U.S. patent application number 10/056414 was filed with the patent office on 2003-01-02 for ribozyme treatment of diseases or conditions related to levels of nf-kappab.
This patent application is currently assigned to Ribozyme Pharmaceuticals, Inc.. Invention is credited to Draper, Kenneth G., McSwiggen, James, Stinchcomb, Dan T..
Application Number | 20030003469 10/056414 |
Document ID | / |
Family ID | 27399854 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003469 |
Kind Code |
A1 |
Stinchcomb, Dan T. ; et
al. |
January 2, 2003 |
Ribozyme treatment of diseases or conditions related to levels of
NF-kappaB
Abstract
Enzymatic RNA molecules which cleave rel A mRNA.
Inventors: |
Stinchcomb, Dan T.;
(Boulder, CO) ; Draper, Kenneth G.; (Boulder,
CO) ; McSwiggen, James; (Boulder, CO) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Assignee: |
Ribozyme Pharmaceuticals,
Inc.
|
Family ID: |
27399854 |
Appl. No.: |
10/056414 |
Filed: |
January 23, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10056414 |
Jan 23, 2002 |
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08777916 |
Dec 23, 1996 |
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08777916 |
Dec 23, 1996 |
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08291932 |
Aug 15, 1994 |
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08291932 |
Aug 15, 1994 |
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08245466 |
May 18, 1994 |
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08245466 |
May 18, 1994 |
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07987132 |
Dec 7, 1992 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 514/44R; 536/23.1 |
Current CPC
Class: |
C12N 15/1138 20130101;
C12N 2310/3517 20130101; C12N 2310/3527 20130101; C12N 2310/122
20130101; C12N 2310/1241 20130101; C12N 2310/321 20130101; C12N
2310/3521 20130101; A61K 48/00 20130101; C12N 15/113 20130101; C12N
15/1137 20130101; C12N 15/1131 20130101; C12N 2310/321 20130101;
C12N 2310/121 20130101; C12N 2310/336 20130101; C12N 2310/3535
20130101; C12N 2310/127 20130101; C12N 15/1135 20130101; C12N
2310/32 20130101; C12N 2310/322 20130101; C12N 2310/332 20130101;
C12N 2310/3513 20130101; C12N 2310/3533 20130101; C12N 2310/334
20130101; C12N 2310/3523 20130101; C12N 2310/317 20130101; C07K
14/4702 20130101; C12N 2310/126 20130101; C12N 15/1136 20130101;
C12N 2310/111 20130101; C12N 2310/315 20130101; C12N 9/6491
20130101; C12N 2310/333 20130101; A61K 38/00 20130101; C12N
2310/123 20130101; C12N 15/101 20130101; C12N 2310/335
20130101 |
Class at
Publication: |
435/6 ;
435/320.1; 435/325; 514/44; 536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; A61K 048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 1993 |
US |
PCT/US93/06313 |
Claims
1. An enzymatic RNA molecule which cleaves rel A mRNA.
2. An enzymatic RNA molecule of claim 1, the binding arms of which
contain sequences complementary to the sequences defined in Table
II.
3. The enzymatic RNA molecule of claim 1, the binding arms of which
contain sequences complementary to the sequences defined in any one
of Tables III, and IV-VII
4. The enzymatic RNA molecule of claim 1, 2, or 3, wherein said RNA
molecule is in a hammerhead motif.
5. The enzymatic RNA molecule of claim 1, 2, or 3, wherein said RNA
molecule is in a hairpin, hepatitis delta virus, group 1 intron, VS
RNA or RNAseP RNA motif.
6. The enzymatic RNA molecule of claim 6, wherein said ribozyme
comprises between 12 and 100 bases complementary to said mRNA.
7. The enzymatic RNA molecule of claim 6, wherein said ribozyme
comprises between 14 and 24 bases complementary to said mRNA.
8. Enzymatic RNA molecule consisting essentially of any sequence
selected from the group of those shown in Tables IV, V, VI, and
VII.
9. A mammalian cell including an enzymatic RNA molecule of claim 1,
2, or 3.
10. The cell of claim 8, wherein said cell is a human cell.
11. An expression vector including nucleic acid encoding an
enzymatic RNA molecule or multiple enzymatic molecules of claim 1,
2, or 3 in a manner which allows expression of that enzymatic RNA
molecule(s) within a mammalian cell.
12. A mammalian cell including an expression vector of claim
11.
13. The cell of claim 13, wherein said cell is a human cell.
14. A method for treatment of a condition related to the level of
NF-.kappa.B activity by administering to a patient an enzymatic
nucleic acid molecule of claim 1, 2, or 3,
15. A method for treatment of a condition related to the level of
NF-.kappa.B activity by administering to a patient an expression
vector of claim 11.
16. The method of claim 14 or 15, wherein said patient is a
human.
17. The method of claim 14 wherein said condition is selected from
the group consisting of restenosis, rheumatoid arthritis, asthma,
inflammatory or autoimmune disorders, and transplant rejection.
18. The method of claim 15 wherein said condition is selected from
the group consisting of restenosis, rheumatoid arthritis, asthma,
inflammatory or autoimmune disorders, and transplant rejection.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of Stinchcomb et
al., "Method and Composition for Treatment of Restenosis and Cancer
Using Ribozymes," filed May 18, 1994, U.S. Ser. No. 08/245,466
which is a continuation-in-part of Draper, "Method and Reagent for
Treatment of a Stenotic Condition", filed Dec. 7, 1992, U.S. Ser.
No. 07/987,132, both hereby incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to therapeutic compositions
and methods for the treatment or diagnosis of diseases or
conditions related to NF-.kappa.B levels, such as restenosis,
rheumatoid arthritis, asthma, inflammatory or autoimmune disorders
and transplant rejection.
BACKGROUND OF THE INVENTION
[0003] The following is a brief description of the physiological
role of NF-.kappa.B. The discussion is not meant to be complete and
is provided only for understanding of the invention that follows.
This summary is not an admission that any of the work described
below is prior art to the claimed invention.
[0004] The nuclear DNA-binding activity, NF-.kappa.B, was first
identified as a factor that binds and activates the immunoglobulin
K light chain enhancer in B cells. NF-.kappa.B now is known to
activate transcription of a variety of other cellular genes (e.g.,
cytokines, adhesion proteins, oncogenes and viral proteins) in
response to a variety of stimuli (e.g., phorbol esters, mitogens,
cytokines and oxidative stress). In addition, molecular and
biochemical characterization of NF-.kappa.B has shown that the
activity is due to a homodimer or heterodimer of a family of DNA
binding subunits. Each subunit bears a stretch of 300 amino acids
that is homologous to the oncogene, v-rel. The activity first
described as NF-.kappa.B is a heterodimer of p49 or p50 with p65.
The p49 and p50 subunits of NF-.kappa.B (encoded by the
nf-.kappa.B2 or nf-.kappa.B1 genes, respectively) are generated
from the precursors NF-.kappa.B1 (p105) or NF-.kappa.B2 (p100). The
p65 subunit of NF-.kappa.B (now termed Rel A) is encoded by the rel
A locus.
[0005] The roles of each specific transcription-activating complex
now are being elucidated in cells (N. D. Perkins, et al., 1992
Proc. Natl. Acad. Sci USA 89, 1529-1533). For instance, the
heterodimer of NF-.kappa.B1 and Rel A (p50/p65) activates
transcription of the promoter for the adhesion molecule, VCAM-1,
while NF-.kappa.B2/RelA heterodimers (p49/p65) actually inhibit
transcription (H. B. Shu, et al., Mol. Cell. Biol. 13, 6283-6289
(1993)). Conversely, heterodimers of NF-.kappa.B2/RelA (p49/p65)
act with Tat-I to activate transcription of the HIV genome, while
NF-.kappa.B1/RelA (p50/p65) heterodimers have little effect (J.
Liu, N. D. Perkins, R. M. Schmid, G. J. Nabel, J. Virol. 1992 66,
3883-3887). Similarly, blocking rel A gene expression with
antisense oligonucleotides specifically blocks embryonic stem cell
adhesion; blocking NF-.kappa.B1 gene expression with antisense
oligonucleotides had no effect on cellular adhesion (Narayanan et
al., 1993 Mol. Cell. Biol. 13, 3802-3810). Thus, the promiscuous
role initially assigned to NF-.kappa.B in transcriptional
activation (M. J. Lenardo, D. Baltimore, 1989 Cell 58, 227-229)
represents the sum of the activities of the rel family of
DNA-binding proteins. This conclusion is supported by recent
transgenic "knock-out" mice of individual members of the rel
family. Such "knock-outs" show few developmental defects,
suggesting that essential transcriptional activation functions can
be performed by more than one member of the rel family.
[0006] A number of specific inhibitors of NF-.kappa.B function in
cells exist, including treatment with phosphorothioate antisense
oliogonucleotide, treatment with double-stranded NF-.kappa.B
binding sites, and over expression of the natural inhibitor MAD-3
(an I.kappa.B family member). These agents have been used to show
that NF-.kappa.B is required for induction of a number of molecules
involved in inflammation, as described below.
[0007] NF-.kappa.B is required for phorbol ester-mediated induction
of IL-6 (I. Kitajima, et al., Science 258, 1792-5 (1992)) and IL-8
(Kunsch and Rosen, 1993 Mol. Cell. Biol. 13, 6137-46).
[0008] NF-.kappa.B is required for induction of the adhesion
molecules ICAM-1 (Eck, et al., 1993 Mol. Cell. Biol. 13,
6530-6536), VCAM-1 (Shu et al., supra), and E-selectin (Read, et
al., 1994 J. Exp. Med. 179, 503-512) on endothelial cells.
[0009] NF-.kappa.B is involved in the induction of the integrin
subunit, CD18, and other adhesive properties of leukocytes (Eck et
al., 1993 supra).
[0010] The above studies suggest that NF-.kappa.B is integrally
involved in the induction of cytokines and adhesion molecules by
inflammatory mediators. Two recent papers point to another
connection between NF-.kappa.B and inflammation: glucocorticoids
may exert their anti-inflammatory effects by inhibiting
NF-.kappa.B. The glucocorticoid receptor and p65 both act at
NF-.kappa.B binding sites in the ICAM-1 promoter (van de Stolpe, et
al., 1994 J. Biol. Chem. 269, 6185-6192). Glucocorticoid receptor
inhibits NF-.kappa.B-mediated induction of IL-6 (Ray and
Prefontaine, 1994 Proc. Natl Acad. Sci USA 91, 752-756).
Conversely, overexpression of p65 inhibits glucocorticoid induction
of the mouse mammary tumor virus promoter. Finally, protein
cross-linking and co-immunoprecipitation experiments demonstrated
direct physical interaction between p65 and the glucocorticoid
receptor (Id.).
SUMMARY OF THE INVENTION
[0011] This invention relates to ribozymes, or enzymatic RNA
molecules, directed to cleave mRNA species encoding Rel A protein
(p65). In particular, applicant describes the selection and
function of ribozymes capable of cleaving this RNA and their use to
reduce activity of NF-.kappa.B in various tissues to treat the
diseases discussed herein. Such ribozymes are also useful for
diagnostic applications.
[0012] Ribozymes that cleave rel A mRNA represent a novel
therapeutic approach to inflammatory or autoimmune disorders.
Antisense DNA molecules have been described that block NF-.kappa.B
activity. See Narayanan et al., supra. However, ribozymes may show
greater perdurance or lower effective doses than antisense
molecules due to their catalytic properties and their inherent
secondary and tertiary structures. Such ribozymes, with their
catalytic activity and increased site specificity (as described
below), represent more potent and safe therapeutic molecules than
antisense oligonucleotides.
[0013] Applicant indicates that these ribozymes are able to inhibit
the activity of NF-.kappa.B 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 rel A encoding
mRNAs may be readily designed and are within the invention.
