U.S. patent application number 11/224524 was filed with the patent office on 2006-06-15 for genetic suppression and replacement.
This patent application is currently assigned to Optigen Patents Limited. Invention is credited to Gwenyth Jane Farrar, Peter Humphries, Paul Francis Kenna.
Application Number | 20060128648 11/224524 |
Document ID | / |
Family ID | 10791494 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060128648 |
Kind Code |
A1 |
Farrar; Gwenyth Jane ; et
al. |
June 15, 2006 |
Genetic suppression and replacement
Abstract
A strategy for suppressing specifically or partially
specifically an endogenous gene and introducing a replacement gene,
said strategy comprising the steps of: 1. providing suppressing
nucleic acids or other suppression effectors able to bind to an
endogenous gene, gene transcript or gene product to be suppressed
and 2. providing genomic DNA or cDNA (complete or partial) encoding
a replacement gene wherein the suppressing nucleic acids are unable
to bind to equivalent regions in the genomic DNA or cDNA to prevent
expression of the replacement gene. The replacement nucleic acids
have modifications in one or more third base (wobble) positions
such that replacement nucleic acids still code for the wild type or
equivalent amino acids.
Inventors: |
Farrar; Gwenyth Jane;
(Monkstown, IE) ; Humphries; Peter; (Cabinteeley,
IE) ; Kenna; Paul Francis; (Dublin, IE) |
Correspondence
Address: |
SULLIVAN & WORCESTER LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Assignee: |
Optigen Patents Limited
Dublin
IE
|
Family ID: |
10791494 |
Appl. No.: |
11/224524 |
Filed: |
September 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09155708 |
Apr 5, 1999 |
|
|
|
11224524 |
Sep 12, 2005 |
|
|
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 48/005 20130101;
C12N 2310/3181 20130101; C12N 2310/111 20130101; A61K 48/00
20130101; C12N 2310/15 20130101; C12N 15/113 20130101; C12N
2310/121 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 2, 1998 |
WO |
PCT/GB97/00929 |
Apr 2, 1996 |
GB |
GB9606961.2 |
Claims
1. A strategy for suppressing or partially suppressing an
endogenous gene and replacing the suppressed gene with a nucleic
acid sequence that differs from the endogenous gene, wherein the
suppressing agent comprises at least one suppressor selected from
the group consisting of an antisense nucleic acid, a peptide
nucleic acid, a nucleic acid capable of forming a triple helix, and
a ribozyme targeted to the endogenous gene or gene transcript,
wherein the replacement nucleic acid sequence encodes at least part
of a gene product and is not suppressed by a suppression agent or
is suppressed less efficiently by a suppression agent, and wherein
the replacement nucleic acid sequence comprises amino acid codons
that encode at least part of the gene product, and have
modifications at one or more wobble sites such that replacement
nucleic acid still encodes the wild type or equivalent amino
acids.
2. A medicament comprising either one or both of a gene suppressing
agent and a nucleic acid encoding at least part of a replacement
gene product, for use in a strategy as claimed in claim 1.
3. A strategy for suppressing or partially suppressing an
endogenous gene and introducing a replacement gene the strategy
comprising the steps of: a. providing a suppression nucleic acid
able to recognise, bind or cleave an endogenous gene, gene
transcript or gene product to be suppressed; and b. providing
complete or partial genomic DNA or cDNA encoding a replacement
gene, wherein the suppression nucleic acid is unable to recognise,
bind or cleave or able to recognise, bind or cleave less
efficiently, equivalent regions in the genomic DNA or cDNA to
prevent suppression of the replacement gene, wherein the coding
sequence of the replacement nucleic acid has been altered to
prevent or reduce efficiency of suppression and wherein the
replacement nucleic acid has modifications in one or more wobble
sites such that the replacement nucleic acid still codes for the
wild type or equivalent amino acids.
4. The use of a strategy as claimed in claim 3 in the preparation
of a medicament for the treatment of an autosomal dominant disease
caused by an endogenous target gene wherein the disease is caused
by different mutations in the same gene in different patients.
5. The use of: a. a vector containing a suppression effector, the
suppression effector able to recognise, bind or cleave a coding
sequence of a target endogenous gene; and b. a vector containing a
replacement nucleic acid in the form of genomic DNA, cDNA or RNA,
which contains altered wobble sites such that the replacement
nucleic acid cannot be recognised, bound or cleaved by the
suppression effector or are recognised, bound or cleaved less
efficiently by the suppression effector, which suppression effector
is targeted towards a coding sequence of the endogenous gene and
provides the wild type gene product and wherein the difference
between the endogenous gene and the replacement gene enables the
expression of the replacement gene; in the preparation of a
medicament for the treatment of an autosomal dominant disease
caused by the endogenous gene wherein the disease is caused by
different mutations in the same gene in different patients.
6. A use as claimed in claim 5 wherein the disease is a polygenic
disorder.
7. A use as claimed in claim 5 wherein the suppressor and/or
replacement gene is administered alone or in a vector chosen from
DNA plasmid vectors, RNA or DNA viral vectors.
8. A use as claimed in claim 5 wherein the suppressor and/or
replacement gene is combined with lipids, polymers or other
derivatives.
9. A use as claimed in claim 5 wherein the replacement gene is
altered from the wild type gene and provides a beneficial effect
when compared to the wild type gene.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/155,708, which was filed under 35 U.S.C. .sctn.371 for, and
claims priority to, PCT/GB97/00929, filed Apr. 2, 1997, which
claims priority to GB9606961.2, filed Apr. 2, 1996, the disclosures
of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a strategy for suppressing
a gene. In particular the invention relates to suppression of a
mutated gene that gives rise to a dominant or deleterious effect,
either monogenically or polygenically.
BACKGROUND OF THE INVENTION
[0003] Studies of degenerative hereditary ocular conditions,
including Retinitis Pigmentosa (RP) and various macular
dystrophies, have resulted in a substantial elucidation of the
molecular basis of these debilitating human retinal degenerations.
Applying the approach of genetic linkage, x-linked RP (xlRP) genes
have been localised to the short arm of the X chromosome (Ott et
al. 1990). Subsequently, the gene involved in one form of xlRP was
identified. Various genes involved in autosomal dominant forms of
RP (adRP) have been localised. The first of these mapped to 3q,
close to the gene encoding the rod photoreceptor protein rhodopsin
(McWilliam et al. 1989; Dryja et al. 1990). Similarly, an adRP gene
was placed on 6p close to the gene encoding the photoreceptor
protein peripherin (Farrar et al. 1991a,b; Kajiwara et al. 1991).
Other adRP genes have been mapped to discrete chromosomal
locations; however the disease genes as yet remain uncharacterised.
As in xlRP and adRP, various genes involved in autosomal recessive
RP (arRP) have been localised and in some cases molecular defects
characterised (Humphries et al. 1992; Farrar et al. 1993; Van Soest
et al. 1994). Similarly, a number of genes involved in macular
dystrophies have been mapped (Mansergh et al. 1995). Genetic
linkage, together with techniques for mutational screening of
candidate genes, enabled identification of causative dominant
mutations in the genes encoding rhodopsin and peripherin. Globally,
about 100 rhodopsin mutations have been found in patients with RP
or congenital stationary night blindness. Similarly, approximately
40 mutations have been characterised in the peripherin gene in
patients with RP or macular dystrophies. Knowledge of the molecular
aetiology of these retinopathies has stimulated the generation of
animal models and the exploration of methods of therapeutic
intervention (Farrar et al. 1995; Humphries et al. 1997).
[0004] Similar to RP, osteogenesis imperfecta (OI) is an autosomal
dominantly inherited human disorder whose molecular pathogenesis is
extremely genetically heterogeneous. OI is often referred to as
`brittle bone disease`, although additional symptoms including
hearing loss, growth deficiency, bruising, loose joints, blue
sclerae and dentinogenesis imperfecta are frequently observed
(McKusick, 1972). Mutations in the genes encoding the two type I
collagen chains (collagen 1A1 and 1A2) comprising the type I
collagen heterodimer have been implicated in OI. Indeed hundreds of
dominantly acting mutations have been identified in OI patients in
these two genes, many of which are single point mutations, although
a number of insertion and deletion mutations have been found
(Willing et al. 1993; Zhuang et al. 1996). Similarly mutations in
these genes have also been implicated in Ehlers-Danlos and Marfan
syndromes (Dalgleish et al. 1986; Phillips et al. 1990; D'Alessio
et al. 1991; Vasan NS et al. 1991).
[0005] Generally, gene therapies utilising viral and non-viral
delivery systems have been used to treat inherited disorders,
cancers and infectious diseases. However, many studies have focused
on recessively inherited disorders, the rationale being that
introduction and expression of the wild type gene may be sufficient
to prevent/ameliorate the disease phenotype. In contrast gene
therapy for dominant disorders requires suppression of the dominant
disease allele. Notably many of the characterised mutations causing
inherited diseases such as RP or OI are inherited in an autosomal
dominant fashion. Indeed there are over 1,000 autosomal dominantly
inherited disorders in man. In addition, there are many polygenic
disorders due to co-inheritance of a number of genetic components
which together give rise to the disease state. Effective gene
therapies for dominant or polygenic diseases may be targeted to the
primary defect and in this case may require suppression of the
disease allele while in many cases still maintaining the function
of the normal allele. This is particularly relevant where disease
pathology is due to a gain of function mutation rather than due to
reduced levels of wild type protein. Alternatively, suppression
therapies may be targeted to secondary effects associated with the
disease pathology, for example, programmed cell death (apoptosis),
which has been observed in many inherited disorders.
[0006] Strategies to differentiate between normal and disease
alleles and to selectively switch off the disease allele using
suppression effectors such as antisense DNA/RNA, PNAs, ribozymes,
or triple helix forming DNA, targeted towards the disease mutation
may be difficult in many cases--frequently disease and normal
alleles differ by only a single nucleotide. A further difficulty
inhibiting development of gene therapies is the heterogeneous
nature of some dominant disorders--many different mutations in the
same gene give rise to a similar disease phenotype. Development of
specific gene therapies for each of these may be prohibitive in
terms of cost.
[0007] Suppression effectors have been used previously to achieve
specific suppression of gene expression. Antisense DNA and RNA has
been used to inhibit gene expression in many instances.
Modifications, such as phosphorothioates, have been made to
oligonucleotides to increase resistance to nuclease degradation,
binding affinity and uptake (Cazenave et al. 1989; Sun et al. 1989;
McKay et al. 1996; Wei et al. 1996). In some instances, using
antisense and ribozyme suppression strategies has led to reversal
of a tumor phenotype by reducing expression of a gene product or by
cleaving a mutant transcript at the site of the mutation (Carter
and Lemoine 1993; Lange et al. 1993; Valera et al. 1994;
Dosaka-Akita et al. 1995; Feng et al. 1995; Quattrone et al. 1995;
Ohta et al. 1996). For example, neoplastic reversion was obtained
using a ribozyme targeted to an H-ras mutation in bladder carcinoma
cells (Feng et al. 1995). Ribozymes have also been proposed as a
means of both inhibiting gene expression of a mutant gene and of
correcting the mutant by targeted trans-splicing (Sullenger and
Cech 1994; Jones et al. 1996). Ribozymes can be designed to elicit
autocatalytic cleavage of RNA targets; however, the inhibitory
effect of some ribozymes may be due in part to an antisense effect
due to the antisense sequences flanking the catalytic core that
specify the target site (Ellis and Rodgers 1993; Jankowsky and
Schwenzer 1996). Ribozyme activity may be augmented by the use of,
for example, non-specific nucleic acid binding proteins or
facilitator oligonucleotides (Herschlag et al. 1994; Jankowsky and
Schwenzer 1996). Multitarget ribozymes (connected or shotgun) have
been suggested as a means of improving efficiency of ribozymes for
gene suppression (Ohkawa et al. 1993). Triple helix approaches have
also been investigated for sequence-specific gene
suppression--triplex forming oligonucleotides have been found in
some cases to bind in a sequence-specific manner (Postel et al.
1991; Duval-Valentin et al. 1992; Hardenbol and Van Dyke 1996;
Porumb et al. 1996). Similarly, peptide nucleic acids have been
shown to inhibit gene expression (Hanvey et al. 1992; Knudson and
Nielsen 1996; Taylor et al. 1997). Minor groove binding polyamides
can bind in a sequence-specific manner to DNA targets and hence may
represent useful small molecules for future suppression at the DNA
level (Trauger et al. 1996). In addition, suppression has been
achieved by interference at the protein level using dominant
negative mutant peptides and antibodies (Herskowitz 1987; Rimsky et
al. 1989; Wright et al. 1989). In some cases suppression strategies
have lead to a reduction in RNA levels without a concomitant
reduction in proteins, whereas in others, reductions in RNA have
been mirrored by reductions in protein.
SUMMARY OF THE INVENTION
[0008] To circumvent difficulties associated with specifically
targeting a disease mutation and with the genetic heterogeneity
present in inherited disorders, a novel strategy for gene
suppression and gene replacement exploiting the degeneracy of the
genetic code is described. The invention allows flexibility in
choice of target sequence for suppression and provides a means of
gene suppression that is independent of the disease mutation.
