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BACKGROUND OF THE INVENTION
The invention encompasses methods and reagents for the diagnosis of a disease
caused by or associated with a transcript mutation giving rise to a frameshi$
mutation within
s an RNA molecule. The methods include the steps of providing a body fluid or
tissue sample
from a patient; and analyzing the sample for the presence of an RNA molecule
having a
frameshift mutation or a protein encoded thereby, wherein the presence of the
mutated RNA
or encoded protein is indicative of the disease.
It is an object of the present invention to provide methods and assays for
detection
1 o and/or treatment of diseases involving transcript mutations, particularly
those diseases relating
to aging, wherein the probability of having the disease increases with the age
of the patient.
The invention contemplates detection and/or treatment of those age-related
diseases which
are due to mutations occurring in the RNA of cells. If the mutations are not
corrected, the
disease may result.
15 Another object of the invention is to treat diseases identified according
to the
invention, by providing to a patient afflicted with the disease or having a
propensity to
develop the disease, a corrective agent such as an enzyme or oligonucleotide.
Yet another object of the invention is to provide a method for identifying age-
related diseases by correlating nucleotide sequence mutation hotspots with the
disease.
a o Other objects of the invention relate to identification, detection and
treatment of
age-related diseases including cancers (especially non-hereditary cancers) and
neurodegenerative diseases, such as Alzheimer's Disease (AD), Downs' syndrome,
frontal
lobe dementia (Pick's Disease), progressive supranuclear palsy (PSP) and other
diseases with
abundant tau-positive filamentous lesions (such as Corticobasal degeneration,
Dementia
z s pugilistica, Dementia with tangles only, Dementia with tangles and
calcification,
Frontotemporal demential with Parkinsonism linked to chromosome 17, Gertsmann-
Strassler-
Scheinker disease with tangles, Myotonic dystrophy, Niemann-Pick disease type
C,
Parkinsonism-dementia complex of Guam, Postencephalitic Parkinsonism and
Subacute
sclerosing panencephalitis), Parkinson's Disease (PD), amyotrophic lateral
sclerosis,
3 o Huntington's Disease, multiple sclerosis, dementia with Lewy bodies,
multisystem atrophy,
other inclusion body diseases associated with ubiquitin (such as Alexander's
disease,
Alcoholic liver disease, lichen amyloidosis, and during aging Marinesco bodies
and Hyaline
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inclusions), diabetes mellitus type II and other degenerative diseases, such
as cardiovascular
diseases and rheumatoid arthritis. Early disease diagnosis is important for
effective treatment.
Alzheimer's Disease is in most cases a disease which is related to aging. AD
is
characterized by atrophy of nerve cells in the cerebral cortex, subcortical
areas, and
hippocampus and the presence of plaques, dystrophic neurites, neuropil threads
and
neurofibrillary tangles. In most cases, it is not known whether AD is caused
by a genetic
abnormality or by environmental factors, or both. The pathogenic mutation is
unknown.
Another object of the invention is to provide a diagnostic test for AD which
enables definitive diagnosis of AD in living patients. Furthermore, as AD is a
progressive
to disease, it is desirable to diagnose AD as early as possible so that
preventative action may be
taken.
A number of diagnostic methods have been previously suggested for AD
diagnosis, most of which have focused on the (3-amyloid precursor protein. See
for example
U.S. Patents 4,666,829, 4,816,416 and 4,933,159. However, (3-amyloid deposits
have been
i5 found in individuals, especially aged persons, who have not shown signs of
dementia (See J.
Biol. Chem., 2~, pp 15977, 1990; and Tables 3-5). Diagnostic tests based on
the ~i-amyloid
protein have therefore been shown to lack specificity for AD.
In U.S. Patent 4,727,041 a diagnostic test for AD is described based on
measuring
levels of somatotropin and somatomedin-C in blood sera following
administration of an L-
2 o dopa proactive test.
In International patent application WO 94/02851, a method is described for
identifying AD by the use of antibodies having affinity for paired helical
filaments in order
to determine the levels of paired helical filaments in cerebral spinal fluid.
The presence of
paired helical filaments is alleged to be indicative of AD.
25 Other diagnostic methods are based on the identification of "disease
specific
marker proteins" in the cerebrospinal fluid. In International patent
application WO 95/05604,
for example, five disease specific proteins are identified by their molecular
weights.
However, the specific identity of the proteins is unknown and their specific
relationship to the
pathogenesis of AD is also unknown. The five "disease specific marker
proteins" may
3 o therefore be present as a result of a more fundamental cellular or
biochemical change.
Another object of the invention is to provide for detection of AD preferably
early
on in the disease state. It is desirable to detect a protein or substance
which is either directly
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responsible for the disease or is involved early on in the pathogenesis of the
disease, or if not
involved is nevertheless generated directly or indirectly by the mechanism
causing the
disease. Such a protein or substance may be the "causative" agent to the
disease or may be
"associated with" the disease state in the sense of being diagnostic of the
disease state.
Recently, Shernngton et al. in Nature, 3~, pp 254-260, 1995, identified a gene
on chromosome 14 bearing missense mutations which are associated with up to
33% of
autosomal dominant early onset AD cases (Table 1). A missense mutation
involves a
nucleotide substitution, usually a single nucleotide substitution, which
results in an amino
acid substitution at the corresponding codon. The missense mutations disclosed
in
io Sherrington et al. are predicted to change the encoded amino acid at the
following positions
(numbering from the first putative initiation codon) Met to Leu at codon 146,
His to Arg at
codon 163, Ala to Glu at codon 246, Leu to Val at codon 286, Cys to Tyr at
codon 410. It has
been proposed that these mutations may be useful in identifying early onset
AD. As stated
earlier, the majority of AD cases are late onset (after 65 years of age; Table
1) and it is
i5 therefore still a problem to identify the majority of individuals having
AD, particularly late
onset AD.
There is no indication that these diseases occur at the RNA level and not at
the
DNA level. Accordingly, the prior art methods of detection are for mutated DNA
or for a
protein encoded by the mutated DNA, and will not give an indication of the
presence of a
a o transcript mutation in an RNA molecule.
Presently, there are a number of substances which are alleged to be useful in
the
treatment of AD. However, so far only limited success has been achieved with
these
substances. Methods for effectively treating and/or preventing AD are still
required (see
Allen and Burns, Journal of Psychopharmacology, 2, pp 43-56, 1995).
SLJMNIARY OF THE INVENTION
The present invention is based on the observation that an RNA molecule
containing a frameshift mutation and encoding a corresponding mutant protein
are correlated
with the presence of a disease.
3 o According to the present invention there is provided a method for the
diagnosis
of a disease caused by or associated with an RNA molecule having one or more
mutations
giving rise to a frameshift mutation comprising: i. providing a biological
sample, such as a
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body fluid or tissue sample, from a patient; and ii. analyzing the sample for
the presence of
an RNA molecule having a frameshift mutation or a mutant protein encoded
thereby, wherein
the presence of the mutated RNA or mutant protein is indicative of the
disease.
A "mutant" protein is a polypeptide encoded by a mutant mRNA at least a part
of
s which is in a reading frame that is shifted relative to the initiation start
codon from that of the
native or wild-type reading frame, and thus will include any protein having an
aberrant
carboxy terminal portion which is encoded by the +1 or +2 reading frame of the
wild type
gene sequence. Thus, the mutant protein will include a hybrid wild-
type/nonsense protein
having an amino terminal amino acid sequence that is encoded by the wild type
(O) reading
io frame and a carboxy terminal amino acid sequence that is encoded by the +1
or +2 reading
frame, and thus the nonsense portion of the mutant protein. The cross-over
point between the
wild type and nonsense amino acid sequences is the codon containing the
frameshift mutation.
The invention is based on the discovery of the presence of such a mutant
protein
or an accumulation of more than one mutant protein in a tissue from a diseased
individual,
and also on identification of the mutant protein as indicative of the disease.
The invention is
also based on the discovery that the mutation that gives rise to the mutant
protein occurs at
the RNA level and not at the DNA level.
The phrase "caused by or associated with" refers to an RNA molecule which is
either fully or partly responsible for the disease, or an RNA molecule which
is not responsible
a o for the disease but is associated with the diseased state in the sense
that it is diagnostic of the
diseased state.
A disease caused by or associated with at least one RNA molecule having one or
more mutations giving rise to a frameshift mutation can be any disease
including non-
hereditary cancers, neurodegenerative diseases such as Alzheimer's Disease
(AD); Downs'
a s syndrome; frontal lobe dementia (Pick's Disease); progressive supranuclear
palsy (PSP) and
other diseases with abundant tau-positive filamentous- lesions such as
Corticobasal
degeneration, Dementia pugilistica, Dementia with tangles only, Dementia with
tangles and
calcification, Frontotemporal dementias with Parkinsonism linked to chromosome
17,
Gertsmann-Strassler-Scheinker disease with tangles, Myotonic dystrophy,
Niemann-Pick
s o disease type C, Parkinsonism-dementia complex of Guam, Postencephalitic
Parkinsonism and
Subacute sclerosing panencephalitis; Parkinson's Disease (PD) amyotrophic
lateral sclerosis;
Huntington's Disease; multiple sclerosis; dementia with Lewy bodies,
multisystem atrophy
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and other inclusion body diseases associated with ubiquitin such as
Alexander's disease,
Alcoholic liver disease, lichen amyloidosis, and during aging Marinesco bodies
and Hyaline
inclusions; and other degenerative diseases such as cardiovascular diseases,
rheumatoid
arthritis and Diabetes mellitus type II. Cancers treatable according to the
invention include,
s but are not limited to, Hodgkin's disease, acute and chronic lymphocytic
leukemias, multiple
myeloma, breast, ovary, lung, and stomach or bladder cancers.
An RNA molecule having a transcript mutation which leads to a frameshift
mutation, and herein referred to as the "mutant RNA", can be any RNA molecule
having at
least one transcript mutation which leads to a frameshift mutation. The RNA
molecule may
i o be any RNA molecule including primary transcripts and messenger RNA
(mRNA).
The term "transcript mutation" refers to a mutation which occurs at the RNA
level
but does not occur in the DNA from which the RNA was transcribed. In order to
identify
transcript mutations a comparison between the RNA and the DNA from which the
RNA was
transcribed has to be made.
15 A "frameshift mutation" refers to a deletion or insertion of one or more
nucleotides within an open reading frame, for example, a single nucleotide or
dinucleotide
deletion or insertion, such that the reading frame of the coding region is
shifted by one or two
nucleotides. Preferably, the frameshift mutation is a nucleotide or
dinucleotide deletion
leading to a + 1 or +2 frameshift mutation. However, any number of nucleotide
deletions can
20 occur provided a frameshift mutation results. Alternatively, the insertion
of one or more
nucleotides may give rise to a frameshift and such mutations also form part of
the present
invention.
Other genetic modifications which give rise to a frameshift also form part of
the
present invention, such as a change in the nucleotide sequence which leads to
translation
s s initiation from a different position or a mutation outside a coding
region, such as within an
Intron (if the RNA molecule is a primary transcript), or a 5' or 3'
untranslated region, which
mutation may result in mis-translation and production of a mutant protein.
It is preferred that the mutation is a nucleotide and more preferably a
dinucleotide
deletion or insertion associated with the nucleotide sequence GAGA or its
complementary
3 o sequence CTCT of the RNA molecule; especially preferred frameshift
mutations are
associated with the nucleotide sequence of the RNA comprising GAGAX or CTCTX,
where
X is one of G, A, U or C, the preferred motifs being GAGAG, GAGAC, GAGAT, and
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GAGAA as well as CTCTC, CTCTG, CTCTA and CTCTT. As used herein, the term "GAGA
mutation" may refer to either a single nucleotide insertion or deletion or a
dinucleotide
insertion or deletion within the GAGA or CTCT motif itself or adj acent to (5'-
or 3'-terminal
to-, and within 5-10 nucleotides of ) the GAGA or CTCT motif.
Preferably, the dinucleotide deletion is a GA deletion within the GAGA motif
or
a GT deletion immediately following (i.e., within 10 nucleotides 3' of) a GAGA
motif and a
CT deletion in the CTCT motif. It is further preferred that the mutant RNA has
one or two
dinucleotide deletions associated with a GAGA, GAGAC, GAGAG, GAGAT or GAGAA,
or with a CTCT, CTCTG, CTCTC, CTCTA or CTCTT, leading to a + 1 or + 2
frameshift
io mutation respectively.
In a preferred embodiment of the invention, the transcript mutations occur in
RNA
molecules of the neuronal system, where the disease is a neurodegenerative
disease.
The "neuronal system" is defined as any cells, RNA molecules, proteins or
substances relating to or forming part of the neuronal system such as nerve
cells, glial cells,
i5 proteins including Tau, (3 amyloid precursor protein, ubiquitin B,
apolipoprotein E4,
neurofilament proteins and microtubule associated protein II, presenilin I,
presenilin II, Big
Tau, glial fibrillary acidic protein (GFAP), Human P53 cellular tumor antigen,
human B-cell
leukemia/lymphoma 2 (BCL-2) protooncogene, semaphorins human homolog of yeast
~-
frameshift protein 1 (HUFF-I), Human Motility Group Protein (HIVIG), neuron
specific
a o protein A (NSP-A) and the RNA molecules encoding the proteins.
Where the disease is a neurodegenerative disease, especially AD, the preferred
mutant RNA molecules of the present invention are those encoding the (3
amyloid precursor
protein, the Tau protein, ubiquitin, apolipoprotein-E4 (Apo-E4), microtubule
associated protein
II (MAP 2), the neurofilament proteins, presenilin I, presenilin II, Big Tau,
GFAP, P53,
2 s BCL2, HUFF-I, HMG and NSP-A, having a deletion, insertion or other
modification leading
to a frameshift mutation. The most preferred mutant RNA molecules of the
present invention
are those encoding (3 amyloid precursor protein, ubiquitin B, MAP 2, the
neurofilament
proteins, presenilin I, presenilin II, Big Tau, GFAP, P53, bcl2 and HUFF-I,
which have a
frameshift mutation.
3 o It is preferred that the mutation is a GA or a GT dinucleotide deletion
associated
with (within or within 10 nucleotides 5' or 3' of) a GAGA or GAGAX sequence
leading to a
frameshift mutation or a CT or CA dinucleotide deletion associated with
(within or within 10
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nucleotides 5' or 3' of) a CTCT or CTCTX sequence leading to a frameshift
mutation. It is
further preferred that the mutant RNA molecule has one or two GA or GT
deletions or one
or two CT or CA deletions, each associated with a GAGA or CTCT sequence or
similar motif,
leading to a + 1 or + 2 frameshift mutation, respectively.
The term "mutant protein" as used herein is defined as the protein encoded by
the
mutant RNA molecule of the present invention.
It is preferred that the methods of the present invention are for the
diagnosis of a
disease caused by or associated with at least one RNA molecule having one or
more transcript
mutations giving rise to a frameshift mutation. A preferred disease for
diagnosis by the
io present invention is AD, except the early onset AD cases found to be linked
to chromosome
1, 14 and 21. It is further preferred that the methods of the present
invention are for the
diagnosis of young and late onset AD, especially non-familial or "sporadic"
late onset AD
cases.
As used herein, "biological sample" refers to a body fluid or body tissue
which
i s contains proteins and/or cells from which nucleic acids and proteins can
be isolated. Preferred
sources include buccal swabs, blood, sperm, epithelial or other tissue, milk,
urine,
cerebrospinal fluid, sputum, fecal matter, lung aspirates, throat swabs,
genital swabs and
exudates, rectal swabs, and nasopharyngeal aspirates.
The body fluid sample can be any body fluid which contains cells having the
a o transcript mutation which gives rise to the frameshift mutation and causes
or is associated
with the diseases. When the disease is a neurodegenerative disease it is
preferred that the
body fluid sample contains cells of the neuronal system or the products of
such cells. When
the disease is a neurodegenerative disease, the preferred body fluid is
cerebral spinal fluid,
which can be obtained after a lumbar puncture (Lannfelt et al., Nature
Medicine, l, pp 829-
25 832, 1995). Another preferred body fluid is blood (including, but not
limited to, venous,
arterial and cord blood), as it is easily obtained and contains lymphocytes
which can be
analyzed for the presence of the mutant RNA molecule or encoded protein.
The tissue sample can be any tissue and is preferably one that can be easily
obtained, such as skin and nose epithelium.
3 o Preferably, when analyzing the sample for a mutant RNA molecule, a nucleic
acid
probe is used. The nucleic acid probe is preferably a nucleotide probe having
a sequence
complementary to part of the mutant RNA molecule encompassing the mutation
giving rise
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to the frameshift mutation.
The probe must be used to detect RNA or DNA reverse transcribed from the RNA,
but must not be used to detect genomic DNA as the genomic DNA will not contain
the
mutation.
The present invention further provides a nucleic acid probe having a sequence
complementary to part of the mutant RNA molecule encompassing the mutation
leading to
the frameshift mutation. The probe is preferably sufficiently complementary to
the mutant
sequence of the RNA molecule so that under stringent conditions the probe only
remains
bound to the mutant sequence, and is able to distinguish under stringent
conditions the mutant
io and corresponding wild-type transcripts. "Stringent" conditions are defined
herein as
RNA:DNA hybridization conditions which may be performed at 65 ° C using
a hybridization
buffer equivalent to SO% formamide and O.1X SSC (see below and Evans et al.
PNAS (1994)
9; 6059-6063, 6060). "Stringent" conditions also preferably include stringent
washes, as
described in Evans et al. (Ibid).
15 The probe may be of any length but is preferably between S and SO
nucleotides
long, more preferably between 10 and 30 nucleotides long. For example, the
probe may be
5, 10, 15, 20, 25, or 30 nucleotides in length.
In a preferred embodiment the probe comprises a sequence complementary to a
GAGA or GAGAX or to a CTCT or CTCTX, having a nucleotide or dinucleotide
deletion or
a o insertion, and nucleotide sequences corresponding to the nucleotide
sequences flanking the
GAGA or CTCT motif in the wild-type RNA molecule. It would be apparent to one
skilled
in the art that if reverse transcribed DNA complementary to the mutant RNA
sequence was
being probed for, a probe comprising a sequence complementary to the
corresponding GAGA
or CTCT motif present in the complementary DNA would have to be used.
2 s Methods of detecting the presence of the mutant RNA molecule include the
reverse transcriptase polymerise chain reaction (RT-PCR) using primers having
a sequence
complementary to the sequence either side of the mutation which gives rise to
the frameshift
mutation. Firstly, one primer is used to reverse transcribe the RNA into DNA,
and secondly,
two primers are used to amplify the DNA, as described hereinbelow.
3 0 ~ The primers used in the above RT-PCR based method can vary in size from
20bp
to 2-3 kb; for example, 20bp, SObp, 100bp, SOObp, 1000bp, 1500bp, 2000bp, or
3000bp. The
primers can be prepared by a number of standard techniques including cloning
the sequences
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flanking the nucleotide region to be amplified or by synthesizing the primers
using
phosphoramidite method.
The present invention further provides primers for use in the above defined RT
PCR based methods for the amplification of the nucleotide region containing
the mutation.
Preferably, when analyzing the sample for the mutant protein of the present
invention an immunological test is employed. The immunological test is
preferably based on
the use an antibody molecule having specificity for the mutant protein of the
present invention
and not the wild-type protein.
The present invention thus further provides an antibody molecule having
to specificity for the mutated protein of the present invention but not for
the wild-type protein.
Preferably, the antibody is specific for the carboxy terminal end of the
mutant protein.
The present invention further provides a method for the diagnosis of a
neurodegenerative disease or other age-related diseases, or a method for the
diagnosis of a
person with a susceptibility for these diseases comprising: i. providing a
body fluid or tissue
i5 sample from a patient; and ii. analyzing the sample for the presence of an
RNA molecule of
the neuronal system having a frameshift mutation or a protein encoded thereby,
wherein the
presence of the mutated RNA molecule is indicative of a neurodegenerative
disease.
Preferably, the neurodegenerative disease is AD and Downs' syndrome.
