 GGUCCCA CUGAUGAG X CGAA GAUACUU C UGGGACC
AAGUAUC
3390 UCAAUGC CUGAUGAG X CGAA GGCCGAU A GCAUUGA
AUCGGCC
3395 GCCCUUC CUGAUGAG X CGAA AUAGCAU U GAAGGGC
AUGCUAU
3436 UUGGGCG CUGAUGAG X CGAA ACGGCCU A CGCCCAA
AGGCCGU
3458 AACCAAG CUGAUGAG X CGAA GGGGCCU A CUUGGUU
25 AGGCCCC
3961 UGCAACC CUGAUGAG X CGAA GCCUACU U GGUUGCA
AGUAGGC
3465 ACAAUGC CUGAUGAG X CGAA ACUUGGU U GCAUUGU
ACCAAGU
3470 UAGUAAC CUGAUGAG X CGAA GUUGCAU U GUUACUA
AUGCAAC
3973 GGCUAGU CUGAUGAG X CGAA GCAUUGU U ACUAGCC
3O ACAAUGC
3474 AGGCUAG CUGAUGAG X CGAA CAUUGUU A CUAGCCU
AACAAUG
3477 GUGAGGC CUGAUGAG X CGAA UGUUACU A GCCUCAC
AGUAACA
3506 CCCCUUC CUGAUGAG X CGAA ACCAGGU C GAAGGGG
ACCUGGU
3549 CAGGAAA CUGAUGAG X CGAA ACACAAU C UUUCCUG
AUUGUGU
3546 GCCAGGA CUGAUGAG X CGAA ACAAUCU U UCCUGGC
AGAUUGU
3547 CGCCAGG CUGAUGAG X CGAA CAAUCUU U CCUGGCG
AAGAUUG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
77
Pos. Ribozyme Substrate
3548 UCGCCAG CUGAUGAGX CGAA AAAGAUUAAUCUUUC CUGGCGA
3563 CACCAUU CUGAUGAGX CGAA ACGCAGGCCUGCGUU
AAUGGUG
S 3564 ACACCAU CUGAUGAGX CGAA AACGCAGCUGCGUUA AUGGUGU
3589 CGUGGAA X CGAA ACGGUCCGGACCGUC UUCCACG
CUGAUGAG
3586 GCCGUGG CUGAUGAGX CGAA AGACGGUACCGUCUU CCACGGC
3587 CGCCGUG CUGAUGAGX CGAA AAGACGGCCGUCUUC CACGGCG
3632 UUUGGGU CUGAUGAGX CGAA AUUGGGCGCCCAAUC ACCCAAA
1~
3643 AUUAGUG CUGAUGAGX CGAA ACAUUUGCAAAUGUA CACUAAU
3648 UCUACAU CUGAUGAGX CGAA AGUGUACGUACACUA AUGUAGA
3653 CUUGGUC CUGAUGAGX CGAA ACAUUAGCUAAUGUA GACCAAG
3665 AGCCGAC CUGAUGAGX CGAA AGGUCUUAAGACCUC GUCGGCU
IS 3668 GCCAGCC CUGAUGAGX CGAA ACGAGGUACCUCGUC GGCUGGC
3720 UCCGAGC CUGAUGAGX CGAA ACCGCAGCUGCGGUA GCUCGGA
3758 CCGGAAU CUGAUGAGX CGAA ACGUCAGCUGACGUC AUUCCGG
3815 AAUAGGA CUGAUGAGX CGAA ACGGGUCGACCCGUC UCCUAUU
2O 3817 CAAAUAG CUGAUGAGX CGAA AGACGGGCCCGUCUC CUAUUUG
3820 CUUCAAA X CGAA AGGAGACGUCUCCUA UUUGAAG
CUGAUGAG
3822 CCCUUCA CUGAUGAGX CGAA AUAGGAGCUCCUAUU UGAAGGG
3823 GCCCUUC CUGAUGAGX CGAA AAUAGGAUCCUAUUU GAAGGGC
3832 ACCCGAA X CGAA AGCCCUUAAGGGCUC UUCGGGU
25 CUGAUGAG
3834 CCACCCG CUGAUGAGX CGAA AGAGCCCGGGCUCUU CGGGUGG
3925 GGGUAUG CUGAUGAGX CGAA AGUCCACGUGGACUU CAUACCC
3926 CGGGUAU CUGAUGAGX CGAA AAGUCCAUGGACUUC AUACCCG
3929 CAACGGG CUGAUGAGX CGAA AUGAAGUACUUCAUA CCCGUUG
3O
3935 UAGACUC CUGAUGAGX CGAA ACGGGUAUACCCGUU GAGUCUA
3990 UUCCAUA CUGAUGAGX CGAA ACUCAACGUUGAGUC UAUGGAA
3942 GUUUCCA CUGAUGAGX CGAA AGACUCAUGAGUCUA UGGAAAC
3951 CGCAUAG CUGAUGAGX CGAA AGUUUCCGGAAACUA CUAUGCG
3954 GACCGCA CUGAUGAGX CGAA AGUAGUUAACUACUA UGCGGUC
3961 GACCGGG CUGAUGAGX CGAA ACCGCAUAUGCGGUC CCCGGUC
CA 02326695 2000-10-26
WO 99/55847 PC'T/US99/090Z7
78
Pos. Ribozyme Substrate
3968 CCGUGAA X CGAA ACCGGGGCCCCGGUC UUCACGG
CUGAUGAG
3970 GUCCGUG CUGAUGAGX CGAA AGACCGGCCGGUCUU CACGGAC
S 3971 UGUCCGU CUGAUGAGX CGAA AAGACCGCGGUCUUC ACGGACA
3982 GGGAGAU CUGAUGAGX CGAA AGUUGUCGACAACUC AUCUCCC
3985 CGGGGGA CUGAUGAGX CGAA AUGAGUUAACUCAUC UCCCCCG
3987 GCCGGGG CUGAUGAGX CGAA AGAUGAGCUCAUCUC CCCCGGC
3998 UCUGCGG CUGAUGAGX CGAA ACGGCCGCGGCCGUA CCGCAGA
1~
4009 CACUUGG CUGAUGAGX CGAA AUGUCUGCAGACAUU CCAAGUG
4010 CCACUUG CUGAUGAGX CGAA AAUGUCUAGACAUUC CAAGUGG
4023 GCGUGUA CUGAUGAGX CGAA AUGGGCCGGCCCAUC UACACGC
4025 GAGCGUG CUGAUGAGX CGAA AGAUGGGCCCAUCUA CACGCUC
IS 4032 CCAGUGG CUGAUGAGX CGAA AGCGUGUACACGCUC CCACUGG
4099 GGACGAG CUGAUGAGX CGAA ACCUUGUACAAGGUA CUCGUCC
4097 UCAGGAC CUGAUGAGX CGAA AGUACCUAGGUACUC GUCCUGA
4100 GGUUCAG CUGAUGAGX CGAA ACGAGUAUACUCGUC CUGAACC
2O 4111 GGCAACA CUGAUGAGX CGAA AUGGGUUAACCCAUC UGUUGCC
4126 AAAACCC CUGAUGAGX CGAA AGGUGGCGCCACCUU GGGUUUU
4131 GCCCCAA X CGAA ACCCAAGCUUGGGUU UUGGGGC
CUGAUGAG
4132 CGCCCCA CUGAUGAGX CGAA AACCCAAUUGGGUUU UGGGGCG
4133 ACGCCCC CUGAUGAGX CGAA AAACCCAUGGGUUUU GGGGCGU
25
4141 AGACAUA CUGAUGAGX CGAA ACGCCCCGGGGCGUA UAUGUCU
4143 UUAGACA CUGAUGAGX CGAA AUACGCCGGCGUAUA UGUCUAA
4147 UGCCUUA CUGAUGAGX CGAA ACAUAUAUAUAUGUC UAAGGCA
4149 UGUGCCU CUGAUGAGX CGAA AGACAUAUAUGUCUA AGGCACA
3O 4161 GGGUCGG CUGAUGAGX CGAA ACCAUGUACAUGGUA CCGACCC
4196 CCGUGGU CUGAUGAGX CGAA AUGGUCCGGACCAUU ACCACGG
4197 CCCGUGG CUGAUGAGX CGAA AAUGGUCGACCAUUA CCACGGG
4214 AGUACGU CUGAUGAGX CGAA AUGGGGGCCCCCAUC ACGUACU
4219 GGUGGAG CUGAUGAGX CGAA ACGUGAUAUCACGUA CUCCACC
4222 AUAGGUG CUGAUGAGX CGAA AGUACGUACGUACUC CACCUAU
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
79
Pos. Ribozyme Substrate
4257 CCCCCAGCUGAUGAGX CGAA ACAUCCAUGGAUGUU CUGGGGG
4258 GCCCCCACUGAUGAGX CGAA AACAUCCGGAUGUUC UGGGGGC
S 4270 GAUAUCACUGAUGAGX CGAA AGGCGCCGGCGCCUA UGAUAUC
4275 AUUAUGACUGAUGAGX CGAA AUCAUAGCUAUGAUA UCAUAAU
4277 AUAUUAUCUGAUGAGX CGAA AUAUCAUAUGAUAUC AUAAUAU
4300 GUCAGUUCUGAUGAGX CGAA AGUGGCAUGCCACUC
AACUGAC
4309 GGUAGUCCUGAUGAGX CGAA AGUCAGUACUGACUC GACUACC
l~
9314 AGGAUGGCUGAUGAGX CGAA AGUCGAGCUCGACUA CCAUCCU
4319 UGCCCAGCUGAUGAGX CGAA AUGGUAGCUACCAUC CUGGGCA
9328 CUGUGCCCUGAUGAGX CGAA AUGCCCAUGGGCAUC GGCACAG
9389 GGAGGCGCUGAUGAGX CGAA AGCGGUGCACCGCUA CGCCUCC
IS 4395 GAUCCCGCUGAUGAGX CGAA AGGCGUAUACGCCUC CGGGAUC
9402 GGUAACCCUGAUGAGX CGAA AUCCCGGCCGGGAUC GGUUACC
9406 GCACGGUCUGAUGAGX CGAA ACCGAUCGAUCGGUU ACCGUGC
4407 GGCACGGCUGAUGAGX CGAA AACCGAUAUCGGUUA CCGUGCC
2O 4427 CCUCCUCCUGAUGAGX CGAA AUAUUUGCAAAUAUU GAGGAGG
4440 UUGGACACUGAUGAGX CGAA AGCCACCGGUGGCUC UGUCCAA
9465 GCCAUAGCUGAUGAGX CGAA AGGGGAUAUCCCCUU CUAUGGC
4966 UGCCAUACUGAUGAGX CGAA AAGGGGAUCCCCUUC UAUGGCA
4968 CUUGCCACUGAUGAGX CGAA AGAAGGGCCCUUCUA UGGCAAG
25
4512 AAAAUGACUGAUGAGX CGAA AUGCCUUAAGGCAUC UCAUUUU
4514 AGAAAAUCUGAUGAGX CGAA AGAUGCCGGCAUCUC AUUUUCU
9517 GGCAGAA X CGAA AUGAGAUAUCUCAUU UUCUGCC
CUGAUGAG
4518 UGGCAGACUGAUGAGX CGAA AAUGAGAUCUCAUUU UCUGCCA
3O
4519 GUGGCAGCUGAUGAGX CGAA AAAUGAGCUCAUUUU CUGCCAC