[0009] In summary, the invention involves gene suppression of
disease and normal alleles targeting coding sequences in a gene
and, when necessary, gene replacement such that the replacement
gene cannot be suppressed. Replacement genes are modified at third
base positions (wobble positions) so that they code for the correct
amino acids but are protected completely or partially from
suppression. The same suppression and replacement steps can be used
for many disease mutations in a given gene. Suppression and
replacement can be undertaken in conjunction with each other or
separately.
[0010] The invention relates to a strategy for suppressing a gene
or disease allele using methods that do not target the disease
allele specifically but instead can be targeted towards a broad
range of sequences in a particular gene. A particular embodiment of
the invention is the use of suppression strategies to target either
the disease or normal alleles alone or to target both disease and
normal alleles. A further embodiment of the invention is the use of
the wobble hypothesis to enable continued expression of a
replacement normal or beneficial gene (a gene modified from the
wild type such that it provides an additional beneficial
effect(s)). The replacement gene will have nucleotide changes from
the endogenous wild type gene but will code for identical amino
acids as the wild type gene. The strategy circumvents the need for
a specific therapy for every mutation within a given gene. In
addition, the invention allows greater flexibility in choice of
target sequence for suppression of a disease allele.
[0011] The invention also relates to a medicament or medicaments
for use in suppressing a deleterious allele that is present in a
genome of one or more individuals or animals.
[0012] Generally, the present invention will be useful where the
gene, which is naturally present in the genome of a patient,
contributes to a disease state. Generally, one allele of the gene
in question will be mutated, that is, will possess alterations in
its nucleotide sequence that affects the function or level of the
gene product. For example, the alteration may result in an altered
protein product from the wild type gene or altered control of
transcription and processing. Inheritance or somatic acquisition of
such a mutation can give rise to a disease phenotype or can
predispose an individual to a disease phenotype. However the gene
of interest could also be of wild type phenotype, but contribute to
a disease state in another way such that the suppression of the
gene would alleviate or improve the disease state or improve the
effectiveness of an administered therapeutic compound.
[0013] Generally, suppression effectors such as nucleic
acids--antisense or sense, ribozymes, peptide nucleic acids (PNAs),
triple helix forming oligonucleotides, peptides and/or antibodies
directed to sequences in a gene, in transcripts or in protein, can
be employed in the invention to achieve gene suppression.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The invention addresses shortcomings of the prior art by
providing a novel approach to the design of suppression effectors
directed to target alleles of a gene carrying a deleterious
mutation. Suppression of every mutation giving rise to a disease
phenotype may be costly and problematic. Disease mutations are
often single nucleotide changes. As a result differentiating
between the disease and normal alleles may be difficult. Some
suppression effectors require specific sequence targets, for
example, hammerhead ribozymes cleave at NUX sites and hence may not
be able to target many mutations. Notably, the wide spectrum of
mutations observed in many diseases adds additional complexity to
the development of therapeutic strategies for such disorders--some
mutations may occur only once in a single patient. A further
problem associated with suppression is the high level of homology
present in coding sequences between members of some gene families.
This can limit the range of target sites for suppression that will
enable specific suppression of a single member of such a gene
family.
[0015] The present invention circumvents shortcomings in the prior
art by utilising the degeneracy of the genetic code. In the
invention suppression effectors are designed specifically to
sequences in coding regions of genes or in gene products.
Typically, one allele of the gene contains a mutation with a
deleterious effect. Suppression targeted to coding sequences
provides greater flexibility in choice of target sequence for
suppression in contrast to suppression directed towards single
disease mutations. Additionally, the invention provides for the
introduction of a replacement gene with modified sequences such
that the replacement gene is protected from suppression. The
replacement gene is modified at third base wobble positions and
hence provides the wild type gene product. Notably, the invention
has the advantage that the same suppression strategy could be used
to suppress, in principle, many mutations in a gene. This is
particularly relevant when large numbers of mutations within a
single gene cause disease pathology. The replacement gene provides
(when necessary) expression of the normal protein product when
required to ameliorate pathology associated with reduced levels of
wild type protein. The same replacement gene could in principle be
used in conjunction with the suppression of many different disease
mutations within a given gene. Target sequences may be found in any
part of the coding sequence. Suppression in coding sequence holds
the advantage that such sequences are present in both precursor and
mature RNAs, thereby enabling suppressors to target all forms of
RNA.
[0016] There is now an armament with which to obtain gene
suppression. This, in conjunction with a better understanding of
the molecular aetiology of disease, results in an ever increasing
number of disease targets for therapies based on suppression. In
many cases, complete suppression of gene expression has been
difficult to achieve. Possibly a combined approach using a number
of suppression effectors may aid in this. For some disorders it may
be necessary to block expression of a disease allele completely to
prevent disease symptoms whereas for others low levels of mutant
protein may be tolerated. In parallel with an increased knowledge
of the molecular defects causing disease has been the realisation
that many disorders are genetically heterogeneous. Examples in
which multiple genes and/or multiple mutations within a gene can
give rise to a similar disease phenotype include osteogenesis
imperfecta, familial hypercholesterolemia, retinitis pigmentosa,
and many others. The utility of the degeneracy of the genetic code
(wobble hypothesis) to enable suppression of one or both alleles of
a gene and the introduction of a replacement gene such that it
escapes suppression has been exploited in the invention.
[0017] According to the present invention there is provided a
strategy for suppressing expression of an endogenous gene with a
deleterious mutation, wherein said strategy comprises providing
suppression effectors such as antisense nucleic acids able to bind
to sequences of a gene to be suppressed, to prevent the functional
expression thereof.
[0018] Generally the term suppression effectors means nucleic
acids, peptide nucleic acids (PNAs), peptides, antibodies or
modified forms of these used to silence or reduce gene expression
in a sequence specific manner.
[0019] Suppression effectors, such as antisense nucleic acids can
be DNA or RNA, can typically be directed to coding sequence;
however suppression effectors can be targeted to coding sequence
and can also be targeted to 5' and/or 3' untranslated regions
and/or introns and/or control regions and/or sequences adjacent to
a gene or to any combination of such regions of a gene. Antisense
nucleic acids including both coding and non-coding sequence can be
used if required to help to optimise suppression. Binding of the
suppression effector(s) prevents or lowers functional expression of
the endogenous gene.
[0020] Generally the term `functional expression` means the
expression of a gene product able to function in a manner
equivalent to, or better than, a wild type product. In the case of
a mutant gene or predisposing gene `functional expression` means
the expression of a gene product whose presence gives rise to a
deleterious effect or predisposes to a deleterious effect. By
deleterious effect is meant giving rise to or predisposing to
disease pathology or altering the effect(s) and/or efficiency of an
administered compound.
[0021] In a particular embodiment of the invention the strategy
further employs ribozymes that can be designed to elicit cleavage
of target RNAs. The strategy further employs nucleotides that form
triple helix DNA. The strategy can employ peptide nucleic acids for
suppression. Nucleic acids for antisense, ribozymes, triple helix
forming DNA and peptide nucleic acids may be modified to increase
stability, binding efficiencies and uptake. Nucleic acids can be
incorporated into a vector. Vectors include naked DNA, DNA plasmid
vectors, RNA or DNA virus vectors, lipids, polymers or other
derivatives and compounds to aid gene delivery and expression.
[0022] The invention further provides the use of antisense
nucleotides, ribozymes, PNAs, triple helix nucleotides, or other
suppression effectors alone or in a vector or vectors, wherein the
nucleic acids are able to bind specifically or partially
specifically to coding sequences of a gene to prevent or reduce the
functional expression thereof, in the preparation of a medicament
for the treatment of an autosomal dominant or polygenic disease or
to increase the utility and/or action of an administered
compound.
[0023] In a further embodiment of the invention, target sequences
for suppression can include non-coding sequences of the gene.
[0024] According to the present invention there is provided a
strategy for suppressing specifically or partially specifically an
endogenous gene and (if required) introducing a replacement gene,
said strategy comprising the steps of: [0025] 1. providing nucleic
acids or other suppression effectors able to bind to an endogenous
gene, gene transcript or gene product to be suppressed and [0026]
2. providing genomic DNA or cDNA (complete or partial) encoding a
replacement gene wherein the nucleic acids are unable to bind to
equivalent regions in the genomic DNA or cDNA to prevent expression
of the replacement gene. The replacement nucleic acids will not be
recognised by suppression nucleic acids or will be recognised less
effectively than the endogenous gene. The coding sequence of
replacement nucleic acids can be altered to prevent or reduce
efficiency of suppression. Replacement nucleic acids have
modifications in one or more third base (wobble) positions such
that replacement nucleic acids still code for the wild type or
equivalent amino acids.
[0027] In a particular embodiment of the invention there is
provided a strategy for gene suppression targeted to coding
sequences of the gene to be suppressed. Suppression will be
specific or partially specific to one allele, for example, to the
allele carrying a deleterious mutation. Suppressors are targeted to
coding regions of a gene or to a combination of coding and
non-coding regions of a gene. Suppressors can be targeted to a
characteristic of one allele of a gene such that suppression is
specific or partially specific to one allele of a gene
(PCT/GB97/00574). The invention further provides for use of
replacement nucleic acids with altered coding sequences such that
replacement nucleic acids will not be recognised (or will be
recognised less effectively) by suppression nucleic acids that are
targeted specifically or partially specifically to one allele of
the gene to be suppressed. Replacement nucleic acids provide the
wild type gene product, an equivalent gene product or an improved
gene product but are protected completely or partially from
suppression effectors targeted to coding sequences.
[0028] In a further embodiment of the invention, replacement
nucleic acids are provided such that replacement nucleic acids will
not be recognised by naturally occurring suppressors found to
inhibit or reduce gene expression in one or more individuals,
animals or plants. The invention provides for use of replacement
nucleic acids that have altered sequences targeted by suppressors
of the gene such that suppression by naturally occurring
suppressors is completely or partially prevented.
[0029] In an additional embodiment of the invention, there is
provided replacement nucleic acids with altered nucleotide sequence
in coding regions such that replacement nucleic acids code for a
product with one or more altered amino acids. Replacement nucleic
acids provide a gene product that is equivalent to or improved
compared with the naturally occurring endogenous wild type gene
product.
[0030] In an additional embodiment of the invention there is
provided a strategy to suppress a gene where the gene transcript or
gene product interferes with the action of an administered
compound.
[0031] The invention further provides the use of a vector or
vectors containing suppression effectors in the form of nucleic
acids, said nucleic acids being directed towards coding sequences
or combinations of coding and non-coding sequences of the target
gene and vector(s) containing genomic DNA or cDNA encoding a
replacement gene sequence to which nucleic acids for suppression
are unable to bind (or bind less efficiently), in the preparation
of a combined medicament for the treatment of an autosomal dominant
or polygenic disease. Nucleic acids for suppression or replacement
gene nucleic acids may be provided in the same vector or in
separate vectors. Nucleic acids for suppression or replacement gene
nucleic acids may be provided as a combination of nucleic acids
alone or in vectors.
[0032] The invention further provides a method of treatment for a
disease caused by an endogenous mutant gene, said method comprising
sequential or concomitant introduction of [0033] (a) nucleic acids
to the coding regions of a gene to be suppressed and/or nucleic
acids to coding regions and any combination of 5' and/or 3'
untranslated regions, intronic regions, control regions or regions
adjacent to a gene to be suppressed [0034] (b) replacement nucleic
acids with sequences that allow the replacement gene to be
expressed.
[0035] The nucleic acid for gene suppression can be administered
before, after or at the same time as the replacement gene is
administered.
[0036] The invention further provides a kit for use in the
treatment of a disease caused by a deleterious mutation in a gene,
the kit comprising nucleic acids for suppression able to bind to
the gene to be suppressed and if required a replacement nucleic
acid to replace the mutant gene having sequence that allows it to
be expressed and completely or partially escape suppression.
[0037] Nucleotides can be administered as naked DNA or RNA.
Nucleotides can be delivered in vectors. Naked nucleic acids or
nucleic acids in vectors can be delivered with lipids or other
derivatives which aid gene delivery. Nucleotides may be modified to
render them more stable, for example, resistant to cellular
nucleases while still supporting RNase H mediated degradation of
RNA or with increased binding efficiencies. Antibodies or peptides
can be generated to target the protein product from the gene to be
suppressed.
[0038] The strategy described herein has applications for
alleviating autosomal dominant diseases. Complete silencing of a
disease allele may be difficult to achieve using antisense, PNA,
ribozyme and triple helix approaches or any combination of gene
silencing approaches. However small quantities of mutant product
may be tolerated in some autosomal dominant disorders. In others a
significant reduction in the proportion of mutant to normal product
may result in an amelioration of disease symptoms. Hence, this
invention may be applied to any autosomal dominantly or
polygenically inherited disease in man where the molecular basis of
the disease has been established or is partially understood. This
strategy enables the same therapy to be used to treat a range of
different disease mutations within the same gene. The development
of such approaches is important to future therapies for autosomal
dominant and polygenic diseases, the key to a general strategy
being that it circumvents the need for a specific therapy for every
mutation causing or predisposing to a disease. This is particularly
relevant in some disorders, for example, rhodopsin linked autosomal
dominant RP, in which to date about one hundred different mutations
in the rhodopsin gene have been observed in adRP patients.