The present invention also relates to methods for preventing and/or treating
the
a o diseases, vectors for preventing and/or treating the diseases and for the
production of
diagnostic reagents, compositions for preventing and/or treating the diseases,
nucleic acid
sequences, probes and antibody molecules for use in the present invention and
transgenic
animals.
Therapies contemplated according to the invention include providing to a cell
as containing a mutant transcript a ribozyme which is capable of selectively
eliminating (i.e.,
cleaving) the mutant transcript, thus rendering the transcript untranslatable.
The therapies also may include providing to a cell which is thus treated with
a
ribozyme a corresponding wild-type transcript which is substantially
uncleavable by the
ribozyme. The wild-type transcript may contain the wild-type sequence
corresponding to the
3 o mutant RNA sequence, except for the GAGA or CTCT permutation, and encoding
the wild-
type protein, and also may include third base (in a codon) silent mutations
which further
differentiate the wild-type RNA from the mutant RNA sequence, and thus further
distinguish
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the sequences with respect to ribozyme recognition and cleavage.
Therapies encompassed by the invention also include providing to cells
containing
a mutant RNA, an RNA or DNA that is complementary to the mutant RNA and able
to form
a duplex with the mutant RNA that is untranslatable in the cell. The
complementary
s sequence may be the entire length of the mutant RNA, but is preferably a
shorter length, for
example, 10, 20, 50 or 100 nucleotides in length. The complementary sequence
thus may be
administered in the form of an oligonucleotide or may be encoded by an
expressible sequence
contained in a vector, wherein the vector is administered to the cell.
The invention therefore encompasses a pharmaceutical composition comprising
1 o a ribozyrne that selectively cleaves a target RNA having a GAGA or CTCT
mutation admixed
with a pharmaceutically acceptable carrier.
The invention also encompasses a pharmaceutical composition comprising a
ribozyme that selectively cleaves a target RNA having a GAGA or CTCT mutation
and a
wild-type analog of an RNA having a GAGA or CTCT sequence giving rise to a
frameshift
is mutation admixed with a pharmaceutically acceptable carrier.
The invention also encompasses a pharmaceutical composition comprising a
wild-type analog of an RNA having a GAGA or CTCT sequence giving rise to a
frameshift
mutation admixed with a pharmaceutically acceptable carrier.
The invention also encompasses a pharmaceutical composition wherein the wild-
z o type analog of an RNA comprises a nucleotide sequence having third base
silent mutations.
The invention also encompasses a pharmaceutical composition comprising a
single stranded nucleic acid having a sequence that is complementary to an RNA
having one
or more GAGA or CTCT mutations giving rise to a frameshift mutation admixed
with a
pharmaceutically acceptable carrier.
25 The invention also encompasses a pharmaceutical composition comprising the
wild-type analog of a mutant protein in admixture with a pharmaceutically
acceptable carrier.
The invention also encompasses a vector comprising an expressible gene
encoding
a ribozyme that selectively cleaves a target RNA having a GAGA or CTCT
sequence.
The invention also encompasses a vector comprising an expressible gene
encoding
s o a sequence complementary to an RNA having a GAGA or CTCT mutation giving
rise to a
frameshift mutation.
The invention also encompasses a host cell containing a vector as described
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herein.
The invention also encompasses a method of treatment and/or prevention of a
disease caused by or associated with an RNA having a GAGA or CTCT mutation
giving rise
to a frameshift mutation, comprising administering the compositions, vectors,
or the host
s cells described above to a patient suffering from or susceptible to the
disease.
The invention also encompasses the use of a vector encoding a ribozyme that
selectively cleaves a target RNA having a GAGA or CTCT mutation under the
control of a
promoter in therapy.
The invention also encompasses the use of a vector encoding a ribozyme under
to the control of a promoter in the manufacture of a composition for the
treatment of a disease
caused by or associated with at least one an RNA having one or more GAGA or
CTCT
mutations giving rise to a frameshift mutation.
The invention also encompasses the use of a vector encoding the sequence
complementary to an RNA having one or more GAGA or CTCT mutations giving rise
to a
is frameshift mutation under the control of a promoter in therapy.
The invention also encompasses the use of more than one of the compositions,
the
vectors, or the host cells described above in any combination in therapy.
The invention also encompasses the use of more than one of the compositions,
the
vectors, or the host cells described herein in any combination in the
treatment and/or
2 o prevention of a disease caused by or associated with at least one an RNA
having one or more
GAGA or CTCT mutations giving rise to a frameshift mutation.
The present invention further provides an early marker for a neurodegenerative
disease. The invention provides a diagnostic kit for diagnosing a disease
caused by or
associated with at least one RNA molecule having one or more transcript
mutations giving
2 s rise to a frameshift mutation comprising: i. a nucleic acid probe having a
sequence
complementary to part of the mutant RNA molecule which encompasses the
mutation which
leads to the frameshift mutation and packaging materials therefor; and ii.
means for detecting
the probe bound to the mutant RNA molecule.
The present invention further provides a diagnostic kit for diagnosing a
disease
3 o caused by or associated with at least one RNA molecule having one or more
transcript
mutations giving rise to a frameshift mutation comprising: i. primers for use
in an RT-PCR
reaction, the primers having a sequence complementary to the sequence either
side of the
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mutation which gives rise to the frameshift mutation, packaging materials
therefor, and
reagents necessary for performing an RT-PCR reaction and amplifying the DNA
sequence
containing the mutation; and ii. means for detecting the amplified DNA
sequence containing
the mutation.
s The present invention further provides a diagnostic kit for diagnosing a
disease
caused by or associated with at least one RNA molecule having one or more
transcript
mutations giving rise to a frameshift mutation comprising: i. an antibody
molecule having
specificity for the mutant protein of the present invention and not the wild-
type protein; and
ii. means for detecting the antibody molecule bound to the mutant protein.
to The antibody molecule and the means for detecting the bound antibody
molecule
are as defined above.
In a further embodiment of the present invention the diagnostic kit described
above additionally comprising: i. an antibody molecule having specificity for
the wild-type
protein; and ii. means for detecting the antibody molecule bound to the wild-
type protein, as
is a control for diagnosing a disease caused by or associated with at least
one RNA molecule
having one or more transcript mutations giving rise to a frameshift mutation.
The present invention further provides an RNA molecule having one or more
transcript mutations giving rise to a frameshift mutation which causes or is
associated with
a disease.
a o The invention further provides several (one or more) RNA molecules
encoding
the same amino acid sequence up to the GAGA or CTCT motif, and thereafter
encoding
different sequences. For example, in a single cell, one RNA molecule encoding
a
frameshifted protein based on, for example, ~3-app, may contain a mutation at
or within the
GAGA motif in exon 9 of the RNA sequence and a second RNA molecule encoding ~3-
app
z s may contain a mutation at or within the GAGA motif in exon 10 of the
sequence.
The present invention further provides a mutated protein encoded by the
mutated
RNA molecule found to be indicative of a disease, the mutant RNA molecule
having one or
more transcript mutations giving rise to a frameshift mutation. Preferably,
the mutant protein
contains an antigenic epitope specific for the diseased state, examples of
which are provided
3 o in Table 9.
In a preferred embodiment of the present invention the mutated RNA molecule
encodes a protein comprising at least part of the sequence designated +1 or +2
in any one of
,y-lFi~ ~H~~'1
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CA 02287084 1999-10-08
r , . , . , . , ~ r
r
- .. .. '. . _ .-. , - r _ ;_ _ r,
r
14
Figures 2 to 19, or an immunologically equivalent fragment thereof.
In a preferred embodiment the mutated protein comprises any one of the
following
individual sequences: RGRTSSKELA [SEQ ID NO: 1]; HGRLAPARHAS [SEQ ID NO:
2]; YADLREDPDRQ [SEQ ID NO: 3J; RQDHHPGSGAQ [SEQ ID NO: 4];
s YADLREDPDRQDHHPGSGAQ [SEQ ID NO: 1400]; GGGAQ [SEQ ID NO: 5],
GAPRLPPAQAA [SEQ ID NO: 6]; KTRFQRKGPS [SEQ ID NO: 7]; PGNRSMGHE [SEQ
ID NO: 8]; EAEGGSRS [SEQ ID NO: 9]; VGAARDSRAA [SEQ ID NO: 10];
HDYPPGGSV [SEQ ID NO: 11]; SIQKFQV [SEQ ID NO: 12]; VEKPGERGGR [SEQ ID
NO: 13]; PLFGRGHKRG [SEQ ID NO: 14J; EDRGDAGWRGH [SEQ ID NO: 15];
to QERGASPRAAPREH [SEQ ID NO: 16]; RQPGDVAPGGQHRPVDD [SEQ ID NO: 17];
AGLLAIPEAK [SEQ ID NO: 18]; YVDVYNGGKFS [SEQ ID NO: 19];
AADERRCHLLHMCGRR [SEQ ID NO: 20; QQATEAGQHYQPGSPLHDHSHV [SEQ
ID NO: 21]; PQEAAARTNR [SEQ ID NO: 22]; RSWVHPAPPYQMCLG [SEQ ID NO:
23]; and GGSRTHPR [SEQ ID NO: 24], especially when the disease is a
neurodegenerative
is disease such as AD.
In a preferred embodiment, the antibody molecule of the present invention has
affinity for the mutant proteins defined above.
The present invention also relates to a method for treating and/or preventing
a
disease caused by or associated with at least one RNA molecule having one or
more transcript
a o mutations giving rise to a frameshift mutation. The finding of mutations
in RNA molecules
which lead to the production of mutant proteins, and which are indicative of a
disease, has led
to a number of ways of treating and/or preventing the disease.
The present invention further provides a method for identifying diseases
caused
by or associated with at least one RNA molecule having one or more transcript
mutations
a s giving rise to a frameshift mutation. The method comprises: i. providing
the sequence of an
RNA molecule suspected of being involved in the pathogenesis of a disease; ii.
identifying
the sequence of the mutant protein encoded by the RNA sequence 3'-terminal to
a frameshift
mutation; iii. preparing a probe to the mutant protein or a fragment thereof;
and iv. probing
a body fluid or tissue sample from a patient having the disease and a patient
not having the
s o disease, in order to find a correlation between the presence of the mutant
protein and the
diseased state.
Preferably, the probe is an antibody molecule as defined herein. It is further
A~it~~~~~~i~ ~~riL~Ei~
CA 02287084 1999-10-08
. . ' ' ~ ~ ~ ~ r '
r . ,. r , ,
. r
preferred that the antibody molecule has affinity for a protein comprising at
least one of the
sequences: RGRTSSKELA [SEQ ID NO: 1]; HGRLAPARHAS [SEQ ID NO: 2];
YADLREDPDRQ [SEQ ID NO: 3J; RQDHHPGSGAQ [SEQ ID NO: 4];
YADLREDPDRQDHHPGSGAQ [SEQ ID NO: 1400]; GGGAQ [SEQ ID NO: 5],
s GAPRLPPAQAA [SEQ )D NO: 6]; KTRFQRKGPS [SEQ ID NO: 7]; PGNRSMGHE [SEQ
ID NO: 8]; EAEGGSRS [SEQ ID NO: 9]; VGAARDSRAA [SEQ ID NO: 10];
HDYPPGGSV [SEQ ID N0: 11]; SIQKFQV [SEQ ID NO: 12]; VEKPGERGGR [SEQ ID
NO: 13J; PLFGRGHKRG [SEQ ID N0: 14]; EDRGDAGWRGH [SEQ ID NO: 15);
QERGASPRAAPREH [SEQ ID NO: 16]; RQPGDVAPGGQHRPVDD [SEQ 117 NO: 17];
to AGLLAIPEAK [SEQ ID NO: 18]; YVDVYNGGKFS [SEQ ID NO: 19];
AADERRCHLLHMCGRR [SEQ )D NO: 20; QQATEAGQHYQPGSPLHDHSHV [SEQ
ID NO: 21]; PQEAAARTNR [SEQ ID NO: 22]; RSWVHPAPPYQMCLG [SEQ ID NO:
23J; and GGSRTHPR [SEQ ID NO: 24J, especially when the disease is a
neurodegenerative
disease such as AD.
1 s Other features and advantages of the invention will be apparent from the
following
description of the preferred embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
The invention is now illustrated in the appended example with reference to the
z o following drawings:
Figure 1 is a copy of a paraffin section (6 ~m thick) of the frontal cortex of
a ~!. ~~~r~ ~
female Alzheimer patient (70 years old, #83002; Table 2) immunocytochemically
incubated
with an antibody against a peptide predicted by the + 1 reading frame of (3APP
(Figure 20).
The hallmarks of A.D., dystrophic neurites (arrowheads) and tangles (arrows),
are clearly
4
as visible in the cortical layer III. RGRTSSKELA [SEQ ID NO: 1] = Amy+' (see
Table 9).
1- ; ~, .1 '
Figure 2 presents the coding nucleotide sequence of the human [i amyloid
precursor protein gene transcript [SEQ ID NO: 25], the amino acid sequence of
the wild-type
protein [SEQ ID NO: 83 and 84J, the mutant + 1 frameshift protein [SEQ ID NO:
50-82] and
the mutant + 2 frameshift protein [SEQ ID NO: 26-49].
3 o Figure 3 presents the coding nucleotide sequence of the human microtubule-
associated protein tau gene transcript [SEQ ID NO: 85], the amino acid
sequence of the wild-
type protein (SEQ 1D NO: 99], the mutant + 1 frameshift protein [SEQ ID NO: 86-
98J and
_.__. _..,..~,.-.w__.. ..... ...-._~._..~._.- ~~~h~~~ y~r~~
CA 02287084 1999-10-08
f .- r . r r
r [ r
i ~ r
r . ~ r . ~ , ~ , - ,
16
the mutant + 2 frameshift protein [SEQ ID NO: 100-112].
Figure 4 presents the coding nucleotide sequence of the human ubiquitin B gene
transcript [SEQ ID NO: 113], the amino acid sequence of the wild-type protein
[SEQ ID
NO: 125 and 126], the mutant + 1 frameshift protein [SEQ ID NO: 114-124] and
the mutant
s + 2 frameshift protein [SEQ ID NO: 127-136J.
Figure 5 presents the coding nucleotide sequence of the human apolipoprotein E
gene transcript [SEQ ID NO: 137], the amino acid sequence of the wild-type
protein [SEQ
ID NO: 144-146], the mutant + 1 frameshift protein [SEQ ID NO: 138-143] and
the mutant
+ 2 frameshift protein [SEQ ID NO: 147-152]. Information concerning
restriction enzyme
1 o sites is also given.
Figure 6 presents the coding nucleotide sequence of the human microtubule-
associated protein 2 transcript [SEQ ID NO: 153], the amino acid sequence of
the wild-type
protein [SEQ ID NO: 154-158], the mutant + 1 frameshift protein [SEQ ID NO:
232-347]
and the mutant + 2 frameshift protein [SEQ ID NO: 159-231).
is Figure 7 presents the coding nucleotide sequence of the human neurofilament
subunit NF-low transcript [SEQ ID NO: 348], the amino acid sequence of the
wild-type
protein [SEQ ID NO: 466-513], the mutant + 1 frameshift protein [SEQ ID NO:
413-465]
and the mutant + 2 frameshift protein [SEQ 117 NO: 349-412J.
Figure 8 presents the coding nucleotide sequence of the human neurofilament
2 o subunit NF-M transcript [SEQ ID NO: 514], the amino acid sequence of the
wild-type protein
[SEQ ID NO: 515-574], the mutant + 1 frameshift protein [SEQ ID NO: 629-695]
and the
mutant + 2 frameshift protein [SEQ ID NO: 575-628].
Figure 9 presents the coding nucleotide sequence of the human neurofilament
subunit NF-H gene transcript [SEQ ID NO: 696], the amino acid sequence of the
wild~type
25 protein [SEQ ID NO: 697-698], the mutant + 1 frameshift protein [SEQ ID NO:
708-710]
and the mutant + 2 frameshift protein [SEQ ID NO: 699-707].
Figure 10 presents the coding mRNA nucleotide sequence [SEQ ID NO: 711 ] and
amino acid sequence of presenilin I expressed in the wildtype [SEQ ID NO:
712], +1 [SEQ
ID NO: 733-753], and +2 [SEQ ID NO: 713-732] reading frames.
3 o Figure 11 presents the coding mRNA nucleotide sequence [SEQ 1D NO: 754]and
amino
acid sequence of presenilin II expressed in the wildtype [SEQ ID NO: 776-787],
+1 [SEQ 1D
NO: 755-775], and +2 [SEQ ID NO: 788-814] reading frames.
,~io ~.
4 ~~L::~ ~ ~;i'!
CA 02287084 1999-10-08
,. ,- r ~ ! o
17
Figure 12 presents the coding mRNA nucleotide sequence [SEQ ID NO: 81 S] and
amino acid sequence of Big Tau expressed in the wildtype [SEQ ID NO: 816-818],
+1 [SEQ
ID NO: 824-834] and +2 [SEQ ID NO: 819-823] reading frames.
Figure 13 presents the coding mRNA nucleotide sequence [SEQ m NO: 835] and
s amino acid sequence of GFAP expressed in the wildtype [SEQ ID NO: 836-852],
+1 [SEQ
ID NO: 883-914], and +2 [SEQ ID NO: 853-882] reading frames.
Figure 14 presents the coding mRNA nucleotide sequence [SEQ ID NO: 915] and
amino acid sequence of P53 expressed in the wildtype [SEQ ID NO: 940-949], +1
[SEQ ID
NO: 916-939] and +2 [SEQ ID NO: 950-965] reading frames.
to Figure 15 presents the coding mRNA nucleotide sequence [SEQ ID NO: 966]
and amino acid sequence of BCL2 expressed in the wildtype [SEQ ID NO: 967-
1015], +1
[SEQ ID NO: 1075-1126] and +2 [SEQ ID NO: 1016-1074] reading frames.
Figure 16 presents the coding mRNA nucleotide sequence [SEQ ID NO: 1127]
and amino acid sequence of Semaphorin III expressed in the wildtype [SEQ ID
NO: 1128
15 1131], +1 [SEQ ID NO: 1162-1212] and +2 [SEQ 1D NO: 1132-1161] reading
frames.
Figure 17 presents the coding mRNA nucleotide sequence [SEQ 1D NO: 1213]
and amino acid sequence of HUFF expressed in the wildtype [SEQ ID NO: 1241-
1244], +1
[SEQ ID NO: 1214-1240] and +2 [SEQ 117.N0: 1245-1281]reading frames.
Figure 18 presents the coding mRNA nucleotide sequence [SEQ ID NO: 1282]
2 o and amino acid sequence of HMG expressed in the wildtype [SEQ ID NO: 1297-
1299], +1
[SEQ ID NO: 1289-1296] and +2 [SEQ ID NO: 1283-1288] reading frames.
Figure 19 presents the coding mRNA nucleotide sequence [SEQ ID NO: 1300]
and amino acid sequence of NSP-A expressed in the wildtype [SEQ ID NO: 1374-
1387],
+1 [SEQ ID NO: 1339-1373), and +2 reading frames [SEQ ID N0: 1301-1338]. --
2 s Figure 20 presents the partial mRNA nucleotide sequence and amino acid !
sequence of two human neuronal proteins ((3 amyloid precursor protein (exons 9
and 10) and i
i ~ ',
Ubiquitin B (exon 2)) expressed in the wildtype and +1 reading frame. 'Number
of GAGAG '° '.'' ~
motifs: 7. PredictedVmolecular weight of truncated protein 38 kDa.[30]. ~~ ~
;., ''
ZNumber of GAGAG motifs: 2. Predicted molecular weight of truncated protein 11
KDa
s o (monomer),and. expressed in brain [31,32].T ~ exon 9/10 junction- ~'
Figure 21: Two examples of novel restriction sites generated by dinucleotide A
, J , ,
deletion in transcripts of (3 amyloid precursor protein and ubiquitin B (wild-
type nucleotide p - J
1,~;i.l ~~~~v
CA 02287084 1999-10-08
r r - f ~.
r , ~ r
r . - . r . ,
18
sequences, [SEQ 117 NO: 25 and 113];~i amyloid precursor protein deletion
sequences [SEQ
ID NO: 1396 and 1397]; ubiquitin deletion sequences [SEQ )D NO: 1398-1399]. In
general,
a mutation in the nucleotide sequence can result in changes in the restriction
enzyme
~.V
recognition sites of the sequence. The GA deletion in exon 9 of ~i-APP and the
GT deletion
s in the first repeat of of Ubi-B do not alter the restriction map of the
sequence down- and
upstream of the deletion. However, due to the GA deletion in exon 10 of (3-APP
an MslI site
is created at the site of the deletion (Figs. 21A and 21B).