[0014] Six basic varieties of naturally-occurring enzymatic RNAs
are known presently. Each can catalyze the hydrolysis of RNA
phosphodiester bonds in trans (and thus can cleave other RNA
molecules) under physiological conditions. Table I summarizes some
of the characteristics 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 a 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 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.
[0015] The enzymatic nature of a ribozyme is advantageous over
other technologies, such as antisense technology (where a nucleic
acid molecule simply binds to a nucleic acid target to block its
translation) since the concentration of ribozyme necessary to
affect a therapeutic treatment is lower than that of an antisense
oligonucleotide. 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 only on the base pairing mechanism of
binding to the target RNA, but also on the mechanism of target RNA
cleavage. Single mismatches, or base-substitutions, near the site
of cleavage can completely eliminate catalytic activity of a
ribozyme. Similar mismatches in antisense molecules do not prevent
their action (Woolf, T. M., et al., 1992, Proc. Natl. Acad. Sci.
USA, 89, 7305-7309). Thus, the specificity of action of a ribozyme
is greater than that of an antisense oligonucleotide binding the
same RNA site.
[0016] In preferred embodiments of this invention, the enzymatic
nucleic acid molecule is formed in a hammerhead or hairpin motif,
but may also be formed in the motif of a hepatitis delta virus,
group I intron or RNaseP RNA (in association with an RNA guide
sequence) or Neurospora VS RNA. Examples of such hammerhead motifs
are described by Rossi et al., 1992, Aids Research and Human
Retroviruses, 8, 183, of hairpin motifs by Hampel et al., "RNA
Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989,
which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed
Sep. 20, 1988, Hampel and Tritz, 1989, Biochemistry, 28, 4929, and
Hampel et al., 1990, Nucleic Acids Res.earch, 18,299, and an
example of the hepatitis delta 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, Neurospora VS RNA
ribozyme motif is described by Collins (Saville and Collins, 1990
Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci.
USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32,
2795-2799) and of the Group I intron by Cech et al., U.S. Pat. No.
4,987,071. 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 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.
[0017] 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 Rel A encoding mRNA 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
vectors that are delivered to specific cells.
[0018] 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
enzymatic nucleic acid motifs (e.g., of the hammerhead or the
hairpin structure) are used for exogenous delivery. The simple
structure of these molecules increases the ability of the enzymatic
nucleic acid to invade targeted regions of the mRNA structure.
However, these catalytic RNA molecules can also be expressed within
cells from eukaryotic promoters (e.g., Scanlon, K. J., et al.,
1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet, M.,
et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic, B., et al.,
1992, J. Virol, 66, 1432-41; Weerasinghe, M., et al., 1991, J.
Virol, 65, 5531-4; Ojwang, J. O., et al., 1992, Proc. Natl. Acad.
Sci. USA, 89, 10802-6; Chen, C. J., et al., 1992, Nucleic Acids
Res., 20, 4581-9; Sarver, H., et al., 1990, Science, 247,
1222-1225)). Those skilled in the art realize that any ribozyme can
be expressed in eukaryotic cells from the appropriate DNA vector.
The activity of such ribozymes can be augmented by their release
from the primary transcript by a second ribozyme (Draper et al.,
PCT WO93/23569, and Sullivan et al., PCT WO94/02595, both hereby
incorporated in their totality by reference herein; Ohkawa, J., et
al., 1992, Nucleic Acids Symp. Ser., 27, 15-6; Taira, K., et al.,
1991, Nucleic Acids Res., 19, 5125-30; Ventura, M., et al., 1993,
Nucleic Acids Res., 21, 3249-55).
[0019] Inflammatory mediators such as lipopolysaccharide (LPS),
interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-.alpha.) act
on cells by inducing transcription of a number of secondary
mediators, including other cytokines and adhesion molecules. In
many cases, this gene activation is known to be mediated by the
transcriptional regulator, NF-.kappa.B. One subunit of NF-.kappa.B,
the relA gene product (termed RelA or p65) is implicated
specifically in the induction of inflammatory responses. Ribozyme
therapy, due to its exquisite specificity, is particularly
well-suited to target intracellular factors that contribute to
disease pathology. Thus, ribozymes that cleave mRNA encoded by rel
A may represent novel therapeutics for the treatment of
inflammatory and autoimmune disorders.
[0020] Thus, in a first aspect, the invention features ribozymes
that inhibit RelA production. 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 RelA encoding
mRNAs, preventing translation and p65 protein accumulation. In the
absence of the expression of the target gene, a therapeutic effect
may be observed.
[0021] By "inhibit" is meant that the activity or level of RelA
encoding mRNA is reduced below that observed in the absense of the
ribozyme, and 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.
[0022] 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 level of NF-.kappa.B activity in
a cell or tissue. By "related" is meant that the inhibition of relA
mRNA and thus reduction in the level of NF-.kappa.B activity will
relieve to some extent the symptoms of the disease or
condition.
[0023] Ribozymes are added directly, or can be complexed with
cationic lipids, packaged within liposomes, or otherwise delivered
to target cells. The RNA or RNA complexes can be locally
administered to relevant tissues ex vivo, or in vivo through
injection or the use of a catheter, infusion pump or stent, with or
without their incorporation in biopolymers. In preferred
embodiments, the ribozymes have binding arms which are
complementary to the sequences in Tables II, III, VI-VII. Examples
of such ribozymes are shown in Tables IV-VII. Examples of such
ribozymes consist essentially of sequences defined in these Tables.
By "consists essentially of" is meant that the active ribozyme
contains an enzymatic center 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.
[0024] In another aspect of the invention, ribozymes that cleave
target molecules and inhibit NF-.kappa.B activity 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 target cells. Once expressed, the ribozymes cleave the
target mRNA. The recombinant vectors are preferably DNA plasmids or
adenovirus vectors. However, other mammalian cell vectors that
direct the expression of RNA may be used for this purpose.
[0025] 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
[0026] The drawings will first briefly be described.
DRAWINGS
[0027] FIG. 1 is a diagrammatic representation of the hammerhead
ribozyme domain known in the art.
[0028] FIG. 2a is a diagrammatic representation of the hammerhead
ribozyme domain known in the art;
[0029] FIG. 2b is a diagrammatic representation of the hammerhead
ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600) into
a substrate and enzyme portion;
[0030] FIG. 2c is a similar diagram showing the hammerhead divided
by Haseloff and Gerlach (1988, Nature, 334, 585-591) into two
portions; and
[0031] FIG. 2d is a similar diagram showing the hammerhead divided
by Jeffries and Symons (1989, Nucl. Acids. Res., 17, 1371-1371)
into two portions.
[0032] FIG. 3 is a representation of the general structure of the
hairpin ribozyme domain known in the art.
[0033] FIG. 4 is a representation of the general structure of the
hepatitis delta virus ribozyme domain known in the art.
[0034] FIG. 5 is a representation of the general structure of the
VS RNA ribozyme domain known in the art.
[0035] FIG. 6 is a schematic representation of an RNAseH
accessibility assay. Specifically, the left side of FIG. 6 is a
diagram of complementary DNA oligonucleotides bound to accessible
sites on the target RNA. Complementary DNA oligonucleotides are
represented by broad lines labeled A, B, and C. Target RNA is
represented by the thin, twisted line. The right side of FIG. 6 is
a schematic of a gel separation of uncut target RNA from a cleaved
target RNA. Detection of target RNA is by autoradiography of
body-labeled, T7 transcript. The bands common to each lane
represent uncleaved target RNA; the bands unique to each lane
represent the cleaved products.
[0036] Ribozymes
[0037] Ribozymes of this invention block to some extent NF-.kappa.B
expression and can be used to treat disease or diagnose such
disease. Ribozymes will be delivered to cells in culture and to
cells or tissues in animal models of restenosis, transplant
rejection and rheumatoid arthritis. Ribozyme cleavage of relA mRNA
in these systems may prevent inflammatory cell function and
alleviate disease symptoms.
[0038] Target Sites
[0039] Targets for useful ribozymes can be determined as disclosed
in Draper et al supra, Sullivan et al., supra, as well as by Draper
et al., "Method and reagent for treatment of arthritic conditions
U.S. Ser. No. 08/152,487, filed Nov. 12, 1993, and hereby
incorporated by reference herein in totality. Rather than repeat
the guidance provided in those documents here, below are provided
specific examples of such methods, not limiting to those in the
art. Ribozymes 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. While specific examples to mouse and human
RNA are provided, those in the art will recognize that the
equivalent human RNA targets described can be used as described
below. Thus, the same target may be used, but binding arms suitable
for targeting human RNA sequences are present in the ribozyme. Such
targets may also be selected as described below.
[0040] The sequence of human and mouse relA mRNA can be screened
for accessible sites using a computer folding algorithm. Potential
hammerhead or hairpin ribozyme cleavage sites were identified.
These sites are shown in Tables II, III, and VI-VII. (All sequences
are 5' to 3' in the tables.) While mouse and human sequences can be
screened and ribozymes thereafter designed, the human targetted
sequences are of most utility. However, as discussed in Stinchcomb
et al. supra, mouse targetted ribozmes are useful to test efficacy
of action of the ribozyme prior to testing in humans. The
nucleotide base position is noted in the Tables as that site to be
cleaved by the designated type of ribozyme. (In Table II, lower
case letters indicate positions that are not conserved between the
Human and the Mouse relA sequences.)
[0041] Hammerhead ribozymes are designed that could bind and are
individually analyzed by computer folding (Jaeger, J. A., et al.,
1989, Proc. Natl. Acad. Sci. USA, 86, 7706-7710) 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 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.
[0042] Referring to FIG. 6, mRNA is screened for accessible
cleavage sites by the method described generally in Draper et al.,
WO/US93/04020 hereby incorporated by reference herein. Briefly, DNA
oligonucleotides representing potential hammerhead ribozyme
cleavage sites are synthesized. A polymerase chain reaction is used
to generate a substrate for T7 RNA polymerase transcription from
human or murine rel A cDNA clones. Labeled RNA transcripts are
synthesized in vitro from the two templates. The oligonucleotides
and the labeled transcripts are annealed, RNAseH is added and the
mixtures are incubated for the designated times at 37.degree. C.
Reactions are stopped and RNA separated on sequencing
polyacrylamide gels. The percentage of the substrate cleaved is
determined by autoradiographic quantitation using a phosphor
imaging system. From these data, hammerhead ribozyme sites are
chosen as the most accessible.
[0043] Ribozymes of the hammerhead motif are designed to anneal to
various sites in the mRNA message. The binding arms are
complementary to the target site sequences described above. The
ribozymes are chemically synthesized. The method of synthesis used
follows the procedure for normal RNA synthesis as described in
Usman, N.; Ogilvie, K. K.; Jiang, M. -Y.; Cedergren, R. J. 1987, J.
Am. Chem. Soc., 109, 7845-7854 and in Scaringe, S. A.; Franklyn,
C.; Usman, N., 1990, Nucleic Acids Res., 18, 5433-5441 and makes
use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
The average stepwise coupling yields were >98%. Inactive
ribozymes were synthesized by substituting a U for G.sub.5 and a U
for A.sub.14 (numbering from (Hertel, K. J., et al., 1992, Nucleic
Acids Res., 20, 3252)). Hairpin ribozymes are synthesized in two
parts and annealed to reconstruct the active ribozyme (Chowrira, B.
M. and Burke, J. M., 1992, Nucleic Acids Res., 20, 2835-2840). All
ribozymes are modified to enhance stability by modification of five
ribonucleotides at both the 5' and 3' ends with 2'-O-methyl groups.
Ribozymes are purified by gel electrophoresis using general methods
or are purified by high pressure liquid chromatography (HPLC; See
Usman et al., Synthesis, deprotection, analysis and purification of
RNA and ribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736
the totality of which is hereby incorporated herein by reference.)
and are resuspended in water.
[0044] The sequences of the chemically synthesized ribozymes useful
in this study are shown in Tables IV-VII. 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 and may be formed
of ribonucleotides or other nucleotides or non-nucleotides. Such
ribozymes are equivalent to the ribozymes described specifically in
the Tables.