Likewise, hundreds of mutations have been identified in the human
type I Collagen 1A1 and 1A2 genes in autosomal dominant
osteogenesis imperfecta. Costs of developing therapies for each
mutation are prohibitive at present. Inventions such as this using
a general approach for therapy will be required. General approaches
may be targeted to the primary defect, as is the case with this
invention, or to secondary effects such as apoptosis.
[0039] This invention may be applied in gene therapy approaches for
biologically important polygenic disorders affecting large
proportions of the world's populations such as age related macular
degeneration, glaucoma, manic depression, cancers having a familial
component and indeed many others. Polygenic diseases require
inheritance of more than one mutation (component) to give rise to
the disease state. Notably an amelioration in disease symptoms may
require reduction in the presence of only one of these components,
that is, suppression of one genotype which, together with others
leads to the disease phenotype, may be sufficient to prevent or
ameliorate symptoms of the disease. In some cases suppression of
more than one component may be required to improve disease
symptoms. This invention may be applied in possible future
interventive therapies for common polygenic diseases to suppress a
particular genotype(s) using suppression and, when necessary,
replacement steps.
[0040] The present invention is exemplified using four genes: human
rhodopsin, mouse rhodopsin, human peripherin and human collagen
1A2. The first of these genes are retinal specific. In contrast,
collagen 1A2 is expressed in a range of tissues including skin and
bone. While these four genes have been used as examples there is no
reason why the invention could not be deployed in the suppression
of many other genes in which mutations cause or predispose to a
deleterious effect. Many examples of mutant genes that give rise to
disease phenotypes are available from the prior art--these genes
all represent targets for the invention. The present invention is
exemplified using hammerhead ribozymes with antisense arms to
elicit RNA cleavage. There is no reason why other suppression
effectors directed towards genes, gene transcripts or gene products
could not be used to achieve gene suppression. Many examples from
the prior art detailing use of suppression effectors such as, inter
alia, antisense RNA/DNA, triple helix forming DNA, PNAs and
peptides to achieve suppression of gene expression are reported.
The present invention is exemplified using hammerhead ribozymes
with antisense arms to elicit sequence specific cleavage of
transcripts from genes implicated in dominant disorders and
non-cleavage of transcripts from replacement genes containing
sequence modifications in wobble positions such that the
replacement gene still codes for wild type protein. The present
invention is exemplified using suppression effectors targeting
sites in coding regions of the human and mouse rhodopsin, human
peripherin and human collagen 1A2 genes. Target sites, which
include sequences from transcribed but untranslated regions of
genes, introns, regions involved in the control of gene expression,
regions adjacent to the gene or any combination of these, could be
used to achieve gene suppression. Multiple suppression effectors,
for example, shotgun ribozymes could be used to optimise efficiency
of suppression when necessary. Additionally, when required,
expression of a modified replacement gene such that the replacement
gene product is altered from the wild type product such that it
provides a beneficial effect may be used to provide gene
product.
Materials and Methods
Cloning vectors
[0041] cDNA templates and ribozymes were cloned into commercial
expression vectors (pCDNA3, pZeoSV or pbluescript) that enable
expression in a test tube from T7, T3 or SP6 promoters or
expression in mammalian cells from CMV or SV40 promoters. DNA
inserts were cloned into the multiple cloning site (MCS) of these
vectors typically at or near the terminal ends of the MCS to delete
most of the MCS and thereby prevent any possible problems with
efficiency of expression subsequent to cloning.
Sequencing Protocols
[0042] Clones containing template cDNAs and ribozymes were
sequenced by ABI automated sequencing machinery using standard
protocols.
Expression of RNAs
[0043] RNA was obtained from clones by in vitro transcription using
a commercially available Ribomax expression system (Promega) and
standard protocols. RNA purifications were undertaken using the
Bio-101 RNA purification kit or a solution of 0.3M sodium acetate
and 0.2% SDS after isolation from polyacrylamide gels. Cleavage
reactions were performed using standard protocols with varying
MgCl.sub.2 concentrations (0-15 mM) at 37.degree. C., typically for
3 hours. Time points were performed at the predetermined optimal
MgCl.sub.2 concentrations for up to 5 hours. Radioactively labelled
RNA products were obtained by incorporating .alpha.-p.sup.32 rUTP
(Amersham) in the expression reactions (Gaughan et al. 1995).
Labelled RNA products were run on polyacrylamide gels before
cleavage reactions were undertaken for the purpose of RNA
purification and subsequent to cleavage reactions to establish if
RNA cleavage had been achieved. Cleavage reactions were undertaken
with 5 mM Tris-HCl pH8.0 and varying concentrations of MgCl.sub.2
at 37.degree. C.
RNA Secondary Structures
[0044] Predictions of the secondary structures of human and mouse
rhodopsin, human peripherin and human collagen 1A2 mRNAs were
obtained using the RNAPlotFold program. Ribozymes and antisense
were designed to target areas of the RNA that were predicted to be
accessible to suppression effectors, for example open loop
structures. The integrity of open loop structures was evaluated
from the 10 most probable RNA structures. Additionally, predicted
RNA structures for truncated RNA products were generated and the
integrity of open loops between full length and truncated RNAs
compared.
Templates and Ribozymes
Human Rhodopsin
Template cDNA
[0045] The human rhodopsin cDNA (SEQ ID NO:1) was cloned into the
HindIII and EcoRI sites of the MCS of pCDNA3 in a 5' to 3'
orientation allowing subsequent expression of RNA from the T7 or
CMV promoter in the vector. The full length 5' UTR sequence was
inserted into this clone using primer driven PCR mutagenesis and a
HindIII (in pCDNA3) to BstEII (in the coding sequence of the human
rhodopsin cDNA) DNA fragment.
cDNA with Altered Sequence at a Wobble Position
[0046] The human rhodopsin hybrid CDNA with a single base
alteration (SEQ ID NO:2), a C-->G change (at nucleotide 271 of
SEQ ID NO:2) was introduced into human rhodopsin CDNA, using a
HindIII to BstEII PCR cassette, by primer directed PCR mutagenesis.
This sequence change occurs at a silent position--it does not give
rise to an amino acid substitution--however it eliminates the
ribozyme cleavage site (GUX -->GUG). The hybrid rhodopsin was
cloned into pCDNA3 in a 5' to 3' orientation allowing subsequent
expression of RNA from the T7 or CMV promoter in the vector.
Rhodopsin cDNA Carrying a Pro23Leu adRP Mutation
[0047] A human rhodopsin adRP mutation, a C-->T change (at codon
23; nucleotide 217 of SEQ ID NO:3) was introduced into human
rhodopsin cDNA, using a HindIII to BstEII PCR cassette by primer
directed PCR mutagenesis. This sequence change results in the
substitution of a Proline for a Leucine residue. Additionally the
nucleotide change creates a ribozyme cleavage site (CCC-->CTC)
(nucleotide 216-218 of SEQ ID NO:3). The mutated rhodopsin nucleic
acid sequence was cloned into the HindIII and EcoRI sites of pCDNA3
in a 5' to 3' orientation allowing subsequent expression of RNA
from the T7 or CMV promoter in the vector (SEQ ID NO:3).
Ribozyme Constructs
[0048] A hammerhead ribozyme (termed Rz10 (SEQ ID NO:29) designed
to target a large conserved open loop structure in the RNA from the
coding regions of the gene was cloned subsequent to synthesis and
annealing into the HindIII and XbaI sites of pCDNA3 again allowing
expression of RNA from the T7 or CMV promoter in the vector (SEQ ID
NO:4). The target site was GUC (the GUX rule) at position 475-477
(nucleotides 369-371 of SEQ ID NO:1) of the human rhodopsin
sequence. Note there is a one base mismatch in one antisense arm of
Rz10. A hammerhead ribozyme (termed Rz20(SEQ ID NO:30) designed to
target an open loop structure in RNA from the coding region of a
mutant rhodopsin gene with a Pro23Leu mutation was cloned
subsequent to synthesis and annealing into the HindIII and XbaI
sites of pCDNA3 again allowing expression of RNA from the T7 or CMV
promoter in the vector (SEQ ID NO:5). The target site was CTC (the
NUX rule) at codon 23 (nucleotides 216-218 of SEQ ID NO:3) of the
human rhodopsin sequence (Accession number: K02281). Antisense
flanks are underlined. TABLE-US-00001 (SEQ ID NO:29; nucleotides
101-137 of SEQ ID NO:4) Rz10: GGTCGGTCTGATGAGTCCGTGAGGACGAAACGTAGAG
(SEQ ID NO:30; nucleotides 104-140 of SEQ ID NO:5) Rz20:
TACTCGAACTGATGAGTCCGTGAGGACGAAAGGCTGC
Mouse Rhodopsin Template cDNA
[0049] The full length mouse rhodopsin CDNA was cloned into the
EcoRI sites of the MCS of pCDNA3 in a 5' to 3' orientation allowing
subsequent expression of RNA from the T7 or CMV promoter in the
vector (SEQ ID NO:6).
cDNA with Altered Sequence at a Wobble Position
[0050] The mouse rhodopsin hybrid cDNA with a single base
alteration, a T-->C change (at position 1460) (nucleotide 190 of
SEQ ID NO:7) was introduced into mouse rhodopsin cDNA, using a
HindIII to Eco47III PCR cassette, by primer directed PCR
mutagenesis. This sequence change occurs at a silent position--it
does not give rise to an amino acid substitution--however it
eliminates the ribozyme cleavage site (TTT-->TCT) (nucleotides
189-191 of SEQ ID NO:7). The hybrid rhodopsin was cloned into
pCDNA3 in a 5' to 3' orientation allowing subsequent expression of
RNA from the T7 or CMV promoter in the vector (SEQ ID NO:7).
Ribozyme Constructs
[0051] A hammerhead ribozyme (termed Rz33) (SEQ ID NO:31) designed
to target a large robust open loop structure in the RNA from the
coding regions of the gene was cloned subsequent to synthesis and
annealing into the HindIII and XbaI sites of pCDNA3 again allowing
expression of RNA from the T7 or CMV promoter in the vector (SEQ ID
NO:8). The target site was TTT (the NUX rule) at position 1459-1461
(nucleotides 405-407 of SEQ ID NO:6) of the mouse rhodopsin
sequence. (Accession number: M55171). Antisense flanks are
underlined. TABLE-US-00002 (SEQ ID NO:31; nucleotides 118-154 of
SEQ ID NO:8) Rz33: GGCACATCTGATGAGTCCGTGAGGACGAAAAAATTGG
Human Peripherin Template cDNA
[0052] The full length human peripherin cDNA was cloned into the
EcoRI sites of the MCS of pCDNA3 in a 5' to 3' orientation allowing
subsequent expression of RNA from the T7 or CMV promoter in the
vector (SEQ ID NO:9).
DNAs with Altered Sequence at a Wobble Position
[0053] A human peripherin hybrid DNA with a single base alteration,
a A-->G change (at position 257) (nucleotide 332 of SEQ ID
NO:10) was introduced into human peripherin DNA by primer directed
PCR mutagenesis (forward 257 mutation
primer--5'CATGGCGCTGCTGAAAGTCA3' (SEQ ID NO:11)--the reverse 257
primer was complementary to the forward primer). This sequence
change occurs at a silent position--it does not give rise to an
amino acid substitution--however it eliminates the ribozyme
cleavage site (CTA-->CTG)(nucleotide 330-332 of SEQ ID NO:10). A
second human peripherin hybrid DNA with a single base alteration, a
A-->G change (at position 359) (nucleotide 468 of SEQ ID NO:13)
was introduced into human peripherin DNA, again by primer directed
PCR mutagenesis (forward 359 mutation
primer--5'CATCTTCAGCCTGGGACTGT3' (SEQ ID NO:12)--the reverse 359
primer was complementary to the forward primer) (SEQ ID NO:12).
Similarly this sequence change occurs at a silent position--it does
not give rise to an amino acid substitution--however again it
eliminates the ribozyme cleavage site (CTA-->CTG) (nucleotides
466-468 of SEQ ID NO:13). The ribozyme cleavage sites at 255-257
(nucleotides 330-332 of SEQ ID NO:10) and 357-359 (nucleotides
466-468 of SEQ ID NO:13) occur at different open loop structures as
predicted by the RNAPlotFold program. Hybrid peripherin DNAs
included the T7 promoter sequence allowing subsequent expression of
RNA.