4520 AGUGGCACUGAUGAGX CGAA AAAAUGAUCAUUUUC UGCCACU
9550 UUGCGGCCUGAUGAGX CGAA AGCUCAUAUGAGCUC GCCGCAA
9569 GAGGCCUCUGAUGAGX CGAA ACAGCUUAAGCUGUC AGGCCUC
9571 UGAUUCCCUGAUGAGX CGAA AGGCCUGCAGGCCUC GGAAUCA
4602 ACGUCAA X CGAA ACCCCGGCCGGGGUC UUGACGU
CUGAUGAG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
Pos. Ribozyme Substrate
4604 ACACGUCCUGAUGAGX CGAA AGACCCCGGGGUCUU GACGUGU
4612 UAUGACGCUGAUGAGX CGAA ACACGUCGACGUGUC CGUCAUA
S 9637 CGAUAACCUGAUGAGX CGAA ACAUCUCGAGAUGUC GUUAUCG
4640 CCACGAUCUGAUGAGX CGAA ACGACAUAUGUCGUU AUCGUGG
9641 GCCACGACUGAUGAGX CGAA AACGACAUGUCGUUA UCGUGGC
4643 UUGCCACCUGAUGAGX CGAA AUAACGAUCGUUAUC GUGGCAA
4659 GUCRUUACUGAUGAGX CGAA AGCGUCUAGACGCUC UAAUGAC
10
4661 CCGUCAUCUGAUGAGX CGAA AGAGCGUACGCUCUA AUGACGG
4684 CGAGUCACUGAUGAGX CGAA AGUCACCGGUGACUU UGACUCG
4685 CCGAGUCCUGAUGAGX CGAA AAGUCACGUGACUUU GACUCGG
4690 GAUCACCCUGAUGAGX CGAA AGUCAAAUUUGACUC GGUGAUC
IS 4715 UCUGGGUCUGAUGAGX CGAA ACACAUGCAUGUGUC ACCCAGA
4727 UGAAAUCCUGAUGAGX CGAA ACUGUCUAGACAGUC GAUUUCA
4731 AAGCUGACUGAUGAGX CGAA AUCGACUAGUCGAUU UCAGCUU
4732 CAAGCUGCUGAUGAGX CGAA AAUCGACGUCGAUUU CAGCUUG
2O 4733 CCAAGCUCUGAUGAGX CGAA AAAUCGAUCGAUUUC AGCUUGG
9738 GGGAUCCCUGAUGAGX CGAA AGCUGAAUUCAGCUU GGAUCCC
4743 AAGGUGGCUGAUGAGX CGAA AUCCAAGCUUGGAUC CCACCUU
4750 AAUGGUACUGAUGAGX CGAA AGGUGGGCCCACCUU UACCAUU
9751 CAAUGGUCUGAUGAGX CGAA AAGGUGGCCACCUUU ACCAUUG
25
4752 UCAAUGGCUGAUGAGX CGAA AAAGGUGCACCUUUA CCAUUGA
4757 UCGUCUCCUGAUGAGX CGAA AUGGUAAUUACCAUU GAGACGA
4824 CCUCCCCCUGAUGAGX CGAA ACCCCUGCAGGGGUA GGGGAGG
4835 ACCUGUACUGAUGAGX CGAA AUGCCUCGAGGCAUC UACAGGU
3O
4837 AAACCUGCUGAUGAGX CGAA AGAUGCCGGCAUCUA CAGGUUU
4843 AGUCACACUGAUGAGX CGAA ACCUGUAUACAGGUU UGUGACU
4899 GAGUCACCUGAUGAGX CGAA AACCUGUACAGGUUU GUGACUC
4851 UCUCCCGCUGAUGAGX CGAA AGUCACAUGUGACUC CGGGAGA
4867 CAUGCCCCUGAUGAGX CGAA AGGGCCGCGGCCCUC GGGCAUG
4876 AGAAUCGCUGAUGAGX CGAA ACAUGCCGGCAUGUU CGAUUCU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
81
Pos. Ribozyme Substrate
4877 AAGAAUCCUGAUGAGXCGAA AACAUGCGCAUGUUC GAUUCUU
4881 ACCGAAGCUGAUGAGXCGAA AUCGAACGUUCGAUU CUUCGGU
S 4882 GACCGAA XCGAA AAUCGAAUUCGAUUC UUCGGUC
CUGAUGAG
9884 AGGACCGCUGAUGAGXCGAA AGAAUCGCGAUUCUU CGGUCCU
4885 CAGGACCCUGAUGAGXCGAA AAGAAUCGAUUCUUC GGUCCUG
4889 CACACAGCUGAUGAGXCGAA ACCGAAGCUUCGGUC CUGUGUG
4903 CGCGUCACUGAUGAGXCGAA AGCACUCGAGUGCUA UGACGCG
1~
5011 UUCCCAGCUGAUGAGXCGAA ACUCCAGCUGGAGUU CUGGGAA
5012 UUUCCCACUGAUGAGXCGAA AACUCCAUGGAGUUC UGGGAAA
5024 CUGUGAA XCGAA ACGCUUUAAAGCGUC UUCACAG
CUGAUGAG
5026 GCCUGUGCUGAUGAGXCGAA AGACGCUAGCGUCUU CACAGGC
IS 5027 GGCCUGUCUGAUGAGXCGAA AAGACGCGCGUCUUC ACAGGCC
5036 UGUGGGUCUGAUGAGXCGAA AGGCCUGCAGGCCUC ACCCACA
5045 GGGCAUCCUGAUGAGXCGAA AUGUGGGCCCACAUA GAUGCCC
5056 GGACAGGCUGAUGAGXCGAA AGUGGGCGCCCACUU CCUGUCC
2O 5057 GGGACAGCUGAUGAGXCGAA AAGUGGGCCCACUUC CUGUCCC
5062 GGUUUGGCUGAUGAGXCGAA ACAGGAAUUCCUGUC CCAAACC
5089 GUAAGGGCUGAUGAGXCGAA AGUUGUCGACAACUU CCCUUAC
5090 GGUAAGGCUGAUGAGXCGAA AAGUUGUACAACUUC CCUUACC
5094 ACCAGGUCUGAUGAGXCGAA AGGGAAGCUUCCCUU ACCUGGU
25
5095 UACCAGGCUGAUGAGXCGAA AAGGGAAUUCCCUUA CCUGGUA
5139 GGAGGUGCUGAUGAGXCGAA AGCCUGAUCAGGCUC CACCUCC
5145 CACGAUGCUGAUGAGXCGAA AGGUGGAUCCACCUC CAUCGUG
5199 AUCCCACCUGAUGAGXCGAA AUGGAGGCCUCCAUC GUGGGAU
3O
5157 CACAUUUCUGAUGAGXCGAA AUCCCACGUGGGAUC AAAUGUG
5172 CGUAUGACUGAUGAGXCGAA ACACUUCGAAGUGUC UCAUACG
5174 GCCGUAUCUGAUGAGXCGAA AGACACUAGUGUCUC AUACGGC
5177 UAAGCCGCUGAUGAGXCGAA AUGAGACGUCUCAUA CGGCUUA
5183 UAGGUUUCUGAUGAGXCGAA AGCCGUAUACGGCUU AAACCUA
5189 GUAGGUUCUGAUGAGXCGAA AAGCCGUACGGCUUA AACCUAC
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
82
Pos. Ribozyme Substrate
5190 UGCAGCG CUGAUGAG X CGAA UAAACCU A CGCUGCA
AGGUUUA
5225 CGGCUCC CUGAUGAG X CGAA AUAGGCU A GGAGCCG
AGCCUAU
S 5234 CAUUUUG CUGAUGAG X CGAA GAGCCGU U CAAAAUG
ACGGCUC
5235 UCAUUUU CUGAUGAG X CGAA AGCCGUU C AAAAUGA
AACGGCU
5246 UGAGGGU CUGAUGAG X CGAA AUGAGAU C ACCCUCA
AUCUCAU
5252 GAUGUGU CUGAUGAG X CGAA UCACCCU C ACACAUC
AGGGUGA
5259 GUUAUGG CUGAUGAG X CGAA CACACAU C CCAUAAC
1~ AUGUGUG
5264 AUUUGGU CUGAUGAG X CGAA AUCCCAU A ACCAAAU
AUGGGAU
5272 CAUGAUG CUGAUGAG X CGAA ACCAAAU U CAUCAUG
AUUUGGU
5273 CCAUGAU CUGAUGAG X CGAA CCAAAUU C AUCAUGG
AAUUUGG
5276 AUGCCAU CUGAUGAG X CGAA AAUUCAU C AUGGCAU
AUGAAUU
1S 5290 GUCGGCC CUGAUGAG X CGAA UGCAUGU C GGCCGAC
ACAUGCA
5349 GCGGCCA CUGAUGAG X CGAA UGCAGCU C UGGCCGC
AGCUGCA
5389 CCACAAU CUGAUGAG X CGAA GUGUGGU C AUUGUGG
ACCACAC
5387 UACCCAC CUGAUGAG X CGAA UGGUCAU U GUGGGUA
AUGACCA
2O 5394 AUGAUCC CUGAUGAG X CGAA UGUGGGU A GGAUCAU
ACCCACA
5402 CGGACAA CUGAUGAG X CGAA GGAUCAU U UUGUCCG
AUGAUCC
5403 CCGGACA CUGAUGAG X CGAA GAUCAUU U UGUCCGG
AAUGAUC
5909 CCCGGAC CUGAUGAG X CGAA AUCAUUU U GUCCGGG
AAAUGAU
5407 CCUCCCG CUGAUGAG X CGAA AUUUUGU C CGGGAGG
25 ACAAAAU
5441 GGUAGAG CUGAUGAG X CGAA GGGAAGU C CUCUACC
ACUUCCC
5449 CCCGGUA CUGAUGAG X CGAA AAGUCCU C UACCGGG
AGGACUU
5446 CUCCCGG CUGAUGAG X CGAA GUCCUCU A CCGGGAG
AGAGGAC
5455 UUCAUCG CUGAUGAG X CGAA CGGGAGU U CGAUGAA
3O ACUCCCG
5456 UUUCAUC CUGAUGAG X CGAA GGGAGUU C GAUGAAA
AACUCCC
5479 GAGGUGU CUGAUGAG X CGAA UGCGCCU C ACACCUC
AGGCGCA
5486 UGUAAGG CUGAUGAG X CGAA CACACCU C CCUUACA
AGGUGUG
5490 UCGAUGU CUGAUGAG X CGAA CCUCCCU U ACAUCGA
AGGGAGG
5991 UUCGAUG CUGAUGAG X CGAA CUCCCUU A CAUCGAA
AAGGGAG
5495 CCUGUUC CUGAUGAG X CGAA CUUACAU C GAACAGG
AUGUAAG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
83
Pos. R,ibozyme Substrate
5513 GCUCGGCCUGAUGAGXCGAA AGCUGCAUGCAGCUCGCCGAGC
5540 GCAACCCCUGAUGAGXCGAA AGUGCCUAGGCACUCGGGUUGC
5545 UUGCAGCCUGAUGAGXCGAA ACCCGAGCUCGGGUUGCUGCAA
5649 GCUGAUGCUGAUGAGXCGAA AGUUCCAUGGAACUUCAUCAGC
5645 CGCUGAUCUGAUGAGXCGAA AAGUUCCGGAACUUCAUCAGCG