[0045] Optimizing Ribozyme Activity
[0046] Ribozyme activity can be optimized as described by
Stinchcomb et al., supra. The details will not be repeated here,
but include altering the length of the ribozyme binding arms (stems
I and III, see FIG. 2c), or chemically synthesizing ribozymes with
modifications that prevent their degradation by serum ribonucleases
(see e.g., Eckstein et al., International Publication No. WO
92/07065; Perrault et al., Nature 1990, 344:565; Pieken et al.,
Science 1991, 253:314; Usman and Cedergren, Trends in Biochem. Sci.
1992, 17:334; Usman et al., International Publication No. WO
93/15187; and Rossi et al., International Publication No. WO
91/03162, as well as Usman, N. et al. U.S. patent application Ser.
No. 07/829,729, and Sproat, B. European Patent Application
92110298.4 which describe various chemical modifications that can
be made to the sugar moieties of enzymatic RNA molecules. All these
publications are hereby incorporated by reference herein.),
modifications which enhance their efficacy in cells, and removal of
stem II bases to shorten RNA synthesis times and reduce chemical
requirements.
[0047] Sullivan, et al., supra, describes the general methods for
delivery of enzymatic RNA molecules. Ribozymes may be administered
to cells by a variety of 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 delivered by direct injection or by use of a catheter,
infusion pump or stent. Other routes of delivery include, but are
not limited to, intrvascular, 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., supra which
have been incorporated by reference herein.
[0048] Another means of accumulating high concentrations of a
ribozyme(s) within cells is to incorporate the ribozyme-encoding
sequences into a DNA expression vector. 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, O. and Moss, B., 1990, Proc. Natl.
Acad. Sci. USA, 87, 6743-7; Gao, X. and Huang, L., 1993, Nucleic
Acids Res., 21, 2867-72; Lieber, A., et al., 1993, Methods
Enzymol., 217, 47-66; Zhou, Y., et al., 1990, Mol. Cell. Biol., 10,
4529-37). Several investigators have demonstrated that ribozymes
expressed from such promoters can function in mammalian cells (e.g.
(Kashani-Sabet, M., et al., 1992, Antisense Res. Dev., 2, 3-15;
Ojwang, J. O., et al., 1992, Proc. Natl. Acad. Sci. USA, 89,
10802-6; Chen, C. J., et al., 1992, Nucleic Acids Res., 20, 4581-9;
Yu, M., et al., 1993, Proc. Natl. Acad. Sci. USA, 96, 6340-4;
L'Huillier, P. J., et al., 1992, Embo J., 11, 4411-8; Lisziewicz,
J., et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90, 8000-4)). 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 vectors), or viral RNA vectors
(such as retroviral vectors).
[0049] In a preferred embodiment of the invention, a transcription
unit expressing a ribozyme that cleaves relA RNA is inserted into a
plasmid DNA vector or an adenovirus DNA viral vector. Both vectors
have been used to transfer genes to the intact vasculature or to
joints of live animals (Willard, J. E., et al., 1992, Circulation,
86, 1-473.; Nabel, E. G., et al., 1990, Science, 249, 1285-1288.)
and both vectors lead to transient gene expression. The adenovirus
vector is delivered as recombinant adenoviral particles. DNA may be
delivered alone or complexed with vehicles (as described for RNA
above). The DNA, DNA/vehicle complexes, or the recombinant
adenovirus particles are locally administered to the site of
treatment, e.g., through the use of an injection catheter, stent or
infusion pump or are directly added to cells or tissues ex
vivo.
EXAMPLE 1
NF-.kappa.B Hammerhead Ribozymes
[0050] By engineering ribozyme motifs we have designed several
ribozymes directed against relA mRNA sequences. These ribozymes are
synthesized with modifications that improve their nuclease
resistance. The ability of ribozymes to cleave relA target
sequences in vitro is evaluated.
[0051] The ribozymes will be tested for function in vivo by
analyzing cytokine-induced VCAM-1, ICAM-1, IL-6 and IL-8 expression
levels. Ribozymes will be delivered to cells by incorporation into
liposomes, by complexing with cationic lipids, by microinjection,
or by expression from DNA vectors. Cytokine-induced VCAM-1, ICAM-1,
IL-6 and IL-8 expression will be monitored by ELISA, by indirect
immunofluoresence, and/or by FACS analysis. Rel A mRNA levels will
be assessed by Northern analysis, RNAse protection or primer
extension analysis or quantitative RT-PCR. Activity of NF-.kappa.B
will be monitored by gel-retardation assays. Ribozymes that block
the induction of NF-.kappa.B activity and/or rel A mRNA by more
than 50% will be identified.
[0052] RNA ribozymes and/or genes encoding them will be locally
delivered to transplant tissue ex vivo in animal models. Expression
of the ribozyme will be monitored by its ability to block ex vivo
induction of VCAM-1, ICAM-1, IL-6 and IL-8 mRNA and protein. The
effect of the anti-rel A ribozymes on graft rejection will then be
assessed. Similarly, ribozymes will be introduced into joints of
mice with collagen-induced arthritis or rabbits with Streptococcal
cell wall-induced arthritis. Liposome delivery, cationic lipid
delivery, or adeno-associated virus vector delivery can be used.
One dose (or a few infrequent doses) of a stable anti-relA ribozyme
or a gene construct that constitutively expresses the ribozyme may
abrogate inflammatory and immune responses in these diseases.
[0053] Uses
[0054] A therapeutic agent that inhibits cytokine gene expression,
inhibits adhesion molecule expression, and mimics the
anti-inflammatory effects of glucocorticoids (without inducing
steroid-responsive genes) is ideal for the treatment of
inflammatory and autoimmune disorders. Disease targets for such a
drug are numerous. Target indications and the delivery options each
entails are summarized below. In all cases, because of the
potential immunosuppressive properties of a ribozyme that cleaves
rel A mRNA, uses are limited to local delivery, acute indications,
or ex vivo treatment.
[0055] *Rheumatoid Arthritis (RA).
[0056] Due to the chronic nature of RA, a gene therapy approach is
logical. Delivery of a ribozyme to inflamed joints is mediated by
adenovirus, retrovirus, or adeno-associated virus vectors. For
instance, the appropriate adenovirus vector can be administered by
direct injection into the synovium: high efficiency of gene
transfer and expression for several months would be expected (B. J.
Roessler, E. D. Allen, J. M. Wilson, J. W. Hartman, B. L. Davidson,
J. Clin. Invest. 92, 1085-1092 (1993)). It is unlikely that the
course of the disease could be reversed by the transient, local
administration of an anti-inflammatory agent. Multiple
administrations may be necessary. Retrovirus and adeno-associated
virus vectors would lead to permanent gene transfer and expression
in the joint. However, permanent expression of a potent
anti-inflammatory agent may lead to local immune deficiency.
[0057] Restenosis.
[0058] Expression of NF-.kappa.B in the vessel wall of pigs causes
a narrowing of the luminal space due to excessive deposition of
extracellular matrix components. This phenotype is similar to
matrix deposition that occurs subsequent to coronary angioplasty.
In addition, NF-.kappa.B is required for the expression of the
oncogene c-myb (F. A. La Rosa, J. W. Pierce, G. E. Soneneshein,
Mol. Cell. Biol. 14, 1039-44 (1994)). Thus NF-.kappa.B induces
smooth muscle proliferation and the expression of excess matrix
components: both processes are thought to contribute to reocclusion
of vessels after coronary angioplasty.
[0059] *Transplantation.
[0060] NF-.kappa.B is required for the induction of adhesion
molecules (Eck et al., supra, K. O'Brien, et al., J. Clin. Invest.
92, 945-951 (1993)) that function in immune recognition and
inflammatory responses. At least two potential modes of treatment
are possible. In the first, transplanted organs are treated ex vivo
with ribozymes or ribozyme expression vectors. Transient inhibition
of NF-.kappa.B in the transplanted endothelium may be sufficient to
prevent transplant-associated vasculitis and may significantly
modulate graft rejection. In the second, donor B cells are treated
ex vivo with ribozymes or ribozyme expression vectors. Recipients
would receive the treatment prior to transplant. Treatment of a
recipient with B cells that do not express T cell co-stimulatory
molecules (such as ICAM-1, VCAM-1, and/or B7 an B7-2) can induce
antigen-specific anergy. Tolerance to the donor's
histocompatibility antigens could result; potentially, any donor
could be used for any transplantation procedure.
[0061] *Asthma.
[0062] Granulocyte macrophage colony stimulating factor (GM-CSF) is
thought to play a major role in recruitment of eosinophils and
other inflammatory cells during the late phase reaction to
asthmatic trauma. Again, blocking the local induction of GM-CSF and
other inflammatory mediators is likely to reduce the persistent
inflammation observed in chronic asthmatics. Aerosol delivery of
ribozymes or adenovirus ribozyme expression vectors is a feasible
treatment.
[0063] Gene Therapy.
[0064] Immune responses limit the efficacy of many gene transfer
techniques. Cells transfected with retrovirus vectors have short
lifetimes in immune competent individuals. The length of expression
of adenovirus vectors in terminally differentiated cells is longer
in neonatal or immune-compromised animals. Insertion of a small
ribozyme expression cassette that modulates inflammatory and immune
responses into existing adenovirus or retrovirus constructs will
greatly enhance their potential.
[0065] Thus, ribozymes of the present invention that cleave rel A
mRNA and thereby NF-.kappa.B activity have many potential
therapeutic uses, and there are reasonable modes of delivering the
ribozymes in a number of the possible indications. Development of
an effective ribozyme that inhibits NF-.kappa.B function is
described above; available cellular and activity assays are number,
reproducible, and accurate. Animal models for NF-.kappa.B function
(Kitajima, et al., supra) and for each of the suggested disease
targets exist and can be used to optimize activity.
[0066] Diagnostic Uses
[0067] Ribozymes of this invention may be used as diagnostic tools
to examine genetic drift and mutations within diseased cells. 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
combinational therapies (e.g., multiple ribozymes targeted to
different genes, ribozymes coupled with known small molecule
inhibitors, or intermittent treatment with combinations of
ribozymes and/or 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 mRNA associated with
an NF-.kappa.B related condition. Such RNA is detected by
determining the presence of a cleavage product after treatment with
a ribozyme using standard methodology.
[0068] 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
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 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 phenotypic changes in target cells.
The expression of mRNA whose protein product is implicated in the
development of the phenotype (i.e., NF-.kappa.B) 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 whether RNA levels are compared
qualitatively or quantitatively.
[0069] Other embodiments are within the following claims.
1TABLE I Characteristics of Ribozymes Group I Introns Size:
.about.200 to >1000 nucleotides. Requires a U in the target
sequence immediately 5' of the cleavage site. Binds 4-6 nucleotides
at 5' side of cleavage site. Over 75 known members of this class.
Found in Tetrahymena thermophila rRNA, fungal mitochondria,
chloroplasts, phage T4, blue-green algae, and others. RNAseP RNA
(M1 RNA) Size: .about.290 to 400 nucleotides. RNA portion of a
ribonucleoprotein enzyme. Cleaves tRNA precursors to form mature
tRNA. Roughly 10 known members of this group all are bacterial in
origin. Hammerhead Ribozyme Size: .about.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. 14 known members of this class. Found in a number of
plant pathogens (virusoids) that use RNA as the infectious agent
(FIGS. 1 and 2 show examples of various manifestations as used in
the art). Hairpin Ribozyme Size: .about.50 nucleotides. Requires
the target sequence GUC immediately 3' of the cleavage site. Binds
4-6 nucleotides at 5' side of the cleavage site and a variable
number to the 3' side of the cleavage site. Only 3 known member 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 as the infectious agent (FIG. 3).
Hepatitis Delta Virus (HDV) Ribozyme Size: 50-60 nucleotides (at
present). Cleavage of target RNAs recently demonstrated. Sequence
requirements not fully determined. Binding sites and structural
requirements not fully determined, although no sequences 5' of
cleavage site are required. Only 1 known member of this class.