Ribozyme Constructs
[0054] Hammerhead ribozymes (termed Rz30 and Rz31)(SEQ ID NOs: 32
and 33, respectively), designed to target robust open loop
structures in the RNA from the coding regions of the gene, were
cloned subsequent to synthesis and annealing into the HindIII and
XbaI sites of pCDNA3 again allowing expression of RNA from the T7
or CMV promoter in the vector (SEQ ID NOS:14 and 15, respectively).
The target sites were both CTA (the NUX rule) at positions 255-257
and 357-359 respectively of the human peripherin sequence.
(Accession number: M73531). Antisense flanks are underlined.
TABLE-US-00003 (SEQ ID NO:32; nucleotides 116-153 of SEQ ID NO:14)
Rz30: ACTTTCAGCTGATGAGTCCGTGAGGACGAAAGCGCCA (SEQ ID NO:33;
nucleotides 112-148 of SEQ ID NO:15) Rz31:
ACAGTCCCTGATGAGTCCGTGAGGACGAAAGGCTGAA
Human Type I Collagen--COL1A2 Template cDNA
[0055] A human type I collagen 1A2 cDNA was obtained from the ATCC
(Accession No: Y00724). A naturally occurring polymorphism has
previously been found in collagen 1A2 at positions 907 of the gene
involving a T-->A nucleotide change (Filie et al. 1993). The
polymorphism occurs in a predicted open loop structure of human
collagen 1A2 RNA. Polymorphic variants of human collagen 1A2 were
generated by PCR directed mutagenesis, using a HindIII to XbaI PCR
cassette. Resulting clones contained the following polymorphism :
collagen 1A2 (A) has a T nucleotide at position 907 (A nucleotide
176 of SEQ ID NO:17, reverse strand). In contrast human collagen
1A2 (B) has an A nucleotide at position 907 (T nucleotide 181 of
SEQ ID NO:16, reverse strand). In collagen 1A2 (A) there is a
ribozyme target site, that is a GTC site (906-908) (nucleotides
175-177 of SEQ ID NO:17, reverse strand), however this cleavage
site is lost in collagen 1A2 (B) as the sequence is altered to GAC
(906-908) (nucleotides 180-182 of SEQ ID NO:16, reverse strand),
thereby disrupting the ribozyme target site.
Ribozyme Constructs
[0056] A hammerhead ribozyme (termed Rz907) (SEQ ID NO:34) was
designed to target a predicted open loop structure in the RNA from
the coding region of the polymorphic variant of the human collagen
1A2 gene. Rz907 oligonucleotide primers were synthesised, annealed
and cloned into the HindIII and XbaI sites of pCDNA3 again allowing
subsequent expression of RNA from the T7 or CMV promoter in the
vector (SEQ ID NO:18). The target site was a GUX site at position
906-908 of the human type I collagen 1A2 sequence (Accession
number: Y00724). Antisense flanks are underlined. TABLE-US-00004
(SEQ ID NO:34; nucleotide 107-141 of SEQ ID NO:18) Rz907:
CGGCGGCTGATGAGTCCGTGAGGACGAAACCAGCA
FIGURE LEGENDS
[0057] FIG. 1:
[0058] pBR322 was cut with MspI, radioactively labelled and run on
a polyacrylamide gel to enable separation of the resulting DNA
fragments. The sizes of these fragments are given in FIG. 1. This
DNA ladder was then used on subsequent polyacrylamide gels (4-8%)
to provide an estimate of the size of the RNA products run on the
gels. However there is a significant difference in mobility between
DNA and RNA depending on the percentage of polyacrylamide and the
gel running conditions--hence the marker provides an estimate of
size of transcripts.
[0059] FIG. 2: [0060] A: Human rhodopsin CDNA (SEQ ID NO:1) was
expressed from the T7 promoter to the BstEII site in the coding
sequence. Resulting RNA was mixed with RZ10RNA in 15 mM magnesium
chloride and incubated at 37.degree. C. for varying times. Lanes
1-4: Human rhodopsin RNA and Rz10RNA after incubation at 37.degree.
C. with 15 mM magnesium chloride for 0, 1 2 and 3 hours
respectively. Sizes of the expressed RNAs and cleavage products are
as expected (Table 1). Complete cleavage of human rhodopsin RNA was
obtained with a small residual amount of intact RNA present at 1
hour. Lane 6 is intact unadapted human rhodopsin RNA (BstEII)
alone. Lane 5 is unadapted human rhodopsin RNA (FspI) alone and
refers to FIG. 2B. From top to bottom, human rhodopsin RNA and the
two cleavage products from this RNA are highlighted with arrows.
[0061] B: The unadapted human rhodopsin cDNA was expressed from the
T7 promoter to the FspI site in the coding sequence. The adapted
human rhodopsin cDNA was expressed from the T7 promoter to the
BstEII site in the coding sequence. Lanes 1-4: Resulting RNAs were
mixed together with Rz10 and 15 mM magnesium chloride and incubated
at 37.degree. C. for varying times (0, 1 , 2 and 3 hours
respectively). The smaller unadapted rhodopsin transcripts were
cleaved by Rz10 while the larger adapted transcripts were protected
from cleavage by Rz10. Cleavage of adapted protected transcripts
would have resulted in products of 564bases and 287bases--the
564bases product clearly is not present--the 287bp product is also
generated by cleavage of the unadapted human rhodopsin transcripts
and hence is resent (FspI). After 3 hours the majority of the
unadapted rhodopsin transcripts has been cleaved by Rz10. Lane 5
contains the intact adapted human rhodopsin RNA (BstEII) alone.
From top to bottom adapted uncleaved human rhodopsin transcripts,
residual unadapted uncleaved human rhodopsin transcripts and the
larger of the cleavage products from unadapted human rhodopsin
transcripts are highlighted by arrows. The smaller 22 bases
cleavage product from the unadapted human rhodopsin transcripts has
run off the gel.
[0062] FIG. 3: [0063] A: Unadapted (SEQ ID NO:l) and adapted (SEQ
ID NO:2) human rhodopsin cDNAs were expressed from the T7 promoter
to the AcyI after the coding sequence and the BstEII site in the
coding sequence respectively. Sizes of expressed RNAs and cleavage
products were as predicted (Table 1). Resulting RNAs were mixed
together with Rz10 RNA at varying magnesium chloride concentrations
and incubated at 37.degree. C. for 3 hours. Lane 1: Intact
unadapted human rhodopsin RNA (AcyI) alone. Lanes 2-5: Unadapted
and adapted human rhodopsin RNAs and Rz10 RNA after incubation at
37.degree. C. with 0, 5, 10 and 15 mM MgCl.sub.2 respectively.
Almost complete cleavage of the larger unadapted human rhodopsin
RNA was obtained with a small residual amount of intact RNA present
at 5 mM MgCl.sub.2. In contrast the adapted human rhodopsin RNA
remained intact. From top to bottom, the unadapted and adapted
rhodopsin RNAs, and two cleavage products from the unadapted human
rhodopsin RNA are highlighted by arrows. Lane 6 is intact adapted
human rhodopsin RNA (BstEII) alone. [0064] B: The adapted human
rhodopsin CDNA was expressed from the T7 promoter to the BstEII
site in the coding sequence. Lanes 1-4: Resulting RNA was mixed
together with Rz10 and 0, 5, 10 and 15 mM magnesium chloride and
incubated at 37.degree. C. for 3 hours respectively. The adapted
rhodopsin transcripts were not cleaved by Rz10. Cleavage of adapted
transcripts would have resulted in cleavage products of 564 bases
and 287 bases which clearly are not present. Lane 5: intact adapted
human rhodopsin RNA (BstEII) alone. Lane 4: RNA is absent--due to a
loading error or degradation. The adapted uncleaved human rhodopsin
RNA is highlighted by an arrow. [0065] C: Unadapted (SEQ ID NO:1)
and adapted (SEQ ID NO:2) human rhodopsin cDNAs were expressed from
the T7 promoter to the AcyI after the coding sequence and the
BstEII site in the coding sequence respectively. Sizes of expressed
RNAs and cleavage products were as predicted (Table 1). Resulting
RNAs were mixed together with Rz10 RNA at varying magnesium
chloride concentrations and incubated at 37.degree. C. for 3 hours.
Lane 1: DNA ladder as in FIG. 1. Lanes 2-5: Unadapted and adapted
human rhodopsin RNAs and Rz10 RNA after incubation at 37.degree. C.
with 0, 5, 10 and 15 mM MgCl.sub.2 respectively. Almost complete
cleavage of the larger unadapted human rhodopsin RNA was obtained
with a small residual amount of intact RNA present at 5 and 10 mM
MgCl.sub.2. In contrast the adapted human rhodopsin RNA remained
intact. Lane 6: Adapted human rhodopsin RNA (BstEII) alone. Lane 7:
Unadapted human rhodopsin RNA (AcyI) alone. Lane 8: DNA ladder as
in FIG. 1. From top to bottom, the unadapted and adapted rhodopsin
RNAs, and two cleavage products from the unadapted human rhodopsin
RNA are highlighted by arrows. Separation of the adapted human
rhodopsin RNA (851 bases) and the larger of the cleavage products
from the unadapted RNA (896 bases) is incomplete in this gel
(further running of the gel would be required to achieve
separation)--however the separation of these two RNAs is
demonstrated in FIG. 3A.
[0066] FIG. 4:
[0067] The mutant (Pro23Leu) (SEQ ID NO:3) human rhodopsin cDNA was
expressed from the T7 promoter to the BstEII in the coding
sequence. Likewise the Rz20 clone was expressed to the XbaI site.
Resulting RNAs were mixed together with 10 mM magnesium chloride
concentrations at 37.degree. C. for varying times. Sizes of
expressed RNAs and cleavage products were as predicted (Table 1).
Lane 1: DNA ladder as in FIG. 1. Lanes 2: Pro23Leu human rhodopsin
RNA alone. Lanes 3-7 Pro23Leu human rhodopsin RNA and Rz20 RNA
after incubation at 37.degree. C. with 10 mM MgCl.sub.2 for 0 mins,
30 mins, 1 hr, 2 hrs and 5 hrs respectively. Almost complete
cleavage of mutant rhodopsin transcripts was obtained with a
residual amount of intact RNA left even after 5 hours. Lane 8: DNA
ladder as in FIG. 1. From top to bottom, intact mutant rhodopsin
RNA and the two cleavage products from the mutant human rhodopsin
RNA are highlighted by arrows.
[0068] FIG. 5:
[0069] The mutant (Pro23Leu) (SEQ ID NO:3) human rhodopsin cDNA was
expressed from the T7 promoter to the BstEII in the coding
sequence. Likewise the Rz10 clone (SEQ ID NO:4) was expressed to
the XbaI site. Resulting RNAs were mixed together with 10 mM
magnesium chloride concentrations at 37.degree. C. for varying
times. Sizes of expressed RNAs and cleavage products were as
predicted (Table 1). Lane 1: DNA ladder as in FIG. 1. Lanes 2:
Pro23Leu human rhodopsin RNA alone. Lanes 3-7 Pro23Leu human
rhodopsin RNA and Rz10 RNA after incubation at 37.degree. C. with
10 mM MgCl.sub.2 for 0 mins, 30 mins, 1 hr, 2 hrs and 5 hrs
respectively. Almost complete cleavage of mutant human rhodopsin
RNA was obtained with a residual amount of intact RNA remaining
even after 5 hours (Lane 7). Lane 8: DNA ladder as in FIG. 1. From
top to bottom, intact mutant rhodopsin RNA and the two cleavage
products from the mutant human rhodopsin RNA are highlighted by
arrows.
[0070] FIG. 6:
[0071] The mouse rhodopsin cDNA clone was expressed in vitro from
the T7 promoter to the Eco47III site in the coding sequence.
Likewise the Rz33 clone was expressed to the XbaI site. A:
Resulting RNAs were mixed together with 10 mM magnesium chloride at
37.degree. C. for varying times. Sizes of expressed RNAs and
cleavage products were as predicted (Table 1). DNA ladder as in
FIG. 1. Lane 1: mouse rhodopsin RNA alone. Lanes 2-5 Mouse
rhodopsin RNA and Rz33 RNA after incubation at 37.degree. C. with
10 mM MgCl.sub.2 at 0, 5, 7.5 and 10 mM MgCl.sub.2 respectively for
3 hours. Cleavage of mouse rhodopsin RNA was obtained after
addition of divalent ions (Lane 3). Residual uncleaved mouse
rhodopsin RNA and the two cleavage products from the mouse
rhodopsin RNA are highlighted by arrows. B: The adapted mouse
rhodopsin cDNA clone with a base change at position 1460
(nucleotide 190 of SEQ ID NO:7) was expressed in vitro from the T7
promoter to the Eco47III site in the coding sequence. Likewise the
Rz33 clone was expressed to the XbaI site. Resulting RNAs were
mixed together with various magnesium chloride concentrations at
37.degree. C. for 3 hours. Sizes of expressed RNAs and cleavage
products were as predicted (Table 1). Lane 1: DNA ladder as in FIG.