5648 UCCCGCUCUGAUGAGXCGAA AUGAAGUACUUCAUCAGCGGGA
5657 AAUACUGCUGAUGAGXCGAA AUCCCGCGCGGGAUACAGUAUU
1~
5662 UGCUAAA XCGAA ACUGUAUAUACAGUAUUUAGCA
CUGAUGAG
5669 CCUGCUACUGAUGAGXCGAA AUACUGUACAGUAUUUAGCAGG
5665 GCCUGCUCUGAUGAGXCGAA AAUACUGCAGUAUUUAGCAGGC
5666 AGCCUGCCUGAUGAGXCGAA AAAUACUAGUAUUUAGCAGGCU
IS 5677 CAGAGUGCUGAUGAGXCGAA AUAAGCCGGCUUAUCCACUCUG
5682 CCAGGCACUGAUGAGXCGAA AGUGGAUAUCCACUCUGCCUGG
5702 GUGAUGCCUGAUGAGXCGAA AUCGCGGCCGCGAUAGCAUCAC
5707 CAUCAGUCUGAUGAGXCGAA AUGCUAUAUAGCAUCACUGAUG
2O 5719 GGCUGUGCUGAUGAGXCGAA AUGCCAUAUGGCAUUCACAGCC
5720 AGGCUGUCUGAUGAGXCGAA AAUGCCAUGGCAUUCACAGCCU
5728 GGUGAUACUGAUGAGXCGAA AGGCUGUACAGCCUCUAUCACC
5730 CUGGUGACUGAUGAGXCGAA AGAGGCUAGCCUCUAUCACCAG
5732 GACUGGUCUGAUGAGXCGAA AUAGAGGCCUCUAUCACCAGUC
25
5739 GUGAGCGCUGAUGAGXCGAA ACUGGUGCACCAGUCCGCUCAC
5744 GGGUGGUCUGAUGAGXCGAA AGCGGACGUCCGCUCACCACCC
5757 AGGAGGGCUGAUGAGXCGAA AUUCUGGCCAGAAUACCCUCCU
5762 UGAACAGCUGAUGAGXCGAA AGGGUAUAUACCCUCCUGUUCA
3O 5774 CCCCUAA XCGAA AUGUUGAUCAACAUCUUAGGGG
CUGAUGAG
5776 UCCCCCUCUGAUGAGXCGAA AGAUGUUAACAUCUUAGGGGGA
5777 AUCCCCCCUGAUGAGXCGAA AAGAUGUACAUCUUAGGGGGAU
5796 GCGAGUUCUGAUGAGXCGAA AGCAGCCGGCUGCUC
AACUCGC
5808 GCACUGGCUGAUGAGXCGAA AGGAGCGCGCUCCUCCCAGUGC
5820 AAGGCCGCUGAUGAGXCGAA AGCAGCAUGCUGCUUCGGCCUU
CA 02326695 2000-10-26
WO 99/55$47 PCT/(JS99/09027
84
Pos. Ribozyme Substrate
5885 UGUCCAC CUGAUGAGXCGAA AGCACCUAGGUGCU UGUGGACA
5899 CCGCCAG CUGAUGAGXCGAA AUGUCCAUGGACAU UCUGGCGG
S 5895 CCCGCCA CUGAUGAGXCGAA AAUGUCCGGACAUU CUGGCGGG
5986 AGGGAGC CUGAUGAGXCGAA AGUUAACGUUAACU UGCUCCCU
5999 GGGAGAG CUGAUGAGXCGAA AUGGCAGCUGCCAU CCUCUCCC
6002 CGGGGGA CUGAUGAGXCGAA AGGAUGGCCAUCCU CUCCCCCG
6101 CGAACGC CUGAUGAGXCGAA AUCAGCCGGCUGAU AGCGUUCG
6112 ACCCCGC CUGAUGAGXCGAA AAGCGAAUUCGCUU CGCGGGGU
6120 ACGUGGU CUGAUGAGXCGAA ACCCCGCGCGGGGU AACCACGU
6128 UGGGGGA CUGAUGAGXCGAA ACGUGGUACCACGU UUCCCCCA
6129 GUGGGGG CUGAUGAGXCGAA AACGUGGCCACGUU UCCCCCAC
IS 6130 CGUGGGG CUGAUGAGXCGAA AAACGUGCACGUUU CCCCCACG
6192 AGGCACG CUGAUGAGXCGAA AGUGCGUACGCACU ACGUGCCU
6173 UCUGAGU CUGAUGAGXCGAA ACACGUGCACGUGU AACUCAGA
6177 AGGAUCU CUGAUGAGXCGAA AGUUACAUGUAACU CAGAUCCU
2O 6182 UGGAGAG CUGAUGAGXCGAA AUCUGAGCUCAGAU CCUCUCCA
6185 GGCUGGA CUGAUGAGXCGAA AGGAUCUAGAUCCU CUCCAGCC
6187 GAGGCUG CUGAUGAGXCGAA AGAGGAUAUCCUCU CCAGCCUC
6194 UGAUGGU CUGAUGAGXCGAA AGGCUGGCCAGCCU CACCAUCA
6200 GCUGAGU CUGAUGAGXCGAA AUGGUGAUCACCAU CACUCAGC
25
6204 AGCAGCU CUGAUGAGXCGAA AGUGAUGCAUCACU CAGCUGCU
6221 ACUGGUG CUGAUGAGXCGAA AGCCUCUAGAGGCU UCACCAGU
6222 CACUGGU CUGAUGAGXCGAA AAGCCUCGAGGCUU CACCAGUG
6233 CCUCAUU CUGAUGAGXCGAA AUCCACUAGUGGAU U
AAUGAGG
6239 UCCUCAU CUGAUGAGXCGAA AAUCCACGUGGAUU AAUGAGGA
6247 UGGCGUG CUGAUGAGXCGAA AGCAGUCGACUGCU C
CACGCCA
6259 CGAGCCG CUGAUGAGXCGAA AGCAUGGCCAUGCU CCGGCUCG
6265 UAGCCAC CUGAUGAGXCGAA AGCCGGAUCCGGCU CGUGGCUA
6272 CAUCCUU CUGAUGAGXCGAA AGCCACGCGUGGCU A
AAGGAUG
6281 AGUCCCA CUGAUGAGXCGAA ACAUCCUAGGAUGU UUGGGACU
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
Pos. Ribozyme Substrate
6282 CAGUCCC CUGAUGAGX CGAA AACAUCCGGAUGUUU GGGACUG
6293 CCGUGCA CUGAUGAGX CGAA AUCCAGUACUGGAUA UGCACGG
S 6304 GUCAGUC CUGAUGAGX CGAA ACACCGUACGGUGUU GACUGAC
6313 GGUCUUG CUGAUGAGX CGAA AGUCAGUACUGACUU CAAGACC
6314 AGGUCUU CUGAUGAGX CGAA AAGUCAGCUGACUUC
AAGACCU
6326 UGGACUG CUGAUGAGX CGAA AGCCAGGCCUGGCUC CAGUCCA
6331 GAGCUUG CUGAUGAGX CGAA ACUGGAGCUCCAGUC CAAGCUC
10
6338 UCGGCAG CUGAUGAGX CGAA AGCUUGGCCAAGCUC CUGCCGA
6349 UCCCGGC CUGAUGAGX CGAA AUUUCGGCCGAAAUU GCCGGGA
6359 AGAAAGG CUGAUGAGX CGAA ACUCCCGCGGGAGUC CCUUUCU
6363 GAGAAGA CUGAUGAGX CGAA AGGGACUAGUCCCUU UCUUCUC
1S 6364 UGAGAAG CUGAUGAGX CGAA AAGGGACGUCCCUUU CUUCUCA
6365 AUGAGAA X CGAA AAAGGGAUCCCUUUC UUCUCAU
CUGAUGAG
6367 GCAUGAG CUGAUGAGX CGAA AGAAAGGCCUUUCUU CUCAUGC
6368 GGCAUGA CUGAUGAGX CGAA AAGAAAGCUUUCUUC UCAUGCC
2O 6370 UUGGCAU CUGAUGAGX CGAA AGAAGAAUUCUUCUC AUGCCAA
6385 UCCCUUG CUGAUGAGX CGAA ACCCGCGCGCGGGUA CAAGGGA
6395 CCCGCCA CUGAUGAGX CGAA ACUCCCUAGGGAGUC UGGCGGG
6446 GUCCGGU CUGAUGAGX CGAA AUUUGUGCACAAAUU ACCGGAC
6447 UGUCCGG CUGAUGAGX CGAA AAUUUGUACAAAUUA CCGGACA
25
6458 CGUUUUU CUGAUGAGX CGAA ACAUGUCGACAUGUC
AAAAACG
6468 CUCAUGG CUGAUGAGX CGAA ACCGUUUAAACGGUU CCAUGAG
6469 CCUCAUG CUGAUGAGX CGAA AACCGUUAACGGUUC CAUGAGG
6479 GCCCAAC CUGAUGAGX CGAA AUCCUCAUGAGGAUC GUUGGGC
3O
6482 UAGGCCC CUGAUGAGX CGAA ACGAUCCGGAUCGUU GGGCCUA
6489 CAGGUUU CUGAUGAGX CGAA AGGCCCAUGGGCCUA
AAACCUG
6520 GAUGGGG CUGAUGAGX CGAA ACGUUCCGGAACGUU CCCCAUC
6521 UGAUGGG CUGAUGAGX CGAA AACGUUCGAACGUUC CCCAUCA
6527 ACGCGUU CUGAUGAGX CGAA AUGGGGAUCCCCAUC
AACGCGU
6535 UGUGGUG CUGAUGAGX CGAA ACGCGUUAACGCGUA CACCACA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
86
Pos. Ribozyme Substrate
6559 CGCCGGGCUGAUGAGXCGAA AGGGUGUACACCCUCCCCGGCG
6610 CUCCACGCUGAUGAGXCGAA ACUCUUCGAAGAGUACGUGGAG
S 6620 CCCGCGUCUGAUGAGXCGAA AUCUCCAUGGAGAUUACGCGGG
6621 ACCCGCGCUGAUGAGXCGAA AAUCUCCGGAGAUUACGCGGGU
6654 GUGGUCACUGAUGAGXCGAA ACCCGUCGACGGGUAUGACCAC
6689 GGGCCGGCUGAUGAGXCGAA ACCUGGCGCCAGGUCCCGGCCC
6781 GACCUGGCUGAUGAGXCGAA AUGUGACGUCACAUUCCAGGUC
1~
6854 UGGAAGUCUGAUGAGXCGAA AGCACUGCAGUGCUCACUUCCA
6858 AGCAUGGCUGAUGAGXCGAA AGUGAGCGCUCACUUCCAUGCU
6859 GAGCAUGCUGAUGAGXCGAA AAGUGAGCUCACUUCCAUGCUC
6866 GGUCGGUCUGAUGAGXCGAA AGCAUGGCCAUGCUCACCGACC
1S 6877 AAUGUGGCUGAUGAGXCGAA AGGGGUCGACCCCUCCCACAUU
6884 CUGCUGUCUGAUGAGXCGAA AUGUGGGCCCACAUUACAGCAG
6885 UCUGCUGCUGAUGAGXCGAA AAUGUGGCCACAUUA
CAGCAGA
6900 CUACGUUCUGAUGAGXCGAA AGCCGUCGACGGCUA
AACGUAG
2O 6945 CUAGCUGCUGAUGAGXCGAA AGAGCUGCAGCUCUUCAGCUAG
6946 GCUAGCUCUGAUGAGXCGAA AAGAGCUAGCUCUUCAGCUAGC
6951 AAUUGGCCUGAUGAGXCGAA AGCUGAAUUCAGCUAGCCAAUU