In the Ubi-B sequence the CT deletion in the second repeat leads to the loss
of a Hin4-I and
BstX-I site and the creation of a Cje-I site upstream and the creation of a
BsR-I and a TspR-I
io site downstream the deletion site (Fig. 21-5-8).
The invention is illustrated by the following nonlimiting examples wherein the
following materials and methods are employed. The entire disclosure of each of
the literature
i5 references cited hereinafter are incorporated by reference herein.
The present invention is based on the discovery that frameshift mutations
occur
in a single RNA molecule or number of RNA molecules whose product or products
are
mutant proteins that are associated with, and indicative of, a disease state.
The invention is
based on the recognition that the presence of a frameshift mutation results in
a new coding
z o sequence for the cell containing the frameshift mutation, and thus a new
polypeptide (herein
termed a mutant protein) which may be correlated with and thus be indicative
of a disease.
According to the present invention, diagnosis and/or identification of a
disease
caused by or associated with at least one RNA molecule having one or more
transcript
mutations which give rise to a frameshift mutation is accomplished as
described herein:
z s According to the present invention, methods for preventing and/or treating
the
diseases, vectors for preventing and/or treating the diseases and for the
production of
diagnostic reagents, compositions for preventing and/or treating the diseases,
nucleic acid
sequences, probes and antibody molecules for use in the present invention and
transgenic
animals are accomplished as described herein.
s o According to the present invention, methods for detecting errors in
transcriptional
mechanisms are accomplished as described herein. The correction of the
mutations found in
the mutant RNA molecules of the present invention is therefore a valuable
method for
AfJ#E~L~~~ ~~~Ef
CA 02287084 1999-10-08
y ,
.. _ ,
19
combatting diseases.
Methods and reagents for disease diagnosis and treatment are described in more
detail hereinbelow.
s Diagnosis of Diseases According to the Invention
The invention relates to methods for diagnosing diseases caused by or
associated
with at least one RNA molecule having one or more transcript mutations which
give rise to
a frameshift mutation. Such diseases include but are not limited to cancers,
Diabetes mellitus
type II and neurodegenerative diseases such as Parkinson's Disease (PD),
Alzheimer's Disease
to (AD), frontal lobe dementia (Pick's Disease), progressive supranuclear
palsy (PSP) and other
diseases with abundant tau-positive filamentous lesions such as Corticobasal
degeneration,
Dementia pugilistica, Dementia with tangles only, Dementia with tangles and
calcification,
Frontotemporal dementias with Parkinsonism linked to chromosome 17, Gertsmann-
Strassler-
Scheinker disease with tangles, Myotonic dystrophy, Niemann-Pick disease type
C,
is Parkinsonism-dementia complex of Guam, Postencephalitic Parkinsonism,
Subacute
sclerosing panencephalitis, amyotrophic lateral sclerosis, Huntington's
Disease, dementia with
Lewy bodies, multisystem atrophy, other inclusion body diseases associated
with ubiquitin
such as Alexander's disease, Alcoholic liver disease, lichen amyloidosis, and
during aging
Marinesco bodies and Hyaline inclusions, multiple sclerosis, Downs' syndrome,
and other
a o degenerative diseases such as cardiovascular diseases rheumatoid
arthritis, and Diabetes
mellitus type II.
Somatic mutations can result in a different gene function and have been
implicated
in diseases associated with aging, such as certain cancers. However, it has
generally been
assumed that non-proliferating cells do not undergo important changes at the
genomic level.
2 5 For example, it was assumed previously that genomic changes are mainly
related to cell
proliferation (Smith, Mutation Research, 2Z2, pp 139-142, 1992) which for non-
proliferating
cells such as most neurons ends during early postnatal life (Rakic, Science,
2.ZZ, pp 1054-
1056, 1985). However, Evans et al., 1994, Proc. Nat. Acad. Sci. 91:6059,
suggested that
somatic mutations do occur in genes of the neuronal system, i.e., in post-
mitotic neurons. The
3 o di/di Brattleboro rat, which suffers from severe diabetes insipidus due to
the absence of the
antidiuretic hormone vasopressin (VP), was the subject of the Evans et al.
paper. It had
previously been established that the VP hormone was absent in the Brattleboro
rat due to a
J~j~~i'e~.'tlJ ~r~'I~CT
CA 02287084 1999-10-08
~ ._ . _ ; ; - ~ . ,
_ _. ,_ ,. ~. ~ ,.. ~ ,. .. - -
deletion of a single G residue in the second exon of the VP gene, resulting in
a mutant VP
precursor with an altered C-terminal amino acid sequence. It had also been
observed that a
small number of neurons in the di/di rat exhibited a heterozygous +/di
phenotype and
expressed an apparently normal VP gene product. In studying the molecular
biology of the
s di/di rat, Evans et al. identified sequence alterations that restored the
reading frame of the
mutant VP precursor mRNA, which were based on a di-nucleotide deletion in a
GAGAG
motif. They correlated the presence of small amounts of normal VP gene product
in single
magnocellular neurons with a reversion of the mutant gene stemming from a
frameshift
mutation. Evans et al. concluded that, because +1 frameshift mutations are
present in VP
io transcripts of both wild-type rats and di/di rats, the events leading to
these mutations are not
caused by the diseased state of the di/di rat per se. Thus, Evans et al. did
not correlate a
mutational GAGAG hotspot with a disease state, or predeliction to a disease.
Furthermore,
there is no suggestion in the prior art that transcript mutations are
occurring and that such
transcript mutations are caused by or associated with a disease. As the
mutations have
i5 previously been considered to occur in DNA, methods of detection have been
unreliable as
there will be no mutation in the genomic DNA and the probing of genomic DNA
will give a
false indication of the absence of the mutation.
In the present invention, the observations of Evans et al., as to reversions
in the
wild-type reading frame at GAGAG hotspots in VP transcripts within single
neurons of the
2 o di/di rat leading to wild-type-like VP gene products, is extended and
developed. According
to the present invention, a human disease which is caused by or associated
with at least one
RNA molecule having one or more transcript mutations occurring at a mutational
hotspot and
which give rise to a frameshift mutation is identified and/or diagnosed. The
nucleotide
sequence of an RNA molecule suspected of being involved in the pathogenesis of
a disease
1S provided, e.g., from published gene sequences or from cloning and
sequencing of a suspect
RNA molecule. The amino acid sequence encoded by the RNA molecule is then
predicted,
as are amino acid sequences of encoded mutant proteins. Mutant protein
sequences are
predicted in +1 and +2 reading frames following a hypothesized frameshift
mutation. The
location of the frameshift mutation may be hypothesized with respect to
certain nucleotide
3 o sequence motifs which are suspected of causing frameshift mutations,
examples of such
motifs present in the RNA molecule including but not limited to those
comprising GAGA,
for example, GAGAC, GAGAG, GAGAT, and GAGAA, or those comprising CTCT, for
~~iu~i~ u~~.~;
CA 02287084 1999-10-08
21
example CTCTC, CTCTG, CTCTA and CTCTT.
A probe is then prepared that is specific for the mutant protein or an
immunogenic
fragment thereof (such probes being described hereinabove for detection of
proteins or protein
fragments). Depending on where the mutation that leads to the frameshift
occurs, part of the
s mutant protein will have the same sequence as the wild-type protein and part
of the protein
will have the sequence of the mutant protein. Furthermore, depending on where
the mutation
occurs the mutant protein will terminate when the nucleotide sequence codes
for a stop codon
(indicated as * in the Figures). Thus, different mutant proteins will be
produced depending
on where the mutation occurs.
to Alzheimer's Disease (AD) is a representative disease diagnosable and
treatable
according to the invention. AD is a neurodegenerative disease characterised by
idiopathic
progressive dementia and is the fourth highest major cause of death in
developed countries.
It affects 5 to 11 % of the population over the age of 65 and as much as 47%
of the population
over the age of 85. At present there are an estimated 4 million patients
suffering from AD in
is the U.S.A. (see Coleman, Neurobiol. of Aging, 1~, Suppl. 2, pp 577-578,
1994), and an
estimated 20 million Alzheimer's patients worldwide.
The clinical criteria for AD diagnosis have been defined (see Reisberg et al.,
Am.
J. Psych. 12, pp 1136-1139, 1982; McKhann et al., Neurology, ~4, pp 939-944,
1984). The
early symptoms of AD vary but generally include depression, paranoia and
anxiety. There
2 o is also a slow degeneration of intellectual function and memory. In
particular, cognitive
dysfunction and specific disturbances of speech (aphasia), motor activity
(apraxia), and
recognition of perception (agnosia) can occur.
There is not yet general consensus in a test for ante mortem diagnosis for AD
due
to the lack of knowledge of the pathogenic mechanisms involved in AD.
Diagnosis of AD
2 s is made by examination of brain tissue. Such diagnosis is usually carned
out on individuals
post mortem. The diagnosis is based on the presence of a large number of
intraneuronal
neurofibrillary tangles and of neuritic plaques in the brain tissue, in
particular in the neocortex
and hippocampus. In order to identify the various types of plaques (e.g.
neuritic plaques),
neuropil threads and neurofibrillary tangles, staining and microscopic
examination of several
3 o brain tissue sections is necessary. Neuritic plaques are believed to be
composed of
degenerating axons (e.g., neuropil threads), nerve terminals and possibly
astrocytic and
microglial elements. It is also often found that neuritic plaques have an
amyloid protein core.
~~.~Lci~ ~'~~~~
CA 02287084 1999-10-08
y
..
22
The neurofibrillary tangles comprise normal and paired helical filaments and
are believed to
consist of several proteins.
There are two major types of AD, late onset (>65 years) and early onset (<65
years). Approximately 85% of all AD cases are late onset and only 15% are
early onset. Of
s the latter group 0.3% consists of the hereditary type of AD linked to
chromosome 21, 2% of
the cases are considered to be linked to chromosome 14, and chromosome 1 has
been
established for juvenile onset (<0.1%), as discussed below. Sporadic cases are
the most
prominent group (40%) in early onset AD.
In the most common late onset group, 40% of cases are considered to be
familial,
i o meaning that Alzheimer was observed in first degree relatives. Of this
familial form only 10%
is autosomal dominant. The remaining late onset cases (60%) are non-familial
or "sporadic"
cases (see Table 1). For these cases relatively little is known and previously
no data was
available which suggested a possible cause of AD.
At present, it is unclear whether the formation of neuritic plaques and/or
i5 neurofibrillary tangles is directly responsible for causing AD. The
formation of neuritic
plaques, neuropil threads and/or neurofibrillary tangles may be a consequence
of a more
fundamental cellular or biochemical change.
Diagnostic methods of the invention will include the detection of nucleic acid
sequences, preferably via procedures which involve formation of a nucleic acid
duplex
2 o between two nucleic acid strands, i.e., a nucleic acid probe and a
complementary sequence
in the mutant RNA or the DNA reverse transcribed from the mutant RNA isolated
from a
biological sample, or detection of a protein, preferably a mutant or hybrid
wild-type/nonsense
protein, as defined herein.
2s 1. Preparation and Detection of RNA for Genetic Screening.
Typically, RNA is prepared from the biological sample by DNA extraction
procedures well-known in the art (see, e.g., Sambrook et al., 1990, A
Laboratory Manual for
Cloning, Cold Spring Harbor Press, CSH, N~, and may be further purified if
desired, e.g.,
by electro-elution, prior to analysis.
3 o Methods of detecting a mutant RNA molecule from a biological sample
include,
but are not limited to the following: (1) reverse transcriptase polymerase
chain reaction (RT-
PCR) followed by sizing gel electrophoresis or hybridization with an allele-
specific (or
A~i~I~~ED ~~i~~T
CA 02287084 1999-10-08
23
sequence-specific) probe; (2) hybridization of the eluted RNA with a nucleic
acid probe that
is complementary to the mutated RNA; (3) the ARMS test, in which one primer
has a
complementary sequence encompassing the mutation which gives rise to the
frameshift
mutation, and amplification only occurs if the mutated sequence is present;
(4) nucleotide
sequencing; (5) RNA amplification via RT-PCR and T7 polymerase; and (6) by a
dinucleotide
deletion in the RNA after RT-PCR a cDNA can be generated with novel
restriction sites
(Figure 21 ).
A nucleic acid probe useful according to the invention is preferably
sufficiently
complementary to the mutant sequence of the RNA molecule so that under
stringent
1 o conditions the probe only remains bound to the mutant sequence (see Evans
et al., Proc. Natl.
Acad. Sci. USA, 91:6059-6063 (1994). The probe is preferably labelled using
any of the
standard techniques known to those skilled in the art, such as radioactively
using 32P or any
other standard isotopes, or using non-radioactive methods including biotin or
DIG labelling.
The labelled probe can then be easily detected by methods well known to those
skilled in the
art.
An alternative method for detecting the presence of the mutant RNA molecule is
via the reverse transcriptase polymerase chain reaction (RT-PCR). Primers
having a sequence
complementary to the sequence either side of the mutation which gives rise to
the frameshift
mutation are used to reverse transcribe the RNA and amplify the reverse
transcribed DNA
2 o containing the mutation. The mutation in the amplified fragment can then
be detected using
the probe described above using standard techniques or by sequencing the
amplified fragment.
The advantages of using the RT-PCR reaction is that less starting material is
required and the
PCR methods allow quantitative as well as qualitative determinations to be
made.
Quantitative determinations allow the number of copies of a mutated RNA
molecule present
2 s in a particular sample to be estimated, and given this information the
severity of the diseased
state can be estimated.
Another alternative method for detecting the presence of the mutant RNA
molecule is one in which one primer has a complementary sequence encompassing
the
mutation which gives rise to the frameshift mutation. Amplification will
therefore only occur
3 o if the mutated sequence is present. Newton et al., Nucl. Acids. Res.
17:2503, 1989. The
method has previously been used in detecting mutations in the gene responsible
for cystic
fibrosis, and one skilled in the art could easily perform this test for the
detection of the mutant
CA 02287084 1999-10-08
r r ~ r r
. , r r . r ~ r r
t~ r
24
RNA or the reverse transcribed DNA corresponding to the mutant RNA of the
present
invention.
An example of analysis method (1) follows. The RNA is reverse transcribed and
the DNA then amplified, e.g., using PCR, prior to analysis. Specific
conditions for any one
s PCR, i.e. a PCR targeting a particular sequence, or for any one multiplex
PCR, i.e. a PCR
targeting a particular set of sequences, may vary but will be known to a
person of ordinary
skill in the art.
Amplification of a mutated or wild-type reverse transcribed DNA sequence can
be accomplished directly from an aliquot of the prepared DNA as follows.
i o 25 ul of DNA is aliquotted into a reaction tube containing 25 ~1 H20, 50
~1 master
mix (see below), 0.5 pl Amplitaq (Perkin Elmer Cetus, Norwalk, CT) and 0.5 ~.1
UNG (Perkin
Elmer Cetus, Norwalk, CT). A 50 pl master mix comprises 20 mM Tris HCI, pH
8.3, 100
mM KCI, S mM MgClz, 0.02 pmoles each of dATP, dGTP, dCTP, 0.04 moles of dUTP,
20
pmoles of each primer (Perkin Elmer Cetus, Norwalk, CT), and 25 ~g gelatin.
is A fragment characteristic of the selected amplification sequence can then
be
visualized under ultraviolet light after ethidium bromide staining a 13%
polyacrylamide gel
in which an aliquot of the amplification has been electrophoresed.
Alternatively,
hybridization with allele-specific probes can identify the presence of
amplified product from
either the normal and/or mutant alleles.
2. Preparation and Detection of Protein for Genetic Screening.
Where the biological molecule to be analyzed is a protein, it may be desirable
to
release the nucleic acid from biological sample cells prior to protein
elution, or to remove
2 s nucleic acid from the sample eluate prior to protein analysis. Thus, the
sample or eluate may
first be treated to release or remove the nucleic acid by mechanical
disruption (such as
freeze/thaw, abrasion, sonication), physical/chemical disruption, such as
treatment with
detergents (e.g., Triton, Tween, or sodium dodecylsulfate), osmotic shock,
heat, enzymatic
lysis (lysozyme, proteinase K, pepsin, etc.), or nuclease treatment, all
according to
3 o conventional methods well known in the art.
Where a biological sample includes a mutant protein, the presence or absence
of
which is indicative of a genetic disease, the protein may be detected using
conventional
A~~E~t?~D ~HE~T
CA 02287084 1999-10-08
detection assays, e.g., using protein-specific probes such as an antibody
probe. Similarly,
where a genetic disease correlates with the presence or absence of an amino
acid or sequence
of amino acids, these amino acids may be detected using conventional means,
e.g., an
antibody which is specific for the native or mutant sequence (see Table 9 for
examples of
s amino acid sequences present in mutant proteins).
Any of the antibody reagents useful in the method of the present invention may
comprise whole antibodies, antibody fragments, polyfunctional antibody
aggregates, or in
general any substance comprising one or more specific binding sites from an
antibody. The
antibody frabalnents may be fragments such as Fv, Fab and F(ab')Z fragments or
any
io derivatives thereof, such as a single chain Fv fragments. The antibodies or
antibody
fragments may be non-recombinant, recombinant or humanized. The antibody may
be of any
immunoglobulin isotype, e.g., IgG, IgM, and so forth. In addition, aggregates,
polymers,
derivatives and conjugates of immunoglobulins or their fragments can be used
where
appropriate.
i5 The immunoglobulin source for an antibody reagent can be obtained in any
manner such as by preparation of a conventional polyclonal antiserum or by
preparation of
a monoclonal or a chimeric antibody. Antiserum can be obtained by well-
established
techniques involving immunization of an animal, such as a mouse, rabbit,
guinea pig or goat,
with an appropriate immunogen.
Preparation of Antibodies
1. Polyclonal antibodies.
The peptide or polypeptide may be conjugated to a conventional carrier (e.g.
thyroglobulin) in order to increases its immunogenicity, and antisera to the
peptide-Garner
conjugate is raised in rabbits. Coupling of a peptide to a Garner protein and
immunizations
are performed as described (Dymecki, S.M., et al., J. Biol. Chem 267:4815-
4823, 1992).
Rabbit antibodies against this peptide are raised and the sera titered against
peptide antigen
by ELISA or alternatively by dot or spot blotting (Boersma and Van Leeuwen,
1994, Jour.
Neurosci. Methods 51:317. At the same time, the antisera may be used in tissue
sections.
3 o The sera is shown to react strongly with the appropriate peptides by
ELISA, following the
procedures of Green et al., Cell, 28, 477-487 (1982). The sera exhibiting the
highest titer is
used in subsequent experiments.
AMENDED SH~~T
CA 02287084 1999-10-08
26
2. Monoclonal antibodies.
Techniques for preparing monoclonal antibodies are, well known, and monoclonal
antibodies of this invention may be prepared using a synthetic peptide,
preferably bound to
a carrier, as described by Arnheiter et al., Nature, 294, 278-280 (1981).
s Monoclonal antibodies are typically obtained from hybridoma tissue cultures
or
from ascites fluid obtained from animals into which the hybridoma tissue was
introduced.
Nevertheless, monoclonal antibodies may be described as being "raised to" or
"induced by"
the synthetic peptides or their conjugates.