1. Lane 2: Adapted mouse rhodopsin RNA alone. Lanes 3-6: Adapted
mouse rhodopsin RNA and Rz33 RNA after incubation at 37.degree. C.
with 0, 5, 7.5 and 10 mM MgCl.sub.2 for 3 hours at 37.degree. C. No
cleavage of the adapted mouse rhodopsin RNA was observed. The
intact adapted mouse rhodopsin RNA is highlighted by an arrow.
[0072] FIG. 7:
[0073] The human peripherin cDNA clone was expressed in vitro from
the T7 promoter to the BglII site in the coding sequence. Likewise
Rz30 (targeting a cleavage site at 255-257) was expressed to the
XbaI site. A: Resulting RNAs were mixed together with 10 mM
magnesium chloride at 37.degree. C. for varying times. Lane 1: DNA
ladder as in FIG. 1. Lane 2: Intact human peripherin RNA alone.
Lanes 3-7: Human peripherin RNA and Rz30 RNA after incubation at
37.degree. C. with 10 mM MgCl.sub.2 for 3 hrs, 2 hrs, 1hr, 30 mins
and 0 mins respectively. Lane 8: DNA ladder as in FIG. 1. From top
to bottom, intact human peripherin RNA and the two cleavage
products from the human peripherin RNA are highlighted by arrows.
B: Resulting RNAs were mixed with Rz30 RNA at varying magnesium
chloride concentrations and incubated at 37.degree. C. for 3 hrs.
Lane 1: DNA ladder as in FIG. 1. Lanes 2-5: Human peripherin RNA
and Rz30 after incubation at 37.degree. C. with 10, 7.5, 5 and 0 mM
magnesium chloride respectively for 3 hrs. Lane 6: Intact human
peripherin RNA alone. Sizes of the expressed RNAs and cleavage
products are as expected (Table 1). Significant cleavage of human
peripherin RNA was obtained with a residual amount of intact RNA
present at each MgCl.sub.2 concentration. From top to bottom, human
peripherin RNA and the two cleavage products from this RNA are
highlighted with arrows. C: The adapted human peripherin DNA with a
single base change at position 257 was expressed from the T7
promoter to the AvrII site in the coding sequence. Resulting RNA
was mixed with Rz30 at various magnesium chloride concentrations
and incubated at 37.degree. C. for 3 hrs. Lane 1: DNA ladder as in
FIG. 1. Lane 2: Intact adapted human peripherin RNA alone. Lanes
3-6: Adapted human peripherin RNA and Rz30 after incubation at
37.degree. C. with 0, 5, 7.5 and 10 mM magnesium chloride
respectively for 3 hrs. Lane 7: DNA ladder as in FIG. 1. D: The
unadapted human peripherin cDNA and the adapted human peripherin
DNA fragment with a single base change at position 257 were
expressed from the T7 promoter to the BglII and AvrII sites
respectively in the coding sequence. Resulting RNAs were mixed with
Rz30 at various magnesium chloride concentrations and incubated at
37.degree. C. for 3 hrs. Lane 1: DNA ladder as in FIG. 1. Lane 2:
Intact unadapted human peripherin RNA alone. Lane 3: Intact adapted
human peripherin RNA alone. Lanes 4-7: Unadapted and adapted human
peripherin RNAs and Rz30 after incubation at 37.degree. C. with 0,
5, 7.5 and 10 mM magnesium chloride respectively for 3 hrs at
37.degree. C. No cleavage of the adapted human peripherin RNA was
observed even after 3 hours whereas a significant reduction in the
unadapted RNA was observed over the same time frame. Lane 8: DNA
ladder as in FIG. 1. From top to bottom, residual unadapted human
peripherin RNA, adapted human peripherin RNA and the two cleavage
products are highlighted by arrows.
[0074] FIG. 8:
[0075] Human peripherin cDNA clone was expressed in vitro from the
T7 promoter to the BglII site in the coding sequence. Likewise the
Rz31 (targeting a cleavage site at 357-359) (nucleotides 466-468 of
SEQ ID NO:13) was expressed to the XbaI site. A: Resulting RNAs
were mixed together with 10 mM magnesium chloride at 37.degree. C.
for varying times. Lane 1: DNA ladder as in FIG. 1. Lanes 2-6:
Human peripherin RNA and Rz31 RNA after incubation at 37.degree. C.
with 10 mM MgCl.sub.2 for 3 hrs, 2 hrs, 1 hr, 30 mins and 0 mins
respectively. Increased cleavage of mouse rhodopsin RNA was
obtained over time--however significant residual intact RNA
remained even after 3 hours (Lane 2). Lane 7: Intact human
peripherin RNA alone. Lane 8: DNA ladder as in FIG. 1. From top to
bottom, intact human peripherin RNA and the two cleavage products
from the human peripherin RNA are highlighted by arrows. B:
Resulting RNAs were mixed with Rz31 RNA at varying magnesium
chloride concentrations and incubated at 37.degree. C. for 3 hrs.
Lane 1: DNA ladder as in FIG. 1. Lanes 2-5: Human peripherin RNA
and Rz31 after incubation at 37.degree. C. with 10, 7.5, 5 and 0 mM
magnesium chloride respectively for 3 hrs. Sizes of the expressed
RNAs and cleavage products are as expected (Table 1). Significant
cleavage of human peripherin RNA was obtained with a residual
amount of intact RNA present at each MgCl.sub.2 concentration
(Lanes 2-4). Lane 6: Intact human peripherin RNA alone. Lane 7: DNA
ladder as in FIG. 1. From top to bottom, human peripherin RNA and
the two cleavage products from this RNA are highlighted with
arrows. C: The adapted human peripherin DNA with a single base
change at position 359 (nucleotide 468 of SEQ ID NO:13) was
expressed from the T7 promoter to the BglII site in the coding
sequence. Resulting RNA was mixed with Rz31 at various magnesium
chloride concentrations and incubated at 37.degree. C. for 3 hrs.
Lane 1: DNA ladder as in FIG. 1. Lane 2: Intact adapted human
peripherin RNA alone. Lanes 3-6: Adapted human peripherin RNA and
RZ31 after incubation at 37.degree. C. with 0, 5, 7.5 and 10 mM
magnesium chloride respectively for 3 hrs. No cleavage of the
adapted human peripherin RNA was observed even after 3 hours. Lane
7: DNA ladder as in FIG. 1.
[0076] FIG. 9: [0077] A: The human collagen 1A2 cDNA clones
containing the A and T alleles of the polymorphism at position 907
were expressed from the T7 promoter to the MvnI and XbaI sites in
the insert and vector respectively. Resulting RNAs were mixed
together with Rz907 and various MgCl.sub.2 concentrations and
incubated at 37.degree. C. for 3 hours. Lane 1: intact RNA from the
human collagen 1A2 (A) containing the A allele of the 907
polymorphism. Lane 2: intact RNA from the human collagen 1A2 (B)
containing the T allele of the 907 polymorphism. Lanes 3-5: Human
collagen 1A2 (A) and (B) representing the A and T allele RNAs and
Rz907 incubated with 0, 5, and 10 mM MgCl.sub.2 at 37.degree. C.
for 3 hours. RNA transcripts from the T allele containing the
906-908 target site are cleaved by Rz907 upon addition of divalent
ions--almost complete cleavage is obtained with a residual amount
of transcript from the T allele remaining (Lane 5). In contrast
transcripts expressed from the A allele (which are smaller in size
to distinguish between the A (MvnI) and T (XbaI) alleles) were not
cleaved by Rz907--no cleavage products were observed. From top to
bottom, RNA from the T allele, the allele and the two cleavage
products from the T allele are highlighted by arrows. Lane 6: DNA
ladder as in FIG. 1. [0078] B: The human collagen 1A2 CDNA (A)+(B)
clones containing the A and T alleles of the polymorphism at 907
were expressed from the T7 promoter to the MvnI and XbaI sites in
the insert and vector respectively. Resulting RNAs were mixed
together with Rz907 and 10 mM magnesium chloride and incubated at
37.degree. C. for varying times. Lane 1: DNA ladder as in FIG. 1.
Lane 2: intact RNA from the human collagen 1A2 (A) with the A
allele of the 907 polymorphism. Lane 3: intact RNA from the human
collagen 1A2 (B) with the T allele of the 907 polymorphism. Lanes
4-9: Human collagen 1A2 A and T allele RNA and Rz907 incubated
with10 mM MgCl.sub.2 at 37.degree. C. for 0, 30 mins, 1 hour, 2
hours, 3 hours and 5 hours respectively. RNA transcripts from the T
allele containing the 906-908 target site are cleaved by
Rz907--almost complete cleavage is obtained after 5 hours. In
contrast transcripts expressed from the A allele (which are smaller
in size to distinguish between the A (MvnI) and T (XbaI) alleles)
were not cleaved by Rz907--no cleavage products were observed. From
top to bottom, RNA from the T allele, the A allele and the two
cleavage products from the T allele are highlighted by arrows. FIG.
10:
[0079] The human rhodopsin cDNA in pcDNA3. (SEQ ID NO: 1).
[0080] FIG. 11:
[0081] The human rhodopsin cDNA in pcDNA3 (SEQ ID NO:2) with a base
change at a silent site (477) (nucleotide 271 of SEQ ID NO:2).
[0082] FIG. 12:
[0083] Mutant (Pro23Leu) (nucleotides 216-218 of SEQ ID NO:3) human
rhodopsin cDNA in pcDNA3 (SEQ ID NO:3).
[0084] FIG. 13:
[0085] Rz10 cloned into pcDNA3 (SEQ ID NO:4). Note there is a one
base mismatch in one antisense arm of Rz10.
[0086] FIG. 14:
[0087] Rz20cloned into pcDNA3 (SEQ ID NO:5).
[0088] FIG. 15:
[0089] The mouse rhodopsin cDNA in pcDNA3 (SEQ ID NO:6).
[0090] FIG. 16:
[0091] The mouse rhodopsin cDNA in pcDNA3 (SEQ ID NO:7) with a base
change at a silent site (1460) (nucleotide 190 of SEQ ID NO:7).
[0092] FIG. 17:
[0093] Rz33 cloned into pcDNA3 (SEQ ID NO:8)
[0094] FIG. 18:
[0095] The human peripherin CDNA in pcDNA3 (SEQ ID NO:9).
[0096] FIG. 19:
[0097] The human peripherin DNA fragment (SEQ ID NO:10) with a base
change at a silent site (257) (nucleotide 332 of SEQ ID NO:10).
[0098] FIG. 20:
[0099] The human peripherin DNA fragment (SEQ ID NO:11) with a base
change at a silent site (359) (nucleotide 468 of SEQ ID NO:13). The
sequence quality was not good in the region of the human peripherin
359 silent change (nucleotide 468 of SEQ ID NO:13)--the sequencing
primer was too far from the target site to achieve good quality
sequence.
[0100] FIG. 21:
[0101] Rz30 cloned into pcDNA3 (SEQ ID NO:12)
[0102] FIG. 22:
[0103] Rz31 cloned into pcDNA3 (SEQ ID NO:13)
[0104] FIG. 23:
[0105] Collagen 1A2 (A) sequence containing the A polymorphism at
position 907. (SEQ ID NO:14) (Note there is an additional
polymorphic site at position 902).
[0106] FIG. 24:
[0107] Collagen 1A2 (B) sequence containing the T polymorphism at
position 907. (SEQ ID NO:15) (Note there is an additional
polymorphic site at position 902).
[0108] FIG. 25:
[0109] Rz907 cloned into pcDNA3 (SEQ ID NO:18)
RESULTS
[0110] Human and mouse rhodopsin, human peripherin and human
collagen 1A2 cDNA clones were expressed in vitro. Ribozymes
targeting specific sites in the human and mouse rhodopsin, human
peripherin and human collagen 1A2 cDNAs were also expressed in
vitro. cDNA clones were cut with various restriction enzymes
resulting in the production of differently sized transcripts after
expression. This aided in differentiating between RNAs expressed
from unadapted and adapted cDNAs. Restriction enzymes used to cut
each clone, sizes of resulting transcripts and predicted sizes of
products after cleavage by target ribozymes are given below in
Table 1. Exact sizes of expression products may vary by a few bases
from that estimated as there may be some ambiguity concerning inter
alia the specific base at which transcription starts.
EXAMPLE 1
A: Human Rhodopsin
[0111] The unadapted human rhodopsin CDNA (SEQ ID NO:1) and the
human rhodopsin CDNA with a single nucleotide substitution in the
coding sequence (SEQ ID NO:2) were cut with BstEII and expressed in
vitro. The single base change occurs at the third base position or
wobble position of the codon (at position 477) (nucleotide 271 of
SEQ ID NO:2) and therefore does not alter the amino acid coded by
this triplet. The Rz10 clone was cut with XbaI and expressed in
vitro. Resulting ribozyme and human rhodopsin RNAs were mixed with
varying concentrations of MgCl.sub.2 to optimise cleavage of
template RNA by Rz10. A profile of human rhodopsin RNA cleavage by
Rz10 over time is given in FIG. 2A. The MgCl.sub.2 curve profile
used to test if adapted human rhodopsin transcripts could be
cleaved by Rz10 is given in FIG. 3B. Unadapted and adapted human
rhodopsin cDNAs were cut with FspI and BstEII respectively,
expressed and mixed together with Rz10 RNA to test for cleavage
(FIG. 2B) over time. Likewise, unadapted and adapted human
rhodopsin cDNAs were cut with AcyI and BstEII respectively, both
were expressed in vitro and resulting transcripts mixed with Rz10
RNA at varying MgCl.sub.2 concentrations to test for cleavage (FIG.