6969 UUCAAGGCUGAUGAGXCGAA AGGCGCAUGCGCCUUCCUUGAA
6970 CUUCAAGCUGAUGAGXCGAA AAGGCGCGCGCCUUCCUUGAAG
25
6973 UGCCUUCCUGAUGAGXCGAA AGGAAGGCCUUCCUUGAAGGCA
6990 UGGUGGGCUGAUGAGXCGAA AGUGCAUAUGCACUACCCACCA
7003 GUCCGGGCUGAUGAGXCGAA AGUCAUGCAUGACUCCCCGGAC
7019 CCUCGAUCUGAUGAGXCGAA AGGUCAGCUGACCUCAUCGAGG
3O
7022 UGGCCUCCUGAUGAGXCGAA AUGAGGUACCUCAUCGAGGCCA
7064 CACGGGUCUGAUGAGXCGAA AUGUUUCGAAACAUCACCCGUG
7078 AUUCUCUCUGAUGAGXCGAA ACUCCACGUGGAGUCAGAGAAU
7086 ACCACCUCUGAUGAGXCGAA AUUCUCUAGAGAAUAAGGUGGU
7094 CCAAAAUCUGAUGAGXCGAA ACCACCUAGGUGGUAAUUUUGG
7097 AGUCCAA XCGAA AUUACCAUGGUAAUUUUGGACU
CUGAUGAG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
87
Pos. Ribozyme Substrate
7098 GAGUCCACUGAUGAGXCGAA AAUUACCGGUAAUUU UGGACUC
7099 AGAGUCCCUGAUGAGXCGAA AAAUUACGUAAUUUU GGACUCU
S 7105 GUCGAAA XCGAA AGUCCAAUUGGACUC UUUCGAC
CUGAUGAG
7107 GGGUCGACUGAUGAGXCGAA AGAGUCCGGACUCUU UCGACCC
7108 CGGGUCGCUGAUGAGXCGAA AAGAGUCGACUCUUU CGACCCG
7109 GCGGGUCCUGAUGAGXCGAA AAAGAGUACUCUUUC GACCCGC
7147 UGCAACGCUGAUGAGXCGAA AUACUUCGAAGUAUC CGUUGCA
1~
7151 CUGCUGCCUGAUGAGXCGAA ACGGAUAUAUCCGUU GCAGCAG
7163 UUCGCAGCUGAUGAGXCGAA AUCUCUGCAGAGAUC CUGCGAA
7174 CUUCUUGCUGAUGAGXCGAA AUUUUCGCGAAAAUC CAAGAAG
7183 GGGGGGGCUGAUGAGXCGAA ACUUCUUAAGAAGUU CCCCCCC
IS 7184 CGGGGGGCUGAUGAGXCGAA AACUUCUAGAAGUUC CCCCCCG
7227 AACAGUGCUGAUGAGXCGAA AGGGUUGCAACCCUC CACUGUU
7290 UUUCCAGCUGAUGAGXCGAA ACUCUAAUUAGAGUC CUGGAAA
7308 GGUAUUGCUGAUGAGXCGAA AGGGCCCGGGCCCUC CAAUACC
2O 7313 GAGGCGGCUGAUGAGXCGAA AUUGGAGCUCCAAUA CCGCCUC
7320 UUCCGUGCUGAUGAGXCGAA AGGCGGUACCGCCUC CACGGAA
7340 UCAGAACCUGAUGAGXCGAA ACCGUCCGGACGGUU GUUCUGA
7343 CUGUCAGCUGAUGAGXCGAA ACAACCGCGGUUGUU CUGACAG
7399 UCUGUCACUGAUGAGXCGAA AACAACCGGUUGUUC UGACAGA
25
7363 GGCAGAA XCGAA ACACGGUACCGUGUC UUCUGCC
CUGAUGAG
7365 AAGGCAGCUGAUGAGXCGAA AGACACGCGUGUCUU CUGCCUU
7366 CAAGGCACUGAUGAGXCGAA AAGACACGUGUCUUC UGCCUUG
7372 CUCCGCCCUGAUGAGXCGAA AGGCAGAUCUGCCUU GGCGGAG
3O
7405 CGAUCCGCUGAUGAGXCGAA AGCUGCCGGCAGCUC CGGAUCG
7446 UGAUCGGCUGAUGAGXCGAA AGGGGCGCGCCCCUC CCGAUCA
7452 GAGGUCUCUGAUGAGXCGAA AUCGGGAUCCCGAUC AGACCUC
7459 GUCGUCACUGAUGAGXCGAA AGGUCUGCAGACCUC UGACGAC
7480 AACGUCACUGAUGAGXCGAA AUUCUUUAAAGAAUC UGACGUU
7487 ACGACUCCUGAUGAGXCGAA ACGUCAGCUGACGUU GAGUCGU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
88
Pos. Ribozyme Substrate
7992 GGAGUACCUGAUGAGX CGAA ACUCAACGUUGAGUCGUACUCC
7495 GGAGGAGCUGAUGAGX CGAA ACGACUCGAGUCGUACUCCUCC
S 7609 CCAUGUGCUGAUGAGX CGAA AGGACAUAUGUCCUACACAUGG
7631 AUGGCGUCUGAUGAGX CGAA AUCAGGGCCCUGAUCACGCCAU
7675 GUUGCUCCUGAUGAGX CGAA ACGCGUUAACGCGUUGAGCAAC
7684 CAGCAGACUGAUGAGX CGAA AGUUGCUAGCAACUCUCUGCUG
7686 CGCAGCA X CGAA AGAGUUGCAACUCUCUGCUGCG
CUGAUGAG
7695 UUGUGGUCUGAUGAGX CGAA ACGCAGCGCUGCGUCACCACAA
7709 UGGCAUACUGAUGAGX CGAA ACCAUGUACAUGGUCUAUGCCA
7711 UGUGGCACUGAUGAGX CGAA AGACCAUAUGGUCUAUGCCACA
7759 CAAAGGUCUGAUGAGX CGAA ACCUUCUAGAAGGUCACCUUUG
IS 7759 UCUGUCACUGAUGAGX CGAA AGGUGACGUCACCUUUGACAGA
7760 GUCUGUCCUGAUGAGX CGAA AAGGUGAUCACCUUUGACAGAC
7802 UCUCCUUCUGAUGAGX CGAA AGCACGUACGUGCUC
AAGGAGA
7825 AACUGUGCUGAUGAGX CGAA ACGCCUUAAGGCGUCCACAGUU
2O 7832 UAGCCUUCUGAUGAGX CGAA ACUGUGGCCACAGUU
AAGGCUA
7833 UUAGCCUCUGAUGAGX CGAA AACUGUGCACAGUUAAGGCUAA
7844 CGGAUAGCUGAUGAGX CGAA AGUUUAGCUAAACUUCUAUCCG
7845 ACGGAUACUGAUGAGX CGAA AAGUUUAUAAACUUCUAUCCGU
7884 UUGGCCGCUGAUGAGX CGAA AUGUGGGCCCACAUUCGGCCAA
25
7885 UUUGGCCCUGAUGAGX CGAA AAUGUGGCCACAUUC
GGCCAAA
7922 GGUUCCGCUGAUGAGX CGAA ACGUCCUAGGACGUCCGGAACC
7931 UGCUGGACUGAUGAGX CGAA AGGUUCCGGAACCUAUCCAGCA
7933 CUUGCUGCUGAUGAGX CGAA AUAGGUUAACCUAUCCAGCAAG
3O
7996 UGUGGUUCUGAUGAGX CGAA AUGGCCUAGGCCAUU
AACCACA
7947 AUGUGGUCUGAUGAGX CGAA AAUGGCCGGCCAUUAACCACAU
8000 UGGUGUCCUGAUGAGX CGAA AUUGGUGCACCAAUUGACACCA
8012 UUGCCAUCUGAUGAGX CGAA AUGGUGGCCACCAUCAUGGCAA
8030 CGCAGAA X CGAA ACUUCACGUGAAGUUUUCUGCG
CUGAUGAG
8031 ACGCAGACUGAUGAGX CGAA AACUUCAUGAAGUUUUCUGCGU
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
89
Pos. Ribozyme Substrate
8032 GACGCAGCUGAUGAGX CGAA AAACUUCGAAGUUUUCUGCGUC
8033 GGACGCACUGAUGAGX CGAA AAAACUUAAGUUUUCUGCGUCC
S 8039 CCGGUUGCUGAUGAGX CGAA ACGCAGAUCUGCGUCCAACCGG
8070 AUAAGGCCUGAUGAGX CGAA AGCUGGCGCCAGCUCGCCUUAU
8081 CUGGGAA X CGAA ACGAUAAUUAUCGUAUUCCCAG
CUGAUGAG
8083 GUCUGGGCUGAUGAGX CGAA AUACGAUAUCGUAUUCCCAGAC
8089 GGUCUGGCUGAUGAGX CGAA AAUACGAUCGUAUUCCCAGACC
1~
8099 AUACACGCUGAUGAGX CGAA ACUCCCAUGGGAGUUCGUGUAU
8100 CAUACACCUGAUGAGX CGAA AACUCCCGGGAGUUCGUGUAUG
8105 UCUCGCACUGAUGAGX CGAA ACACGAAUUCGUGUAUGCGAGA
8121 UCGUAAA X CGAA AGCCAUUAAUGGCUCUUUACGA
CUGAUGAG
IS 8123 CGUCGUACUGAUGAGX CGAA AGAGCCAUGGCUCUU.UACGACG
8124 ACGUCGUCUGAUGAGX CGAA AAGAGCCGGCUCUUUACGACGU
8125 CACGUCGCUGAUGAGX CGAA AAAGAGCGCUCUUUACGACGUG
8135 GGGUGGACUGAUGAGX CGAA ACCACGUACGUGGUCUCCACCC
2O 8137 AAGGGUGCUGAUGAGX CGAA AGACCACGUGGUCUCCACCCUU
8144 CCUGAGGCUGAUGAGX CGAA AGGGUGGCCACCCUUCCUCAGG
8145 GCCUGAGCUGAUGAGX CGAA AAGGGUGCACCCUUCCUCAGGC
8148 ACGGCCUCUGAUGAGX CGAA AGGAAGGCCUUCCUCAGGCCGU
8164 GUACGAGCUGAUGAGX CGAA AGCCCAUAUGGGCUCCUCGUAC
25
8167 UCCGUAGCUGAUGAGX CGAA AGGAGCCGGCUCCUCGUACGGA
8177 AGUACUGCUGAUGAGX CGAA AAUCCGUACGGAUUCCAGUACU
8185 CCCAGGACUGAUGAGX CGAA AGUACUGCAGUACUCUCCUGGG
8291 AAGCCCACUGAUGAGX CGAA AGGGCUUAAGCCCUAUGGGCUU
3O
8248 AUACGAGCUGAUGAGX CGAA AGCCCAUAUGGGCUUCUCGUAU
8249 CAUACGACUGAUGAGX CGAA AAGCCCAUGGGCUUCUCGUAUG
8251 GUCAUACCUGAUGAGX CGAA AGAAGCCGGCUUCUCGUAUGAC
8259 GGUGUCACUGAUGAGX CGAA ACGAGAAUUCUCGUAUGACACC
8269 UGAGUCACUGAUGAGX CGAA AGCAGCGCGCUGCUUUGACUCA
8270 UUGAGUCCUGAUGAGX CGAA AAGCAGCGCUGCUUUGACUCAA
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
Pos. Ribozyme Substrate
8275 GACUGUU CUGAUGAGXCGAA AGUCAAAUUUGACUC
AACAGUC