Particularly preferred immunological tests rely on the use of either
monoclonal
io orpolyclonal antibodies and include enzyme linked immunoassays (ELISA),
immunoblotting,
immunoprecipitation and radioimmunoassays. See Voller, A., Diagnostic Horizons
2:1-7,
1978, Microbiological Associates Quarterly Publication, Walkersville, MD;
Voller, A. et al.,
J. Clin. Pathol. 31:507-520 (1978); U.S. Reissue Pat. No. 31,006; UK Patent
2,019,408;
Butler, J.E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme
Immunoassay,
15 CRC Press, Boca Raton, FL, 1980) or radioimmunoassays (RIA) (Weintraub, B.,
Princi
of radis~~mmun~aas~, Seventh Training Course on Radioligand Assay Techniques,
The
Endocrine Society, March 1986, pp. 1-5, 46-49 and 68-78). For analyzing
tissues for the
presence of the mutant protein of the present invention, immunohistochemistry
techniques are
preferably used. It will be apparent to one skilled in the art that the
antibody molecule will
2 o have to labelled to facilitate easy detection of mutant protein.
Techniques for labelling
antibody molecules are well known to those skilled in the art (see Harlour and
Lane,
Antibodies, Cold Spring Harbour Laboratory, pp 1-726, 1989).
Alternatively, sandwich hybridization techniques may be used, e.g., an
antibody
specific for a given protein. In addition, an antibody specific for a haptenic
group conjugated
25 to the binding protein can be used. Another sandwich detection system
useful for detection
is the avidin or streptavidin system, where a protein specific for the
detectable protein has
been modified by addition of biotin. In yet another embodiment, the antibody
may be
replaced with a non-immunoglobulin protein which has the property of binding
to an
immunoglobulin molecule, for example Staphylococcal protein A or Streptococcal
protein G,
3 o which are well-known in the art. The protein may either itself be
detectable labeled or may
be detected indirectly by a detectable labeled secondary binding protein, for
example, a
second antibody specific for the first antibody. Thus, if a rabbit-anti-hybrid
wild-
k~t~:~~~~r~u ~NE~T
CA 02287084 1999-10-08
27
type/nonsense protein antibody serves as the first binding protein, a labeled
goat-anti-rabbit
immunoglobulin antibody would be a second binding protein.
In another embodiment, the signal generated by the presence of the hybrid wild-
type/nonsense protein is amplified by reaction with a specific antibody for
that fusion protein
s (e.g., an anti-~3-galactosidase antibody) which is detectably labeled. One
of ordinary skill in
the art can devise without undue experimentation a number of such possible
first and second
binding protein systems using conventional methods well-known in the art.
Alternatively, other techniques can be used to detect the mutant proteins,
including chromatographic methods such as SDS PAGE, isoelectric focusing,
Western
to blotting, HPLC and capillary electrophoresis.
Identification of Diseases According to the Invention
The invention provides methods for identifying diseases caused by or
associated
with at least one RNA molecule having one or more transcript mutations which
give rise to
is a frameshift mutation.
Diseases are identified according to the invention as follows. The nucleotide
sequence of an RNA molecule suspected of being involved in the pathogenesis of
a disease
is provided, e.g., from published gene sequences or from cloning and
sequencing of a suspect
gene. The amino acid sequence encoded by the RNA is then predicted, as are
amino acid
2 o sequences of encoded mutant proteins. Mutant protein sequences are
predicted in +1 and +2
reading frames following a hypothesized frameshift mutation. The location of
the frameshift
mutation may be hypothesized with respect to certain nucleotide sequence
motifs in the RNA
molecule, examples of such motifs including, but not limited to, GAGA, for
example,
GAGAC, GAGAG, GAGAT, and GAGAA, or CTCT, for example CTCTG, CTCTC,
2 s CTCTA and CTCTT.
A probe is then prepared that is specific for the mutant protein or an
immunogenic
fragment thereof (such probes being described hereinabove for detection of
proteins or protein
fragments). Depending on where the mutation that leads to the frameshift
occurs, part of the
mutant protein will have the same sequence as the wild-type protein and part
of the protein
3 o will have the sequence of the mutant protein. Furthermore, depending on
where the mutation
occurs the mutant protein will terminate when the nucleotide sequence codes
for a stop codon
(indicated as * in the Figures). Thus, different mutant proteins will be
produced depending
A~~~'~~.~D S~E~3
CA 02287084 1999-10-08
r; _
28
on where the mutation occurs.
The simplest method of probing for the presence of a particular mutant protein
is
to make an antibody to that protein or an immunogenic portion thereof. An
immunogenic
fragment may be synthesized corresponding to the C-terminus of the predicted
mutant
s proteins because even if the mutation occurred at another position in the
sequence, the
probability that the derived mutant protein contains the peptide sequence is
increased. For
example, in the ~3-App encoding RNAs, two different transcript modifications
have occurred
(i.e., at two different GAGA motifs) which result in two frameshifted proteins
having
identical C-terminal sequences. Furthermore, the C-terminal region of a
protein is more likely
io to form an epitope than other regions of the protein.
Once a probe is made, a biological sample from a patient having the disease
and
a biological sample from a patient not having the disease is probed for the
presence or absence
of the mutant protein, also as described above. Alternatively, several probes
may be prepared
and the combination of probes used to probe the tissue sample. The presence of
the mutant
is protein in a biological sample from a patient having the disease and the
absence of said
mutant protein in a biological sample from a patient not having the disease
indicates that the
mutant protein is a marker for the disease or susceptibility to the disease.
Treatment of Diseases According to the Invention
2 o The invention also relates to methods for preventing and/or treating
diseases,
vectors for preventing and/or treating the diseases, and compositions such as
nucleic acid
sequences and proteins for preventing and/or treating the diseases, which
methods and
compositions are useful in gene and protein therapies.
The invention includes methods of treatment and/or prevention of a disease
caused
2 s by or associated with an RNA having a mutation in GAGA or CTCT giving rise
to a
frameshi$ mutation in which a ribozyme, a wild-type RNA, or both, an RNA or
DNA that is
complementary to the mutant RNA and capable of forming a hybrid with the
mutant RNA,
or a vector comprising a sequence encoding any of these sequences, or the wild-
type form
of a mutant protein, is administered to a patient suffering from or
susceptible to the disease.
Preferred diseases which are treated according to the invention include but
are not
limited to cancer or a neurodegenerative disease, especially AD, the preferred
mutant RNAs
!~~~,~;~~;t1 u~iE~'
CA 02287084 1999-10-08
29
of the present invention are those encoding the (3 amyloid precursor protein,
the Tau protein,
ubiquitin B, apolipoprotein-E4 (Apo-E~), micro-tubule associated protein II
(MAP 2), the
neurofilament proteins (L, M, H), presenilin I, presenilin II, Big Tau, GFAP,
P53, BCL2,
semaphorin III, HUFF, HMG and NSP-A, having a deletion, insertion or other
modification
s in the RNA leading to a frameshift mutation.
Ribozymes useful in treatment according to the present invention are
preferably
hammerhead ribozymes.
A pharmaceutical composition according to the invention will include a
therapeutically effective amount of a ribozyme, the wild-type analog of the
mutant RNA, or
io both, or a DNA or RNA that is complementary to the mutant RNA and capable
of forming
a hybrid untranslatable sequence in vivo, in admixture with a carrier. A
therapeutically
effective amount is considered that amount which, when administered to a
patient, provides
a therapeutic benefit to the patient. Such amounts will generally be in the
range of 10 ug-100
mg of therapeutic proteinlkg body weight of the patient, preferably 50 ug-10
mg, and most
i5 preferably 100 ug-1 mg.
Where vectors are useful according to the invention, the vector may be of
linear
or circular configuration and may be adapted for episomal or integrated
existence in the host
cell, as set out in the extensive body of literature known to those skilled in
the art. The
vectors may be delivered to cells using viral or non-viral delivery systems.
The choice of
2 o delivery system will depend on whether the substance is to be delivered to
a selected central
nervous system or neuronal cell type or generally to these cells.
Vectors of the present invention additionally may comprise further control
sequences such as enhancers or locus control regions (LCS), in order to lead
to more
controlled expression of the encoded gene or genes. LCS are described in EP-A-
0332667.
z s The inclusion of a locus control region (LC), is particularly preferred as
it ensures the DNA
is inserted in an open state at the site of integration, thereby allowing
expression of the gene
or genes contained in the vector. The vectors of the present invention have a
wide range of
applications in ex vivo and in vivo gene therapy.
3 o Animal Models for Disease Diagnosis
and Treatment According to the Invention
The invention also includes stable cell lines and transgenic animals for use
as
p~A~~!n=n ~~~Fp
CA 02287084 1999-10-08
disease models for testing or treatment.
A stable cell line or transgenic animal according to the invention will
contain a
recombinant gene or genes, also known herein as a transgene, encoding one or
more
mutations giving rise to a frameshift mutation which causes or is associated
with a disease.
s The recombinant gene will encode an RNA encoding a mutated protein found to
be indicative of a disease. Preferably, the mutant protein will contain an
antigenic epitope
specific for the diseased state. The recombinant gene may encode a protein
comprising at least
part of the sequence designated +1 or +2 in any one of Figures 2 to 9, or an
immunologically
equivalent fragment thereof.
i o A cell line containing a transgene encoding a mutant protein, as described
herein,
is made by introducing the transgene into a selected cell line according to
any one of several
procedures known in the art for introducing a foreign gene into a cell.
A transgenic animal containing such a transgene includes a rodent, such as a
rat
or mouse, or other mammals, such as a goat, a cow, etc. and may be made
according to
1 s procedures well-known in the art.
Transgenic animals are useful according to the invention as disease models for
the
purposes of research into diseases caused by or associated with at least one
gene encoding an
RNA containing one or more mutations giving rise to a frameshift mutation, and
therapies
therefore. By specifically expressing one or more mutant genes, as defined
above, the effect
a o of such mutations on the development of the disease can be studied.
Furthermore, therapies
including gene therapy and various drugs can be tested on the transgenic
animals.
Recombinant genes introduced into an animal to make a transgenic animal useful
in the invention will include those genes specifically disclosed herein,
containing a
dinucleotide deletion or insertion relative to the wildtype sequence of the
gene, the
2 5 dinucleotide deletion or insertion being associated with the nucleotide
sequence GAGA or
CTCT; for example GAGAX or CTCTX, where X is one of G, A, T or C; such as
GAGAG,
GAGAC, GAGAT and GAGAA or CTCTG, CTCTC, CTCTT and CTCTA. Such transgenes
will preferably contain a dinucleotide deletion which is an AG deletion or a
GT deletion just
adjacent to GAGAG (Figure 20), for example, one or two dinucleotide deletions
associated
3 o with a GAGA, GAGAG, GAGAC, GAGAT, GAGAA leading to a + 1 or + 2 frameshift
mutation respectively. In a similar manner, CTCTX can undergo the same
deletion process
(ACT).
~ric,.ucu ~nt,'tT
CA 02287084 1999-10-08
31
Recombinant transgenes containing such a mutation which are particularly
useful
in animal models of disease include those associated with neurodegenerative
diseases,
especially Alzheimer's disease, and include but are not limited to mutant gene
sequences
disclosed herein encoding mutant (3 amyloid precursor protein, the Tau
protein, ubiquitin B,
s apolipoprotein-E4 (Apo-E4), microtubule associated protein II (MAP 2), the
neurofilament
proteins (L, M, H), presenilin I, presenilin II, Big Tau, GFAP, P53, BCL2,
semaphorin III,
HUFF, HMG and NSP-A (see also Tables 2-8).
It also is contemplated that transgenic animals of the invention may contain
transgenes that are controlled via a regulatable and/or a regulated promoter
such that the
1 o corresponding wildtype protein is expressed during selected stages of
development and
maturity of the animal and in a selected tissue, and the mutant gene is turned-
on when desired.
This is particularly desirable where the animal model is of Alzheimer's
disease, wherein the
mutant protein begins to be expressed later in life of the animal. Thus, if
the mutant gene is
under the control of a brain-specific inducible promoter, e.g., a
neurofilament, aldolase or
is modified Thy-1 promoter, then onset of the disease may be controlled via
expression of the
mutant gene. Transgenic animals according to the invention may be generated
to over-express a) human ~3 amyloid precursor protein +1, b) human ubiquitin
+1 proteins, c)
human neurofilament proteins.
z o Described below is an embodiment of the invention involving identification
of
transcript frameshift mutations in RNA molecules encoding proteins which are
present in
neuronal tissue, and how such mutations are useful in diagnosis of certain
disease states.
The cDNA sequences coding for the human (3 amyloid precursor protein, Tau,
ubiquitin, apolipoprotein E4, MAP 2, the neurofilament subunits low, medium
and high,
2s presenilin I, presenilin II, Big Tau, GFAP, P53, BCL2, semaphorin III, HUFF-
I, HMG and
NSP-A were obtained from various gene sequence databases.
Using the sequence data, the various GAGA or CTCT motifs in the sequences
were identified, and deletions were hypothesized and the sequences of the
derived mutant
proteins predicted, as shown in Figures 2-19. Both the sequences of the +1 and
+2 frameshift
3 o mutant proteins were predicted.
By examining the sequences of the hypothesized mutant proteins, a peptide
corresponding to the C-terminus of the hypothesized mutant proteins was
synthesized. The
AA~~~a~n ar~r~-
CA 02287084 1999-10-08
32
peptides were synthesized using standard techniques known to those skilled in
the art. The
peptides having the following sequences were synthesized: RGRTSSKELA [SEQ ID
NO:
1]; HGRLAPARHAS [SEQ ID N0: 2); YADLREDPDRQ [SEQ ID NO: 3];
RQDHHPGSGAQ [SEQ ID NO: 4]; YADLREDPDRQDHHPGSGAQ [SEQ ID NO: 1400];
s GGGAQ [SEQ ID NO: 5], GAPRLPPAQAA [SEQ ID NO: 6]; KTRFQRKGPS [SEQ ID
NO: 7]; PGNRSMGHE [SEQ ID NO: 8]; EAEGGSRS [SEQ 1D NO: 9]; VGAARDSRAA
[SEQ ID NO: 10]; HDYPPGGSV [SEQ ID NO: 11]; SIQKFQV [SEQ ID NO: 12];
VEKPGERGGR [SEQ 117 NO: 13J; PLFGRGHKRG [SEQ ID NO: 14]; EDRGDAGWRGH
[SEQ ID NO: 15); QERGASPRAAPREH [SEQ ID NO: 16]; RQPGDVAPGGQHRPVDD
i o [SEQ ID NO: 17); AGLLAIPEAK [SEQ ID NO: 18]; YVDVYNGGKFS [SEQ ID NO: 19];
AADERRCHLLHMCGRR [SEQ ID NO: 20; QQATEAGQHYQPGSPLHDHSHV [SEQ
ID NO: 21]; PQEAAARTNR [SEQ ID NO: 22]; RSWVHI'APPYQMCLG [SEQ ID NO:
23]; and GGSRTHPR [SEQ ID NO: 24].
Depending on where the mutation that leads to the frameshift occurs, part of
the
15 mutant protein will have the same sequence as the wild-type protein and
part of the protein
will have the sequence of the mutant protein. Furthermore, depending on where
the mutation
occurs the mutant protein will terminate when the nucleotide sequence codes
for a stop codon
(indicated as * in the Figures). Thus different mutant proteins will be
produced depending
on where the mutation occurs.
2 o It is predicted that mutations will occur at GAGA or CTCT motifs in the
cDNA
and the sequences of the mutant proteins predicted accordingly.
Peptides were synthesized corresponding to the C-terminus of the predicted
mutant proteins because even if the mutation occurred at another position in
the sequence the
probability that the derived mutant protein contains the peptide sequence is
increased.
2 s Furthermore, the C-terminus region of a protein is more likely to form an
epitope than other
regions of the protein.
The uniqueness of the synthesized peptides was confirmed by a gene sequence
database search.
Each synthesized peptide was then injected into a rabbit and an antibody
having
3 o affinity for the peptide purified. The techniques used to obtain the
antibodies are standard
techniques known to those skilled in the art.
The antibodies obtained were then tested on autopsy material of frontal
cortex,
pi~t~Ut:u ~ri
CA 02287084 1999-10-08
r r r , .
r , r r r r .. ~. ,
.. r . -
33
temporal lobe and hippocampus of neuropathologically confirmed AD cases and
control non-
AD cases. The presence of the antibodies is determined using standard
detection methods
known to those skilled in the art.
Figure 1 shows the presence of the ~3 amyloid precursor mutant protein
((3APP+')
s in the frontal cortex of an Alzheimer patient identified using an antibody
against a peptide
predicted by the +1 reading frame of ~3 amyloid precursor protein. The
antibody used had
affinity for a peptide having the following sequence RGRTSSKELA [SEQ ID NO:
1].
The results of other immunoreactive tests performed using the antibodies
against
the predicted peptides are shown in Tables 2-5.
io It can be seen that the presence of the mutant protein can be detected and
correlates with the subject having AD. The presence of one or more of the
mutant proteins
can therefore be seen to be indicative of AD.
Table 6 summarizes the immunoreactivity results within the frontal cortex
(area
11), temporal cortex (area 38) and the hippocampus.
15 Other diseases also may be correlated with the presence of mutant proteins,
as
defined herein. For example, seven patients with Downs' syndrome were tested
according
to the invention. Downs' syndrome is trisomy of chromosome 21 which leads to
over-
expression of (3-amyloid precursor protein. We noted that the frontal and
temporal cerebral
cortex and hippocampus of these patients contained plaques and neurofibrillary
tangles, and
a o hypothesized that such over-expression may promote accumulation of
transcript mutations
in neurons, by frameshift mutations at a GAGAG motif in the over-expressed (3-
amyloid gene.
After immunocytochemical staining of tissue from frontal and temporal cerebral
cortex from
the Downs' patients with the above-described antibody specific for the amyloid
+1 carboxy
terminal peptide, immunoreactivity was observed in the neurofibrillary tangles
in six of seven
as patients. Staining was absent in the frontal cortex of the matched
controls. Therefore, the
mutant amyloid protein is correlated not only with Alzheimer's disease, but
also with other
diseases, such as Downs', involving Alzheimer's neuropathology.
It has been found that a number of the mutations occur at GAGA or CTCT motifs.
Table 7 shows the presence of the complementary GAGA motifs in various cDNAs
of the
3 o neuronal system. The motif or, as can be seen from the sequences of Tau
and apolipoprotein
E4, similar motifs such as GAGAG GAGAC, GAGAA, and GAGAT (in the cDNA) may be
associated with the frameshift mutations that lead to or are associated with
the disease. The
~r~t~w
;~r: ~ ~ h
CA 02287084 1999-10-08
34
presence of the motif or similar motifs in other RNA molecules may indicate
that they are
relevant to a disease. It is also possible that other mutations occur that are
not associated with
such motifs but still lead to frameshift mutations that cause or are
associated with a disease.
Table 8 shows the presence of GAGAC motifs in particular RNA molecules of
s the neuronal system, namely ~iAPP, Tau and Ubiquitin. This table also
indicates, inter alia,
the chromosomal location of the genes from which the mutant RNA molecules are
transcribed
and the molecular weight of the longest polypeptide forms encoded by the RNA
molecules
and the predicted size of the aberrant +1 peptide with its C-terminus against
which the
antibodies were raised. These peptides were revealed in a Western blot and
also identified
io with a different antibody recognizing an epitope on the unaffected wild-
type N-terminus.
Synthetic polypeptides corresponding in sequence to a portion of a mutant
protein
(whether such peptides are chemically synthesized or are chemically or
recombinantly
15 generated fragments of a protein), as described herein, will be usefi.~l
according to the
invention as antigenic peptides for generation of antibodies specific for a
mutant protein,
provided they possess the following characteristics. The synthetic peptide
will include a
minimum of 8 and preferably 12-15 amino acid residues, and an optimum length
of 20-21
amino acids. The hydrophilicity and antigenic index of the amino acid sequence
of the hybrid
2 o wild-type/nonsense protein may be determined by Analytical Biotechnology
Sciences,
Boston, MA, using computer programming. Potential synthetic peptides useful
according to
the invention include a stretch of 12-20 amino acids preferably within the
carboxy terminal
100-150 amino acids of the hybrid wild-type/nonsense protein.
The amino acid sequence of a selected peptide is searched in a computer
database
as of sequences (e.g., GenBank) to preclude the possibility that at reasonable
concentrations,
antisera to any one peptide would specifically interact with any protein of a
lrnown sequence.