3A, 3C). In all cases expressed RNAs were the predicted size.
[0112] Similarly in all cases unadapted transcripts were cleaved
into products of the predicted size. Cleavage of nadapted human
rhodopsin RNA was almost complete--little residual uncleaved RNA
remained. In all cases adapted human rhodopsin RNAs with a single
base change at a silent site remained intact, that is, they were
not cleaved by Rz10. Clearly, transcripts from the unadapted human
rhodopsin cDNA are cleaved by Rz10 while transcripts from the
adapted replacement gene which has been modified in a manner which
exploits the degeneracy of the genetic code are protected from
cleavage. It is worth noting that AcyI enzyme cuts after the stop
codon and therefore the resulting RNA includes the complete coding
sequence of the gene.
B: Human Rhodopsin
[0113] Rz20 was cut with XbaI and expressed in vitro. Similarly the
rhodopsin cDNA containing a Pro23Leu mutation was cut with BstEII
and expressed in vitro. Resulting RNAs were mixed and incubated at
37.degree. C. with 10 mM MgCl.sub.2 for varying times. Rz20 was
designed to elicit mutation specific cleavage of transcripts
containing a Pro23Leu rhodopsin mutation. All expressed products
and cleavage products were the correct size. FIG. 4 demonstrates
mutation specific cleavage of the mutant RNA over time incubated at
37.degree. C. with 10 mM MgCl.sub.2. Cleavage of mutant rhodopsin
transcripts by Rz10 which targets a ribozyme cleavage site 3' of
the site of the Pro23Leu mutation in rhodopsin coding sequence was
explored. The mutant rhodopsin cDNA and Rz10 clones were cut with
BstEII and XbaI respectively and expressed in vitro. Resulting RNAs
were mixed and incubated with 10 mM MgCl.sub.2 for varying times
(FIG. 5). All expressed products and cleavage products were the
correct size. Rz10 cleaved mutant rhodopsin transcripts. Using a
replacement gene with a sequence change around the Rz10 cleavage
site which is at a wobble position we demonstrated in Example 1A
that transcripts from the replacement gene remain intact due to
absence of the Rz10 target site (FIGS. 2B, 3A and 3B). Hence Rz10
could be used to cleave mutant transcripts in a manner independent
of the disease mutation itself (that is, using this site) while
transcripts from the replacement gene which code for the correct
protein would remain intact and therefore could supply the wild
type protein.
Example 2
Mouse Rhodopsin
[0114] Rz33 was cut with XbaI and expressed in vitro. Similarly the
mouse rhodopsin cDNA was cut with Eco47III and expressed in vitro.
Resulting RNAs were mixed and incubated with varying concentrations
of MgCl.sub.2. All expressed products and cleavage products were
the correct size. FIG. 6A demonstrates specific cleavage of the
mouse rhodopsin RNA over various MgCl.sub.2 concentrations
incubated at 37.degree. C. for 3 hours. Using a replacement gene
with a sequence change around the Rz33 cleavage site (TTT-->TCT)
(nucleotides 189-191 of SEQ ID NO:7) which is at a wobble position
we demonstrated that transcripts from the replacement gene remain
intact due to absence of the Rz33 target site (FIG. 6B). Hence Rz33
could be used to cleave mutant transcripts in a manner independent
of the disease mutation itself (that is, using this site) while
transcripts from the replacement gene which code for the correct
protein would remain intact and therefore could supply the wild
type protein.
Example 3
Human Peripherin
[0115] The unadapted human peripherin cDNA and two human peripherin
DNA fragments generated by PCR mutagenesis with a single nucleotide
substitution in the coding sequence were cut with BglII and AvrII
respectively and expressed in vitro. The single base changes in the
adapted DNAs occur at third base positions or wobble positions of
the codon (at position 257 and 359) (nucleotide 468 of SEQ ID NO:13
and nucleotide 332 of SEQ ID NO:10, respectively) and therefore do
not alter the amino acid coded by these triplets. The Rz30 and Rz31
clones were cut with XbaI and expressed in vitro. Resulting
ribozymes and unadapted human rhodopsin RNAs were mixed with
varying concentrations of MgCl.sub.2 to optimise cleavage of
template RNA by Rz30 and Rz31 . Profiles of human peripherin RNA
cleavage by Rz30 over various MgCl.sub.2 concentrations and over
time are given in FIG. 7. Similarly profiles of human peripherin
RNA cleavage by Rz31 over various MgCl.sub.2 concentrations and
over time are given in FIG. 8. In all cases expressed RNAs were the
predicted size. Similarly in all cases unadapted transcripts were
cleaved into products of the predicted size. Adapted human
rhodopsin RNAs were mixed together with Rz30 and Rz31 RNA over
various MgCl.sub.2 concentrations to test if adapted human
peripherin transcripts could be cleaved by Rz30 and Rz31 (FIGS. 7
and 8). Expressed RNAs were the predicted size. In all cases
adapted human peripherin RNAs with single base changes at silent
sites remained intact, that is, they were not cleaved by Rz30 or
Rz31. Clearly, transcripts from the unadapted human peripherin cDNA
are cleaved by Rz30 and Rz31 while transcripts from the adapted
replacement DNAs which have been modified in a manner which
exploits the degeneracy of the genetic code are protected from
cleavage.
Example 4
Human Collagen 1A2
[0116] Rz907 clones targeting a polymorphic site in human collagen
1A2 sequence was cut with XbaI and expressed in vitro. The human
collagen 1A2 cDNA clones (A and B) containing two allelic forms of
a polymorphism in the coding sequence of the gene at positions 907
were cut with MvnI and XbaI respectively, expressed in vitro and
RNAs mixed together with Rz907 RNA to test for cleavage of
transcripts by this ribozyme. All expressed transcripts were of the
predicted sizes. RNAs were mixed with varying concentrations of
MgCl.sub.2 to optimise cleavage of RNAs by Rz907 (FIG. 9). Notably
the majority of the RNA transcripts from human collagen 1A2 (A)
which has a T nucleotide at position 907 (A nucleotide 176 of SEQ
ID NO:17, reverse strand) is cleaved by Rz907 (FIG. 9).
[0117] This allelic form of the gene has a ribozyme cleavage site
at 906-908. Notably the situation is reversed with transcripts from
human collagen 1A2 (B) where in this allelic form of the gene due
to the nature of the polymorphism present at position 907 the
ribozyme cleavage site has been lost.
[0118] In contrast to transcripts from human collagen (A),
transcripts from human collagen (B) were protected from cleavage by
Rz907 due to the alteration in the sequence around the ribozyme
cleavage site (FIG. 9). Cleavage of collagen 1A2 (A) by Rz907 was
efficient which is consistent with 2-D predictions of RNA open loop
structures for the polymorphism--in the allele containing the Rz907
ribozyme cleavage site, the target site is found quite consistently
in an open loop structure. This polymorphism found in an open loop
structure of the transcript clearly demonstrates the feasibility
and utility of using the degeneracy of the genetic code in the
suppression of an endogenous gene (either suppressing both alleles
or a single allele at a polymorphic site) and restoration of gene
expression using a gene which codes for the same protein but has
sequence modifications at third base wobble positions which protect
the replacement gene from suppression. TABLE-US-00005 TABLE 1
Restriction Cleavage Enzyme RNA Size Products Example 1 Human
rhodopsin BstEII 851 bases 287 + 564 bases AcyI 1183 bases 287 +
896 bases FspI 309 bases 287 + 22 Adapted Human BstEII 851 bases
rhodopsin Human rhodopsin BstEII 851 bases 170 + 681 (Rz20) Pro-Leu
Human rhodopsin BstEII 851 bases 287 + 564 (Rz10) Pro-Leu Rz10 XbaI
52 bases Rz20 XbaI 52 bases (Table 1; SEQ ID NOS: 1-5; FIGS. 1-5)
Example 2 Mouse rhodopsin Eco47III 774 bases 400 + 374 Adapted
mouse Eco47III 774 bases rhodopsin Rz33 XbaI 52 bases (Table 1; SEQ
ID NOS: 6-9; FIG. 6) Example 3 Human peripherin BglII 545 bases 315
+ 230 (Rz30) Human peripherin BglII 545 bases 417 + 128 (Rz31)
Adapted human AvrII 414 bases peripherin Adapted human BglII 545
bases peripherin Rz30 XbaI 52 bases Rz31 XbaI 52 bases (Table 1;
SEQ ID NOS: 10, 13-16; FIGS. 7 and 8) Example 4 Human Collagen 1A2
XbaI 888 bases 690 + 198 bases (B) -Rz907 Human Collagen MvnI 837
bases 1A2 (A) Rz907 XbaI 52 bases (Table 1; SEQ ID NOS: 16-18; FIG.
9)
[0119] TABLE-US-00006 TABLE 2 A: Rhodopsin mutations tested to
assess if the predicted open loop RNA structure containing the Rz10
target site (475-477) remains intact in mutant transcripts.
Rhodopsin mutation RNA open loop targeted by Rz10 Pro 23 Leu Intact
Gly 51 Val Intact Thr 94 Ile Intact Gly 188 Arg Intact Met 207 Arg
Intact Ile del 255 Intact B: Utilisation of the degeneracy of the
genetic code. Ribozyme cleavage sites are underlined uz,1/11 Human
rhodopsin Unadapted sequence 475-477 TAC GTC ACC GTC CAG (SEQ ID
NO:19) Val Adapted sequence 475-477 TAC GTG ACC GTC CAG (SEQ ID
NO:20) Val Mouse rhodopsin Unadapted sequence 1459-1461 AAT TTT TAT
GTG CCC (SEQ ID NO:21) Phe Adapted sequence 1459-1461 AAT TTC TAT
GTG CCC (SEQ ID NO:22) Phe Human peripherin Unadapted sequence
255-257 GCG CTA CTG AAA GTC (SEQ ID NO:23) Leu Adapted sequence
255-257 GCG CTG CTG AAA GTC (SEQ ID NO:24) Leu Unadapted sequence
357-359 AGC CTA GGA CTG TTC (SEQ ID NO:25) Leu Adapted sequence
357-359 AGC CTG GGA CTG TTC (SEQ ID NO:26) Leu Human type I
collagen 1A2 Sequence (A) 906-908 GCT GGT CCC GCC GGT (SEQ ID
NO:27) Gly Sequence (B) 906-908 GCT GGA CCC GCC GGT (SEQ ID NO:28)
Gly
Discussion
[0120] In the examples outlined above, RNA was expressed from cDNAs
coding for four different proteins: human and mouse rhodopsin,
human peripherin and human type I collagen 1A2. Rhodopsin and
peripherin have been used to exemplify the invention for
retinopathies such as adRP--suppression effectors have been
targeted to the coding sequences of these genes. In the case of the
human collagen 1A2 gene a naturally occurring polymorphism has been
used to demonstrate the invention and the potential use of the
invention for disorders such as OI--however non-polymorphic regions
of the collagen 1A2 gene could be used to achieve suppression. The
suppression effectors of choice in the invention have been
hammerhead ribozymes with antisense flanks to define sequence
specificity. Hammerhead ribozymes require NUX cleavage sites in
open loop structures of RNA. Notably, other suppression effectors
could be utilised in the invention and may lead to a more flexible
choice of target sequences for suppression. Transcripts expressed
from all four genes have been significantly attacked in vitro using
suppression effectors directed towards target cleavage sites. In
all four examples the ribozymes directed to cleavage sites were
successful in cleaving target RNAs in the predicted manner.
Antisense complementary to sequences surrounding the cleavage sites
was used successfully to elicit binding and cleavage of target RNAs
in a sequence specific manner. Additionally, transcripts from
replacement genes, modified using the degeneracy of the genetic
code so that they code for wild type protein, were protected fully
from cleavage by ribozymes.
[0121] The utility of an individual ribozyme designed to target an
NUX site in an open loop structure of transcripts from a gene will
depend in part on the robust nature of the RNA open loop structure
when various deleterious mutations are also present in the
transcript. To evaluate this, we analysed RNAPlotFold data for six
different adRP causing mutations in the rhodopsin gene. For each of
these, the large RNA open loop structure which is targeted by Rz10
was predicted to be maintained in the mutant transcripts (Table
2A). This is clearly demonstrated in example 1B (FIG. 4) using a
Pro23Leu rhodopsin mutation. Rz10 clearly cleaves the mutant
transcript effectively in vitro. The Pro23Leu mutation creates a
ribozyme cleavage site and can be cleaved in vitro by Rz20 a
ribozyme specifically targeting this site--however this is not the
case for many mutations. In contrast we have shown that the Rz10
ribozyme cleavage site is available for different mutant rhodopsins
and could potentially be used to suppress multiple mutations using
a suppression and replacement approach.