8282 UCUCAGU CUGAUGAGXCGAA ACUGUUGCAACAGUCACUGAGA
8297 CAACACG CUGAUGAGXCGAA AUGUCGCGCGACAUCCGUGUUG
8303 ACUCCUC CUGAUGAGXCGAA ACACGGAUCCGUGUUGAGGAGU
8311 GUAGAUU CUGAUGAGXCGAA ACUCCUCGAGGAGUC
AAUCUAC
8315 AUUGGUA CUGAUGAGXCGAA AUUGACUAGUCAAUCUACCAAU
8317 ACAUUGG CUGAUGAGXCGAA AGAUUGAUCAAUCUACCAAUGU
10
8325 AAGUCAC CUGAUGAGXCGAA ACAUUGGCCAAUGUUGUGACUU
8332 GGGGGCC CUGAUGAGXCGAA AGUCACAUGUGACUUGGCCCCC
8400 UUUGAAU CUGAUGAGXCGAA AGUCAGGCCUGACUAAUUCAAA
8403 CCUUUUG CUGAUGAGXCGAA AUUAGUCGACUAAUUCAAAAGG
IS 8409 CCCUUUU CUGAUGAGXCGAA AAUUAGUACUAAUUC
AAAAGGG
8472 GUGAGGG CUGAUGAGXCGAA AUUGCCGCGGCAAUACCCUCAC
8977 AGCAUGU CUGAUGAGXCGAA AGGGUAUAUACCCUCACAUGCU
8485 UUUCAAG CUGAUGAGXCGAA AGCAUGUACAUGCUACUUGAAA
2O 8488 GGCUUUC CUGAUGAGXCGAA AGUAGCAUGCUACUUGAAAGCC
8565 UCACAGA CUGAUGAGXCGAA AACGACAUGUCGUUAUCUGUGA
8567 UUUCACA CUGAUGAGXCGAA AUAACGAUCGUUAUCUGUGAAA
8606 AGACUCG CUGAUGAGXCGAA AGGCUCGCGAGCCUACGAGUCU
8612 CCGUGAA XCGAA ACUCGUAUACGAGUCUUCACGG
25 CUGAUGAG
8619 CUCCGUG CUGAUGAGXCGAA AGACUCGCGAGUCUUCACGGAG
8615 CCUCCGU CUGAUGAGXCGAA AAGACUCGAGUCUUCACGGAGG
8625 CUAGUCA CUGAUGAGXCGAA AGCCUCCGGAGGCUAUGACUAG
8631 GAGUACC CUGAUGAGXCGAA AGUCAUAUAUGACUAGGUACUC
3O
8635 GGCAGAG CUGAUGAGXCGAA ACCUAGUACUAGGUACUCUGCC
8677 CAACUCC CUGAUGAGXCGAA AGUCGUAUACGACUUGGAGUUG
8683 UGUUAUC CUGAUGAGXCGAA ACUCCAAUUGGAGUUGAUAACA
8687 AUGAUGU CUGAUGAGXCGAA AUCAACUAGUUGAUAACAUCAU
8692 GGAGCAU CUGAUGAGXCGAA AUGUUAUAUAACAUCAUGCUCC
8710 CGCGACC CUGAUGAGXCGAA ACACGUUAACGUGUCGGUCGCG
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
91
Pos. Ribozyme Substrate
8714 CGUGCGC CUGAUGAG X CGAA ACCGACAUGUCGGU C GCGCACG
8743 GAGGUAG CUGAUGAG X CGAA ACACUCUAGAGUGU A CUACCUC
8746 AGUGAGG CUGAUGAG X CGAA AGUACACGUGUACU A CCUCACU
8750 CACGAGU CUGAUGAG X CGAA AGGUAGUACUACCU C ACUCGUG
8759 GGAUCAC CUGAUGAG X CGAA AGUGAGGCCUCACU C GUGAUCC
8760 GUGGUGG CUGAUGAG X CGAA AUCACGAUCGUGAU C CCACCAC
8799 GUGUGUC CUGAUGAG X CGAA AGCUGUCGACAGCU A GACACAC
1~
8808 UUGACUG CUGAUGAG X CGAA AGUGUGUACACACU C CAGUCAA
8813 AGGAGUU CUGAUGAG X CGAA ACUGGAGCUCCAGU C AACUCCU
8818 UAGCCAG CUGAUGAG X CGAA AGUUGACGUCAACU C CUGGCUA
8825 UGUUGCC CUGAUGAG X CGAA AGCCAGGCCUGGCU A GGCAACA
1S 8839 ACAUGAU CUGAUGAG X CGAA AUGUUGCGCAACAU C AUCAUGU
8837 CAUACAU CUGAUGAG X CGAA AUGAUGUACAUCAU C AUGUAUG
8870 UCAUCAA CUGAUGAG X CGAA AUCAUCCGGAUGAU U UUGAUGA
8872 AGUCAUC CUGAUGAG X CGAA AAAUCAUAUGAUUU U GAUGACU
2O 8884 GGAGAAG CUGAUGAG X CGAA AGUGAGUACUCACU U CUUCUCC
8885 UGGAGAA CUGAUGAG X CGAA AAGUGAGCUCACUU C UUCUCCA
8887 GAUGGAG CUGAUGAG X CGAA AGAAGUGCACUUCU U CUCCAUC
8888 GGAUGGA CUGAUGAG X CGAA AAGAAGUACUUCUU C UCCAUCC
8890 AAGGAUG CUGAUGAG X CGAA AGAAGAAUUCUUCU C CAUCCUU
25
8894 CUAGAAG CUGAUGAG X CGAA AUGGAGAUCUCCAU C CUUCUAG
8897 GGGCUAG CUGAUGAG X CGAA AGGAUGGCCAUCCU U CUAGCCC
8898 UGGGCUA CUGAUGAG X CGAA AAGGAUGCAUCCUU C UAGCCCA
8900 CCUGGGC CUGAUGAG X CGAA AGAAGGAUCCUUCU A GCCCAGG
3O 8915 CCUUUUC CUGAUGAG X CGAA AGCUGUUAACAGCU U GAAAAGG
8952 AUGGAGU CUGAUGAG X CGAA ACAGGCCGGCCUGU U ACUCCAU
8953 AAUGGAG CUGAUGAG X CGAA AACAGGCGCCUGUU A CUCCAUU
8956 CUCAAUG CUGAUGAG X CGAA AGUAACAUGUUACU C CAUUGAG
8960 GUGGCUC CUGAUGAG X CGAA AUGGAGUACUCCAU U GAGCCAC
8969 GUAGGUC CUGAUGAG X CGAA AGUGGCUAGCCACU U GACCUAC
CA 02326695 2000-10-26
WO 99/55847 PC'f/US99/09027
92
Pos. Ribozyme Substrate
8975 UCUGAGGCUGAUGAGX CGAA AGGUCAAUUGACCUA CCUCAGA
8979 AUGAUCUCUGAUGAGX CGAA AGGUAGGCCUACCUC AGAUCAU
S 8984 GUUGAAUCUGAUGAGX CGAA AUCUGAGCUCAGAUC AUUCAAC
8987 GUCGUUGCUGAUGAGX CGAA AUGAUCUAGAUCAUU CAACGAC
8988 AGUGGUUCUGAUGAGX CGAA AAUGAUCGAUCAUUC
AACGACU
8996 GACCAUGCUGAUGAGX CGAA AGUCGUUAACGACUC CAUGGUC
9003 GCGCUAA X CGAA ACCAUGGCCAUGGUC UUAGCGC
I~ CUGAUGAG
9005 AUGCGCUCUGAUGAGX CGAA AGACCAUAUGGUCUU AGCGCAU
9006 AAUGCGCCUGAUGAGX CGAA AAGACCAUGGUCUUA GCGCAUU
9013 GAGUGAGCUGAUGAGX CGAA AUGCGCUAGCGCAUU CUCACUC
9014 GGAGUGACUGAUGAGX CGAA AAUGCGCGCGCAUUC UCACUCC
IS 9016 AUGGAGUCUGAUGAGX CGAA AGAAUGCGCAUUCUC ACUCCAU
9020 AACUAUGCUGAUGAGX CGAA AGUGAGAUCUCACUC CAUAGUU
9024 GAGUAACCUGAUGAGX CGAA AUGGAGUACUCCAUA GUUACUC
9027 GGAGAGUCUGAUGAGX CGAA ACUAUGGCCAUAGUU ACUCUCC
2O 9028 UGGAGAGCUGAUGAGX CGAA AACUAUGCAUAGUUA CUCUCCA
9031 ACCUGGACUGAUGAGX CGAA AGUAACUAGUUACUC UCCAGGU
9033 UCACCUGCUGAUGAGX CGAA AGAGUAAUUACUCUC CAGGUGA
9044 CCCUAUUCUGAUGAGX CGAA AUCUCACGUGAGAUC
AAUAGGG
9048 GCCACCCCUGAUGAGX CGAA AUUGAUCGAUCAAUA GGGUGGC
2S
9057 AGGCAUGCUGAUGAGX CGAA AGCCACCGGUGGCUU CAUGCCU
9058 GAGGCAUCUGAUGAGX CGAA AAGCCACGUGGCUUC AUGCCUC
9105 CUGGCCCCUGAUGAGX CGAA AUGUCUCGAGACAUC GGGCCAG
9169 GAAGAGGCUGAUGAGX CGAA ACUUGCCGGCAAGUA CCUCUUC
3O
9173 AGUUGAA X CGAA AGGUACUAGUACCUC UUCAACU
CUGAUGAG
9175 CCAGUUGCUGAUGAGX CGAA AGAGGUAUACCUCUU CAACUGG
9176 CCCAGUUCUGAUGAGX CGAA AAGAGGUACCUCUUC
AACUGGG
9188 UGGUCCUCUGAUGAGX CGAA ACUGCCCGGGCAGUA AGGACCA
9200 UGAGUUUCUGAUGAGX CGAA AGCUUGGCCAAGCUC
AAACUCA
9206 UUGGAGUCUGAUGAGX CGAA AGUUUGAUCAAACUC ACUCCAA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
93
Pos. Ribozyme Substrate
9210 GGGAUUG CUGAUGAGX CGAA AGUGAGUACUCACUC CAAUCCC
9215 CGGCCGG CUGAUGAGX CGAA AUUGGAGCUCCAAUC CCGGCCG
9261 CCGCUGU CUGAUGAGX CGAA ACCAGCAUGCUGGUU ACAGCGG
9262 CCCGCUG CUGAUGAGX CGAA AACCAGCGCUGGUUA CAGCGGG
9299 CGGGCAC CUGAUGAGX CGAA AGACAGGCCUGUCUC GUGCCCG
9313 CCACAUA CUGAUGAGX CGAA ACCAGCGCGCUGGUU UAUGUGG
9314 ACCACAU CUGAUGAGX CGAA AACCAGCGCUGGUUU AUGUGGU
9315 CACCACA CUGAUGAGX CGAA AAACCAGCUGGUUUA UGUGGUG
9409 AAAAGGG CUGAUGAGX CGAA AUGGCCUAGGCCAUC CCCUUUU
9414 AAAAAAA X CGAA AGGGGAUAUCCCCUU UUUUUUU
CUGAUGAG
Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992
Nucleic Acids Res. 20:
3252). The length of stem II may be 2 base-pairs.