Preferred sequences are those which are determined not to have a close homolog
(i.e., "close"
meaning 80-100% identity).
3 o Detection of "Mutant" Prr,te~
Another embodiment of this invention relates to an assay for the presence of
the
"mutant" or mutant protein in a given tissue as indicative of a disease state.
Here, an above-
pi~~~v~7't' U ~~~
CA 02287084 1999-10-08
described antibody is prepared. The antibody or idiotype-containing polyamide
portion
thereof is then admixed with candidate tissue and an indicating group. The
presence of the
naturally occurnng amino acid sequence is ascertained by the formation of an
immune
reaction, as signalled by the indicating group. Candidate tissues include any
tissue or cell line
5 or bodily fluid to be tested for the presence of the mutant protein, as
described hereinabove.
Expression of a given hybrid wild-type/nonsense protein may be investigated
using antiserum prepared in rabbits against a peptide corresponding to a
carboxy terminal
stretch of amino acids in the hybrid wild-type/nonsense protein as follows.
CMK cells or U3T3 cells are metabolically labeled with 35S-methionine and
io extracts are immunoprecipitated with antiserum. If the hybrid wild-
type/nonsense protein is
present in the cells, then a protein species of corresponding molecular weight
will be detected
in CMK and U3T3 cells. The protein may be localized to the membrane, nucleus
or
cytoplasm by Western blot analysis of the nuclear, membrane and cytoplasmic
fractions, as
generally described in Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350-
4354 (1979). This
i5 localization may be confirmed by immunofluorescence analysis to be
associated mainly with
the plasma membrane.
Metabolic labeling immunoprecipitation, and immunolocalization assays are
performed as described previously (Furth, M.E., et al., Oncogene 1:47-58,
1987; Laemmli,
U.K., Nature 227:680-685, 1970; Yarden, Y., et al., EMBO J. 6:3341-3351, 1987;
Konopka,
ao J.B., et al., Mol. Cell. Biol. 5:3116-3123, 1985). For immunoblot analysis,
total lysates are
prepared (using Fruth's lysis buffer) (Fruth, M.E., et al., Oncogene, 1:47-58,
1987). Relative
protein concentrations are determined with a colorimetric assay kit (Bio-Rad)
with bovine
serum albumin as the standard. A protein of lysate containing approximately
0.05 mg of
protein was mixed with an equal volume of 2 x SDS sample buffer containing 2
a 5 mercaptoethanol, boiled for S min., fractioned on 10% polyacrylamide-SDS
gels (Konopka,
J.B., et al., J. Virol., 51:223-232, 1984) and transferred to immunobilon
polyvinyldine
difluoride (Millipore Corp., Bedford, MA) filters. Protein blots were treated
with specific
antipeptide antibodies (see below). Primary binding of the specific antibodies
may be
detected using anti-IgG second antibodies conjugated to horseradish peroxidase
and
3 o subsequent chemiluminescence development ECL Western blotting system
(Amersham
International).
For metabolic labeling, 106 cells are labeled with 100 pCi of 35S-methionine
in 1
A~ttivJt:u ~i~t~T
CA 02287084 1999-10-08
36
ml of Dulbecco's modified Eagles medium minus methionine (Amersham Corp.) for
16 h.
Immunoprecipitation of protein from labeled cells with antipeptide antiserum
is performed
as described (Dymecki, S.M., et al., J. Biol. Chem 267:4815-4823, 1992).
Portions of lysates
containing 10' cpm of acid-insoluble ~Smethionine were incubated with 1 ue of
the
s antiserum in 0.5 ml of reaction mixture. Immunoprecipitation samples were
analyzed by SDS-
polylarcylamide gel electrophoresis and autoradiography.
For immunolocalization studies, 10' CMK cells are resuspended in 1 ml of
sonication buffer (60 mM Tris-HCI, pH 7.5, 6 mM EDTA, 1 S mM EGTA, 0.75 M
sucrose,
0.03% leupeptin 12 mM phenylmethylsulfonyl fluoride, 30 mM 2-mercaptoethanol).
Cells
io are sonicated 6 times for 10 seconds each and centrifuged at 25,000 xg for
10 min at 4°C.
The pellet is dissolved in 1 ml of sonication buffer and centrifuged at 25,000
x g for 10 min
at 4°C.
The pellet (nucleus fraction) is resuspended in 1 ml of sonication buffer and
added
to an equal volume of 2 x SDS sample buffer. The supernatant obtained above
(after the first
i5 sonication) is again centrifuged at 100,000 x g for 40 min at 4°C.
The supernatant (cytosolic
fraction) is removed and added to an equal volume of 2 x concentrated SDS
sample buffer.
The remaining pellet (membrane fraction) is washed and dissolved in sonication
buffer and
SDS sample buffer as described above. Protein samples are analyzed by
electrophoresis on
10% polyacrylamide gels, according to the Laemmli method (Konopka, J.B., et
al., Mol. Cell.
a o Biol. 5:3116-3123, 1985). The proteins are transferred from the gels on a
0.45-pm
polyvinylidine difluoride membrane for subsequent immunoblot analysis. Primary
binding
of antibodies is detected using anti-IgG second antibodies conjugated to
horseradish
peroxidase.
For immunohistochemical localization of a given protein, if desired, CMK cells
z s or U3T3 are grown on cover slips to approximately 50% confluence and are
washed with PBS
(pH 7.4) after removing the medium. The cells are prefixed for 1 min at
37°C in 1%
paraformaldehyde containing 0.075% Triton X-100, rinsed with PBS and then
fixed for 10
min with 4% paraformaldehyde. After the fixation step, cells are rinsed in
PBS, quenched in
PBS with 0.1 and finally rinsed again in PBS. For antibody staining, the cells
are first
3o blocked with a blocking solution (3% bovine serum albumin in PBS) and
incubated for 1 h
at 37°C. The cells are then incubated for 1 h at 37°C with
antiserum (1:100 dilution or with
preimmune rabbit serum (1:100) (see below). After the incubation with the
primary antibody,
AFvfE~JDtD SH~fT
CA 02287084 1999-10-08
37
the cells are washed in PBS containing 3% bovine and serum albumin and 0.1%
Tween 20
and incubated for 1 hour at 37°C in a fluorescein-conjugated donkey
anti-rabbit IgG (Jackson
Immunoresearch, Maine), diluted 1:100 in blocking solution.
The coverslips are washed in PBS (pH 8.0), and glycerol is added to each
s coverslip before mounting on glass slides and sealing with clear nail
polish. All glass slides
were examined with a Zeiss Axiophot microscope.
The above methods for detection of a given mutant protein or nucleic acid are
io applicable to analyses involving tissues, cell lines and bodily fluids
(e.g. cerebrospinal liquor
or blood, including but not limited to venous, arterial and cord blood)
suspected of containing
the marker protein.
For example, a sample of CNS tissue suspected of being in a diseased state may
be analyzed, it having been previously observed according to the invention
that tissue of that
is particular diseased state contains detectable levels of hybrid wild-
type/nonsense proteins
relative to healthy tissue.
An aliquot of the suspect sample and a healthy control sample are provided and
admixed with an effective amount of an antibody specific for the hybrid wild-
type/nonsense
protein, as herein described, and an indicating group. The admixture is
typically incubated,
z o as is known, for a time sufficient to permit an immune reaction to occur.
The incubated
admixture is then assayed for the presence of an immune reaction as indicated
by the
indicating group. The relative levels of the hybrid wild-type/nonsense protein
in the suspect
sample and the control sample are then compared, allowing for diagnosis of a
diseased or
healthy state in the suspect sample.
2 s The above types of analyzing for the presence of the hybrid wild-
type/nonsense
protein may, of course, be performed using analysis for the coding RNA, e.g.,
via Northern
blot or RNA dot blot analysis, both of which are conventional and known in the
art.
3 o Disease treatment according to the invention contemplates eliminating
mutant
transcripts. Evidence supporting the presence of transcript mutant RNA
molecules is that in
homozygous Brattleboro hypothalamus cells, vasopressin cDNAs having the
frameshift
p~AE'~D~D ~EEf
CA 02287084 1999-10-08
38
mutation were observed in 1 in 100 colonies, where as genomic vasopressin DNA
having the
frameshift mutation was not identified.
In the human hypothalamus no age related increase in the number of vasopressin
+1 immunoreactive cells is observed (contrary to that in rat). However, in the
fetal period
s (29-42 weeks of gestation) an enormous increase in the number of +1
immunoreactive cells
containing the +1 vasopressin protein is detected. After birth, the number of
these cells falls
back to just a few. In Downs' syndrome, where (3app gene expression is very
high (5-fold
higher than normal), the highest levels of ~iapp and +1 mutant proteins were
also observed,
higher than in AD where (iapp gene expression is not found to be increased
over normal
io levels. It also has been found that ~iAPP+1 and UbiB+1 mutant proteins
coexist (and are
present in tangles and dystrophic neurites) in the same cell. Accordingly, is
unlikely that the
transient increase is due to a genomic event.
Once an RNA molecule containing a frameshift mutation (i.e., a frameshifted
transcript), or a mutant protein is correlated with a disease state, the
disease is treatable
i5 according to the invention as follows: by administering to a patient in
need thereof enzymes
which serve to selectively eliminate frameshifted RNA via cleavage, e.g.,
ribozymes; by
administering the wild-type version of the mutant RNA, preferably in
substantially
uncleavable form, by administering the wild-type version of the hybrid wild-
type/nonsense
protein; or by administering oligonucleotides or sequences encoding
oligonucleotides
a o complementary to a mutant RNA to a cell in order to form a nucleic acid
duplex which
renders the mutant RNA untranslatable.
A patient in need thereof will include a patient exhibiting symptoms of the
disease, even those patients suspected of developing the disease, i.e., who
are monitored
according to the invention by measuring the a tissue sample, e.g., the
cerebrospinal fluid, for
25 the presence of frameshifted peptides (e.g. peptides having an amino acid
sequence in the +1
or +2 reading frame).
According to the invention, a ribozyme may be delivered to affected or
susceptible
cells leading to the cleavage of the mutant RNA and resultant inability of the
cell to translate
the mutant RNA into mutant protein. The wild-type protein, if not akeady
produced by the
3 o cell, may be provided in protein form or via administering the wild-type
RNA to the cell
along with the ribozyme. The wild-type RNA may be engineered so as to contain
a sequence
that is distinguishable from the mutant RNA sequence other than simply at the
level of the
r'~'D~-(? ~H~~1'
CA 02287084 1999-10-08
39
GAGA or CTCT mutation. For example, the mutant RNA may contain third base
silent
mutations, i.e., which do not change the coding sequence of the RNA, but which
render the
wild-type RNA less or substantially unsusceptible to cleavage by the ribozyme.
Without being bound by any one theory, it is suggested that decreasing the
s percentage of mutant RNA and increasing the percentage of the correct
protein produced in
relation to the hybrid wild-type/nonsense protein will reduce or prevent
further progression
of the disease, and possibly reverse the diseased state. In addition, it is
possible that not
every mutant transcript results in a mutant protein that is directly toxic to
the neuronal tissue.
For example, the mutant protein may be routed to the proteasomal and/or
lysosomal system
io or just secreted (e.g. by the constitutive or regulated pathway)and
degraded elsewhere.
However, sometimes the mutant protein will be accumulated in the membranes of
organelles,
for instance in the endoplasmic reticulum, thus disrupting the normal
processes of the cellular
machinery.
The wild-type version of the mutated RNA encodes the correct protein. When
15 the disease is a neurodegenerative disease, preferred wild-type sequences
include the RNAs
encoded by the ~i amyloid precursor protein gene, the Tau gene, the ubiquitin
B gene, the
apolipoprotein-E4 gene, the microtubule associated protein II (MAP2) gene, the
neurofilament
protein genes (L, M and H), the presenilin I and II genes, Big Tau, GFAP, P53,
BCL2,
Semaphorin III, HUFF-1, HMG and NSP-A. The sequences of these genes are
provided
a o herein in the figures. Other preferred wild-type RNAs are encoded by the
alpha and beta
tubulin genes, the sequences of which are found in Cowan et al., Mol. Cell.
Biol., 3, 1738-
1745(1983) and Lewis et al., J. Mol. Biol. 182, 11-20(1985), respectively.
When the disease is a non-hereditary cancer, preferred wild-type RNAs are
encoded by gene sequences which include but are not limited to the human p53
gene and the
2s BCL-2 gene. Mammalian phosphoprotein p53 has been shown to play an
essential role in
regulation of cell division and is required for the transition from phase GO
to G1 of the cell
cycle. P53 is normally present in very low levels in normal cells and is
believed to be a tumor
suppressor gene; when present at high levels, p53 has been shown to play a
role in
transformation and malignancy. P53 gene alleles from normal and malignant
tissues have
3 o been shown to contain BgIII site polymorphism (Buchman et al., 1988, Gene
70:245). The
p53 coding region contains several GAGA motifs, e.g., GAGAC at position 1476
of the
sequence published in Buchman et al., GAGA at position 1498; GAGA at position
1643; and
AIVt~~;a~~ ~NEET
CA 02287084 1999-10-08
GAGA at position 1713, which motifs present candidate sites for frameshift
RNAs according
to the invention. A frameshift mutation within a p53 RNA thus may lead to loss
of the natural
p53 tumor suppressor function. Detection of such a mutation in p53 may be
diagnostic of
pre-malignancy or malignancy, and treatment as described herein which results
in correction
s of p53 function may restore tumor suppressor function.
In Diabetes mellitus type II, which occurs with increased frequency in aged
persons, the islands of Langerhans degenerate possibly as a result of
frameshift mutations in
various transcripts (e.g., the ubiquitin transcript).
The invention also encompasses methods of combatting diseases caused by at
to least one an RNA having one or more GA, GT or CT deletions giving rise to a
frameshift
mutation by targeting the RNA transcript. Thus, it is also contemplated
according to the
invention that a frameshift mutation within an RNA may be corrected at the
level of the
frameshifted RNA via cleavage using a ribozyme having specificity for the
mutant RNA
sequence (see Denman et al., Arch. Of Biochem. Biophysics. 323,71-78,1995),
and
is eliminating the mutant mRNA. The disease associated with the frameshi$ed
RNA is thus
treated by administering an appropriate ribozyme, or sequences encoding the
ribozyme, to the
patient.
Ribozymes of selected specificities may be made as described by Sullenger &
Cech, Nature 371: 619-622, 1994), herein incorporated by reference. Ribozymes
and
a o sequences encoding ribozymes may be prepared as described by Tuschl et
al., Curr. Opin.
Struc. Biol. 5:296, 1995 and Wahl et al., Curr. Opin. Struc. Biol. 5:282,
1995.
The invention also encompasses methods of treating diseases caused by or
associated with at least one an RNA having one or more GAGA or CTCT mutations
giving
rise to a frameshift by delivery of complementary oligonucleotides or
sequences encoding
a s complementary oligonucleotides to a target cell containing a frameshifted
RNA. The
oligonucleotides will have a mutant sequence with respect to the region of the
mutant RNA
containing the GA, GT or CT deletion, and thus may serve to form a hybrid in
vivo with the
mutant RNA, rendering it untranslatable. Oligonucleotides with strong target
site binding
affinity, i.e., with full target site homology are preferred. Also preferred
are oligonucleotides
3 o between 10 - 30 nucleotides in length and containing a CTCT, CTCTG, CTCTC,
CTCTT or
CTCTA motif or a GAGA, GAGAG, GAGAC, GAGAT or GAGAA motif.
Disease treatment according to the invention is described below and includes
i'dt)CU ~t'~EtT ,
CA 02287084 1999-10-08
41
preparation of the administered substance and administration of the substance
to a patient
suffering from a disease according to the invention. As used herein
"substance" refers to any
one of the following: a ribozyme, a nucleic acid sequence encoding a ribozyme,
a wild-type
transcript, an antisense mutant RNA or DNA or a nucleic acid sequence encoding
an antisense
s sequence, a wild type protein encoded by the wild type gene, an antibody
specific for the
frameshifted (nonsense) protein.
Disease treatment according to the invention may be accomplished as follows.
In Example 5, treatment using ribozymes according to the invention is
described.
In Example 6, administration of vectors is described. In Example 7,
1 o administration of proteins, ribozymes, or nucleic acid vectors using
liposomes is described.
In Example 8, delivery of these substances across the blood-brain barrier is
described. Lastly,
in Example 9, methods ofdelivering cells comprising a protein, ribozyme or
other nucleic
acid, such as a vector bearing a gene expression construct, are described.
EXAMPLE 5
is Treatment According to the Invention king Ribo~
Preparation and delivery of a ribozyme or nucleic acid sequence encoding a
ribozyme which effect or facilitate selective removal of the frameshifted RNA
is carried out
as follows.
selective Elimination of m ~ ant ran~cy~,~Accordinz to the Invention.
a o The invention thus also encompasses methods of treating diseases caused by
the
translation of frameshifted mRNA's which are the result of transcriptional
infidelity occurring
at or adjacent to GAGA or CTCT motifs in the (3-APP and ubiquitin B genes. It
is believed
that accumulation of aberrant proteins encoded by these messages contributes
to the
progression of Alzheimer's disease; therefore, elimination of the mutant
transcripts is of
2 s therapeutic value. It is contemplated according to the invention that the
mutant transcripts
described herein are rendered untranslatable in a cell via ribozyme-mediated
cleavage using
ribozymes designed and administered as described herein. In addition, it may
be
advantageous in certain circumstances to replace the ribozyme-cleaved messages
with an
exogenous transcript encoding the wild-type protein, which transcript is
cleavage-resistant and
3 0 ~ the synthesis of which is therefore not subj ect to transcriptional
errors or post-transcriptional
modification such as those that produced the mutant transcripts described
herein.
Treatment strategies are described below for selective elimination of the
ubiquitin
~~I~:~~~rC1 ~~~~
CA 02287084 1999-10-08
42
B and (3-APP mutant transcripts in cells. The invention, however, also
contemplates selective
removal of other mutant transcripts, whether disclosed herein or later-
discovered, according
to the methods described hereinbelow.
Ribozymes of the hammerhead class are the smallest known, and lend themselves
s both to in vitro synthesis and delivery to cells (summarized by Sullivan, J.
Invest. Dermatol.
103: 855-985, 1994; Usman et al., Curr. Opin. Struct. Biol. 6: 527-533, 1996).
It is required
of hammerhead cleavage targets that they comprise the sequence motif UH,
wherein H
denotes the ribonucleotides A, U, or C, but not G; the sequence is cleaved
following the H.
The core functional unit of the hammerhead ribozyme is a tripartite structure
made up of helix
to I, which hybridizes to mRNA sequences 3' of the cleavage site, helix II, a
22 ribonucleotide
catalytic domain which mediates the cleavage reaction, and helixIII, which
hybridizes to
sequences 5' of and including the "U" of the UH cleavage motif (Haseloff and
Gerlach,
Nature 334: 585-591, 1988; Ruffner et al., Biochemistry 33: 10695-10702,
1990). Studies
have shown that the lengths of helices I and III are proportional to the
efficiency with which
is ribozymes both bind the area surrounding the cleavage site and release
themselves from it
following cleavage; the former is critical for target recognition, while the
latter is important
for maintaining kinetics that indicate true catalytic activity, namely,
raising the ratio of target
molecules inactivated to ribozymes above the 1:1 stoichiometric ratio observed
with
antisense-RNA-mediated inactivation. Ideally, helix I is 3 to 5
ribonucleotides in length,
2 o relative to 9 to 13 ribonucleotides for helix III (Tabler et al., Nzzcleic
Acids Res. 22: 3958-
3965, 1994; Hendry and McCall, Nucleic Acids Res. 24: 2679-2684, 1996). Other
factors,
such as stem loop formation in the unbound ribozyme and target mRNA also play
a role in
reaction kinetics and ribozyme stability, and in order to predict and/or
compensate for such
interactions, molecular modeling studies and in vitro trials of numerous
ribozyme designs are
2 s often undertaken (Sioud et al., Nucleic Acids Res. 22: 5571-5575, 1994;
Gavin and Gupta, J.
Biol. Chem. 272: 1461-1472, 1997).