Found in human HDV (FIG. 4). Neurospora VS RNA Ribozyme Size:
.about.144 nucleotides (at present) Cleavage of target RNAs
recently demonstrated. Sequence requirements not fully determined.
Binding sites and structural requirements not fully determined.
Only 1 known member of this class. Found in Neurospora VS RNA (FIG.
5).
[0070]
2TABLE II Mouse rel A HH Target sequence nt. HH Target Seq. ID nt.
HH Target Seq. Pos. Sequence No. Pos. Sequence ID No. 19 AAUGGCU a
caCaGgA 7 467 cCAGGCU c cuguUCg 108 22 aGCUCcU a cGUgGUG 8 469
AaGCcAU u AGcCAGC 109 26 CcUCcaU u GcGgACa 9 473 UuUgAGU C AGauCAg
110 93 GAuCUGU U uCCCCUC 10 481 AGCGaAU C CAGACCA 111 94 AuCUGUU u
CCCCUCA 11 501 AACCCCU U uCAcGUU 112 100 UuCCCCU C AUCUUuC 12 502
ACCCCUU u CAcGUUC 113 103 CCCUCAU C UuuCCcu 13 508 UuCAcGU U
CCUAUAG 114 105 CUCAUCU U uCCcuCA 14 509 uCAcGUU C CUAUAGA 115 106
UCAUCUU u CccuCAG 15 512 cGUUCCU A UAGAgGA 116 129 CAGGCuU C
UGGgCCu 16 514 UUCCUAU A GAgGAGC 117 138 GGgCCuU A UGUGGAG 17 534
GGGGACU A uGACuUG 118 148 UGGAGAU C AucGAaC 18 556 UGCGcCU C
UGCUUCC 119 151 AGAUCAU c GaaCAGC 19 561 CUCUGCU U CCAGGUG 120 180
AUGCGaU U CCGCUAu 20 562 UCUGCUU C CAGGUGA 121 181 UGCGaUU C
CGCUAuA 21 585 aAgCCAU u AGcCAGc 122 186 UUCCGCU A uAAaUGC 22 598
GGCCCCU C CuCCUGa 123 204 GGGCGCU C aGCGGGC 23 613 CcCCUGU C
CUcuCaC 124 217 GCAGuAU U CcuGGCG 24 616 CUGUCCU c uCaCAUC 125 239
CACAGAU A CCACCAA 25 617 gucCCUU C CUCAgCC 126 262 CCACCAU C
AAGAUCA 26 620 CCUUCCU C AgCCaug 127 268 UCAAGAU C AAUGGCU 27 623
UCCUgcU u CCAUCUc 128 276 AAUGGCU A CACAGGA 28 628 AUCCGAU U
UUUGAUA 129 301 UuCGaAU C UCCCUGG 29 630 CCgAUuU U UGAuAAc 130 303
CGaAUCU C CCUGGUC 30 631 CgAUuUU U GAuAAcC 131 310 CCCUGGU C
ACCAAGG 31 638 UGgCcAU u GUGuuCC 132 323 GGcCCCU C CUCcuga 32 661
CCGAGCU C AAGAUCU 133 326 uCCaCCU C ACCGGCC 33 667 UCAAGAU C
UGCCGAG 134 335 CCGGCCU C AuCCaCA 34 687 CGgAACU C UGGgAGC 135 349
AuGAaCU U GugGGgA 35 700 GCUGCCU C GGUGGGG 136 352 AGaUcaU c
GaAcAGc 36 715 AUGAGAU C UUCuUgC 137 375 GAUGGCU a CUAUGAG 37 717
GAGAUCU U CuUgCUG 138 376 AUGGucU C UccGgaG 38 718 AGAUCUU C
uUgCUGU 139 378 GGCUaCU A UGAGGCU 39 721 UucUCCU c CauUGcG 140 391
CUGAcCU C UGCCCaG 40 751 AaGACAU U GAGGUGU 141 409 GCaGuAU C
CauAGcU 41 759 GAGGUGU A UUUCACG 142 416 CCgCAGU a UCCAuAg 42 761
GGUGUAU U UCACGGG 143 417 CAuAGcU U CCAGAAC 43 762 GUGUAUU U
CACGGGA 144 418 AuAGcUU C CAGAACC 44 763 UGUAUUU C ACGGGAC 145 433
UGGGgAU C CAGUGUG 45 792 CGAGGCU C CUUUUCu 146 795 GGCUCCU U
UUCuCAA 46 1167 GAUGAGU U UuCCcCC 147 796 GCUCCUU U UcuCAAG 47 1168
AUGAGUU U uCCcCCA 148 797 CUCCUUU U CuCAAGC 48 1169 UGAGUUU u
CCcCCAU 149 798 UCCUUUU C uCAAGCU 49 1182 AUGcUGU U aCCaUCa 150 829
UGGCCAU U GUGUUCC 50 1183 UGcUGUU a CCaUCaG 151 834 AUUGUGU U
CCGGACu 51 1184 GGccccU C CUcCUGa 152 835 UUGUGUU C CGGACuC 52 1187
GUccCuU c CUcAGCc 153 845 GACuCCU C CgUACGC 53 1188 UUaCCaU C
aGGGCAG 154 849 CCUCCgU A CGCcGAC 54 1198 GGgAGuU u AGuCuGa 155 872
cCAGGCU C CUGUuCG 55 1209 CAGcCCU a caCCUUc 156 883 UuCGaGU C
UCCAUGC 56 1215 cuGGCCU U aGCaCCG 157 885 CGaGUCU C CAUGCAG 57 1229
GGuCCCU u CCucAGc 158 905 GCGGCCU U CuGAuCG 58 1237 CCCAgcU C
CUGCCCC 159 906 CGGCCUU C uGAuCGc 59 1250 CCAGcCU C CAGgCUC 160 919
GcGAGCU C AGUGAGC 60 1268 CCCaGCU C CuGCCcc 161 936 AUGGAgU U
CCAGUAC 61 1279 CCAUGGU c cCuuCcu 162 937 UGGAgUU C CAGUACu 62 1281
gUGGgcU C AGCUgcG 163 942 UUCCAGU A CuUGCCA 63 1286 AUgAGuU u
UccCCCA 164 953 GCCucAU c CacAuGA 64 1309 CuCCUGU u CgAGUCu 165 962
AGAuGAU C GcCACCG 65 1315 cCCCAGU u CUAaCCC 166 965 CagUacU u
gCCaGAc 66 1318 CAGUuCA A aCCCCgG 167 973 ACCGGAU U GaaGAGA 67 1331
gGGuCCU C CcCAGuC 168 986 GAgACcU u cAAGagu 68 1334 CuuUuCU C
AaGCUGa 169 996 AGGACcU A UGAGACC 69 1389 ACGCUGU C gGAaGCC 170
1005 GAGACCU U CAAGAGu 70 1413 CUGCAGU U UGAUGcU 171 1006 AGACCUU C
AAGAGuA 71 1414 UGCAGUU U GAUGcUG 172 1015 AGAGuAU C AUGAAGA 72
1437 GGGGCCU U GCUUGGC 173 1028 GAAGAGU C CUUUCAa 73 1441 CCUUGCU U
GGCAACA 174 1031 GAGUCCU U UCAauGG 74 1467 GgaGUGU U CACAGAC 175
1032 AGUCCUU U CaauGGA 75 1468 gaGUGUU C ACAGACC 176 1033 GUCCUUU C
AauGGAC 76 1482 CUGGCAU C uGUgGAC 177 1058 CCGGCCU C CaaCcCG 77
1486 CuUCgGU a GggAACU 178 1064 UaCACCU u GaucCAa 78 1494 GACAACU C
aGAGUUU 179 1072 GgCGuAU U GCUGUGC 79 1500 UCaGAGU U UCAGCAG 180
1082 UGUGCCU a CCCGaAa 80 1501 CaGAGUU U CAGCAGC 181 1083 aaGCCUU C
CCGaAGu 81 1502 aGAGUUU C AGCAGCU 182 1092 CGaAaCU C AaCUUCU 82
1525 gGuGCAU c CCUGUGu 183 1097 CUCAaCU U CUGUCCC 83 1566 AUGGAGU A
CCCUGAa 184 1098 UCAaCUU C UGUCCCC 84 1577 UGAaGCU A UAACUCG 185
1102 CUUCUGU C CCCAAGC 85 1579 AaGCUAU A ACUCGCC 186 1125 CAGCCCU A
caCCUUc 86 1583 UAUAACU C GCCUgGU 187 1127 GCCaUAU a gCcUUAC 87
1588 CUCuCCU A GaGAggG 188 1131 cAUCCCU c agCacCA 88 1622 CCCAGCU C
CUGCcCC 189 1132 AcaCCUU c cCagCAU 89 1628 UCCUGCU u CggUaGG 190
1133 UCCaUcU c CagCuUC 90 1648 CGGGGCU u CCCAAUG 191 1137 UUUACuU u
AgCgCgc 91 1660 cUGaCCU C ugccCAG 192 1140 cCagCAU C CCUcAGC 92
1663 cuCUgCU U cCAGGuG 193 1153 GCACCAU C AACUuUG 93 1664 uCUgCUU c
CAGGuGA 194 1158 AUCAACU u UGAUGAG 94 1665 CUCgcUU u cGGAGgU 195
1680 GAAGACU U CUCCUCC 95 1681 AAGACUU C UCCUCCA 96 1683 GACUUCU C
CUCCAUU 97 1686 UUCUCCU C CAUUGCG 98 1690 CCUCCAU U GCGGACA 99 1704
AUGGACU U CUCuGCu 100 1705 UGGACUU C UCuGCuC 101 1707 GACUUCU C
uGCuCUu 102 1721 uuUGAGU C AGAUCAG 103 1726 GUCAGAU C AGCUCCU 104
1731 AUCAGCU C CUAAGGu 105 1734 AGCUCCU A AGGuGcU 106 1754 CaGugCU
C CCaAGAG 107
[0071]
3TABLE III Human rel A HH Target Sequences nt. HH Target Seq. ID
nt. HH Target Seq. ID Pos. Sequence No. Pos. Sequence No. 19
AAUGGCU C GUCUGUA 196 467 GCAGGCU A UCAGUCA 297 22 GGCUCGU C
UGUAGUG 197 469 AGGCUAU C AGUCAGC 298 26 CGUCUGU A GUGCACG 198 473
UAUCAGU C AGCGCAU 299 93 GAACUGU U CCCCCUC 199 481 AGCGCAU C
CAGACCA 300 94 AACUGUU C CCCCUCA 200 501 AACCCCU U CCAAGUU 301 100
UCCCCCU C AUCUUCC 201 502 ACCCCUU C CAAGUUC 302 103 CCCUCAU C
UUCCCGG 202 508 UCCAAGU U CCUAUAG 303 105 CUCAUCU U CCCGGCA 203 509
CCAAGUU C CUAUAGA 304 106 UCAUCUU C CCGGCAG 204 512 AGUUCCU A
UAGAAGA 305 129 CAGGCCU C UGGCCCC 205 514 UUCCUAU A GAAGAGC 306 138
GGCCCCU A UGUGGAG 206 534 GGGGACU A CGACCUG 307 148 UGGAGAU C
AUUGAGC 207 556 UGCGGCU C UGCUUCC 308 151 AGAUCAU U GAGCAGC 208 561
CUCUGCU U CCAGGUG 309 180 AUGCGCU U CCGCUAC 209 562 UCUGCUU C
CAGGUGA 310 181 UGCGCUU C CGCUACA 210 585 GACCCAU C AGGCAGG 311 186
UUCCGCU A CAAGUGC 211 598 GGCCCCU C CGCCUGC 312 204 GGGCGCU C
CGCGGGC 212 613 CGCCUGU C CUUCCUC 313 217 GCAGCAU C CCAGGCG 213 616
CUGUCCU U CCUCAUC 314 239 CACAGAU A CCACCAA 214 617 UGUCCUU C
CUCAUCC 315 262 CCACCAU C AAGAUCA 215 620 CCUUCCU C AUCCCAU 316 268
UCAAGAU C AAUGGCU 216 623 UCCUCAU C CCAUCUU 317 276 AAUGGCU A
CACAGGA 217 628 AUCCCAU C UUUGACA 318 301 UGCGCAU C UCCCUGG 218 630
CCCAUCU U UGACAAU 319 303 CGCAUCU C CCUGGUC 219 631 CCAUCUU U