25
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
94
Table VII: HCV Hairpin (I~) Ribozyme and Target Sequence
Pos. Ribozyme Substrate
Sequence
CCCCCA AGAA ACCAGAGAAACAX CCCCCGAUUGGGGG
GGGG GUACAUUACCUGGUA
59 CGUGAA ACCAGAGAAACAX CUACUGUCUUCACG
AGAA GUACAUUACCUGGUA
GUAG
109 CCUGGA AGAA ACCAGAGAAACAX GUGCAGCCUCCAGG
GCAC GUACAUUACCUGGUA
209 GCAUUG AGAA ACCAGAGAAACAX AACCCGCUCAAUGC
1~ GGUU GUACAUUACCUGGUA
290 CUAUCA AGAR ACCAGAGAAACAX GUACUGCCUGAUAG
GUAC GUACAUUACCUGGUA
390 GUGGGC AGAR ACCAGAGAAACAX CUACCGCCGCCCAC
GUAG GUACAUUACCUGGUA
393 CCUGUG AGAA ACCAGAGAAACAX CCGCCGCCCACAGG
GCGG GUACAUUACCUGGUA
427 CCAACG AGAR ACCAGAGAAACAX GGUCAGAUCGUUGG
GACC GUACAUUACCUGGUA
505 GGUUGC AGAA ACCAGAGAAACAX GAACGGUCGCAACC
GUUC GUACAUUACCUGGUA
599 CCUCGG AGAA ACCAGAGAAACAX UCGCCGACCCGAGG
GCGA GUACAUUACCUGGUA
574 UACCCA AGAA ACCAGAGAAACAX GCUCAGCCUGGGUA
GAGC GUACAUUACCUGGUA
645 GCCGGG AGAA ACCAGAGAAACAX CCGCGGCUCCCGGC
GCGG GUACAUUACCUGGUA
2O 652 CAACUA AGAA ACCAGAGAAACAX UCCCGGCCUAGUUG
GGGA GUACAUUACCUGGUA
671 CCGGGG AGAA ACCAGAGAAACAX CCACGGACCCCCGG
GUGG GUACAUUACCUGGUA
726 CGGCGA AGAA ACCAGAGAAACAX GUGCGGCUUCGCCG
GCAC GUACAUUACCUGGUA
734 CAUGAG AGAR ACCAGAGAAACAX UCGCCGACCUCAUG
GCGA GUACAUUACCUGGUA
754 CCGACG AGAA ACCAGAGAAACA GUACAUUACCUGGUAAUUCCGCUCGUCGG
GAAU X
852 AAGAGC AGAR ACCAGAGAAACAX GCCCGGUUGCUCUU
GGGC GUACAUUACCUGGUA
883 CAGGAC AGAR ACCAGAGAAACAX GCCCUGCUGUCCUG
GGGC GUACAUUACCUGGUA
886 AAACAG AGAA ACCAGAGAAACAX CUGCUGUCCUGUUU
GCAG GUACAUUACCUGGUA
891 UGGUCA AGAA ACCAGAGAAACAX GUCCUGUUUGACCA
3O GGAC GUACAUUACCUGGUA
905 AGCGGA AGAA ACCAGAGAAACAX UCCCAGCUUCCGCU
GGGA GUACAUUACCUGGUA
911 CUGAUA AGAA ACCAGAGAAACAX CUUCCGCUUAUCAG
GAAG GUACAUUACCUGGUA
960 AGUUGG AGAR ACCAGAGAAACAX UGACUGCUCCAACU
GUCA GUACAUUACCUGGUA
1050CCCAAC AGAA ACCAGAGAAACAX CUCCCGUUGUUGGG
GGAG GUACAUUACCUGGUA
1145GAAAGC AGAR ACCAGAGAAACAX GGGCGGCCGCUUUC
GCCC GUACAUUACCUGGUA
1198ACAGAA ACCAGAGAAACAX CGGCCGCUUUCUGU
AGAA GUACAUUACCUGGUA
GCCG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
Pos. Ribozyme Substrate
Sequence
1155UGGCGG AGAA X GUACAUUACCUGGUAUUUCUGUUCCGCCA
GAAA
ACCAGAGAAACA
1185AAACGG AGAA ACCAGAGAAACAX GUACAUUACCUGGUACUGCGGAUCCGUUU
GCAG
1190GAGGAA ACCAGAGAAACAX GUACAUUACCUGGUAGAUCCGUUUUCCUC
5 AGAA
GAUC
1207GUGAAC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUCCCAGUUGUUCAC
GGGA
1331CACUAG AGAR ACCAGAGAAACAX GUACAUUACCUGGUACAACAGCCCUAGUG
GUUG
1357UGUGGG AGAA ACCAGAGAAACAX GUACAUUACCUGGUACUCCGGAUCCCACA
GGAG
1370AUCCAC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAAAGCUGUCGUGGAU
1~ GCUU
1562UCUCUG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGGCCGGCCCAGAGA
GGCC
1576UUUAUG AGAR ACCAGAGAAACAX GUACAUUACCUGGUAAUCCAGCUCAUAAA
GGAU
1596UGUGCC AGAR ACCAGAGAAACA GUACAUUACCUGGUAUGGCAGCUGGCACA
GCCA X
1616GUUCAG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGGACUGCCCUGAAC
GUCC
IS 1663GCGUAG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGCACUGUUCUACGC
GUGC
1692CUGGGC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGUCCGGAUGCCCAG
GGAC
1713AGCUGC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGGCCAGCUGCAGCU
GGCC
1719CGAUGG AGAR ACCAGAGAAACAX GUACAUUACCUGGUACUGCAGCUCCAUCG
GCAG
2O 1797AAUGCC AGAA GUACAUUACCUGGUAUUACUGCUGGCAUU
GUAA
ACCAGAGAAACA
X
1863GGGUGA AGAR ACCAGAGAAACAX GUACAUUACCUGGUAGUACUGUUUCACCC
GUAC
1880CACUAC AGAR ACCAGAGAAACAX GUACAUUACCUGGUAGCCCUGUUGUAGUG
GGGC
1898GGACCG AGAR ACCAGAGAAACAX GUACAUUACCUGGUACGACCGAUCGGUCC
GUCG
1903GCACCG AGAA ACCAGAGAAACA GUACAUUACCUGGUAGAUCGGUCCGGUGC
25 GAUC X
1993CAGCAC AGAR ACCAGAGAAACAX GUACAUUACCUGGUAAGACAGAUGUGCUG
GUCU
1951UUGAGA AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGUGCUGCUUCUCAA
GCAC
1969UGUGGC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAACGCGGCCGCCACA
GCGU
2082CCGUGG AGAR ACCAGAGAAACAX GUACAUUACCUGGUAGACCUGCCCCACGG
3O GGUC
2090AAAGCA AGAA ACCAGAGAAACAX GUACAUUACCUGGUACCACGGAUUGCUUU
GUGG
2316GCUCCG AGAR ACCAGAGAAACA GUACAUUACCUGGUAGGACAGAUCGGAGC
GUCC X
2328GCAGCG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGCUCAGCCCGCUGC
GAGC
2332AGCAGC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAAGCCCGCUGCUGCU
GGCU
2335GACAGC AGAR ACCAGAGAAACAX GUACAUUACCUGGUACCGCUGCUGCUGUC
GCGG
2338GUGGAC AGAA ACCAGAGAAACAX GUACAUUACCUGGUACUGCUGCUGUCCAC
GCAG
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
96
Pos. Ribozyme Substrate
Sequence
2341GUCGUG AGAA ACCAGAGAAACAX GUACAUUACCUGGUACUGCUGUCCACGAC
GCAG
2370UGAAGG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUCCCUGUUCCUUCA
GGGA
2390GGACAG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUACCGGCUCUGUCC
S GGUA
2395CCAGUG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGCUCUGUCCACUGG
GAGC
2465GGAGAC AGAA ACCAGAGAAACAX GUACAUUACCUGGUACAGCGGUUGUCUCC
GCUG
2522GCGCGC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUGGC GCGCGC
GCCA GGAC
2541UCCACA AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUGCCUGCUUGUGGA
GGCA
2557GCUAUC AGAR ACCAGAGAAACA GUACAUUACCUGGUAAUGCUGCUGAUAGC
GCAU X
2579CUCUAG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAAGGCCGCCCUAGAG
GCCU
2627AAUGCC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGAGCGGAUGGCAUU
GCUC
2663GUACCA AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGUGCCGCCUGGUAC
GCAC
IS 2725AGGAGC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUGGCCGCUGCUCCU
GCCA
2728AGCAGG AGAA ACCAGAGAAACAX GUACAUUACCUGGUACCGCUGCUCCUGCU
GCGG
2734AGCAGG AGAR ACCAGAGAAACAX GUACAUUACCUGGUACUCCUGCUCCUGCU
GGAG
2740AACGCC AGAA ACCAGAGAAACAX GUACAUUACCUGGUACUCCUGCUGGCGUU
GGAG
2O 2978UGGGUG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGUGCGGCCCACCCA
GCAC
3016AUGGCG AGAR ACCAGAGAAACAX GUACAUUACCUGGUACUCCUGCUCGCCAU
GGAG
3030UGAGCG AGAR GUACAUUACCUGGUAUCUCGGUCCGCUCA
GAGA
ACCAGAGAAACA
X
3034ACCAUG AGAA ACCAGAGAAACA GUACAUUACCUGGUAGGUCCGCUCAUGGU
GACC X
3260GAAGAC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAAGCCCGUCGUCUUC
2S GGCU
3340GAGACG AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGGACUGCCCGUCUC
GUCC
3394GGCGGA AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUGCCCGUCUCCGCC
GGCA
3350CCUUCG AGAR ACCAGAGAAACAX GUACAUUACCUGGUAUCUCCGCCCGAAGG
GAGA
3383GCUAUC AGAA ACCAGAGAAACAX GUACAUUACCUGGUAGACCGGCCGAUAGC
3O GGUC
3431GGCGUA AGAA ACCAGAGAAACAX GUACAUUACCUGGUAUCACGGCCUACGCC
GUGA
3581GUGGAA ACCAGAGAAACAX GUACAUUACCUGGUAGGACCGUCUUCCAC
AGAA
GUCC
3597UCUUUG AGAA ACCAGAGAAACAX GUACAUUACCUGGUACGCCGGCUCAAAGA
GGCG
3615CUUUUG AGAA ACCAGAGAAACA GUACAUUACCUGGUAAGCCGGCCCAAAAG
GGCU X
3669CAUGCC AGAA ACCAGAGAAACAX GUACAUUACCUGGUACGUCGGCUGGCAUG
GACG
3725AUAGAG AGAR ACCAGAGAAACAX GUACAUUACCUGGUAGCUCGGACCUCUAU
GAGC
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
97
Pos. Ribozyme Substrate
Sequence
3752 AAUGACAGAA ACCAGAGAAACAX GUACAUUACCUGGUAAUGCUGACGUCAUU