Mutant ubiquitin B transcripts can be removed from cells via the application
of
hammerhead ribozymes delivered to these cells in liposome vectors. The
invention comprises
use of these ribozymes to recognize and cleave mutant transcripts at a site 5'
to the GAGA or
3 o GGT-containing site that is the source of polymerase slippage during
transcription, thereby
ridding the cells of the frame-shifted portion of the translated products of
these defective
messages.
i ~~~1='~
~sfi~.~~ri>1~~
CA 02287084 1999-10-08
43
The sequence immediately preceding the GAGA motif in the ubiquitin transcript
is GCGUCU, which includes the cleavage recognition motif UC. Given the length
considerations posed above, the sequence ideally bound by helix I for a
particular mutant is
GAG; however, in that such a ribozyme would be expected to bind the mutant and
wild-type
s transcripts with equal efficiency, helix I must be lengthened to include
four more nucleotides,
such that all seven bases will hybridize to the mutant transcript, while
sufficient mismatch to
destabilize binding to the wild-type sequence will result. This strategy is
more efficient in
cases in which the mutant transcript has arisen via deletion rather than
insertion, since in the
latter, the effect of the absolute length of helix I on target release becomes
a concern;
io however, delivery of a pool of differentially-designed ribozymes
complementary to various
mutant sequences that can result from imprecise transcription of the GAGA
motif or GGT
sequence in the case of ubiquitin and of sufficient mismatch with the wild-
type sequence to
inhibit efficient binding of a ribozyme to it should eliminate translation of
the frame shifted
products of a large proportion of defective messages.
15 Such a strategy is practical in situations in which the cleavage site is 1
to (at most)
bases to the 5' side of the cleavage site; however, longer distances require
accordingly
longer helix I binding domains which, combined with the need to create 3'
mismatches for
differentiation between mutant and wild-type transcripts, make such an
approach inadequate
for dealing with certain mutations. This is true of GAGA- defective
transcripts of ~3-APP.
a o While one GAGA motif is separated from the cleavage site by a single base,
the remaining
four motifs are between 7 and 20 bases from the nearest 5' cleavage site. In
such a case,
cleavage of both of the wild-type transcript via hammerhead ribozymes may be
unavoidable,
and its replacement with a cleavage-resistant transcript must be undertaken in
concert with
removal of mutant transcripts (see Table 10 for possible sequence
substitutions resulting in
2 s a cleavage-resistant transcript); here, the ribozyme is designed to cleave
both types of message
at any UH site S' of the first GAGA motif.
It may be advantageous to replace the (3-APP transcript in all cells in which
it is
needed; therefore, co-delivery of an expression vector bearing a spliced (3-
APP minigene
driven by ~3-APP promoter sequences may be employed. Numerous studies of this
promoter
3 o have been undertaken (among them, Lahiri and Robakis, Brain Res. Mol.
Brain Res. 9: 253-7,
1991; Bourbonniere and Nalbantoglu, Brain Res. Mol. Brain Res. 19: 246-250,
1993; Lukiw
et al., Brain Res. Mol. Brain Res. 22: 121-131, 1994; Lahiri and Nall, Brain
Res. Mol. Brain
:.:;~:'-.".i',i>3i'i.a '-'':~"k
CA 02287084 1999-10-08
44
Res. 32: 233-40, 1995; Bourbonniere and Nalbantoglu, Brain Res. Mol. Brain
Res. 35: 304-
308, 1996; Quitschke et al., J. Biol. Chem. 271: 22231-9, 1996), and it has
been demonstrated
that 96 base pairs 5' to the transcriptional start site are sufficient for
cell-type-specific
promoter activity in tissue culture (Quitschke and Goldgaber, J. Biol. Chem.
267: '17362-
17368, 1992). The 96 base pairs can be fused to a minigene engineered such
that alterations
are made in the ribozyme recognition site to prevent cleavage of the
replacement ~3-APP
transcript and in the GAGA motifs to inhibit slippage of the transcriptional
machinery such
as produces the mutant transcripts in the first place; these replacements
should be performed
such that translationally "silent" mutations are introduced in each case.
Examples of such
i o changes are shown in Table 10.
Sequences encoding ribozymes or a wild-type version of a mutant RNA, or an
antisense (complementary) mutant oligonucleotide sequence may be cloned into
an
appropriate vector for expression in a desired mammalian cell. The vector will
include a
promoter that is expressed in the target cell type, and also may include an
enhancer and locus
control region, as selected for expression in a given cell type. Examples of
vectors useful
according to the invention include but are not limited to any vector which
results in successful
transfer of the coding sequences to the target mammalian cell. A nucleic acid
may be
s o transfected for use in the invention using a viral (e.g. adenoviral or
retroviral) or non-viral
DNA or RNA vector, where non-viral vectors include, but are not limited to,
plasmids, linear
nucleic acid molecules, artificial chromomosomes and episomal vectors.
Expression of
heterologous genes has been observed after injection of plasmid DNA into
muscle (Wolff J.
A. et al., 1990, Science, 247: 1465-1468; Carson D.A. et al., US Patent No.
5,580,859),
thyroid (Sykes et al., 1994, Human Clene Ther, 5: 837-844), melanoma (Vile et
al., 1993,
C'.ancer Rep., 53: 962-967), skin (Hengge et al., 1995, Namr . r n . ., 10:
161-166), liver
(Hickman et al., 1994, Human Cent, h ranv, 5: 1477-1483) and after exposure of
airway
epithelium (Meyer et al., 1995, gene TheranT, 2: 450-460).
For example, the retroviral gene transfer vector SAX (Kantoff et al., Proc.
Nat.
3 o Aca. Sci. 83:6563, 1986) may be used to insert a selected coding sequence
into a target cell.
SAX is a moloney virus based vector with the neon gene promoted from the
retroviral LTR
and the human ADA gene promoted from an internal SV40 promoter. Thus, the SAX
vector
CA 02287084 1999-10-08
may be engineered by one of skill in the art to contain the coding sequence
for a ribozyme,
or a wild-type RNA, or a selected antisense sequence, identified as described
herein, e.g., by
substituting the desired coding region for the hADA coding region in the SAX
vector.
Expression vectors are known in the art which encode, or may be engineered to
5 encode, a selected ribozyme. Yuyama et al., Nucl. Acids Res. 22:5060, 1994,
describe a
multifunctional expression vector encoding several ribozymes. This vector may
be adapted
to encoded a ribozyme of a selected specificity by substituting one or both
ribozyme
sequences in the vector for a selected ribozyme sequence. Zhou et al., Gene
149:33, 1994,
and Yamada et al., Virology 205;121, 1994, describe retroviral transduction of
ribozyme
i o sequences into T cells. These retroviral vectors may be adapted to encode
a selected ribozyme
sequence. Liu et al., Gene Therapy 1:32, 1994, and Lee et al., Gene Therapy
2:377, 1995,
describe expression vectors which are adaptable for use in expression of any
nucleic acid
sequence contemplated according to the invention.
Generally, nucleic acid molecules are administered in a manner compatible with
15 the dosage formulation, and in such amount as will be prophylactically
and/or therapeutically
effective. When the end product (e.g. an antisense RNA molecule or ribozyme)
is
administered directly, the dosage to be administered is directly proportional
to the the amount
needed per cell and the number of cells to be transfected, with a correction
factor for the
efficiency of uptake of the molecules. In cases in which a gene must be
expressed from the
2 o nucleic acid molecules, the strength of the associated transcriptional
regulatory seuqences also
must be considered in calculating the number of nucleic acid molecules per
target cell that
will result in adequate levels of the encoded product. Suitable dosage ranges
are on the order
of, where a gene expression construct is administered, 0.5- to leg, or 1-
l0,ug, or optionally
10- 100 pg of nucleic acid in a single dose. It is conceivable that dosages of
up to lmg may
25 be advantageously used. Note that the number of molar equivalents per cell
vary with the size
of the construct, and that absolute amounts of DNA used should be adjusted
accordingly to
ensure adequate gene copy number when large constructs are injected.
Nucleic acid molecules to be administered according to the invention may, for
example, be formulated in a physiologically acceptable diluent such as water,
phosphate
3 o buffered saline, or saline, and further may include an adjuvant; however,
it is contemplated
that other formulations may advantageously be employed. Adjuvants such as
incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are
materials well
APv~EIVL~D ~~~~~ ~
CA 02287084 1999-10-08
46
known in the art. Administration of a nucleic acid molecule as described
herein may be
either localized or systemic. Methods for both localized and systemic
administration of a
pharmacological composition are well known in the art.
Nucleic acid constructs of use in the invention can be given in a single- or
s multiple dose. A multiple dose schedule is one in which a primary course of
administration
can include 1-10 separate doses, followed by other doses given at subsequent
time intervals
required to maintain and or reinforce the cellular level of the transfected
nucleic acid. Such
intervals are dependent on the continued need of the recipient for the
therapeutic nucleic acid,
the ability of a given nucleic acid to self replicate in a mammalian cell if
it does not become
i o integrated into the recipient's genome and the half life of a non-
renewable nucleic acid (e.g.
a molecule that will not self replicate). Preferably, when the medical needs
of the recipient
mammal dictate that a nucleic acid or a product thereof will be required
throughout its
lifetime, or at least over an extended period of time, such as a year or more,
a nucleic acid
may be encoded by sequences of a vector that will self replicate in the target
cells. The
Zs efficacy of transfection and subsequent maintenance of the nucleic acid
molecules may be
assayed either by monitoring the activity of a marker gene, which may
additionally be
comprised by the transfected construct, or by the direct measurement of either
the protein
product encoded by the gene of interest or the reduction in the levels of a
protein the
production of which it is designed to inhibit. The assays can be performed
using conventional
2 o molecular and biochemical techniques, such as are known to one skilled in
the art.
The success of treatment using nucleic acid molecules in the invention may be
determined by the assessment of known clinical indicators (e.g., for a
neurodegenerative
disease, loss of cognitive or motor function). The progression or (if
treatment is undertaken
prophylactically on an patient believed to be at risk of disease) development
of such
2s symptoms in a treated individual is compared to those observed in untreated
control subjects;
if an improvement in the treated patient's condition is observed relative to
that of control
subjects, treatment is judged to be effective. The making of such an
assessment is well within
the knowledge of one of skill in the art.
3 o T_.inosomal Delivenr According to the Invemrion
Substances may be administered according to the invention using any delivery
means known in the art. Described below is liposomal delivery. Liposomes which
are used
A~~~;i)~f~ 4~r~.
CA 02287084 1999-10-08
r . , . ~ ,. r ,. r , r
r a r . r . r
r r r r. r ,
r , ~. ~ ,-rr r r , rr
' ~ r r r
47
to administer the substances described herein, e.g., a ribozyme can be of
various types and can
have various compositions. The primary restrictions are that the liposomes
should not be
toxic to the living cells and that they should deliver their contents into the
interior of the cells
being treated.
s The use of pH sensitive liposomes to mediate the cytoplasmic delivery of
calcein
and FITC dextran has been described (see Straubinger et al., Cell 32:1069-
1079, 1983; and
Straubinger et al., FEBS Letters 179:148-154, 1985. Other discussions of pH
sensitive
liposomes can be found in chapter 11 of the book CELL FUSION, edited by A.E.
Sowers,
entitled "Fusion of Phospholipid Vesicles Induced by Divalent Cations and
Protons" by Nejat
to Duzgunes et al., Plenum Press, N.Y., 1987, 241-267. See also Ellens et al.,
Biochemistry,
23:1532-1538, 1984, and Bentz et al., Biochemistry 26:2105-2116, 1987.
The liposomes may be of various sizes and may have either one or several
membrane layers separating the internal and external compartments. The most
important
elements in liposome structure are that a sufficient amount of enzyme or
nucleic acid be
i5 sequestered so that only one or a few liposomes are required to enter each
cell for delivery of
the substance, and that the liposome be resistant to disruption. Liposome
structures include
small unilamellar vesicles (SUVs, less than 250 angstroms in diameter), large
unilamellar
vesicles (LUVs, greater than 500 angstroms in diameter), and multilamellar
vesicles (MLs).
In the example presented below, although SUVs are used to administer a
ribozyme, the
a o methods are applicable to administration of any substance described
herein.
SUVs can be isolated from other liposomes and unincorporated enzyme by
molecular weight can be isolated from other liposomes and unincorporated
enzyme by
molecular sieve chromatography, which is precise but time consuming and
dilutes the
liposomes, or differential centrifugation, which is rapid but produces a wider
range of
2 s liposome sizes.
The liposomes may be made from natural and synthetic phospholipids,
glycolipids, and other lipids and lipid congeners; cholesterol, cholesterol
derivatives and other
cholesterol congeners; charged species which impart a net charge to the
membrane; reactive
species which can react after liposome formation to link additional molecules
to the liposome
3 o membrane; and other lipid soluble compounds which have chemical or
biological activity.
The liposomes useful according to the invention may be prepared, for example,
as described in U.S. Patent No. 5,296,231, which describes preparation of
liposomes
:~~~~~J~~~~ ~~~
CA 02287084 1999-10-08
. ,. ,. . r r r -
r r r r
r . ~ , c . . ,
r .. - , r . r - . ,-.
r r ._
48
containing a ribozyme, although it should be borne in mind that liposomes
useful according
to the invention may contain any one of the substances as herein described.
Briefly, by
combining a phospholipid component with an aqueous component containing the
ribozyme
(or desired substance) under conditions which will result in vesicle
formation. The
s phospholipid concentration must be sufficient to form lamellar structures,
and the aqueous
component must be compatible with biological stability of the enzyme. Methods
for
combining the phospholipids onto glass and then vesicles will form include:
drying the
phospholipids onto glass and then dispersing them in the aqueous component;
injecting
phospholipids dissolved in a vaporizing or non-vaporizing organic solvent into
the aqueous
1 o component which has previously been heated; and dissolving phospholipids
in the aqueous
phase with detergents and then removing the detergent by dialysis. The
concentration of the
ribozyme in the aqueous component can be increased by lyophilizing the enzyme
onto dried
phospholipids and then rehydrating the mixture with a reduced volume of
aqueous buffer.
SIIV's can be produced from the foregoing mixtures either by sonication or by
dispersing the
i5 mixture through either small bore tubing or through the small orifice of a
French Press.
Ribozymes incorporated into liposomes can be administered to living cells
internally or topically. Internal administration to animals or humans requires
that the
liposomes be pyrogen-free and sterile. To eliminate pyrogens, pyrogen-free raw
materials,
including all chemicals, enzymes, and water, are used to form the liposomes.
Sterilization can
ao be performed by filtration of the liposomes through 0.2 micron filters. For
injection, the
liposomes are suspended in a sterile, pyrogen-free buffer at a physiologically
effective
concentration. Topical administration also requires that the liposome
preparation be pyrogen-
free, and sterility is desirable. In this case, a physiologically effective
concentration of
liposomes can be suspended in a buffered polymeric glycol gel for even
application to the
a s skin. In general, the gel should not include non-ionic detergents which
can disrupt liposome
membranes. Other vehicles can also be used to topically administer the
liposomes.
The concentration of the substance in the final preparation can vary over a
wide
range, a typical concentration being on the order of 50 ug/ml. In the case of
pH sensitive
liposomes, lower concentrations of the substance can be used, e.g., on the
order of 0.01 to 1.0
3 o ug/ml for liposomes administered to cells internally. In case of topical
application, higher
liposome concentrations used, e.g., ten or more times higher.
AR~~l~~ttG' ~~~~
CA 02287084 1999-10-08
~_
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49
Where it is desired according to the invention to administer a substance as
described herein or its coding sequence, or liposomes containing such
substances, to an
s individual such that the administered material crosses the blood-brain
barrier, several methods
are known in the art.
For example, a substance to be administered, whether it be protein or nucleic
acid
or liposome, may be co-administered with a polypeptide, for example a
lipophilic polypeptide
that increases permeability at the blood-brain barrier. Examples of such
polypeptides include
io but are not limited to bradykinin and receptor mediated permeabilizers,
such as A-7 or its
conformational analogues, as described in U.S. Patent Nos. 5,112,596 and
5,268,164. The
permeabilizing polypeptide allows the co-administered ribozyme, coding
sequence or
liposome to penetrate the blood-brain barrier and arnve in the cerebrospinal
fluid
compartment of the brain, where the ribozyme, or coding sequence may then
reach and enter
i5 a target neuronal cell. Alternatively, the substance to be administered may
be coupled to a
steroidal estrogel or androgel to increase binding to steroid receptors and
thus access to the
brain
Another exemplary method for administering a substance such as a ribozyme,
antibody, nucleic acids, or liposomes containing such molecules, according to
the invention
a o includes forming a complex between the substance to be administered and an
antibody that
is reactive with a transferrin receptor, as described in U.S. Patent No.
5,182,107. The
complex may include a cleavable or non-cleavable linker and is administered
under conditions
whereby binding of the antibody to a transferrin receptor on a brain capillary
endothelial cell
occurs and the substance is transferred across the blood-brain barner in
active form.
2s FXAMP . .9
It is possible to administer a therapeutic nucleic acid for use not only in in
vivo
therapy (i.e., that in which a nucleic acid is administered directly to a
patient for uptake by-
and subsequent expression in cells in situ) but also in ex vivo therapy (i.e.,
that in which a
3 o nucleic acid is administered to cultured or explanted cells in vitro,
which transfected cells are
subsequently transplanted into the clinical patient in order to supply a
therapeutic product).
Methods of ex vivo gene therapy are described in detail herein. By these
methods, a plasmid
fiiv#~i'~~ttJ ~.iiEEl~
CA 02287084 1999-10-08
r r . . . r ,
,. ,. , r .. r ~ r ; - . r: r ~ n
,. _ .. ~ ;. ~ ;r r c
r . - r r r . r r r r ,
which continues to be maintained in a transformed or transfected cell after
such a cell has
been administered (e.g. via transplantation) to a multicellular host, such as
a mammal,
delivers a gene product to that individual. It is contemplated that a gene of
interest,
particularly a therapeutic gene, will be expressed by the transplanted cell,
thereby providing
5 the recipient organism, particularly a human, with a needed RNA (e.g., an
antisense RNA or
ribozyme) or protein.
A cell type may be used according to the invention which is amenable to
methods
of nucleic acid transfection such as are known in the art. Such cells may
include cells of an
organism of the same species as the recipient organism, or even cells
harvested from the
io recipient organism itself for ex vivo nucleic acid transfection prior to re-
introduction. Such
autologous cell transplants are known in the art. One common example is that
of bone
marrow transplantation, in which bone marrow is drawn either from a donor or
from a clinical
patient (for example, one who is about to receive a cytotoxic treatment, such
as high doses of
ionizing radiation), and then transplanted into the patient via injection,
whereupon the cells
i5 re-colonize bones and other organs of the hematopoietic system.
a. Cell dosage
The number of transfected cells which are administered to a recipient organism
is determined by dividing the absolute amount of therapeutic or other gene
product required
by the organism by the average amount of such an agent which is produced by a
transfected
2 o cell. Note that steady-state plasmid copy number varies depending on the
strength of its
origin of replication as well as factors determined by the host cell
environment, the
availability of nucleotides and replicative enzyme complexes, as does the
level of expression
of the gene of interest encompassed by the plasmid, which level likewise is
determined by the
strength of its associated promoter and the availability of nucleotides and
transcription factors
z s in a given host cell background. As a result, the level of expression per
cell of a given gene
of interest must be determined empirically prior to administration of cells to
a recipient.