[0122] In some cases lowering RNA levels may lead to a parallel
lowering of protein levels however this may not always be the case.
In some situations mechanisms may prevent a significant decrease in
protein levels despite a substantial decrease in levels of RNA.
However in many instances suppression at the RNA level has been
shown to be effective (see prior art). In some cases it is thought
that ribozymes elicit suppression not only by cleavage of RNA but
also by an antisense effect due to the antisense arms of the
ribozyme surrounding the catalytic core.
[0123] In all examples provided ribozymes were designed to cleave
at specific target sites. Target sites for four of the ribozymes
utilised were chosen in open loop structures in the coding regions
of transcripts from three retinal genes (human and mouse rhodopsin
and human peripherin). In all cases sequence specific cleavage was
obtained at the target cleavage sites (FIGS. 1-7). Target sites
were chosen in open loop structures to optimise cleavage.
Additionally target sites were chosen such that they could be
obliterated by single nucleotide changes at third base wobble
positions and therefore would code for the same amino acid (Table
2B). In turn this enabled the generation of replacement genes with
single nucleotide alterations which code for wild type protein. In
all cases tested transcripts from replacement genes were protected
from cleavage by ribozymes. Further modifications could be made to
replacement genes in wobble positions, for example, to limit the
binding ability of the antisense arms flanking the ribozyme
catalytic core. The examples provided for rhodopsin and peripherin
involve suppression of expression of both disease and wild type
alleles of a retinal gene and restoration of the wild type protein
using a replacement gene. However, there may be situations where
single alleles can be targeted specifically or partially
specifically (PCT/GB97/00574).
[0124] In one example, human collagen 1A2, Rz907 was used to target
a naturally occurring polymorphic site at amino acid 187, (GGA
(glycine) -->GGT (glycine), located in an open loop structure
from the predicted 2-D conformations of the transcript (FIG. 9,
Table 2B). The ribozyme Rz907 cleaved transcripts containing the
GGT sequence but transcripts with GGA were protected from cleavage.
Transcripts from both alleles of individuals homozygous for the GGT
polymorphism could be cleaved by Rz907 whereas in the case of
heterozygotes cleavage could be directed to single alleles (in
particular to alleles containing deleterious mutations
PCT/GB97/00574). In both situations replacement genes could have
the sequence GGA and therefore would be protected from cleavage by
Rz907. The presence of many such naturally occurring silent
polymorphisms highlights that replacement genes could be modified
in a similar fashion in wobble positions and should produce in most
cases functional wild type protein. Multiple modifications could be
made to replacement genes at wobble positions which would augment
protection from suppression effectors. For example, in situations
where antisense nucleic acids were used for suppression,
transcripts from replacement genes with multiple modifications at
third base positions would be protected partially or completely
from antisense binding.
[0125] In all four examples provided transcripts from cDNA clones
were cleaved in vitro in a sequence specific manner at ribozyme
cleavage sites. Additionally one base of the ribozyme cleavage site
occurs at a wobble position and moreover can be altered so as to
eliminate the cleavage site. Ribozyme cleavage sites in the
examples given were destroyed by changing nucleotide sequences so
that the consensus sequence for ribozyme cleavage sites was broken.
However it may be that in some cases the cleavage site could be
destroyed by altering the nucleotide sequence in a manner that
alters the 2-D structure of the RNA and destroys the open loop
structure targeted by the ribozyme. cDNAs or DNA fragments with
altered sequences in the regions targeted by ribozymes were
generated. RNAs expressed from these cDNAs or DNA fragments were
protected entirely from cleavage due to the absence of the ribozyme
cleavage site for each of the ribozymes tested. Of particular
interest is the fact that a single nucleotide alteration can
obliterate a ribozyme target site, thereby preventing RNA cleavage.
Although ribozymes have been used in the demonstration of the
invention, other suppression effectors could be used to achieve
gene silencing. Again replacement genes with altered sequences (at
third base wobble positions) could be generated so that they are
protected partially or completely from gene silencing and provide
the wild type (or beneficial) gene product.
[0126] As highlighted before in the text, using the invention the
same method of suppression (targeting coding sequences of a gene)
and where necessary gene replacement (using a replacement gene with
a sequence modified in third base positions to restore gene
expression) may be used as a therapeutic approach for many
different mutations within a given gene. Given the continuing
elucidation of the molecular pathogenesis of dominant and polygenic
diseases the number of targets for this invention is rapidly
increasing.
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Sequence CWU 1
1
34 1 617 DNA Homo sapiens modified_base (7)..(7) a, c, g, or t 1
tcccttntgn tagattgcan nncccaataa aanaaggncc cgcttaaagg cttatcgaaa
60 ttaatacgac tcactatang gagacccaag cttagagtca tccagctgga
gccctgagtg 120 gctgagctca ggccttcgca gcattcttgg gtgggagcag
ccacgggtca gccacaaggg 180 ccacagccat gaatggcaca gaaggcccta
acttctacgt gcccttctcc aatgcgacgg 240 gtgtggtacg cagccccttc
gagtacccac agtactacct ggctgagcca tggcagttct 300 ccatgctggc
cgcctacatg tttctgctga tcgtgctggg cttccccatc aacttcctca 360
cgctctacgt caccgtccag cacaagaagc tgcgcacgcc tctcaactac atcctggctc
420 aacctagccg tggctgaact cttcatggtc ctangtggct tcaccagcac
ctctacanct 480 ctctgcatgg atactcgtct tcgggcccac aggatgcaat
tgganggctc tttgcacctg 540 gngggaaatt gcctgtggtc ctngtggtcn
ggncaccaac gtactggtng tgtntanccc 600 agaacaactc cgctccc 617 2 639
DNA Artificial Sequence Description of Artificial Sequence
Synthetic human rhodopsin hybrid cDNA 2 ggnnnnttgg gtcgcgcatt
naagaactca nggncccgca gcattcttgg gtgggagcag 60 ctacgggtca
gccacaaggg ccacagccat gaatggcaca gaangcccta acttctacgt 120
gcccttctcc aatgcgacgg gtgtggtacg cagccccttc gagtacccac agtactacct
180 ggctgagcca tggcagttct ccatgctggc cgcctacatg tttctgctga
tcgtgctggg 240 cttccccatc aacttcctca cgctctacgt gaccgtccag
cacaagaagc tgcgcacgcc 300 tctcaactac atcctgctca acctanccgt
ggntgaactc ttcatggtcc taggtggctt 360 caccancaac ctctanacct
ctctgcatgg anacttcntc ttccggccca caggatgcaa 420 tttggaaggn
ttcctttaac acccgggggg ggaaaattgc ctgtggtcct tggtggtccg 480
gncancnaac ggtacttgtg gtntttaanc cataaacaat tccgcttcgg gaaaaacatg
540 ccancntggg gtttccttca ctnggttang ggcnggctgc ccccacccca
atcccnggtn 600 gtcaantaat cccaagggcn nantgncntt ttaaacaaa 639 3 686
DNA Artificial Sequence Description of Artificial Sequence
Synthetic human rhodopsin adRP mutant sequence 3 nnnttagggn
cggatgtcna tataagcaga nctctctggg ctaactaana agaacccact 60
ggcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag cttccggaaa
120 gcctgagctc agccacaagg gccacagcca tgaatggcac agaaagccct
aacttctacg 180 tgcccttctc caatgcgacg ggtgtggtac gcagcctctt
cgagtaccca cagtactacc 240 tggctgagcc atggcagttc tccatgctgg
ccgcctacat gtttctgctg atcgtgctgg 300 gcttccccat caacttcctc
acgctctacg tcaccgtcca gcacaagaag ctgcgcacgc 360 ctctcaacta
catcctgctc aacctanccg tggctgaact cttcatggtc ctangtggct 420
tcaccancac cctctacacc tctctgcatg gatacttcgt cttccgggcc acaggatgca
480 atttggaagg cttctttgca ncctgggncg ggaaattgcc tgtngtcctg
gtggtcctgg 540 ccatcaacng tacttgttgt ntnttaccca tnaacaattc
cgctccggga aaacatgcac 600 atgggnttgc ctcactnggt ctggggcngg
cnccccaccc cacccccggt ggtcanttat 660 cccanggcgn aatgcctttn annaaa
686 4 787 DNA Artificial Sequence Description of Artificial
Sequence Synthetic hammerhead ribozyme sequence 4 cngcncgttg
aaatataagc agaccctctg gntaactana ataaccactg cttactggct 60
tatcgaaatt aatacgactc actatangga gaccaagctt ggtcggtctg atgagtccgt
120 gaggacgaaa cgtanantct anagggccct attctatagt gtcacctaaa
tgctaganct 180 cgctgatcag cctcgactgt gccttctagt tgccagccat
ctgttgtttg cccctccccc 240 gtgccttcct tgancctgga aggtgccact
cccactgtcc tttcctaata aaatgagnaa 300 ttgcntctca ttgtctgagt
agtgtcatcc aatctggggg tgggtggggc agnacacnag 360 gggaagatgg
gaaaacatac aggcatgctg gggangccgt ggntctatgn ctcngaggcg 420
aaaaaacact ggggnctagg ggtaccccac cccctgtacg gccataacnc gnggtttgtg
480 gtacccacta acgtanntgc accctacccg ncttcnttct cctcttncca
tttccggttc 540 cctcaccnaa cgggccttng tcatatctng gnccaccaaa
tanagtagtc tttgccccca 600 aagtccctna tgacctntaa gaccttcann
ancccccctt ntttnaaana nccnnnnnnn 660 nnnnannnnc cngnaaaaan
aacaactaat tttgggaacc ccccccnana aaccctttcc 720 ntnttccccc
natttaatnt tnnnntnccc cccccccccc ccccnntttt tnncnccccn 780 nnannng
787 5 665 DNA Artificial Sequence Description of Artificial
Sequence Synthetic hammerhead ribozyme sequence 5 cnccccgccc
ntttnaaana anccnagcct ctggcnaact ananaaccac tgcttactgg 60
cttatcnaaa ttaatacgac tcactatagg gagacccaag ctttactcga actgatgagt
120 ccgtgaggac gaaangctgc tctananggc cctattctat antgtcacct
aaatgctaga 180 gctcgctgat cagcctcgac tgtgccttct aattgccagc
catctgttgt ttgcccctcc 240 cccgtgcctt ccttgaccct ggaaggtgcc
actcccactg tcctttccta ataaaatgaa 300 gatnttncat cncattgtct
gagtaagtgt cattctattc tggggggtgg ggtggggcac 360 gacancaang
gggaagattg ggaaaaaata ncaggcntgc tggggatncc gtgggctcta 420
tngcttctga agcggaaaaa acaactgggg ctctangggg tatccccccc cccctgtaac
480 gngcattaaa cncgggggtg ttgtggttac cccaacttaa cgctancttg
caacgcccna 540 acgccccncc tttcctttct cccttccttc ncccactttc
cgggttcccn tcaacccnaa 600 tcggggcccc ttaggtccaa ttatgcttcg
gccccncccn aaactaatag gtnggttctt 660 tngcc 665 6 74 DNA Mus
musculus 6 tcagtgcctg gagttgcgct gtgggagccg tcagtggctg agctcgccaa
gcagccttgg 60 tctctgtcta cgaa 74 7 630 DNA Artificial Sequence
Description of Artificial Sequence Synthetic mouse rhodopsin hybrid
cDNA 7 nnnntcttcc nctttcgttt gttgnanant cannaaanan aggcgncccg
gaaggtgtca 60 gtgcctggag ttgcgctgtg ggacccgtca ntggctgagc