GCAU
3771 CACCGCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGCGCCGACGCGGUG
GCGC
3783 UCCCCCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUGACGGUCGGGGGA
GUCA
3799 CUGGGGAGAR ACCAGAGAAACAX GUACAUUACCUGGUACUACUGUCCCCCAG
GUAG
3807 AGACGGAGAA ACCAGAGAAACAX GUACAUUACCUGGUACCCCAGACCCGUCU
GGGG
3812 AUAGGAAGAR ACCAGAGAAACAX GUACAUUACCUGGUAGACCCGUCUCCUAU
GGUC
3847 GGGCAGAGAA X GUACAUUACCUGGUACCACUGCUCUGCCC
IO GUGG
ACCAGAGAAACA
3852 CCGAAGAGAR ACCAGAGAAACAX GUACAUUACCUGGUAGCUCUGCCCUUCGG
GAGC
3887 GCACACAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGGGCUGCUGUGUGC
GCCC
3932 AGACUCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUACCCGUUGAGUCU
GGUA
3958 ACCGGG ACCAGAGAAACAX GUACAUUACCUGGUAAUGCGGUCCCCGGU
AGAA
GCAU
IS 3965 CGUGAA ACCAGAGAAACAX GUACAUUACCUGGUACCCCGGUCUUCACG
AGAA
GGGG
3992 CGGUACAGAA ACCAGAGAAACAX GUACAUUACCUGGUACCCCGGCCGUACCG
GGGG
9069 GUACGCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAUGCCGGCUGCGUAC
GGCA
4076 CCCUUGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAACGCAGCCCAAGGG
GCGU
2O 9112 GGCGGCAGAA ACCAGAGAAACAX GUACAUUACCUGGUACAUCUGUUGCCGCC
GAUG
4163 GUUGGGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGUACCGACCCCAAC
GUAC
4244 UCCACCAGAA X GUACAUUACCUGGUAUUGCCGACGGUGGA
GCAA
ACCAGAGAAACA
4304 AGUCGAAGAR ACCAGAGAAACAX GUACAUUACCUGGUACAACUGACUCGACU
GUUG
4334 GUCCAGAGAR ACCAGAGAAACAX GUACAUUACCUGGUAGCACAGUCCUGGAC
25 GUGC
4355 CGCUCCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAAGACGGCUGGAGCG
GUCU
9366 ACGACGAGAR ACCAGAGAAACAX GUACAUUACCUGGUAGCGCGGCUCGUCGU
GCGC
4491 GUGUUGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGCUCUGUCCAACAC
GAGC
4621 CCGCUAAGAR ACCAGAGAAACAX GUACAUUACCUGGUAAUACCGACUAGCGG
GURU
3O 4652 UAGAGCAGAA ACCAGAGAAACA GUACAUUACCUGGUACAACAGACGCUCUA
GUUG X
4?24 GAAAUCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAAGACAGUCGAUUUC
GUCU
9734 GAUCCAAGAA X GUACAUUACCUGGUAUUUCAGCUUGGAUC
GAAA
ACCAGAGAAACA
9861 CCCGAGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGAACGGCCCUCGGG
GUUC
4886 ACACAGAGAR ACCAGAGAAACAX GUACAUUACCUGGUACUUCGGUCCUGUGU
GAAG
4937 AGUCUCAGAR ACCAGAGAAACAX GUACAUUACCUGGUACGCCCGCUGAGACU
GGCG
CA 02326695 2000-10-26
WO 99/55847 PCTNS99/09027
98
Pos. Ribozyme Substrate
Sequence
9988 CUGGCAAGAR ACCAGAGAAACAXGUACAUUACCUGGUAUGCC CGUCUGCCAG
GGCA
5059 GUUUGGAGAA XGUACAUUACCUGGUAUUCC UGUCCCAAAC
GGAA
ACCAGAGAAACA
5179 GGUUUAAGAR ACCAGAGAAACAXGUACAUUACCUGGUAAUAC GGCUUAAACC
GUAU
5212 CUAUACAGAR ACCAGAGAAACAXGUACAUUACCUGGUACCCC UGCUGUAUAG
GGGG
5231 AUUUUGAGAR ACCAGAGAAACAXGUACAUUACCUGGUAGAGC CGUUCAAAAU
GCUC
5291 CAGGUCAGAA ACCAGAGAAACAXGUACAUUACCUGGUAUGUC GGCCGACCUG
GACA
5299 CUCCAGAGAA ACCAGAGAAACAXGUACAUUACCUGGUACGGC CGACCUGGAG
1~ GCCG
5345 GGCCAGAGAA XGUACAUUACCUGGUAUUGC AGCUCUGGCC
GCAA
ACCAGAGAAACA
5417 AACAACAGAR ACCAGAGAAACAXGUACAUUACCUGGUAGGCC GGCUGUUGUU
GGCC
5920 GGGAACAGAA ACCAGAGAAACAXGUACAUUACCUGGUACGGC UGUUGUUCCC
GCCG
5509 UCGGCGAGAA ACCAGAGAAACAXGUACAUUACCUGGUAAUGC AGCUCGCCGA
GCAU
IS 5521 UGCUUGAGAA ACCAGAGAAACAXGUACAUUACCUGGUAGAGC AGUUCAAGCA
GCUC
5576 GGGAGCAGAA ACCAGAGAAACAXGUACAUUACCUGGUAAGGC CGCUGCUCCC
GCCU
5579 CACGGGAGAR ACCAGAGAAACAXGUACAUUACCUGGUACCGC UGCUCCCGUG
GCGG
5683 UUCCCAAGAA ACCAGAGAAACAXGUACAUUACCUGGUAACUC UGCCUGGGAA
GAGU
2~ 5710 AAUGCCAGAA ACCAGAGAAACAXGUACAUUACCUGGUAUCAC UGAUGGCAUU
GUGA
5723 GAUAGA ACCAGAGAAACAXGUACAUUACCUGGUAUCAC AGCCUCUAUC
AGAA
GUGA
5736 UGAGCGAGAR ACCAGAGAAACAXGUACAUUACCUGGUACACC AGUCCGCUCA
GGUG
5740 GUGGUGAGAR ACCAGAGAAACAXGUACAUUACCUGGUAAGUC CGCUCACCAC
GACU
5764 AUGUUGAGAA ACCAGAGAAACAXGUACAUUACCUGGUACUCC UGUUCAACAU
25 GGAG
5792 GAGUUGAGAA ACCAGAGAAACAXGUACAUUACCUGGUAUGGC UGCUCAACUC
GCCA
5816 GGCCGAAGAR ACCAGAGAAACAXGUACAUUACCUGGUAGUGC UGCUUCGGCC
GCAC
5822 CACGAA ACCAGAGAAACAXGUACAUUACCUGGUACUUC GGCCUUCGUG
AGAR
GAAG
5966 GUCCUCAGAA ACCAGAGAAACAXGUACAUUACCUGGUACCUC CGCCGAGGAC
GAGG
3O 6099 GCUAUCAGAA ACCAGAGAAACAXGUACAUUACCUGGUAAACC GGCUGAUAGC
GGUU
6178 GAGAGGAGAA ACCAGAGAAACAXGUACAUUACCUGGUAACUC AGAUCCUCUC
GAGU
6189 UGGUGAAGAA ACCAGAGAAACAXGUACAUUACCUGGUACUCC AGCCUCACCA
GGAG
6205 UUCAGCAGAR ACCAGAGAAACAXGUACAUUACCUGGUAACUC AGCUGCUGAA
GAGU
6208 CUCUUCAGAR ACCAGAGAAACAXGUACAUUACCUGGUACAGC UGCUGAAGAG
GCUG
6243 GCGUGGAGAR ACCAGAGAAACAXGUACAUUACCUGGUAGGAC UGCUCCACGC
GUCC
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
99
Pos. Ribozyme Substrate
Sequence
6261 GCCACGAGAA ACCAGAGAAACAX CUCCGGCUCGUGGC
GGAG GUACAUUACCUGGUA
6308 CUUGAA ACCAGAGAAACAX UGACUGACUUCAAG
AGAR GUACAUUACCUGGUA
GUCA
6328 AGCUUGAGA.A ACCAGAGAAACAX CUCCAGUC
GGAG GUACAUUACCUGGUA CAAGCU
6340 AAUUUCAGAA ACCAGAGAAACAX CUCCUGCCGAAAUU
GGAG GUACAUUACCUGGUA
6426 CACAUGAGAA ACCAGAGAAACAX CACCUGCCCAUGUG
GGUG GUACAUUACCUGGUA
6965 UCAUGGAGAR ACCAGAGAAACAX AAACGGUUCCAUGA
GUUU GUACAUUACCUGGUA
6599 CUCUUCAGAA ACCAGAGAAACAX UGGCUGCUGAAGAG
lO GCCA GUACAUUACCUGGUA
6692 UUCGGGAGAR ACCAGAGAAACAX UCCCGGCCCCCGAA
GGGA GUACAUUACCUGGUA
6727 CUGUGCAGAA ACCAGAGAAACAX GUGCGGUUGCACAG
GCAC GUACAUUACCUGGUA
6753 GGAGAGAGAA ACCAGAGAAACAX GUGCAGACCUCUCC
GCAC GUACAUUACCUGGUA
6817 CAUGGGAGAA ACCAGAGAAACA GUACAUUACCUGGUAUCACAGCUCCCAUG
GUGA X
IS 6839 UGCCACAGAA ACCAGAGAAACAX AACCGGAUGUGGCA
GGUU GUACAUUACCUGGUA
6869 GGAGGGAGAR ACCAGAGAAACAX UCACCGACCCCUCC
GUGA GUACAUUACCUGGUA
6939 CUGAAGAGAA ACCAGAGAAACAX GGCCAGCUCUUCAG
GGCC GUACAUUACCUGGUA
7007 GUCAGCAGAR ACCAGAGAAACAX CCCCGGACGCUGAC
GGGG GUACAUUACCUGGUA
ZO 7013 GAUGAGAGAA ACCAGAGAAACAX ACGCUGACCUCAUC
GCGU GUACAUUACCUGGUA
7114 GCUCGAAGAR ACCAGAGAAACAX GACCCGCUUCGAGC
GGUC GUACAUUACCUGGUA
7148 UGCUGCAGAA ACCAGAGAAACAX UAUCCGUUGCAGCA
GAUA GUACAUUACCUGGUA
7214 GUUGUAAGAA ACCAGAGAAACA GUACAUUACCUGGUAGCCCGGAUUACAAC
GGGC X
7253 GACGUAAGAA ACCAGAGAAACAX GUCCGGACUACGUC
25 GGAC GUACAUUACCUGGUA
7291 GUGGUAAGAA GUACAUUACCUGGUAUUGCCGCCUACCAC
GCAA
ACCAGAGAAACA
X
7315 CGUGGAAGAA ACCAGAGAAACAX AUACCGCCUCCACG
GUAU GUACAUUACCUGGUA
7337 CAGAACAGAA ACCAGAGAAACAX GGACGGUUGUUCUG
GUCC GUACAUUACCUGGUA
7367 CGCCAA ACCAGAGAAACAX CUUCUGCCUUGGCG
AGAA GUACAUUACCUGGUA
GAAG
3O 7901 AUCCGGAGAR ACCAGAGAAACAX CGGCAGCUCCGGAU
GCCG GUACAUUACCUGGUA
7407 CCGACGAGAA ACCAGAGAAACAX CUCCGGAUCGUCGG
GGAG GUACAUUACCUGGUA
7415 GUCAACAGAR ACCAGAGAAACAX CGUCGGCCGUUGAC
GACG GUACAUUACCUGGUA
7418 GCUGUCAGAA ACCAGAGAAACAX CGGCCGUUGACAGC
GCCG GUACAUUACCUGGUA
7439 GGGAGGAGAR ACCAGAGAAACAX CGACCGCCCCUCCC
GUCG GUACAUUACCUGGUA
7448 GGUCUGAGAA ACCAGAGAAACAX CUCCCGAUCAGACC
GGAG GUACAUUACCUGGUA
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
100
Pos. Ribozyme Substrate
Sequence
7453 UCAGAGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGAUCAGACCUCUGA
GAUC
7460 ACCGUCAGAA ACCAGAGAAACAX GUACAUUACCUGGUACCUCUGACGACGGU
GAGG
7481 CUCAACAGAR ACCAGAGAAACAX GUACAUUACCUGGUAAAUCUGACGUUGAG
S GAUU