While efficient methods of cell transfection and transplantation are known in
the
art, they do not ensure that the transfected cell is immortal. In addition,
the requirements of
the recipient organism for the product encoded by the transgene may change
over time. In
3 0 light of these considerations, it is contemplated that cells may be
administered in a single dose
or in multiple doses, as needed. A multiple dose schedule is one in which a
primary course
of administration can include 1-10 separate doses, followed by other doses
given at
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CA 02287084 1999-10-08
r r f r f , f
r ~ r' t r . f r r
r r r , r , r .
r r , . r
51
subsequent time intervals required to maintain and or reinforce the cellular
level of the
transfected nucleic acid. Such intervals are dependent on the continued need
of the recipient
for the therapeutic gene product. Preferably, when the medical needs of the
recipient mammal
dictate that a gene product will be required throughout its lifetime, or at
least over an extended
period of time, such as a year or more, the transfected cells will be
replenished on a regular
schedule, such as monthly or semi-monthly, unless such cells are able to
colonize the recipient
patient in permanent fashion, such as is true in the case of a successful bone-
marrow cell
transplant.
b. Nucleic acid dosage
1 o Provided a nucleic acid vector capable of replication in the transfected
cell is used,
the absolute amount of nucleic acid which is transfected into cells prior to
transplantation is
not critical, since in cells receiving at least one copy of such a vector, the
vector will replicate
until an equilibrium copy-number is achieved. As a first approximation, an
amount of vector
equivalent to between 1 and 10 copies thereof per cell to be transfected may
be used; one of
skill in the art may adjust the ratio of plasmid molecules to cells as is
necessary to optimize
vector uptake. Of particular used in the invention are vectors or transfection
techniques which
result in the stable integration of the gene of interest into the chromosome
of the transfected
cell, so as to avoid the need to maintain selection for cells bearing the
vector following
transplantation into a recipient multicellular organism, such as a human.
z o c. Administration of autologous or syngeneic cells
A cell type which is commonly transplanted between individuals of a single
species (or, even, from an individual to a cell culture system and back to the
same individual)
is that of hematopoietic stem cells (HSCs), which are found in bone marrow;
such cells have
the advantage that they are amenable to nucleic acid transfection while in
culture, and are,
therefore, well suited for use in the invention. Cultures of HSCs are
transfected with a
minimal plasmid comprising an operator sequence and a gene of interest and the
transfected
cells administered to a recipient mammal in need of the product of this gene.
Transfection
of hematopoietic stem cells is described in Mannion-Henderson et al., 1995, ,
23: 1628; Schiffmann et al., 1995, Blood, 86: 1218; Williams, 1990, Bone
Marrow
3o Transplant, 5: 141; Boggs, 1990, Int. J Cell .loping, 8: 80; Martensson et
al., 1987, Eur..
I~ImmunQL, 17: 1499; Okabe et al., 1992, FLr. 1. Immunol., 22: 37-43; and
Banerji et al.,
1983, ~~11, 33: 729. Such methods may advantageously be used according to the
present
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CA 02287084 1999-10-08
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r . r r r r - r r r
n . r r r ~- r r
r , .- ~ r
52
invention. Administration of transfected cells proceeds according to methods
established for
that of non-transfected cells, as described below.
The transplantation of hematopoietic cells, such as in a bone marrow
transplant,
is commonly performed in the art by procedures such as those described by
Thomas et al.
(1975, New .ngland J. Med , 292: 832-843) and modifications thereof. Such a
procedure is
briefly summarized: In the case of a syngeneic graft or of a patient suffering
from an
immunological deficiency, no immunosuppressive pre-treatment regiment is
required;
however, in cases in which a cells of a non-self donor are to be administered
to a patient with
a responsive immune system, an immunosuppressive drug must be administered,
e.g.
io cyclophosphamide (50 mg/kg body weight on each of four days, with the last
does followed
36 hours later by the transplant). Leukemic patients routinely receive a 1000-
rad midline dose
of total-body irradiation in order to ablate cancerous blood cells; this
irradiation also has an
immune-suppressive effect. Following pre-treatment, bone marrow cells (which
population
comprises a small number of pluripotent hematopoietic stem cells, or HSCs),
are administered
i5 via injection, after which point they colonize the hematopoietic system of
the recipient host.
Success of the graft is measured by monitoring the re-appearance of the
numerous adult blood
cell types by the immunological and molecular methods which are well known in
the art.
While as few as 1-10 HSCs are, in theory, able to colonize and repopulate a
lethally-irradiated
recipient mammal over time, it is advantageous to optimize the rate at which
repopulation
z o occurs in a human bone marrow transplant patient; therefore, a
transplanted bone marrow
sample comprising 10 to 100, or even 100 to 1000 HSCs should be administered
in order to
be therapeutically effective.
It is contemplated that both lymphoid and parenchyma) cells are of use in the
invention. Such parenchyma) cells include those of the islets of Langerhans,
the thyroid, the
a 5 adrenal cortex, muscles, cartilagenous- or other synovial tissue, the
kidneys, epithelial tissues
(both external and internal, particularly that of the intestinal lumen, lung,
heart, liver, kidney,
neurons and synovial cells) and, in particular, the nervous system.
To render the transplanted cells resistant, at least collectively, to immune
rejection by the
recipient organism, it is contemplated that transplanted cells expressing a
high level of
3 o activated NFxB (a high NFKB "set point"), while still subject to
destruction by autoimmune
host lymphocytes, would enjoy the advantage of robust proliferative capacity
in order to
multiply at a rate surpassing that of cell killing, thereby providing a long-
lived population of
rzx~t".';~,!i'~~.t'~ n4~F
CA 02287084 1999-10-08
~~ ;
53
therapeutic cells to the recipient organism. Such cells may be transfected
with gene
expression constructs which result in the production of high levels of
activated NFxB, or may
be cells obtained from a donor selected for high endogenous NFxB activity, as
may be
determined in an in vitro transcription assay or DNA/protein binding assay by
methods well
s known in the art, using protein extracts drawn from such a donor, which may,
itself, be a
transgenic mammal.
d. Administration of xenogeneic and allogeneic cells
While transfection and subsequent tranplantation of cells which are obtained
from
an individual or cell culture system of like species with the recipient
organism may be
io performed, it is equally true that the invention may be practised using
cells of another
organism (such as a well-characterized eukaryotic microorganism, e.g. yeast,
in which
appropriate processing of proteins encoded by therapeutic genes is likely and
in which useful
origins of replication are known). In such a case, certain concerns must be
addressed.
First, when a protein is encoded by the gene of interest, the transplanted
cells must
15 produce the protein in a form that may is of use to the recipient organism.
Post-translational
processing (including, but not limited to, cleavage and patterns of
glycosylation) must be
consistent with proper function in the recipient. In addition, either a
protein or an RNA
molecule of interest must be made available to the recipient after synthesis,
such as by
secretion, excretion or exocytosis from the transplanted cell. To address the
former, the
a o protein produced by the transfected cells may be qualitatively compared to
the native protein
produced by an individual of the same species as the recipient organism by
biochemical
methods well known in the art of protein chemistry. The latter, release of the
protein of
interest by the cells to be transplanted, may be assayed by isolating protein
from culture
medium which has been decanted from the transfected cells or from which such
cells have
zs been separated (i.e. by centrifugation or filtration), and performing
Western analysis using an
antibody directed at the protein of interest. Antibodies against many proteins
are
commercially available; techniques for the production of antibody molecules
are well known
in the art.
Second, the cells must be shielded from immune rejection by the recipient
3 0 organism. It is contemplated that such cells may be transfected with
constructs expressing
cell-surface markers (e.g. MHC antigens) characteristic of the recipient
patient so as to
provide them with biochemical camoflage.
Af~~lvUril 4~EE1'
CA 02287084 1999-10-08
54
In addition, methods for the encapsulation of living cultures of cells for
growth
either in an artificial growth environment, such as in a fermentor, or in a
recipient organism
have been developed, and are also of use in the administration of cells
transfected according
to the invention. Such an encapsulation system renders the cell invisible to
immune detection
s and, in addition, allows for the free exchange of materials (e.g. the gene
product of interest,
oxygen, nutrients and waste materials) between the transplanted cells and the
environment of
the host organism.
Methods and devices for cell encapsulation are disclosed in numerous U.S.
Patents; among these are Nos. 4,353,888; 4,409,311; 4,673,566; 4,744,933;
4,798,786;
io 4,803,168; 4,892,538; 5,011,472; 5,158,881; 5,182,111; 5,283,187;
5,474,547; 5,498,401
(which is particularly directed to the encapsulation of bacterial and yeast
cells in chitosan);
5,550,050; 5,573,934; 5,578,314; 5,620,883; 5,626,561; 5,653,687; 5,686,115;
5,693,513; and
5,698,413, the contents of which are fully incorporated by reference herein.
Typically
required for the successful culture of encapsulated cells is a selectively-
permeable outer
15 covering or 'skin' which is biocompatible (i.e., tolerated by both the
encapsulated cells and
the recipient host), and, optionally, a matrix in- or upon which cells are
distributed such that
the matrix provides structural support and a substrate to which anchorage-
dependent cells may
attach themselves. As relates to encapsulation devices applicable to use in
the invention, the
term "selectively-permeable" refers to materials comprising openings through
which small
a o molecules (including molecules of up to about 50,000 M.W. - 100,000 M.W.)
may pass, but
from which larger molecules, such as antibodies (approximately 150,000 M.W.),
are
excluded. Suitable covering materials include, but are not limited to, porous
and/or polymeric
materials such as polyaspartate, polyglutamate, polyacrylates (e.g., acrylic
copolymers or
RL~, Monsanto Corporation), polyvinylidene fluoride, polyvinylidienes,
polyvinyl chloride,
2 5 polyurethanes, polyurethane isocyanates, polystyrenes, polyamides,
cellulose-based polymers
(e.g. cellulose acetates and cellulose nitrates), polymethyl-acrylate,
polyalginate,
polysulfones, polyvinyl alcohols, polyethylene oxide, polyacrylonitriles and
derivatives,
copolymers and/or mixtures thereof, stretched polytetrafluoroethylene (U.S.
Pat. Nos.
3,953,566 and 4,187,390, both incorporated herein by reference), stretched
polypropylene,
3 o stretched polyethylene, porous polyvinylidene fluoride, woven or non-woven
collections of
fibers or yarns, such as "Angel Hair" (Anderson, Science, 246: 747-749;
Thompson et al.,
1989, Proc Natl Acad S i T ~ A , 86: 7928-7932), fibrous matrices (see U.S.
Pat. No.
~,~~Ft~~ED v'~EEf
CA 02287084 1999-10-08
f r . . . , . . ~ ,
f . . , ~ . r . ~ . . ~ ,
5,387,237, incorporated herein by reference), either alone or in combination,
or silicon-
oxygen-silicon matrices (LT.S. Patent No. 5,693,513). Polylysine having a
molecular weight
of 10,000 to 30,000, preferably 15,000 to 25,000 and most preferably 17,000 is
also of use
in the invention (see U.S. Patent No. 4,673,566). Alternatively, the matrix
material,
5 comprising the transfected cells of the invention, is exposed to conditions
that induce it to
form its own outer covering, as discussed below.
As described in U.S. Patent No. 5,626,561, the selective permeability of such
a
covering may be varied by impregnating the void spaces of a porous polymeric
material (e.g.,
stretched polytetrafluoroethylene) with a hydrogel material. Hydrogel material
can be
io impregnated in substantially all of the void spaces of a porous polymeric
material or in only
a portion of the void spaces. For example, by impregnating a porous polymeric
material with
a hydrogel material in a continuous band within the material adjacent to
and/or along the
interior surface of a porous polymeric material, the selective permeability of
the material is
varied sharply from an outer cross-sectional area of the material to an inner
cross-sectional
i5 area of the material. The amount and composition of hydrogel material
impregnated in a
porous polyhmeric material depends in large part on the particular porous
polymeric material
used to encapsulate cells for transplant. Examples of suitable hydrogel
materials include, but
are not limited to, HYPAN~ Structural Hydrogel (Hymedix International, Inc.;
Dayton, NJ),
non-fibrogenic alginate, as taught by Dorian in PCT/US93/05461, which is
incoIporated
z o herein by reference, agarose, alginic acid, carrageenan, collagen,
gelatin, polyvinyl alcohol,
poly(2-hydroxyethyl methacrylate), poly(N-vinyl-2-pyrrolidone) or gellan gum,
either alone
or in combination. The matrix typically has a high surface-area:volume ratio,
comprising pores or other spaces in- or on which cells may grow and through
which fluids
may pass; in addition, suitable matrix materials are stable following
transplantation into a
2 s recipient organism. Preferably, the matrix comprises an aggregation of
multiple particles,
fibers or laminae. Alternatively, a matrix may comprise an aqueous solution,
such as a
physiological buffer or body fluid from the recipient organism (see U.S.
Patent No.
5,011,472). Suitable matrix materials include liquid, gelled, polymeric, co-
polymeric or
particulate formulations of aminated glucopolysachharides (e.g., deacetylated
chitin, or
s o "chitosan", which is prepared from the pulverized shells of crabs or other
crustaceans, and is
commercially available as a dry powder; Cat. # C 3646, Sigma, St. Louis, MO),
alginate (LJ.S.
Patent No. 4,409,331), poly-~3-1~5-N-acetylglucosamine (p-GIcNAc)
polysaccharide species
~! may..r~ ns 1
CA 02287084 1999-10-08
- . . r
56
(either alone of formulated as co-polymer with collagen; see U.S. Patent No.
5,686,115),
reconstituted extracellular matrix preparations (e.g. Matrigel~; Collaborative
Research, Inc,
Lexington, MA; Babensee et al., 1992, J. Biomed. Matr R ~ , 26: 1401),
proteins,
polyacrylamide, agarose and others.
s Methods by which cells become encapsulated using such materials are both
numerous and varied. Encapsulation devices comprising a semi-permeable
membrane
material, as described above, may be pre-formed, filled with cells (e.g. by
injection or other
manual means) and then sealed (U.S. Patent Nos. 4,892,538; 5,011,472;
5,626,56; and
5,653,687); such sealing may be effectively permanent (e.g. by the use of heat-
sealing), semi-
io permanent (e.g. by the use of a biocompatible adhesive, such as an epoxy,
which will not
dissolve or degrade in an aqueous environment) or temporary (e.g. by the use
of a removable
cap or plug, or by shutting of a valve or stopcock). Methods of permanent and
semi-
permanent sealing are disclosed in U.S. Patent No. 5,653,687. As an
alternative to the use of
a pre-formed, semi-permeable cell reservoir, methods by which cells suspended
in matrix
i 5 material and the substance which is to form the outer covering of the
encapsulation device are
co-extruded under conditions which cause the cell/matrix mixture, which may be
in liquid or
semi-liquid (i.e., gelled) form to be encased in a continuous tube of the semi-
permeable
polymer, which either forms, or becomes crosslinked, under the extrusion
conditions; such
an extrusion procedure may lead to the formation of capsules which have only
one cell
ao reservoir (LJ.S. Patent No. 5,283,187) or which are divided into multiple,
discrete
compartments (U.S. Patent No. 5,158,881). As an alternative to both types
ofprocedure, a
liquid or semi-liquid (i.e., gelled) cell/matrix mixture droplet is suspended
either in an agent
which induces 'curing' or crosslinking of the outer layer of matrix material
to form a semi-
permeable barrier (U.S. Patent Nos. 4,798,786 and 5,489,401) or in a solution
of polymeric
2 5 material (or monomers thereof), which will polymerize and/or crosslink
upon contact with the
cell/matrix droplet such that a semi-permeable membrane is deposited thereon
(U.S. Patent
Nos. 4,353,888; 4,673,566; 4,744,933; 5,620,883; and 5,693,513).
One of skill in the art is well able to select the appropriate matrix and semi-
permeable membrane materials and to construct a cell-encapsulation device as
described
3 o above.
Implantation of such a device is achieved surgically, via standard techniques,
to
a site at or near the anatomical location to which the product encoded by the
gene on the gene
AMEt~DrD SI~Er"T
CA 02287084 1999-10-08
57
of interest is to be delivered, as is deemed safest and most expedient. Such a
device may take
a convenient shape, including, but not limited to, that of a sphere, pellet or
other capsule
shape, disk, rod or tube; often, the shape of the device is determined by its
method of
synthesis. For example, one which is formed by co-extrusion of a cell
suspension and a
polymeric covering material is typically tubular, while one formed by the
deposition of a
covering on droplets comprising cells in matrix material might be spherical.
As discussed
above, the number of cells which must be implanted (and, therefore,
encapsulated) is
dependent upon the requirements of the recipient organism for the product of
the transfected
gene. The encapsulation devices described above are typically small (most
usefully, l0um
io to lmm in diameter, so as to permit efficient diffusion of substances back
and forth between
the outer covering and the cells most deeply embedded in the matrix), and it
is contemplated
that such devices may carry between 10 and 10'° cells each. Should the
need for larger
numbers, of cells be anticipated, a plurality (2, 10 or even 100 or more) of
such in vivo
culturing devices may be made and implanted in a given recipient organism.
is An encapsulated cell device may be intended for permanent installation;
alternatively, retrieval of the device may be desirable, whether to terminate
delivery of the
product of the gene of interest to the recipient organism at the discretion of
one of skill in the
art, such as a physician (who must determine on a case-by-case basis the
length of time for
which a given cell implant is beneficial to the recipient organism) or to
replenish the device
z o with fresh cells after long-term use (i.e. months to years). To the latter
end, an implantation
device may usefully comprise a retrieval aid, such as a guidewire, and a cap
or other port,
such as may be opened and re-sealed in order to gain access to the cell
reservoir, both as
described in U.S. Patent No. 4,892,538.
Live cultures of encapsulated cells have been used successfully to deliver
gene
25 products to tissues of a recipient animal. U.S. Patent No. 4,673,566
discloses successful
maintenance of normal blood sugar levels in a diabetic rat into which
encapsulated rat islet
of Langerhans cells were implanted; two administrations of 3,000 cells each
together were
effective for six months, while a single dose of 1,000 cells was effective for
two months.
Similarly, heterospecific transplantation of encapsulated islet cells has been
3 o demonstrated to treat diabetes successfully (dog islet cells to a mouse
recipient, U.S. Patent
No. 5.578,314; porcine islet cells to a mouse recipient, Sun et al., 1992,
A~ATO T., 38: 124).
It is believed that such an approach is promising for the clinical treatment
of diabetes mellitus
AivtwJrD ~I~cET
CA 02287084 1999-10-08
r r !~ r
r r r
r , r ~ r r. r
r r . . r r , r r ~ ,
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58
in humans (Calafiore, 1992, A~,~Qy~ 3g; 34).
It is contemplated that these techniques, which have been applied successfully
to
untransfected cells, may be utilized advantageously with cells that are
transfected with
therapeutic nucleic acid molecules of use in the invention.
s e. Assay of efficacy of transplanted cells in a recipient organism
The efficacy of the transfected cells so administered and their subsequent
maintenance in the recipient host may be assayed either by monitoring the
activity of a marker
gene, which may additionally be comprised by the transfected construct, or by
the direct
measurement of either the product (e.g. a protein) encoded by the gene of
interest or the
i o reduction in the levels of a protein the production of which it (an
antisense message or
ribozyme) is designed to inhibit. The assays can be performed using
conventional molecular
and biochemical techniques, such as are known to one skilled in the art, or
may comprise
histological sampling (i.e., biopsy) and examination of tranplanted cells or
organs.
In addition to direct measurements of protein or nucleic acid levels in blood
or
i5 target tissues encoded by the gene of interest borne by the vector in
transfected/transplanted
cells, it is possible to monitor changes in the disease state in patients
receiving gene transfer
via transplantation of cells in which the gene of interest is maintained and
compare them to
the progression or persistence of disease in patients receiving comparable
cells transfected
with vector constructs lacking the gene of interest.
Other DoyQe~ and Modes of AdminiStrahion
A patient that is subject to a disease state which is associated with a
frameshift
mutation may be treated in accordance with the invention, as described above,
via inin vivo, ~
2s vivo or in vitro methods. For example in in vivo treatments, a ribozyme or
a nucleic acid
vector encoding a ribozyme, a wild-type RNA, an antisense RNA or DNA or a
sequence
encoding the antisense RNA, or a wild-type version of a hybrid wild-
type/nonsense protein,
can be administered to the patient, preferably in a pharmaceutically
acceptable delivery
vehicle and a biologically compatible solution, by ingestion, injection,
inhalation or any
3 o number of other methods. The dosages administered will vary from patient
to patient; an
"effective dose" will be determined by the level of enhancement of function of
the transferred
genetic material balanced against any risk of deleterious side effects.