tcgccaagca gccttggtct 120 ctgtctacga agagcccgtg gggcagcctc
gagagccgca gccatgaacg gcacagaggg 180 ccccaatttc tatgtgccct
tctccaacgt cacaggcgtg gtgcggagcc ccttcgancn 240 tccgcagtac
tacctggcgg aaccatggca gttctccatg ctggcagcgt acatgttcct 300
gctcatcgtg ctgggcttcc ccatcaactt cctcacgctc tacgtcaccg tacagcacaa
360 gaagctgcgc acaccccctc aactacatcc tggctcaact tgggccgntg
ggnttggaac 420 ctccttccca ttgggtcntt cccggaangg antncaccaa
ccacccctct aacacatcaa 480 ctcccatggg ctacttcgtt cttttggggc
ccncaggctg ttaatctcga agggcttctt 540 tgccacacct tggaagtgaa
atcnccctgt ggttccctgg tggtcntggc cattaacgct 600 acttgtggtc
ctgcaaccca ataacaattc 630 8 649 DNA Artificial Sequence Description
of Artificial Sequence Synthetic hammerhead ribozyme sequence 8
tcccctnntt tttgtagcnc tgccaanaaa aaaggccagc tcacaggana antananaac
60 ccactgctta ctggcttanc naaattaata cgactcacta tagggagacc
caagcttggc 120 acatctgatg agtccgtgag gacgaaaaaa ttggtctaca
gggccctatt ctataatgtc 180 acctaaatgc tanagctcgc tgatcatcct
cnactgtgcc ttctacttgc cagccntctn 240 ttgtttgccc ctcccccgtg
ccttccttga ccctggaagg tgccactccc actgtccttt 300 cctaataaaa
tgaggaaatt gcatcgcatt gtctgagtaa gtgtcattct attctggggg 360
gtggggtggg gcaggacnnc aaaggggaag attgggaaat acaatancca aggancnctc
420 ccccngggta attgcggatt nggctctntc gcttccttaa ggcngaaana
aacaactngg 480 gcgctncggg gtttcccccn cccnccctnt tagcngcgca
ttantcgccg cgggtgttgt 540 tgttactccc cacctnaacg ctacanttgc
cagcgcctaa cgccccccct tnctnttctt 600 ccctcctttc tcncacttcc
ccggctttcc ccnccaancc naaatcngg 649 9 681 DNA Homo sapiens
modified_base (1)..(2) a, c, g, or t 9 nnttgttggt ncagtnggat
gtctatataa gcagagnctc tggctaacta gnagaaccca 60 ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 120
gagctcngat ccactagtaa cggccgccag tgtgctggaa ttcttcagcg cccacgacca
180 gtgactatcc cctgctcaag ctgtgattcc gagacccctg ccaccactac
tgcattcacg 240 ggggatccca ngctaatggg actcgacatg ggttgccccc
acggcanctc cctacanctt 300 gggccanctn cacttttccc aaagncctaa
atctccgcct ctcggctcnt taangttngg 360 ggtgggganc tgtgctgtgg
gaaacaaccc agaananact tgggcagcat ggngctactg 420 aaagtncatt
ttgaacagaa naaacggtcc antttggccc aaggnncnng ntcctaaant 480
ggttctccnt ntttggtngn ntccncnctt tccncctngg aatgttcctg aaaaattnaa
540 cnccaaaaaa gaacaaattg aaaaatantt ctnaaaaccc ttttgttncc
ccccccccna 600 aaagggaagg ggnnggnncc tttttnttcc ccccccgggg
ggggaaaatt ttnnnnaanc 660 cccccccccc ccnttttttn a 681 10 682 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
human peripherin hybrid DNA sequence 10 ttatacnaca cactatangg
agaccaagct tggtaccgag ctcggatcca ctagtaacgg 60 ccgccagtgt
gctggaattc ttcancgccc aggaccagga ctatcccctg ctcaagctgt 120
gattccgaga cccctgccac cactactgca ttcacggggg atcccaggct agtgggacnc
180 gacatgggta tcccccaggg cagctcccta cagcttgggc catctgcact
tttcccaagg 240 ccctaagtct ccgcctctgg gctcgttaan gtntggggtg
ggagctgtgc tgtgggaaac 300 aacccggact acacttggca agcatggcgc
tgctgaaagt caagtttgaa cagaaaaaan 360 gggtcaagtt ggcccaaggg
ctctggctca gggaaactgg gttncccncc nngttttngg 420 tttggntgca
tcanctncca aaaanannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnn nn 682 11 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
primer 11 catggcgctg ctgaaagtca 20 12 20 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 12 catcttcagc
ctgggactgt 20 13 610 DNA Artificial Sequence Description of
Artificial Sequence Synthetic human peripherin hybrid DNA sequence
13 tttttntggn tntcnaatta atacgactca ctatagggag acccaagctt
ggtaccgagc 60 tcggatccac tagtaacggc cgccagtgtg ctggaattct
tcancgccca ggaccaggac 120 tatcccctgc tcaagctgtg attccgagac
ccctgccacc actactgcat tcacggggat 180 cccaggctag tgggactcga
catgggtagc ccccagggca gctccctaca gcttgggcca 240 tctgcacttt
tcccaaggcc ctaagtctcc gcctctgggc tcgttaaggt ttggggtggg 300
agctgtgctg tgggaagcaa cccggactac acttggcaag catggcgcta ctgaaagtca
360 agtttgacca gaaaaancgg gtcaagttgg gcccaagggc tctgggctcn
atgnaaacct 420 nggtttcccc ccccctnttt gggctgggca tcatcatctt
tcagcctggg antgttcctg 480 aanattgaac tcccaaagag ancgatgtga
tgaataattc tgaaanccat tttgtgcccc 540 actcattgan aaggangggg
tgnatcctgt ttcttcactc cctgntggaa aatgctacaa 600 nccctgaacc 610 14
680 DNA Artificial Sequence Description of Artificial Sequence
Synthetic hammerhead ribozyme sequence 14 cnttggtggt nctgtcggnt
gtctatataa gcagagctct ctggctaact agaagaaccc 60 actgcttact
ggcttatcga aattaatacg actcactata gggagaccca agcttacttt 120
cagctgatga gtccgtgang gacgaaagcg ccatctagag ggccctattc tatagtgtca
180 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc
agccatctgt 240 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
gccactccca ctgtcctttc 300 ctaataaaat gatgaaattg catcgcattg
tctgagtagg tgtcattcta ttctgggggg 360 tgggtggggc angacancaa
gggggaagat tgggaaaaca atncccgcct gctggggatg 420 cggtgggctc
tatggcttct gaggcgaaan aacnnctggg gtctnggggg ttcccncccc 480
cctgtnncgg ccttnanncg ggggttttgt gntccccccn cttancnntn nttnnnnnnc
540 cnncccccnn cnntncnntt nntccnnnnn ntncncnnnt tnnnnngnnt
ccnnnnnnnn 600 tnnnnngggg cncnnnngnt ccnntnnnnc cncnnnnnnc
nnncnnnnnn nnntntgnng 660 gcccnnnncn nnnnnncncn 680 15 691 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
hammerhead ribozyme sequence 15 nntttntcct acgnccgttt taaananaac
cagaccctct gganaattan atnnccactg 60 cttactggct tatcgaaatc
aatacgactc actatangga gacccaagct tacagtccct 120 gatgagtccg
tgaggacgaa aggctgaatc tanagggccc tattctatag tgtcacctaa 180
atgctagagc tcgctgatca gcctcgactg tgccttctaa ttgccagcca tctgttgttt
240 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
ctntcctaat 300 aaaatgatga nnttgcatcg cattgtctga gtaagtgtca
ntctattctg gggggtgggg 360 tggggcanga cancaagggg gaagattggg
aaaaacattn cacgcatgcc gggggatgcg 420 gtgggctctn ttngcntcng
aaggcngaaa aaaacnactg gggccctang ggtnncccnn 480 tcccccntgt
aacngncctt naacncgggg gtttgtggtt nnccnanctt ancnctnaac 540
ttccnncccc nnncccccnc tcttcccttt ttcctccatc tccncntttn cccgntctcc
600 cttncactna aatgggggcc cctacngggn ctntntntct cttnnnnccn
ccncccnana 660 natatnctng ntnnttcncc tctcggcccc t 691 16 805 DNA
Unknown Organism Description of Unknown Organism Mammalian
nucleotide sequence 16 ntcncgncat ttaancaggc caggnctacc gcnnggtcca
ngtaggccgg gagccccagc 60 aacgccggga aggccagcag cacccttggc
accagtaagg ccgtttgctc caggattacc 120 angaggtcca acggggccgg
agaggcctgg aanaccactt caccacgggg aaccggcggg 180 tccagtagga
ccagcgttac caacagctcc aatttcaccc ttggggccag gggcacctgg 240
gaagcctgga nggccagcag accaatggga ccagcaggac cacggaccac acttccatca
300 ctgctttngc ncagctgggc aagggcacaa cacttctctc tcacangaac
ccacggctcc 360 tgtttnactg aattccattt cacagggcac agttcacctt
cacacaagaa cacggntgtc 420 cttcatcatc agacatgttt ccctaatgct
tgagcagant cagattcagg aaacacacac 480 ctttgtccac atctctncac
agtctcggtt tcaggtacac tcccacctgc agaggcactg 540 accaacctga
gacattgaca ttncagncca cagtctgaac tgagcgggca cgccatggcn 600
agtcatacct gtcagnatca tcttctctta ncattcccaa ngggcagaat gaaagctgac
660 tccccaatgt cttattttta annanggttt naaanaannn nnnnnnnnnn
nnnnnnnnnc 720 cccccccctt tngggtttat tatctatncn ncccntngga
tatctttncc ccnttncccc 780 ctnaaanttt tnttnttttt tnnnn 805 17 797
DNA Unknown Organism Description of Unknown Organism Mammalian
nucleotide sequence 17 ccctttaaaa canggccagg aataccgcgg ggtccaggga
ggccgggacc ccancaacgc 60 cgggaangcc cagcagcacc cttggcacca
gtaangccgt ttgctccagg attaccagga 120 ggtccaacgg ggccggagan
gcctggaaga ccacttcacc acggggaacg gcgggaccag 180 cangaccagc
gttaccaaca gctccaattt cacccttggg gccaggggca cctgggaagc 240
ctgganggcc agcagaccaa tgggancagc aggaccacgg gaccacactt ccatcnctgc
300 cnctggcacc agctgggcaa gggcacaaca cttctctctc acnaagaacc
cacggntcct 360 gtttaactga attccatttc acagggcaca gttcaccttc
anacagaaca cgggtgtcct 420 tcatcatcaa acatntttcc tatnccttga
gcagaatcag attcaggaac acacactttg 480 tcacatctcc tcacagtctc
ggtttcaggt aacactcnca cctgcagagg cactgacnaa 540 nctcaganat
ttanattccn ctccncagtt tgaacttagg cgggccctnn catttggntt 600
gtcctaacct ntngggggtt ttncttnnnn nnnnnnnttt nacnantccc aanggggana
660 ananagntga ctcctatgtc ttnttntnaa aaggtttttn aaaaattaac
cccccccctn 720 ttgggttatt tatttttttt nncccccctt ttgngaancn
tnnccccntt ttccccnnna 780 aanttttttn ttttttt 797 18 697 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
hammerhead ribozyme sequence 18 nctttcnntc tnatncatan aagcaggccc
tctnnaaaaa ctanantttc cactgcttac 60 tggcttatcg aaancaatac
gactcactat agggagaccc aagcttcggc ggctgatgag 120 tccgtgagga
cgaaaccagc atctagaggg ccctattcta tagtgtcacc taaatgctag 180
agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
240 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
aataaaatga 300 ngaaattgca tcgcattgtc tgagtangtg tcattctatt
ctggggggtg gggtggggca 360 ngacancaag ggggaagatt gggaanacaa
taacaggcat gctggggatg cggtgggctc 420 tatggcttct gaggcggaaa
gaaccaactg gggctctang gggtatcccc acncccctgt 480 taccggcgca
ttaancgcgg gggtgttgtg gttacccnca acttaacgct acacttgcca 540
cgcctaacgc ccctcctttc gcttcttcct tccttctccc acttccccgn tttcccttca
600 actctaatcg gggcncctta ggtccaatta atcttacggn cncacccaaa
actnataggt 660 aagtccttnt ggccccccaa aaaggttccc ctaaatg 697 19 15
DNA Homo sapiens 19 tacgtcaccg tccag 15 20 15 DNA Homo sapiens 20
tacgtgaccg tccag 15 21 15 DNA Mus musculus 21 aatttttatg tgccc 15
22 15 DNA Mus musculus 22 aatttctatg tgccc 15 23 15 DNA Homo
sapiens 23 gcgctactga aagtc 15 24 15 DNA Homo sapiens 24 gcgctgctga
aagtc 15 25 15 DNA Homo sapiens 25 agcctaggac tgttc 15 26 15 DNA
Homo sapiens 26 agcctgggac tgttc 15 27 15 DNA Homo sapiens 27
gctggtcccg ccggt 15 28 15 DNA Homo sapiens 28 gctggacccg ccggt 15
29 37 DNA Artificial Sequence Description of Artificial Sequence
Synthetic hammerhead ribozyme sequence 29 ggtcggtctg atgagtccgt
gaggacgaaa cgtagag 37 30 37 DNA Artificial Sequence Description of
Artificial Sequence Synthetic hammerhead ribozyme sequence 30
tactcgaact gatgagtccg tgaggacgaa aggctgc 37 31 37 DNA Artificial
Sequence Description of Artificial Sequence Synthetic hammerhead
ribozyme sequence 31 ggcacatctg atgagtccgt gaggacgaaa aaattgg 37 32
37 DNA Artificial Sequence Description of Artificial Sequence
Synthetic hammerhead ribozyme sequence 32 actttcagct gatgagtccg
tgaggacgaa agcgcca 37 33 37 DNA Artificial Sequence Description of
Artificial Sequence Synthetic hammerhead ribozyme sequence 33
acagtccctg atgagtccgt gaggacgaaa ggctgaa 37 34 35 DNA Artificial
Sequence Description of Artificial Sequence Synthetic hammerhead
ribozyme sequence 34 cggcggctga tgagtccgtg aggacgaaac cagca 35
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