GACAAUC 320 310 CCCUGGU C ACCAAGG 220 638 UGACAAU C GUGCCCC 321 323
GGACCCU C CUCACCG 221 661 CCGAGCU C AAGAUCU 322 326 CCCUCCU C
ACCGGCC 222 667 UCAAGAU C UGCCGAG 323 335 CCGGCCU C ACCCCCA 223 687
CGAAACU C UGGCAGC 324 349 ACGAGCU U GUAGGAA 224 700 GCUGCCU C
GGUGGGG 325 352 AGCUUGU A GGAAAGG 225 715 AUGAGAU C UUCCUAC 326 375
GAUGGCU U CUAUGAG 226 717 GAGAUCU U CCUACUG 327 376 AUGGCUU C
UAUGAGG 227 718 AGAUCUU C CUACUGU 328 378 GGCUUCU A UGAGGCU 228 721
UCUUCCU A CUGUGUG 329 391 CUGAGCU C UGCCCGG 229 751 AGGACAU U
GAGGUGU 330 409 GCUGCAU C CACAGUU 230 759 GAGGUGU A UUUCACG 331 416
CCACAGU U UCCAGAA 231 761 GGUGUAU U UCACGGG 332 417 CACAGUU U
CCAGAAC 232 762 GUGUAUU U CACGGGA 333 418 ACAGUUU C CAGAACC 233 763
UGUAUUU C ACGGGAC 334 433 UGGGAAU C CAGUGUG 234 792 CGAGGCU C
CUUUUCG 335 795 GGCUCCU U UUCGCAA 235 1167 GAUGAGU U UCCCACC 336
796 GCUCCUU U UCGCAAG 236 1168 AUGAGUU U CCCACCA 337 797 CUCCUUU U
CGCAAGC 237 1169 UGAGUUU C CCACCAU 338 798 UCCUUUU C GCAAGCU 238
1182 AUGGUGU U UCCUUCU 339 829 UGGCCAU U GUGUUCC 239 1183 UGGUGUU U
CCUUCUG 340 834 AUUGUGU U CCGGACC 240 1184 GGUGUUU C CUUCUGG 341
835 UUGUGUU C CGGACCC 241 1187 GUUUCCU U CUGGGCA 342 845 GACCCCU C
CCUACGC 242 1188 UUUCCUU C UGGGCAG 343 849 CCUCCCU A CGCAGAC 243
1198 GGCAGAU C AGCCAGG 344 872 GCAGGCU C CUGUGCG 244 1209 CAGGCCU C
GGCCUUG 345 883 UGCGUGU C UCCAUGC 245 1215 UCGGCCU U GGCCCCG 346
885 CGUGUCU C CAUGCAG 246 1229 GGCCCCU C CCCAAGU 347 905 GCGGCCU U
CCGACCG 247 1237 CCCAAGU C CUGCCCC 348 906 CGGCCUU C CGACCGG 248
1250 CCAGGCU C CAGCCCC 349 919 GGGAGCU C AGUGAGC 249 1268 CCCUGCU C
CAGCCAU 350 936 AUGGAAU U CCAGUAC 250 1279 CCAUGGU A UCAGGUC 351
937 UGGAAUU C CAGUACC 251 1281 AUGGUAU C AGCUCUG 352 942 UUCCAGU A
CCUGCCA 252 1286 AUCAGCU C UGGCCCA 353 953 GCCAGAU A CAGACGA 253
1309 CCCCUGU C CCAGUCC 354 962 AGACGAU C GUCACCG 254 1315 UCCCAGU C
CUAGCCC 355 965 CGAUCGU C ACCGGAU 255 1318 CAGUCCU A GCCCCAG 356
973 ACCGGAU U GAGGAGA 256 1331 AGGCCCU C CUCAGGC 357 986 GAAACGU A
AAAGGAC 257 1334 CCCUCCU C AGGCUGU 358 996 AGGACAU A UGAGACC 258
1389 ACGCUGU C AGAGGCC 359 1005 GAGACCU U CAAGAGC 259 1413 CUGCAGU
U UGAUGAU 360 1006 AGACCUU C AAGAGCA 260 1414 UGCAGUU U GAUGAUG 361
1015 AGAGCAU C AUGAAGA 261 1437 GGGGCCU U GCUUGGC 362 1028 GAAGAGU
C CUUUCAG 262 1441 CCUUGCU U GGCAACA 363 1031 GAGUCCU U UCAGCGG 263
1467 GCUGUGU U CACAGAC 364 1032 AGUCCUU U CAGCGGA 264 1468 CUGUGUU
C ACAGACC 365 1033 GUCCUUU C AGCGGAC 265 1482 CUGGCAU C CGUCGAC 366
1058 CCGGCCU C CACCUCG 266 1486 CAUCCGU C GACAACU 367 1064 UCCACCU
C GACGCAU 267 1494 GACAACU C CGAGUUU 368 1072 GACGCAU U GCUGUGC 268
1500 UCCGAGU U UCAGCAG 369 1082 UGUGCCU U CCCGCAG 269 1501 CCGAGUU
U CAGCAGC 370 1083 GUGCCUU C CCGCAGC 270 1502 CGAGUUU C AGCAGCU 371
1092 CGCAGCU C AGCUUCU 271 1525 AGGGCAU A CCUGUGG 372 1097 CUCAGCU
U CUGUCCC 272 1566 AUGGAGU A CCCUGAG 373 1098 UCAGCUU C UGUCCCC 273
1577 UGAGGCU A UAACUCG 374 1102 CUUCUGU C CCCAAGC 274 1579 AGGCUAU
A ACUCGCC 375 1125 CAGCCCU A UCCCUUU 275 1583 UAUAACU C GCCUAGU 376
1127 GCCCUAU C CCUUUAC 276 1588 CUCGCCU A GUGACAG 377 1131 UAUCCCU
U UACGUCA 277 1622 CCCAGCU C CUGCUCC 378 1132 AUCCCUU U ACGUCAU 278
1628 UCCUGCU C CACUGGG 379 1133 UCCCUUU A CGUCAUC 279 1648 CGGGGCU
C CCCAAUG 380 1137 UUUACGU C AUCCCUG 280 1660 AUGGCCU C CUUUCAG 381
1140 ACGUCAU C CCUGAGC 281 1663 GCCUCCU U UCAGGAG 382 1153 GCACCAU
C AACUAUG 282 1664 CCUCCUU U CAGGAGA 383 1158 AUCAACU A UGAUGAG 283
1665 CUCCUUU C AGGAGAU 384 1680 GAAGACU U CUCCUCC 284 1681 AAGACUU
C UCCUCCA 285 1683 GACUUCU C CUCCAUU 286 1686 UUCUCCU C CAUUGCG 287
1690 CCUCCAU U GCGGACA 288 1704 AUGGACU U CUCAGCC 289 1705 UGGACUU
C UCAGCCC 290 1707 GACUUCU C AGCCCUG 291 1721 GCUGAGU C AGAUCAG 292
1726 GUCAGAU C AGCUCCU 293 1731 AUCAGCU C CUAAGGG 294 1734 AGCUCCU
A AGGGGGU 295 1754 CUGCCCU C CCCAGAG 296
[0072]
4TABLE IV Mouse rel A HH Ribozyme Sequences nt. Seq. HH Ribozyme
Sequence Seq. ID No. 19 UCCUGUG CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 385
22 CACCACG CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 386 26 UGUCCGC
CUGAUGAGGCCGAAAGGCCGAA AUGGAGG 387 93 GAGGGGA
CUGAUGAGGCCGAAAGGCCGAA ACAGAUC 388 94 UGAGGGG
CUGAUGAGGCCGAAAGGCCGAA AACAGAU 389 100 GAAAGAU
CUGAUGAGGCCGAAAGGCCGAA AGGGGAA 390 103 AGGGAAA
CUGAUGAGGCCGAAAGGCCGAA AUGAGGG 391 105 UGAGGGA
CUGAUGAGGCCGAAAGGCCGAA AGAUGAG 392 106 CUGAGGG
CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 393 129 AGGCCCA
CUGAUGAGGCCGAAAGGCCGAA AAGCCUG 394 138 CUCCACA
CUGAUGAGGCCGAAAGGCCGAA AAGGCCC 395 148 GUUCGAU
CUGAUGAGGCCGAAAGGCCGAA AUCUCCA 396 151 GCUGUUC
CUGAUGAGGCCGAAAGGCCGAA AUGAUCU 397 180 AUAGCGG
CUGAUGAGGCCGAAAGGCCGAA AUCGCAU 398 181 UAUAGCG
CUGAUGAGGCCGAAAGGCCGAA AAUCGCA 399 186 GCAUUUA
CUGAUGAGGCCGAAAGGCCGAA AGCGGAA 400 204 GCCCGCU
CUGAUGAGGCCGAAAGGCCGAA AGCGCCC 401 217 CGCCAGG
CUGAUGAGGCCGAAAGGCCGAA AUACUGC 402 239 UUGGUGG
CUGAUGAGGCCGAAAGGCCGAA AUCUGUG 403 262 UGAUCUU
CUGAUGAGGCCGAAAGGCCGAA AUGGUGG 404 268 AGCCAUU
CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 405 276 UCCUGUG
CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 406 301 CCAGGGA
CUGAUGAGGCCGAAAGGCCGAA AUUCGAA 407 303 GACCAGG
CUGAUGAGGCCGAAAGGCCGAA AGAUUCG 408 310 CCUUGGU
CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 409 323 UCAGGAG
CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 410 326 GGCCGGU
CUGAUGAGGCCGAAAGGCCGAA AGGUGGA 411 335 UGUGGAU
CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 412 349 UCCCCAC
CUGAUGAGGCCGAAAGGCCGAA AGUUCAU 413 352 GCUGUUC
CUGAUGAGGCCGAAAGGCCGAA AUGAUCU 414 375 CUCAUAG
CUGAUGAGGCCGAAAGGCCGAA AGCCAUC 415 376 CUCCGGA
CUGAUGAGGCCGAAAGGCCGAA AGACCAU 416 378 AGCCUCA
CUGAUGAGGCCGAAAGGCCGAA AGUAGCC 417 391 CUGGGCA
CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 418 391 CUGGGCA
CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 428 409 AGCUAUG
CUGAUGAGGCCGAAAGGCCGAA AUACUGC 419 416 CUAUGGA
CUGAUGAGGCCGAAAGGCCGAA ACUGCGG 420 417 GUUCUGG
CUGAUGAGGCCGAAAGGCCGAA AGCUAUG 421 418 GGUUCUG
CUGAUGAGGCCGAAAGGCCGAA AAGCUAU 422 433 CACACUG
CUGAUGAGGCCGAAAGGCCGAA AUCCCCA 423 467 CGAACAG
CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 424 469 GCUGGCU
CUGAUGAGGCCGAAAGGCCGAA AUGGCUU 425 473 CUGAUCU
CUGAUGAGGCCGAAAGGCCGAA ACUCAAA 426 481 UGGUCUG
CUGAUGAGGCCGAAAGGCCGAA AUUCGCU 427 501 AACGUGA
CUGAUGAGGCCGAAAGGCCGAA AGGGGUU 428 502 GAACGUG
CUGAUGAGGCCGAAAGGCCGAA AAGGGGU 429 508 CUAUAGG
CUGAUGAGGCCGAAAGGCCGAA ACGUGAA 430 509 UCUAUAG
CUGAUGAGGCCGAAAGGCCGAA AACGUGA 431 512 UCCUCUA
CUGAUGAGGCCGAAAGGCCGAA AGGAACG 432 514 GCUCCUC
CUGAUGAGGCCGAAAGGCCGAA AUAGGAA 433 534 CAAGUCA
CUGAUGAGGCCGAAAGGCCGAA AGUCCCC 434 556 GGAAGCA
CUGAUGAGGCCGAAAGGCCGAA AGGCGCA 435 561 CACCUGG
CUGAUGAGGCCGAAAGGCCGAA AGGAGAG 436 562 UCACCUG
CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 437 585 GCUGGCU