Monitoring gene
~i~~~~:~~)w~l ~~~!_~
CA 02287084 1999-10-08
59
expression and/or the presence or levels of the encoded mutant RNA or protein
or its
corresponding "sense" protein will assist in selecting and adjusting the
dosages administered.
Generally, a composition including a nucleoprotein such as a ribozyme will be
administered
in single or multiple doses, as determined by the physician, in the range of
50 ug - 1 mg, or
within the range of 100 ug - 500 ug. A composition including an
oligonucleotide will be
administered in a single dose in the range of 5 ng - 10 ug, or within the
range of 100 ng - S00
ng. A composition including a wild-type RNA or a vector will be administered
in a single
dose in the range of 10 ng - 100 ug/kg body weight, preferably in the range of
100 ng - 10
ug/kg body weight, such that at least one copy of the sequence is delivered to
each target cell.
i o A composition including a protein, e.g., a wild-type version of a hybrid
wild-type/nonsense
protein, will be administered in single or multiple doses, as determined by
the physician, in
the range of 10 ug - 1 mg, or within the range of 100 ug - 50 ug. Any of the
above dosages
may be administered according to the body weight of the patient, as determined
by the
physician.
i5 Ex vivo transduction is also contemplated within the present invention.
Cell
populations can be removed from the patient or otherwise provided, transduced
with a vector
in accordance with the invention, then reintroduced into the patient. The
number of cells
reintroduced into the patient will depend upon the efficiency of vector
transfer, and will
generally be in the range of 104 - 106 transduced cells/patient.
z o The cells targeted for ex vivo gene transfer in accordance with the
invention
include any cells to which the delivery of the vector is desired, for example,
neuronal cells or
stem cells.
Protein, nucleic acid, or cells administered according to the invention is
preferably
administered in admixture with a pharmaceutically acceptable carrier
substance, e.g.,
a 5 magnesium carbonate, lactose, or a phospholipid to form a micelle, the
carrier and protein,
nucleic acid or cell together can form a therapeutic composition, e.g., a
pill, tablet, capsule
or liquid for oral administration to the mammal. Other forms of compositions
are also
envisioned, e.g., a liquid capable of being administered nasally as drops or
spray, or a liquid
capable of intravenous, parenteral, subcutaneous, or intraperitoneal
administration. The
3 o substance administered may be in the form of a biodegradable sustained
release formulation
for intramuscular administration. For maximum efficacy, where zero order
release is
desirable, e.g., an implantable or external pump, e.g., an InfusaidTM pump
(Infusaid Coip,
~~~'eu9::IJ ~~.~~,~,~.:1
CA 02287084 1999-10-08
r , ' r .
r
r ~ r ~ '
r
MA), may be used.
Kits I Tie ~l A ording to the Invention
The invention encompasses kits for diagnosis or treatment of diseases
according
s to the invention.
A diagnostic kit includes suitable packaging materials and one or more of the
following reagents: a nucleic acid probe as defined hereinabove, and
optionally means for
detecting the probe when bound to its complementary sequences. For example,
the nucleic
acid probe may be labeled, e.g., radiolabeled, fluorescently labeled, etc., or
may be detected
io via indirect labeling techniques, e.g., using a biotin/avidin system, well
known in the art.
A diagnostic system, preferably in kit form, comprises yet another embodiment
of this invention. This system is useful for assaying the presence of a hybrid
wild-
type/nonsense protein or its derivative in cells by the formation of an immune
complex. This
system includes at least one package that contains an antibody of this
invention. Optionally,
i5 a kit also may include a positive tissue sample control.
Antibodies are also utilized along with an "indicating group" also sometimes
referred to as a "label". The indicating group or label is utilized in
conjunction with the
antibody as a means for determining whether an immune reaction has taken
place, and in
some instances for determining the extent of such a reaction.
a o The terms "indicating group" or "label" are used herein to include single
atoms
and molecules that are linked to the antibody or used separately, and whether
those atoms or
molecules are used alone or in conjunction with additional reagents. Such
indicating groups
or labels are themselves well-known in immunochemistry and constitute a part
of this
invention only insofar as they are utilized with otherwise novel antibodies,
methods and/or
a s systems.
For example, an antigen-specific antibody or antibody fragment is detectably
labeled by linking the same to an enzyme and use it in an EIA, or enzyme-
linked
immunosorbent assay (ELISA). This enzyme, in turn, when later exposed to a
substrate in
such a mariner as to produce a chemical moiety which can be detected, for
example, by
3 o spectrophotometric, flourometric or, most preferably, by visual means. The
substrate may be
a chromogenic substrate which generates a, reaction product visible to the
naked eye.
Enzymes which can be used to detectably label the binding protein which is
~~I~DED SI~~T
CA 02287084 1999-10-08
61
specific for the desired detectable mutant protein, include, but are not
limited to, alkaline
phosphatase, horseradish peroxidase, glucose-6-phosphate dehydrogenase,
staphylococcal
nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-
glycerophosphate
dehydrogenase, triose phosphate isomerase, asparaginase, ribonuclease, urease,
catalase,
s glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
By radioactively labeling the binding protein, for example, the antibody, it
is
possible to detect the antigen bound to a solid support through the use of a
radioimmunoassay
(RIA). The radioactive isotope can be detected by such means as the use of a
gamma counter
or a scintillation counter or by autoradiography. Isotopes which are
particularly useful for the
to purpose of the present invention are: 3H, ~3'I, ~4C, and preferably i2sl.
It is also possible to label the first or second binding protein with a
fluorescent
compound. When the fluorescently labeled antibody is exposed to light of the
proper wave
length, its presence can then be detected due to fluorescence. Among the most
commonly
used fluorescent labelling compounds are fluorescein isothiocyanate,
rhodamine,
15 phycoerythrin, phycocyanin, allophycocyanin, Q-phthaldehyde and
fluorescamine.
The first or second binding protein also can be detectably labeled by coupling
it
to a chemiluminescent compound. The presence of the chemiluminescent-tagged
antibody
is then determined by detecting the presence of luminescence that arises
during the course of
a chemical reaction. Examples of particularly useful chemiluminescent labeling
compounds
z o are luminol, isoluminol, theromatic acridinium ester, imidazole,
acridinium salt and oxalate
ester.
Likewise, a bioluminescent compound may be used to label the first or second
binding protein. Bioluminescence is a type of chemiluminescence found in
biological
systems in which a catalytic protein increases the efficiency of the
chemiluminescent reaction.
25 The presence of a bioluminescent protein is determined by detecting the
presence of
luminescence. Important bioluminescent compounds for purposes of labeling are
luciferin,
luciferase and aequorin.
The invention also includes diagnostic reagents for use in the present
invention,
such as nucleic acid sequences, probes and antibody molecules, and/or positive
tissue
a o controls, as described above, and kits including such reagents for use in
diagnosing or treating
a disease.
An indicating group or label is preferably supplied along with the antibody
and
AM~~~DED SHfET
CA 02287084 1999-10-08
r r. r ~ ., r
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62
may be packaged therewith or packaged separately. Additional reagents such as
hydrogen
peroxide and diaminobenzidine, and nickel ammonium sulfate may also be
included in the
system when an indicating group such as HRP is utilized. Such materials are
readily available
in commerce, as are many indicating groups, and need not be supplied along
with the
s diagnostic system. In addition, some reagents such as hydrogen peroxide
decompose on
standing, or are otherwise short-lived like some radioactive elements, and are
better supplied
by the end-user.
OTHER EMBODIMENTS
1 o It will be understood that the invention is described by way of
illustration only.
Many other embodiments of the present invention in addition to those herein
described will
be apparent to those skilled in the art from the description herein given
without departing
from the scope of the present invention as defined in the appended claims.
~AfJIE~~:D~D SHfET
CA 02287084 1999-10-08
..
r r.
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63
Table 1
EARLY ONSET
10-20% of total number of AD cases
familial
60% 90% unknown (54)
33% PS1
0% autosomal dominant (6) 5% APP
<1% PS2
60% unknown
40% sporadic
LATE ONSET
80-90% of total number of AD cases
familial
40% 90% unknown (36)
~0% autosomal dominant* (4)
60% slporadic
Based upon a population-based cross-section study of dementia, 72% of all
demented
people suffer from Alzheimer's disease (AD), 16% from vascular dementia, 6%
from
Parkinson's disease dementia and 6% of other dementias (Ott et al., 1995).
Early (<65
years) and late (>65 years) onset (EGAD and LOAD) forms of Alzheimer's disease
are
distinguished. Familial means that AD was observed in relatives of the first
degree. This
study is based upon Ott et al., 1995; Van Broeckhoven, 1995; Cruts et al.,
1998.
In familial EOAD the majority (54%) is not yet linked to a chromosome, whereas
6% is
inherited in a an autosomal dominant way and linked to chromosome 1 (PS2, <1
%), 14
(PS1, 33%), 19 (APP, 5%), whereas 60% of the autosomal dominant forms is still
not linked.
In familial LOAD, the majority (90%) is not yet linked to a chromosome,
whereas 10% is
inherited in an autosomal dominant way. A subset may be linked to chromosome
12
(Pericak-Vance et al., 1997) and ApoE4 nuclear families.
Risk factors: 65% of all EOAD and 25% of all LOAD cases display ApoE4
polymorphism
(one or two E4 alleles). ApoE4 data in early onset AD are based upon a study
by Van
Broeckhoven and Cruts (n = 102 patients). Other risk factors for late onset AD
are
butyrylcholinesterase and cytochrome c oxidase.
References
Cruts, M. et al. (1998) Hum. Mol. Genetics 7, 43-51
Davis, R.E. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4526-4531
Lehmann, D.J. ef aL (1997) Human Mol. Gen. 6, 1933-1936
Ott, A. et al. (1995) Br. Med. J. 310, 970-973
Pericak-Vance, M.A. et aL (1997) JAMA 278, 1237-1241
Van Broeckhoven, C.L. (1995) Eur. Neurology 35, 8-19
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CA 02287084 1999-10-08
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66
Table 3
Immunoreactivities in the human frontal cortex (Brodman area 11 ) for mutant p
amyloid precursor
+otein and ubiquitin-B, the mRNA of which is expressed in the +1 reading frame
(pAPP" and Ubi
). Tissues were obtained from controls and neuropathologically confirmed
Alzheimer and Down
_.._a__...... ..____
NBB
autopsy age sex neurop atho- pAPP"' Ubi-B"
no. (years) (m/f) logical state'
TIPS tangles
Non-demented controls
89003 34 m _ _ _ _
81021 43 . m _ _ _ _
94119 51 f _ _ _ _
94125 51 m _ _ _ _
88037 58 m _ _ _
90073 65 f _ _ _ _
90079 72 m +a _ _ _
91026 80 f - _ _ _
91027 82 f _ _ _
90080 85 m + +a _ . _
81007 90 f +a _ _
90083 90 f + _ _ _
!~ oOS. staining
Alzheimer cases
89057 40 m + + _ +
86055 45 m + + _ +
90102 49 m + + _ +
91092 54 f + + _ +
85013 56 f + + _ _
92054 61 m +~ +~ _ +
88073 66 m + + _ +
83002 70 f +~ + + +
93047 70 m + +~ + _
91094 73 f +~ + - +
90118 77 m +~ +~ - +
93044 77 m + +e _ +
93087 81 m +a + - +
90015 81 f + +a + +
93045 83 f +a + - +
91081 85 f +a +a _ _
88028 85 f + + _ +
90117 86 m + +~ _ +
91086 88 m +a +a - +
86002 90 f + +a + +
93048 92 f + _ _ _
DOS. Staining 19% 80%
Downs' syndrome
93162 54 f +~ + + +
92080 58 f + + + +
89005 59 f + + + +
93161 62 f +~ +~ + +
96015 63 f _ _ _
94058 64 m + + + +
93028 67 f +~ + + +
% nns staininn Qaoi flaoi
NBB = Netherlands Brain Bank, 'Number of plaques (all types) [1j and tangles
as revealed by Congo
Red and Bodian silver staining: a) few, b) moderate, c) many.
~d;~~u 4:~~~E:
CA 02287084 1999-10-08
r f n r
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67
Table 4
Immunoreactivities in the human temporal cortex (Brodman area 38) for mutant ~
amyloid precursor
~~rotein and ubiquitin-B the mRNA of which is expressed in the +1 reading
frame (~iAE~P" and Ubi-B
). Tissues were obtained from controls and neuropathologically confirmed
Alzheimer cases. Down
C\IYII~I/\mtf n~~ie~.~~n eW,w.....J AI-L_~... .
NBB
notopsy (years) mx/f) logical state' LAPP" Ubi-B"
( i,LqPS IangiPs
Non-demented
controls
89003 34 m _ _ _
81021 43 m _ _ _
94119 51 f - _
94125 51 m _ _ _
-
88037 58 m _ _
-
90073 65 f _ _ _
_
90079 72 m _ _
_
91026 80 f - _ _
91027 82 f _
90080 85 m + + _ +
81007 90 f +a +a _
90083 90 f + _ _ _
oos_ staining p%
Alzheimer
cases
8905740 m + + _ +
8605545 m + +~ + +
9010249 m + + _ +
9109254 f + +~ _ +
8501356 f + + _ +
9205461 m + + + +
8807366 m +a +a + +
8300270 f + + + +
9304770 m + + + +
9109473 f + ~ + - +
9011877 m + + _ +
9304477 m +a + _ +
9308781 m +e + + +
9001581 f + + + +
9304583 f + + + +
8802885 f +a +~ _ +
9108185 f + + - +
9011786 m +~ +~ _ +
9108688 m + +a _ +
8600290 f +~ +~ + +
9304892 f +~ +a _ _
oo~. tainino 4~o/n 45%
a
Downs'syndrome
9316254 f + + + +
9208058 f + + + +
8900559 f + +~ + +
9316162 f +~ + + +
9601563 f +a
9405864 m + + + +
9302867 f + + + +
NBB = Netherlands Brain Bank, *Number of plaques (all types) [1] and tangles
as revealed by Congo
Red and Bodian silver staining: a) few, b) moderate, c) many.
~i~~i~~D ~~F~.-'~~
CA 02287084 1999-10-08
r ~~
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68 ~f r.
Table 5
Immunoreactivities in the human hippocampus for mutant amyloid precursor
protein and ubiquitin-B,
the mRNA of which is expressed in the +1 readin frame. (APP" and Ubi-B").
Tissues were obtained
NBB
notopsy ( 9 ars) m/f) logical pAPP" Ubi-B"
( fu state'
4uP~ tan9i
~
Non-demented controls
89003 34 m _ _ _ _
81021 43 m _ _ _ _
94119 51 f _ _ _
94125 51 m _ _
88037 58 n1 _ +a _
90073 65 f
90079 72 m + +~ _ +
91026 80 f + +~ _ +
91027 82 f + _ +
90080 85 m + + _ +
81007 90 f + + _ +
90083 90 f _ +a _ +
oos. staining
Alzheimer cases
89057 40 m +a + _ +
86055 45 m + +~ _ +
90102 49 m + +~ _ +
91092 54 f + +~ + +
85013 56 f + +a _ +
92054 61 m + + + +
88073 66 f +~ +~ _ +
83002 70 f + + _ . + +
93047 70 m + + + _
91094 73 f + + - +
90118 77 m + + + +
93044 77 m + + + +
93087 81 m + +~ + +
93045 83 f + + - +
88028 85 f +a + - +
91081 85 f + + _ +
90117 86 m + + + +
91086 88 m + + + +
86002 90 f + + _ +
93048 92 f + + + +
ogs. staining 50% A5%
Downs' syndrome
93162 54 f + + + +
92080 58 f + +~ + +
89005 59 f + + + +
93161 62 f +~ +~ + +
96015 63 f _ _ _
94058 64 m + + + +
93028 67 f +~ + _ +
positive staining 71 % 86%
NBB = Netherlands Brain Bank, * Number of plaques all types [11 and tangles as
revealed by Congo
Red and Bodian silver staining: a) few, b) moderate, c~ many. n tf~a absence
of hippocampal tissue,
patient #90015 was not studied.
~~~~~~ ~e~f
CA 02287084 1999-10-08
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CA 02287084 1999-10-08
r r ~- r - r
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7~
Table 7
GAGAG MOTIFS
BASE PAIRS EXPECTED 1 ACTUAL
(CODING SEQUENCE NUMBER NUMBER
OF LONGEST FORM) (1:1024)
(iAPP 2234 2,2 7
Tau 1096 1.1 _
Ubiquitin B 687 0.7 2
Apolipoprotein g53 O.g -
E4
MAP2b 5475 5.3 11
NF-low (68 K) 582 0.6 3
NF-medium (145 2748 2,7 3
K)
NF-H (200 K) 3063 3,1 2
Presenilin I 1392 1,4 3
Presenilin II 1346 1.3 3
Big Tau 2058 2 5
GFAP l2gg 1,3 6
P53 1239 1.2 2
BCL2 717 0.7 1
Semaphorin III 2313 2.3 4
HUPF-I 3351 3,3 3
HMG 327 0.3 1
NSP-A 2268 2.3 2
.;y
_~L1~?li~:rt~.~ ;~!.ilawE~~~~
CA 02287084
1999-10-08
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CA 02287084 1999-10-08
f r
f :- r , f ~ r r. f .. r ~ ~. .
r f , I , f r r ~ r ~ .
f r c ~ r
f r r ~ r
r . ~ ~ ' , ~. r
72
Table 9
+1 protein sequences
(right) predicted
by a dinucleotide
deletion iri
an mRNA molecule ing for different proteins
encod (left)
aAPP" RGRTSSKELA SEQ 1
" ID
NO:
Tau HGRLAPARHAS SEQ ID 2
Ubi NO:
B"
- YADLREDPDRQ SEQ ID 3
Ubi NO:
B"
- GGGAQ SEQ ID 5
Ubi NO:
B"
- RQDHHPGSGAQ SEQ ID 4
Ubi NO:
B"
- YADLREDPDRQDHHPGSGAQ SEQ ID 1400]
NO:
A~ po-E GAPRLPPAQAA SEQ ID 6
+' NO:
MAP2B KTRFQRKGPS SEQ ID 7
Neurofilament-L" PGNRSMGHE NO:
SEQ ID 8
Neurofilament-M" EAEGGSRS NO: 9
' SEQ ID
NO:
Neurofilament-H' VGAARDSRAA SEQ ID 1
" NO:
Presenilin I HDYPPGGSV SEQ ID 11
" NO:
Presenilin I SIQKFQV SEQ ID 12
NO:
Presenilin II" VEKPGERGGR SEQ ID 13
NO:
Big Tau" PLFGRGHKRG SEQ ID 14
" NO:
GFAP EDRGDAGWRGH SEQ ID 15
P53" NO:
QERGASPRAAPREH SEQ ID 16
BCL2+' NO:
RQPGDVAPGGQHRPVDD SEQ ID 17
+, NO:
Semaphorin III AGLLAIPEAK SEQ ID 18
NO:
Semaphorin III" YVDVYNGGKFS SEQ ID 19
" NO:
HUPF-I AADERRCHLLHMCGRR SEQ ID 20
HUPF NO:
I"
- QQATEAGQHYQPGSPLHDHSHV SEQ ID 21
HMG" NO:
PQEAAARTNR SEQ ID 22
NSP NO:
A"
- RSWVHPAPPYQMCLG SEQ ID 23
NSP NO:
A+'
- GGSRTHPR SEQ ID 24
NO:
pN!~~fD~D SffEET
CA 02287084 1999-10-08
t r r r
f t. C f . f f- , f .
f f . f f '
f r I. , I , I' r
~' r f f
f ~ I
. . f .
73
Table 10
~''t b altered
561 565 561 565
GAG AGG GAA CGU
Glu Arg Glu Arg
1107 1112 1107 1112
GAG AGG GAG AGG
Glu Arg Glu Arg
1128 1133 1128 1133
CGA GAG CGU GAA
Arg Glu Arg Glu
1149 1157 1149
1157
AUG AGA GAA AUG CGC GAA
Met Arg Glu Met Arg Glu
1266 1271 1266
1271
GAG AGA GAA CGC
Glu Arg Glu Arg