7535 GCUGAGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAACCCUGAUCUCAGC
GGGU
7593 UUGAGCAGAR ACCAGAGAAACAX GUACAUUACCUGGUACGUCUGCUGCUCAA
GACG
7596 ACAUUGAGAR ACCAGAGAAACAX GUACAUUACCUGGUACUGCUGCUCAAUGU
GCAG
7627 GGCGUGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGCCCUGAUCACGCC
1~ GGGC
7660 UUGAUGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAAAGCUGCCCAUCAA
GCUU
7687 UGACGCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUCUCUGCUGCGUCA
GAGA
7769 CUUGCAAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUGACAGACUGCAAG
GUCA
7870 GGGGGCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAAAGCUGACGCCCCC
GCUU
IS 7956 ACACGGAGAA ACCAGAGAAACAX GUACAUUACCUGGUACAUCCGCUCCGUGU
GAUL
7975 UCUUCCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGACCUGCUGGAAGA
GGUC
8066 AAGGCGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAAGCCAGCUCGCCUU
GGCU
8087 UCCCAGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAUCCCAGACCUGGGA
GGGA
2O 8172 ACUGGAAGAR ACCAGAGAAACAX GUACAUUACCUGGUAGUACGGAUUCCAGU
GUAC
8262 CAAAGCAGAA ACCAGAGAAACAX GUACAUUACCUGGUACACCCGCUGCUUUG
GGUG
8265 AGUCAA ACCAGAGAAACAX GUACAUUACCUGGUACCGCUGCUUUGACU
AGAA
GCGG
8374 AUGUAGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGAGCGGCUCUACAU
GCUC
8395 GAAUUAAGAA ACCAGAGAAACAX GUACAUUACCUGGUACCCCUGACUAAUUC
2S GGGG
_
8452 CUAGUCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGUGCUGACGACUAG
GCAC
8501 UCGACAAGAR ACCAGAGAAACAX GUACAUUACCUGGUACUGCGGCCUGUCGA
GCAG
8505 CAGCUCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGGCCUGUCGAGCUG
GGCC
8639 GGGGGGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAACUCUGCCCCCCCC
3O GAGU
8656 GGUUGGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGACCCGCCCCAACC
GGUC
8711 GUGCGCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAUGUCGGUCGCGCAC
GACA
8911 UUUUCAAGAA ACCAGAGAAACAX GUACAUUACCUGGUAGAACAGCUUGAAAA
GUUC
8935 CCGUAGAGAA ACCAGAGAAACAX GUACAUUACCUGGUAUGUCAGAUCUACGG
GACA
8980 UGAAUGAGAA ACCAGAGAAACAX GUACAUUACCUGGUACCUCAGAUCAUUCA
GAGG
9082 CGCAAGAGAR ACCAGAGAAACAX GUACAUUACCUGGUAGUACCGCCCUUGCG
GUAC
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
101
Pos. Ribozyme Substrate
Sequence
9133 CCUUGGAGAA ACCAGAGAAACAX GUACAUUACCUGGUACUACUGUCCCAAGG
GUAG
9218 GGACGCAGAA ACCAGAGAAACAX GUACAUUACCUGGUAUCCCGGCCGCGUCC
GGGA
9229 AAGUCCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUCCCAGCUGGACUU
GGGA
9243 CGAACCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAGUCCAGCUGGUUCG
GGAC
9285 GAGACAAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUCACAGCCUGUCUC
GUGA
9289 GCACGAAGAR ACCAGAGAAACAX GUACAUUACCUGGUAAGCCUGUCUCGUGC
GGCU
9300 AGCGGGAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUGCCCGACCCCGCU
lO GGCA
9306 UAAACCAGAR ACCAGAGAAACAX GUACAUUACCUGGUAACCCCGCUGGUUUA
GGGU
9358 UUGGGGAGAR ACCAGAGAAACAX GUACAUUACCUGGUAUACCUGCUCCCCAA
GGUA
Where "X" represents stem IV region of a HI' ribozyme (Beczal-Herranz et al.,
1993, EMBO.J. 12,
15 2567). The length of stem IV may be 2 base-pairs.
25
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
102
Table VIII: Additional HCV Conserved Hammerhead ribozyme and target sequence
Nos. Name* Pos.T Riboryme Substrate
S 1 HCV.C-4$278 UUGGUGUCUGAUGAGX CGAA ACGUUUGCAAACGUAACACCAA
2 HCV.C-60290 UGUGGGCCUGAUGAGX CGAA ACGGUUGCAACCGUCGCCCACA
3 HCV.C-175405 AGGUUGUCUGAUGAGX CGAA ACCGCUCGAGCGGUCACAACCU
9 HCV.3-1189418 AAAAAAA X CGAA AAAAAAAUUUUUUUUUUUUUUU
CUGAUGAG
HCV.3-1459495 UAAGAUGCUGAUGAGX CGAA AGCCACCGGUGGCUCCAUCUUA
6 HCV.3-1499449 GGGCUAA X CGAA AUGGAGCGCUCCAUCUUAGCCC
CUGAUGAG
7 HCV.3-1519451 UAGGGCUCUGAUGAGX CGAA AGAUGGAUCCAUCUUAGCCCUA
8 HCV.3-1529452 CUAGGGCCUGAUGAGX CGAA AAGAUGGCCAUCUUAGCCCUAG
9 HCV.3-1589458 CCGUGACCUGAUGAGX CGAA AGGGCUAUAGCCCUAGUCACGG
IS 10 HCV.3-1619961 UAGCCGUCUGAUGAGX CGAA ACUAGGGCCCUAGUCACGGCUA
11 HCV.3-1689468 UCACAGCCUGAUGAGX CGAA AGCCGUGCACGGCUAGCUGUGA
12 HCV.3-1819481 GCUCACGCUGAUGAGX CGAA ACCUUUCGAAAGGUCCGUGAGC
Where "X" represents stem II region of a HH ribozyme (Hertel et al., 1992
Nucleic Acids Res. 20:
3252). The length of stem II may be 2 base-pairs.
Core Reference Sequence for Nos. 1-3 = HPCCOPR (Acc#L38318) 1-600 by
*-Nucleotide 231 (8 nucleotide upstream of the initiator ATG) has been
designated as "1" for the
purpose of numbering ribozyme sites in the core protein coding region.
3'-NCR Reference Sequence for Nos. 4-12= D85S16 (Acc#D8SS16) 9301-9535 by
*- Nucleotide 9301 has been designated as "1" for the purpose of numbering
ribozyme sites in the
3'NCR.
t- position number reflects the reference sequence from HPCCOPR.
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
103
ro a~ ~r
ro
U ro ro
~ ro ~ U
C0 0.1 ~ ro
P7 00 W al frla1 !t1W ~ C'J CU'J
~ U U U CU7
N U ro ro U U ro U ~ '~U'iU7U U
ro ~ p C7
U tT ~ U b~ ro ro U tn ~ ~ ~ U U
tr U U ~ CT U U U U b~ U
t0 ro U U ro b ro CT bi tT V U '
:y ~ b U U U U b~ U H ~ ~ U U
ro U U ro rO U ro ro ~ Nro ro ~ ~ C~.7
CT N ~ tr~~ U W 0 ~ U' C7 ~
3 ~ U U U ro b~ U ro U U ~ U U
rd ro ro ro r0 G~ N b~ C7 U U ~ C7
C7 CT b~ CT tT CT O~ b~ U b~ b~
O. y U U U U U U U U U CT b~ V
ca ~ a~ tr~rn tn a~ a~ a~ ro ro V U
U U U U U U U U ro ~
C7 tn CT tn CT CT CT b~ ~ tT CT b
U U
D ro ro ro ro ro b ro O~ U U b U
~ ~ ~ ~ ~ ~ ~ U U U ~
U U U U U U U U b U ro
ro ro ro ro ro ro ro ~ CT tr ~
~ U U U U U U U U ro N t0 ~ ro
~C c~ ~ ~ ~ c~ c7 ~ c~ ~ c~ c~ ro
r~ ro ro ro ro ro c0 ro [~ r.~ ~ V ~ b'
U U U U U U U U b b~
C~ b~ kn b~ b~ o~ O~ O~ ~ ~ ~ ro d' a~
cr o~ a ~ ~ tr tn U U U V
p D D D ~ ~ ~ D D U ~' ~ rt ~'
FC r.CrC FC FC ~ r~ ~C U U ~' b' tn U
to ~ c7 c~ c~ c~ ~ ~ ", ~, ~, ~ ,~ ro r0
D ~ ~ ~ ~ ~ ~ ~ U ~ U b' U ~
Q" b ~ ro ~ ~ ~ % ~ ~ ro ~ ro
U U b~ ~ ro U CT U ~ U U b~
G~ U ro U U U ~ ro ro ~ U ~ ~ U ~
'..)U ro b~ U ~ U U o, v~ m U
C/~ N ro U CT U ~ ~ tT ro ~ CT ~ ~ U
M M ~- ~ N N M r- N e- ~ h~ f~ O Cp
O O O O O O O O M O O O O ~ '-
O O O O O G7 O O O O O CO O O O
r
N M ~ tn O CflL17N O ~ ~ Op f~ N
.'nr'~"~ N ~- ~- i- ~ ~ e- e- e- oO O N M ~ (D
p O O O O O O O O ~- O r- e- O O O
_
U~ ~ O
~"".G O 00 0~ o co t~ o ~ o o C m n o
U U
o a M M v ~t m f~ ~n M ~ ~ 0 0 0
~ f~.~ f~-1~ 1~ h Q M M ~"'I~ h f~
w z ~ ~ ~ ~ ~ ~ ~ ~ p
1 > ~ r- r- ~- e- ~- ~ N N ~ i- ~ ~-
.. ~ N
C
y .~ v_-~. ;,_~ .~ y- ,~ - - _
w _ ...':~.w. .._.
O O p
O O O O
~ CL'~ ~' ~ Q_'Q'
v7 O .-~-~ O
N
CA 02326695 2000-10-26
WO 99/55847 PCT/US99/09027
104
00
U II7
U U
ro
ro
aT sT
~ c7
c~ c~
U U
C7 C9
U U
U U
U C7
C~ c.7
U
U U
U U
U U
ro ro
ro ro
ro ro
~ :S
ro ro
tr~~
U U
ro U
D ro
U U
v CT U
<T ro
U ~
ro tT
G U ~
C/~ ~ CT
O Q
O O
iii
3
0
; ~
_
0 0
U
b N
(0
G ed ~ ~ C
. rn N N
a~o0~0~ ~ ~ N U
.v 0 N (~O .ilk
N U ~'
q) ~ ~ 'o
m v~
a.
N C ~ O ~ ~
- - w C
v o U ~ ~
O O ~ L ~ '~ a~
O CV Q Q >
O
.C
O .~ O II II
U ~ 00 ~ C7~n m
a a
I I
I
° ~ o
N