CUGAUGAGGCCGAAAGGCCGAA AUGGCUU 438 598 UCAGGAG
CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 439 613 GUGAGAG
CUGAUGAGGCCGAAAGGCCGAA ACAGGGG 440 616 GAUGUGA
CUGAUGAGGCCGAAAGGCCGAA AGGACAG 441 617 GGCUGAG
CUGAUGAGGCCGAAAGGCCGAA AAGGGAC 442 620 CAUGGCU
CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 443 623 GAGAUGG
CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 444 628 UAUCAAA
CUGAUGAGGCCGAAAGGCCGAA AUCGGAU 445 630 GUUAUCA
CUGAUGAGGCCGAAAGGCCGAA AAAUCGG 446 631 GGUUAUC
CUGAUGAGGCCGAAAGGCCGAA AAAAUCG 447 638 GGAACAC
CUGAUGAGGCCGAAAGGCCGAA AUGGCCA 448 661 AGAUCUU
CUGAUGAGGCCGAAAGGCCGAA AGCUCGG 449 667 CUCGGCA
CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 450 687 GCUCCCA
CUGAUGAGGCCGAAAGGCCGAA AGUUCCG 451 700 CCCCACC
CUGAUGAGGCCGAAAGGCCGAA AGGCAGC 452 715 GCAAGAA
CUGAUGAGGCCGAAAGGCCGAA AUCUCAU 453 717 CAGCAAG
CUGAUGAGGCCGAAAGGCCGAA AGAUCUC 454 718 ACAGCAA
CUGAUGAGGCCGAAAGGCCGAA AAGAUCU 455 721 CGCAAUG
CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 456 751 ACACCUC
CUGAUGAGGCCGAAAGGCCGAA AUGUCUU 457 759 CGUGAAA
CUGAUGAGGCCGAAAGGCCGAA ACACCUC 458 761 CCCGUGA
CUGAUGAGGCCGAAAGGCCGAA AUACACC 459 762 UCCCGUG
CUGAUGAGGCCGAAAGGCCGAA AAUACAC 460 763 GUCCCGU
CUGAUGAGGCCGAAAGGCCGAA AAAUACA 461 792 AGAAAAG
CUGAUGAGGCCGAAAGGCCGAA AGCCUCG 462 795 UUGAGAA
CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 463 796 CUUGAGA
CUGAUGAGGCCGAAAGGCCGAA AAGGAGC 464 797 GCUUGAG
CUGAUGAGGCCGAAAGGCCGAA AAAGGAG 465 798 AGCUUGA
CUGAUGAGGCCGAAAGGCCGAA AAAAGGA 466 829 GGAACAC
CUGAUGAGGCCGAAAGGCCGAA AUGGCCA 467 834 AGUCCGG
CUGAUGAGGCCGAAAGGCCGAA ACACAAU 468 835 GAGUCCG
CUGAUGAGGCCGAAAGGCCGAA AACACAA 469 845 GCGUACG
CUGAUGAGGCCGAAAGGCCGAA AGGAGUC 470 849 GUCGGCG
CUGAUGAGGCCGAAAGGCCGAA ACGGAGG 471 872 CGAACAG
CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 472 883 GCAUGGA
CUGAUGAGGCCGAAAGGCCGAA ACUCGAA 473 885 CUGCAUG
CUGAUGAGGCCGAAAGGCCGAA AGACUCG 474 905 CGAUCAG
CUGAUGAGGCCGAAAGGCCGAA AGGCCGC 475 906 GCGAUCA
CUGAUGAGGCCGAAAGGCCGAA AAGGCCG 476 919 GCUCACU
CUGAUGAGGCCGAAAGGCCGAA AGCUCGC 477 936 GUACUGG
CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 478 937 AGUACUG
CUGAUGAGGCCGAAAGGCCGAA AACUCCA 479 942 UGGCAAG
CUGAUGAGGCCGAAAGGCCGAA ACUGGAA 480 953 UCAUGUG
CUGAUGAGGCCGAAAGGCCGAA AUGAGGC 481 962 CGGUGGC
CUGAUGAGGCCGAAAGGCCGAA AUCAUCU 482 965 GUCUGGC
CUGAUGAGGCCGAAAGGCCGAA AGUACUG 483 973 UCUCUUC
CUGAUGAGGCCGAAAGGCCGAA AUCCGGU 484 986 ACUCUUG
CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 485 1005 ACUCUUG
CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 486 1006 UACUCUU
CUGAUGAGGCCGAAAGGCCGAA AAGGUCU 487 1015 UCUUCAU
CUGAUGAGGCCGAAAGGCCGAA AUACUCU 488 1028 UUGAAAG
CUGAUGAGGCCGAAAGGCCGAA ACUCUUC 490 1031 CCAUUGA
CUGAUGAGGCCGAAAGGCCGAA AGGACUC 491 1032 UCCAUGG
CUGAUGAGGCCGAAAGGCCGAA AAGGACU 492 1033 GUCCAUU
CUGAUGAGGCCGAAAGGCCGAA AAAGGAC 493 1058 CGGGUUG
CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 494 1064 UUGGAUC
CUGAUGAGGCCGAAAGGCCGAA AGGUGUA 495 1072 GCACAGC
CUGAUGAGGCCGAAAGGCCGAA AUACGCC 496 1082 UUUCGGG
CUGAUGAGGCCGAAAGGCCGAA AGGCACA 497 1083 ACUUCGG
CUGAUGAGGCCGAAAGGCCGAA AAGGCUU 498 1092 AGAAGUU
CUGAUGAGGCCGAAAGGCCGAA AGUUUCG 499 1097 GGGACAG
CUGAUGAGGCCGAAAGGCCGAA AGUUGAG 500 1098 GGGGACA
CUGAUGAGGCCGAAAGGCCGAA AAGUUGA 501 1102 GCUUGGG
CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 502 1125 GAAGGUG
CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 503 1127 GUAAGGC
CUGAUGAGGCCGAAAGGCCGAA AUAUGGC 504 1131 UGGUGCU
CUGAUGAGGCCGAAAGGCCGAA AGGGAUG 505 1132 AUGCUGG
CUGAUGAGGCCGAAAGGCCGAA AAGGUGU 506 1133 GAAGCUG
CUGAUGAGGCCGAAAGGCCGAA AGAUGGA 507 1137 GCGCGCU
CUGAUGAGGCCGAAAGGCCGAA AAGUAAA 508 1140 GCUGAGG
CUGAUGAGGCCGAAAGGCCGAA AUGCUGG 509 1153 CAAAGUU
CUGAUGAGGCCGAAAGGCCGAA AUGGUGC 510 1158 CUCAUCA
CUGAUGAGGCCGAAAGGCCGAA AGUUGAU 511 1167 GGGGGAA
CUGAUGAGGCCGAAAGGCCGAA ACUCAUC 512 1168 UGGGGGA
CUGAUGAGGCCGAAAGGCCGAA AACUCAU 513 1169 AUGGGGG
CUGAUGAGGCCGAAAGGCCGAA AAACUCA 514 1182 UGAUGGU
CUGAUGAGGCCGAAAGGCCGAA ACAGCAU 515 1183 CUGAUGG
CUGAUGAGGCCGAAAGGCCGAA AACAGCA 516 1184 UCAGGAG
CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 517 1187 GGCUGAG
CUGAUGAGGCCGAAAGGCCGAA AAGGGAC 518 1188 CUGCCCU
CUGAUGAGGCCGAAAGGCCGAA AUGGUAA 519 1198 UCAGACU
CUGAUGAGGCCGAAAGGCCGAA AACUCCC 520 1209 GAAGGUG
CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 521 1215 CGGUGCU
CUGAUGAGGCCGAAAGGCCGAA AGGCCAG 522 1229 GCUGAGG
CUGAUGAGGCCGAAAGGCCGAA AGGGACC 523 1237 GGGGCAG
CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 524 1250 GAGCCUG
CUGAUGAGGCCGAAAGGCCGAA AGGCUGG 525 1268 GGGGCAG
CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 526 1279 AGGAAGG
CUGAUGAGGCCGAAAGGCCGAA ACCAUGG 527 1281 CGCAGCU
CUGAUGAGGCCGAAAGGCCGAA AGCCCAC 528 1286 UGGGGGA
CUGAUGAGGCCGAAAGGCCGAA AACUCAU 529 1309 AGACUCG
CUGAUGAGGCCGAAAGGCCGAA ACAGGAG 530 1315 GGGUUAG
CUGAUGAGGCCGAAAGGCCGAA ACUGGGG 531 1318 CCGGGGU
CUGAUGAGGCCGAAAGGCCGAA AGAACUG 532 1331 GACUGGG
CUGAUGAGGCCGAAAGGCCGAA AGGACCC 533 1334 UCAGCUU
CUGAUGAGGCCGAAAGGCCGAA AGAAAAG 534 1389 GGCUUCC
CUGAUGAGGCCGAAAGGCCGAA ACAGCGU 535 1413 AGCAUCA
CUGAUGAGGCCGAAAGGCCGAA ACUGGAG 536 1414 CAGCAUC
CUGAUGAGGCCGAAAGGCCGAA AACUGCA 537 1437 GCCAAGC
CUGAUGAGGCCGAAAGGCCGAA AGGCCCC 538 1441 UGUUGCC
CUGAUGAGGCCGAAAGGCCGAA AGCAAGG 539 1467 GUCUGUG
CUGAUGAGGCCGAAAGGCCGAA ACACUCC 540 1468 GGUCUGU
CUGAUGAGGCCGAAAGGCCGAA AACACUC 541 1482 GUCCACA
CUGAUGAGGCCGAAAGGCCGAA AUGCCAG 542 1486 AGUUCCC
CUGAUGAGGCCGAAAGGCCGAA ACCGAAG 543 1494 AAACUCU
CUGAUGAGGCCGAAAGGCCGAA AGUUGUC 544 1500 CUGCUGA
CUGAUGAGGCCGAAAGGCCGAA ACUCUGA 545 1501 GCUGCUG
CUGAUGAGGCCGAAAGGCCGAA AACUCUG 546 1502 AGCUGCU
CUGAUGAGGCCGAAAGGCCGAA AAACUCU 547 1525 ACACAGG
CUGAUGAGGCCGAAAGGCCGAA AUGCACC 548 1566 UUCAGGG
CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 549 1577 CGAGUUA
CUGAUGAGGCCGAAAGGCCGAA AGCUUCA 550 1579 GGCGAGU
CUGAUGAGGCCGAAAGGCCGAA AUAGCUU 551 1583 ACCAGGC
CUGAUGAGGCCGAAAGGCCGAA AGUUAUA 552 1588 CCCUCUC
CUGAUGAGGCCGAAAGGCCGAA AGGAGAG 553 1622 GGGGCAG
CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 554 1628 CCUACCG
CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 555 1648 CAUUGGG
CUGAUGAGGCCGAAAGGCCGAA AGCCCCG 556 1660 CUGGGCA
CUGAUGAGGCCGAAAGGCCGAA AGGUCAG 557 1663 CACCUGG
CUGAUGAGGCCGAAAGGCCGAA AGCAGAG 558 1664 UCACCUG
CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 559 1665 ACCUCCG
CUGAUGAGGCCGAAAGGCCGAA AAGCGAG 560 1680 GGAGGAG
CUGAUGAGGCCGAAAGGCCGAA AGUCUUC 561 1681 UGGAGGA
CUGAUGAGGCCGAAAGGCCGAA AAGUCUU 562 1683 AAUGGAG
CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 563 1686 CGCAAUG
CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 564 1690 UGUCCGC
CUGAUGAGGCCGAAAGGCCGAA AUGGAGG 565 1704 AGCAGAG
CUGAUGAGGCCGAAAGGCCGAA AGUCCAU 566 1705 GAGCAGA
CUGAUGAGGCCGAAAGGCCGAA AAGUCCA 567 1707 AAGAGCA
CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 568 1721 CUGAUCU
CUGAUGAGGCCGAAAGGCCGAA ACUCAAA 569 1726 AGGAGCU
CUGAUGAGGCCGAAAGGCCGAA AUCUGAC 570 1731 ACCUUAG
CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 571 1734 AGCACCU
CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 572 1754 CUCUUGG
CUGAUGAGGCCGAAAGGCCGAA AGCACUG 573
[0073]
5TABLE V Human rel A HH Ribozyme Sequences nt. Sequence HH Ribozyme
Sequence SEQ ID NO. 19 UACAGAC CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 574
22 CACUACA CUGAUGAGGCCGAAAGGCCGAA ACGAGCC 575 26 CGUGCAC
CUGAUGAGGCCGAAAGGCCGAA ACAGACG 576 93 GAGGGGG
CUGAUGAGGCCGAAAGGCCGAA ACAGUUC 577 94 UGAGGGG
CUGAUGAGGCCGAAAGGCCGAA AACAGUU 578 100 GGAAGAU
CUGAUGAGGCCGAAAGGCCGAA AGGGGGA 579 103 CCGGGAA
CUGAUGAGGCCGAAAGGCCGAA AUGAGGG 580 105 UGCCGGG
CUGAUGAGGCCGAAAGGCCGAA AGAUGAG 581 106 CUGCCGG
CUGAUGAGGCCGAAAGGCCGAA AAGAUGA 582 129 GGGGCCA
CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 583 138 CUCCACA
CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 584 148 GCUCAAU
CUGAUGAGGCCGAAAGGCCGAA AUCUCCA 585 151 GCUGCUC
CUGAUGAGGCCGAAAGGCCGAA AUGAUCU 586 180 GUAGCGG
CUGAUGAGGCCGAAAGGCCGAA AGCGCAU 587 181 UGUAGCG
CUGAUGAGGCCGAAAGGCCGAA AAGCGCA 588 186 GCACUUG
CUGAUGAGGCCGAAAGGCCGAA AGCGGAA 589 204 GCCCGCG
CUGAUGAGGCCGAAAGGCCGAA AGCGCCC 590 217 CGCCUGG
CUGAUGAGGCCGAAAGGCCGAA AUGCUGC 591 239 UUGGUGG
CUGAUGAGGCCGAAAGGCCGAA AUCUGUG 592 262 UGAUCUU
CUGAUGAGGCCGAAAGGCCGAA AUGGUGG 593 268 AGCCAUU
CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 594 276 UCCUGUG
CUGAUGAGGCCGAAAGGCCGAA AGCCAUU 595 301 CCAGGGA
CUGAUGAGGCCGAAAGGCCGAA AUGCGCA 596 303 GACCAGG
CUGAUGAGGCCGAAAGGCCGAA AGAUGCG 597 310 CCUUGGU
CUGAUGAGGCCGAAAGGCCGAA ACCAGGG 598 323 CGGUGAG
CUGAUGAGGCCGAAAGGCCGAA AGGGUCC 599 326 GGCCGGU
CUGAUGAGGCCGAAAGGCCGAA AGGAGGG 600 335 UGGGGGU
CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 601 349 UUCCUAC
CUGAUGAGGCCGAAAGGCCGAA AGCUCGU 602 352 CCUUUCC
CUGAUGAGGCCGAAAGGCCGAA ACAAGCU 603 375 CUCAUAG
CUGAUGAGGCCGAAAGGCCGAA AGCCAUC 604 376 CCUCAUA
CUGAUGAGGCCGAAAGGCCGAA AAGCCAU 605 378 AGCCUCA
CUGAUGAGGCCGAAAGGCCGAA AGAAGCC 606 391 CCGGGCA
CUGAUGAGGCCGAAAGGCCGAA AGCUCAG 607 409 AACUGUG
CUGAUGAGGCCGAAAGGCCGAA AUGCAGC 608 416 UUCUGGA
CUGAUGAGGCCGAAAGGCCGAA ACUGUGG 609 417 GUUCUGG
CUGAUGAGGCCGAAAGGCCGAA AACUGUG 610 418 GGUUCUG
CUGAUGAGGCCGAAAGGCCGAA AAACUGU 611 433 CACACUG
CUGAUGAGGCCGAAAGGCCGAA AUUCCCA 612 467 UGACUGA
CUGAUGAGGCCGAAAGGCCGAA AGCCUGC 613 469 GCUGACU
CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 614 473 AUGCGCU
CUGAUGAGGCCGAAAGGCCGAA ACUGAUA 615 481 UGGUCUG
CUGAUGAGGCCGAAAGGCCGAA AUGCGCU 616 501 AACUUGG
CUGAUGAGGCCGAAAGGCCGAA AGGGGUU 617 502 GAACUUG
CUGAUGAGGCCGAAAGGCCGAA AAGGGGU 618 508 CUAUAGG
CUGAUGAGGCCGAAAGGCCGAA ACUUGAA 619 509 UCUAUAG
CUGAUGAGGCCGAAAGGCCGAA AACUUGG 620 512 UCUUCUA
CUGAUGAGGCCGAAAGGCCGAA AGGAACU 621 514 GCUCUUC
CUGAUGAGGCCGAAAGGCCGAA AUAGGAA 622 534 CAGGUCG
CUGAUGAGGCCGAAAGGCCGAA AGUCCCC 623 556 GGAAGCA
CUGAUGAGGCCGAAAGGCCGAA AGCCGCA 624 561 CACCUGG
CUGAUGAGGCCGAAAGGCCGAA AGCAGAG 625 562 UCACCUG
CUGAUGAGGCCGAAAGGCCGAA AAGCAGA 626 585 CCUGCCU
CUGAUGAGGCCGAAAGGCCGAA AUGGGUC 627 598 GCAGGCG
CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 628 613 GAGGAAG
CUGAUGAGGCCGAAAGGCCGAA ACAGGCG 629 616 GAUGAGG
CUGAUGAGGCCGAAAGGCCGAA AGGACAG 630 617 GGAUGAG
CUGAUGAGGCCGAAAGGCCGAA AAGGACA 631 620 AUGGGAU
CUGAUGAGGCCGAAAGGCCGAA AGGAAGG 632 623 AAGAUGG
CUGAUGAGGCCGAAAGGCCGAA AUGAGGA 633 628 UGUCAAA
CUGAUGAGGCCGAAAGGCCGAA AUCGGAU 634 630 AUUGUCA
CUGAUGAGGCCGAAAGGCCGAA AGAUGGG 635 631 GAUUGUC
CUGAUGAGGCCGAAAGGCCGAA AAGAUGG 636 638 GGGGCAC
CUGAUGAGGCCGAAAGGCCGAA AUUGUCA 637 661 AGAUCUU
CUGAUGAGGCCGAAAGGCCGAA AGCUCGG 638 667 CUCGGCA
CUGAUGAGGCCGAAAGGCCGAA AUCUUGA 639 687 GCUGCCA
CUGAUGAGGCCGAAAGGCCGAA AGUUUCG 640 700 CCCCACC
CUGAUGAGGCCGAAAGGCCGAA AGGCAGC 641 715 GUAGGAA
CUGAUGAGGCCGAAAGGCCGAA AUCUCAU 642 717 CAGUAAG
CUGAUGAGGCCGAAAGGCCGAA AGAUCUC 643 718 ACAGUAG
CUGAUGAGGCCGAAAGGCCGAA AAGAUCU 644 721 CACACAG
CUGAUGAGGCCGAAAGGCCGAA AGGAAGA 645 751 ACACCUC
CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 646 759 CGUGAAA
CUGAUGAGGCCGAAAGGCCGAA ACACCUC 647 761 CCCGUGA
CUGAUGAGGCCGAAAGGCCGAA AUACACC 648 762 UCCCGUG
CUGAUGAGGCCGAAAGGCCGAA AAUACAC 649 763 GUCCCGU
CUGAUGAGGCCGAAAGGCCGAA AAAUACA 650 792 CGAAAAG
CUGAUGAGGCCGAAAGGCCGAA AGCCUCG 651 795 UUGCGAA
CUGAUGAGGCCGAAAGGCCGAA AGGAGCC 652 796 CUUGCGA
CUGAUGAGGCCGAAAGGCCGAA AAGGAGC 653 797 GCUUGCG
CUGAUGAGGCCGAAAGGCCGAA AAAGGAG 654 798 AGCUUGC
CUGAUGAGGCCGAAAGGCCGAA AAAAGGA 655 829 GGAACAC
CUGAUGAGGCCGAAAGGCCGAA AUGGCCA 656 834 GGUCCGG
CUGAUGAGGCCGAAAGGCCGAA ACACAAU 657 835 GGGUCCG
CUGAUGAGGCCGAAAGGCCGAA AACACAA 658 845 GCGUAGG
CUGAUGAGGCCGAAAGGCCGAA AGGGGUC 659 849 GUCUGCG
CUGAUGAGGCCGAAAGGCCGAA AGGGAGG 660 872 CGCACAG
CUGAUGAGGCCGAAAGGCCGAA AGCCUGC 661 883 GCAUGGA
CUGAUGAGGCCGAAAGGCCGAA ACACGCA 662 885 CUGCAUG
CUGAUGAGGCCGAAAGGCCGAA AGACACG 662 905 CGGUCGG
CUGAUGAGGCCGAAAGGCCGAA AGGCCGC 664 906 CCGGUCG
CUGAUGAGGCCGAAAGGCCGAA AAGGCCG 665 919 GCUCAGU
CUGAUGAGGCCGAAAGGCCGAA AGCUCCC 666 936 GUACUGG
CUGAUGAGGCCGAAAGGCCGAA AUUCCAU 667 937 GGUACUG
CUGAUGAGGCCGAAAGGCCGAA AAUUCCA 668 942 UGGCAGG
CUGAUGAGGCCGAAAGGCCGAA ACUGGAA 669 953 UCGUCUG
CUGAUGAGGCCGAAAGGCCGAA AUCUGGC 670 962 CGGUGAC
CUGAUGAGGCCGAAAGGCCGAA AUCGUCU 671 965 AUCCGGU
CUGAUGAGGCCGAAAGGCCGAA ACGAUCG 672 973 UCUCCUC
CUGAUGAGGCCGAAAGGCCGAA AUCCGGU 673 986 GUCCUUU
CUGAUGAGGCCGAAAGGCCGAA AGGUUUC 674 996 GGUCUCA
CUGAUGAGGCCGAAAGGCCGAA AUGUCCU 675 1005 GCUCUUG
CUGAUGAGGCCGAAAGGCCGAA AGGUCUC 676 1006 UGCUCUU
CUGAUGAGGCCGAAAGGCCGAA AAGGUCU 677 1015 UCUUCAU
CUGAUGAGGCCGAAAGGCCGAA AUGCUCU 678 1028 CUGAAAG
CUGAUGAGGCCGAAAGGCCGAA ACUCUUC 679 1031 CCGCUGA
CUGAUGAGGCCGAAAGGCCGAA AGGACUC 680 1032 UCCGCUG
CUGAUGAGGCCGAAAGGCCGAA AAGGACU 681 1033 GUCCGCU
CUGAUGAGGCCGAAAGGCCGAA AAAGGAC 682 1058 CGAGGUG
CUGAUGAGGCCGAAAGGCCGAA AGGCCGG 683 1064 AUGCGUC
CUGAUGAGGCCGAAAGGCCGAA AGGUGGA 684 1072 GCACAGC
CUGAUGAGGCCGAAAGGCCGAA AUGCGUC 685 1082 CUGCGGG
CUGAUGAGGCCGAAAGGCCGAA AGGCACA 686 1083 GCUGCGG
CUGAUGAGGCCGAAAGGCCGAA AAGGCAC 687 1092 AGAAGCU
CUGAUGAGGCCGAAAGGCCGAA AGCUGCG 688 1097 GGGACAG
CUGAUGAGGCCGAAAGGCCGAA AGCUGAG 689 1098 GGGGACA
CUGAUGAGGCCGAAAGGCCGAA AAGCUGA 690 1102 GCUUGGG
CUGAUGAGGCCGAAAGGCCGAA ACAGAAG 691 1125 AAAGGGA
CUGAUGAGGCCGAAAGGCCGAA AGGGCUG 692 1127 GUAAAGG
CUGAUGAGGCCGAAAGGCCGAA AUAGGGC 693 1131 UGACGUA
CUGAUGAGGCCGAAAGGCCGAA AGGGAUA 694 1132 AUGACGU
CUGAUGAGGCCGAAAGGCCGAA AAGGGAU 695 1133 GAUGACG
CUGAUGAGGCCGAAAGGCCGAA AAAGGGA 696 1137 CAGGGAU
CUGAUGAGGCCGAAAGGCCGAA ACGUAAA 697 1140 GCUCAGG
CUGAUGAGGCCGAAAGGCCGAA AUGACGU 698 1153 CAUAGUU
CUGAUGAGGCCGAAAGGCCGAA AUGGUGC 699 1158 CUCAUCA
CUGAUGAGGCCGAAAGGCCGAA AGUUGAU 700 1167 GGUGGGA
CUGAUGAGGCCGAAAGGCCGAA ACUCAUC 701 1168 UGGUGGG
CUGAUGAGGCCGAAAGGCCGAA AACUCAU 702 1169 AUGGUGG
CUGAUGAGGCCGAAAGGCCGAA AAACUCA 703 1182 AGAAGGA
CUGAUGAGGCCGAAAGGCCGAA ACACCAU 704 1183 CAGAAGG
CUGAUGAGGCCGAAAGGCCGAA AACACCA 705 1184 CCAGAAG
CUGAUGAGGCCGAAAGGCCGAA AAACACC 706 1187 UGCCCAG
CUGAUGAGGCCGAAAGGCCGAA AAGAAAC 707 1188 CUGCCCA
CUGAUGAGGCCGAAAGGCCGAA AAGGAAA 708 1198 CCUGGCU
CUGAUGAGGCCGAAAGGCCGAA AUCUGCC 709 1209 GAAGGCC
CUGAUGAGGCCGAAAGGCCGAA AGGCCUG 710 1215 CGGGGCC
CUGAUGAGGCCGAAAGGCCGAA AGGCCGA 711 1229 ACUUGGG
CUGAUGAGGCCGAAAGGCCGAA AGGGGCC 712 1237 GGGGCAG
CUGAUGAGGCCGAAAGGCCGAA ACUUGGG 713 1250 GGGGCUG
CUGAUGAGGCCGAAAGGCCGAA AGCCUGG 714 1268 AUGGCUG
CUGAUGAGGCCGAAAGGCCGAA AGCAGGG 715 1279 GAGCUGA
CUGAUGAGGCCGAAAGGCCGAA ACCAUGG 716 1281 CAGAGCU
CUGAUGAGGCCGAAAGGCCGAA AUACCAU 717 1286 UGGGCCA
CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 718 1309 GGACUGG
CUGAUGAGGCCGAAAGGCCGAA ACAGGGG 719 1315 GGGCUAG
CUGAUGAGGCCGAAAGGCCGAA ACUGGGA 720 1318 CUGGGGC
CUGAUGAGGCCGAAAGGCCGAA AGGACUG 721 1331 GCCUGAG
CUGAUGAGGCCGAAAGGCCGAA AGGGCCU 722 1334 ACAGCCU
CUGAUGAGGCCGAAAGGCCGAA AGGAGGG 723 1389 GGCCUCU
CUGAUGAGGCCGAAAGGCCGAA ACAGCGU 724 1413 AUCAUCA
CUGAUGAGGCCGAAAGGCCGAA ACUGCAG 725 1414 CAUCAUC
CUGAUGAGGCCGAAAGGCCGAA AACUGCA 726 1437 GCCAAGC
CUGAUGAGGCCGAAAGGCCGAA AGGCCCC 727 1441 UGUUGCC
CUGAUGAGGCCGAAAGGCCGAA AGCAAGG 728 1467 GUCUGUG
CUGAUGAGGCCGAAAGGCCGAA ACACAGC 729 1468 GGUCUGU
CUGAUGAGGCCGAAAGGCCGAA AACACAG 730 1482 GUCGACG
CUGAUGAGGCCGAAAGGCCGAA AUGCCAG 731 1486 AGUUGUC
CUGAUGAGGCCGAAAGGCCGAA ACGGAUG 732 1494 AAACUCG
CUGAUGAGGCCGAAAGGCCGAA AGUUGUC 733 1500 CUGCUGA
CUGAUGAGGCCGAAAGGCCGAA ACUCGGA 734 1501 GCUGCUG
CUGAUGAGGCCGAAAGGCCGAA AACUCGG 735 1502 AGCUGCU
CUGAUGAGGCCGAAAGGCCGAA AAACUCG 736 1525 CCACAGG
CUGAUGAGGCCGAAAGGCCGAA AUGCCCU 737 1566 CACAGGG
CUGAUGAGGCCGAAAGGCCGAA ACUCCAU 738 1577 CGAGUUA
CUGAUGAGGCCGAAAGGCCGAA AGCCUCA 739 1579 GGCGAGU
CUGAUGAGGCCGAAAGGCCGAA AUAGCCU 740 1583 ACCAGGC
CUGAUGAGGCCGAAAGGCCGAA AGUUAUA 741 1588 CUGUCAC
CUGAUGAGGCCGAAAGGCCGAA AGGCGAG 742 1622 GGAGCAG
CUGAUGAGGCCGAAAGGCCGAA AGCUGGG 743 1628 CCCAGUG
CUGAUGAGGCCGAAAGGCCGAA AGCAGGA 744 1648 CAUUGGG
CUGAUGAGGCCGAAAGGCCGAA AGCCCCG 745 1660 CUGAAAG
CUGAUGAGGCCGAAAGGCCGAA AGGCCAU 746 1663 CUCCUGA
CUGAUGAGGCCGAAAGGCCGAA AGGAGGC 747 1664 UCUCCUG
CUGAUGAGGCCGAAAGGCCGAA AAGGAGG 748 1665 AUCUCCU
CUGAUGAGGCCGAAAGGCCGAA AAAGGAG 749 1680 GGAGGAG
CUGAUGAGGCCGAAAGGCCGAA AGUCUUC 750 1681 UGGAGGA
CUGAUGAGGCCGAAAGGCCGAA AAGUGUU 751 1683 AAUGGAG
CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 752 1686 CGCAAUG
CUGAUGAGGCCGAAAGGCCGAA AGGAGAA 753 1690 UGUCCGC
CUGAUGAGGCCGAAAGGCCGAA AUGGAGG 754 1704 GGCUGAG
CUGAUGAGGCCGAAAGGCCGAA AGUCCAU 755 1705 GGGCUGA
CUGAUGAGGCCGAAAGGCCGAA AAGUCCA 756 1707 CAGGGCU
CUGAUGAGGCCGAAAGGCCGAA AGAAGUC 757 1721 CUGAUCU
CUGAUGAGGCCGAAAGGCCGAA ACUCAGC 758 1726 AGGAGCU
CUGAUGAGGCCGAAAGGCCGAA AUCUGAC 759 1731 CCCUUAG
CUGAUGAGGCCGAAAGGCCGAA AGCUGAU 760 1734 ACCCCCU
CUGAUGAGGCCGAAAGGCCGAA AGGAGCU 761 1754 CUCUGGG
CUGAUGAGGCCGAAAGGCCGAA AGGGCAG 762
[0074]
6TABLE VI Human rel A Hairpin Ribozyme/Target Sequences nt. Seq ID
Seq ID Position Hairpin Ribozyme sequence No. Substrate No. 90
UGAGGGGG AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 763 GAACU
GUU CCCCCUCA 778 156 GCUGCUUG AGAA GCUC
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 764 GAGCA GCC CAAGCAGC 779 362
GCCAUCCC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 765 GGACU
GCC GGGAUGGC 780 413 GUUCUGGA AGAA GUGG
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 766 CCACA GUU UCCAGAAC 781 606
GAAGGACA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 767 CUGCC
GCC UGUCCUUC 782 652 UUGAGCUC AGAA GUGU
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 768 ACACU GCC GAGCUCAA 783 695
CCCACCGA AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 769 CAGCU
GCC UCGGUGGG 784 853 AGGCUGGG AGAA GCGU
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 770 ACGCA GAC CCCAGCCU 785 900
GGUCGGAA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 771 CGGCG
GCC UUCCGACC 786 955 UGACGAUC AGAA GUAU
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 772 AUACA GAC GAUCGUCA 787
1037 GUCGGUGG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 773
CAGCG GAC CCACCGAC 788 1045 GGCCGGGG AGAA GUGG
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 774 CCACC GAC CCCCGGCC 789
1410 CAUCAUCA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 775
CUGCA GUU UGAUGAUG 790 1453 ACAGCUGG AGAA GUGC
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 776 GCACA GAC CCAGCUGU 791
1471 GAUGCCAG AGAA GUGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 777
UCACA GAC CUGGCAUC 792
[0075]
7TABLE VII Mouse rel A Hairpin Ribozyme/Target Sequences nt. Seq.
ID Seq. ID Position Hairpin Ribozyme sequence No. Substrate No. 137
GUUGCUUC AGAA GUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 793 GAACA
GCC GAAGCAAC 812 273 GAGAUUCG AGAA GUUC
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 794 GAACA GUU CGAAUCUC 813 343
GCCAUCCC AGAA GUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 795 GGACU
GCC GGGAUGGC 814 366 GGGCAGAG AGAA GCCU
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 796 AGGCU GAC CUCUGCCC 815 633
UUGAGCUC AGAA GUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 797 ACACU
GCC GAGCUCAA 816 676 CCCACCGA AGAA GCUC
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 798 GAGCU GCC UCGGUGGG 817 834
AGGCUGGG AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 799 ACGCC
GAC CCCAGCCU 818 881 GAUCAGAA AGAA GCCG
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 800 CGGCG GCC UUCUGAUC 819
1100 AGGUGUAG AGAA GCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 801
CCGCA GCC CUACACCU 820 1205 GGGCAGAG AGAA GUGC
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 802 GCACC GUC CUCUGCCC 821
1361 GGGCUUCC AGAA GCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 803
ACGCU GUC GGAAGCCC 822 1385 CAGCAUCA AGAA GCAG
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 804 CUGCA GUU UGAUGCUG 823
1431 ACUCCUGG AGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 805
GCACA GAC CCAGGAGU 824 1449 GAUGCCAG AGAA GUGA
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 806 UCACA GAC CUGGCAUC 825
1802 AAGUCGGG AGAA GCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 807
CAGCU GCC CCCGACUU 826 2009 UGGCUCCA AGAA GUCC
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 808 GGACA GAC UGGAGCCA 827
2124 UGGUGUCG AGAA GCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 809
GUGCU GCC CGACACCA 828 2233 AUUCUGAA AGAA GCCA
ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 810 UGGCC GCC UUCAGAAU 829
2354 UCAGUAAA AGAA GUCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 811
AGACA GCC UUUACUGA 830
[0076]
Sequence CWU 1
1
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